2. Interoperability and reusability: Although the AIRR Standards provide mechanisms to enable reuse of AIRR-seq data, challenges remain. These standards have varying levels of rigor applied to the AIRR fields. For example, many fields are defined as ontology terms, coming from well-known and widely used ontologies such as the NCBI Taxonomy (https://www.ncbi. nlm.nih.gov/taxonomy) and the Disease Ontology (https:// disease-ontology.org). Other terms come from AIRR defined controlled vocabularies (e.g., pcr\_target\_locus, as used above in the ADC query API, comes from a controlled vocabulary of IGH, IGI, IGK, IGL, TRA, TRB, TRD, TRG). Such rigor makes it possible to be very precise about sharing, exploring, and reusing data across studies. In particular, it is possible to compare such metadata computationally, taking away the need for an expert to interpret such fields.

At the same time, it is not possible to be so precise about all fields. The AIRR-seq world is evolving rapidly, and agreement around rigor on all terms in the standard is not feasible and in many cases is not desirable. Researchers need flexibility to describe parts of their study in ways that we are not currently able to capture precisely. As the domain matures, more rigor will be applied to more fields in the standard, but in some cases, it is still challenging to compare two repertoires interoperable way. The most prominent fields where this is prevalent is in the fields involving data processing. Given the complexities discussed around preprocessing, annotation, and analysis of AIRR-seq data, standardization around fields and their contents are yet to be determined. Although the MiAIRR data standard has fields to capture this information, the community is working toward a concise specification for describing these processes in detail.

3. Knowing where to look for data: In working from the ADC API level to find and access data, one challenge when searching for data across the ADC is knowing which repositories are actually part of the ADC. Currently, there is no central registry where one can get a list of the repositories in the ADC; at present, the main central resource providing this information is the AIRR Community ADC documentation page (https:// docs.airr-community.org/en/stable/resources/adc\_support. html) with the AIRR Community also maintaining a registry of repositories on Faisharing.org (https://fairsharing.org/ biodbcore/?q¼AIRR). This page provides links to the existing repository providers. If searching for data in the ADC using a programmatic interface (Python, R), then it is necessary for the end user to manage the list of repositories and send queries to the appropriate repositories as required.

Web-based user interfaces, such as the iReceptor Gateway, typically hide the fact that there are multiple repositories being queried, and these platforms maintain an internal registry so the end user does not need to worry about where the data resides.

The AIRR Community is working on the specification and establishment of a central registry that will provide programmatic access to a list of repositories known to support the ADC API.

4. The time and cost of data sharing: As evidenced by the nuances discussed in both this and the "AIRR-Community Guide: Planning and Performing AIRR-Seq Experiments" chapter, the process of defining and performing a study involving AIRR-seq data is incredibly complex. The MiAIRR and other AIRR Standards are designed to guide researchers in capturing study/data processing features important for data reuse. Because of this completeness, the standards necessarily contain a lot of detail. Although the AIRR Community encourages researchers to be as complete as possible in documenting their study design and process, the more critical aspect of data sharing and re-use is that when one does document a part of a study, it is done in a standard's compliant way. Although the MiAIRR Standard has a large number of fields, all of them considered "important," only a small subset of these fields are "essential/required" for a study to be AIRR-compliant.

There is a balance to be struck between the time it takes to capture study metadata at an appropriate level, both for internal requirements to perform the study and external requirements for data sharing and reuse. The MiAIRR Standard was created to help on both of these fronts, providing researchers with a comprehensive list of metadata fields that they might consider when designing a study and fields that enable comparison of study methods and guide decisions on data reuse. One of the main reasons that it is currently costly in terms of time and effort to reuse data (see below) is because the data has not been curated in a manner that facilitates this reuse.

5. The challenges of starting with FASTA/FASTQ: Although the INSDC sequence archive repositories (SRA, ENA, etc.) are a critical resource for long term storage of raw AIRR-seq data, it can be challenging for some researchers to reuse this data. This challenge comes from the need to transform data as stored in SRA/ENA to data that can be reused in analysis. As discussed in the "AIRR Community Guide: TR and IG Gene Annotation" chapter, the transformation of raw sequence data to annotated data, which is in turn the basis for data reuse in analysis, can be complex. If all data reuse requires starting from FASTA/FASTQ sequence files, each case of reuse requires the reannotation of the data, including the expertise and time to run the annotation pipelines. Even for experienced bioinformaticians, lack of data preprocessing information (e.g. primer/barcode sequences or thresholds for trimming/ filtering) adds significant time to data reannotation and can potentially impact reproducibility of results. In some cases, researchers may need to contact the data generator directly to obtain critical details such as barcodes for sample demultiplexing; depending on the level of collaboration, this could pose a barrier to data reuse. In addition, unless the AIRR-to-NCBI pipeline (Subheading 3.1.2) was used to process study metadata, it is unlikely that the study, subject, and sample metadata will be stored in a standard-based and reusable format. Mapping study metadata and rerunning annotation pipelines is error prone, and this process needs to be redone by each researcher wanting to reuse a data set obtained from INSDC sequence archives.

The AIRR Community has attempted to minimize this cost, firstly by providing a process to store the critical study, subject, and sample metadata in the INSDC repositories and secondly by providing a mechanism—the AIRR Standards and the AIRR Data Commons—by which researchers can curate and share annotated AIRR-seq data. Through storing AIRRseq data in a standard-based curated and annotated form, each AIRR-seq data set can be annotated and stored once, and then reused by many others without the costly overheads of reannotation. Because the AIRR Standard supports the ability to curate the annotation process, it is possible to have a data set from a single study, annotated multiple times using different annotation tools and have the user differentiate and choose the annotation that they think is the most appropriate for their data reuse.

Regarding differences in experimental protocols, each of the decision points outlined in the "AIRR Community Guide to TR and IG Gene Annotation" chapter, Fig. 1 (single cell versus bulk sequencing, gDNA versus mRNA as the starting molecule, and whether UMIs were used), as well as differences in sequencing depth, sequencing error rates, read length, and the primers used can impact the data in ways that can make certain conclusions invalid. For example, differences in hybridization and amplification efficiencies between primers in a primer set can influence gene usage estimates. Thus, combining or comparing gene usage estimates between studies that used different primer sets can result in gene usage differences that are attributable to experimental artifacts and do not reflect true repertoire differences. The potential impact of specific experimental protocol choices on analysis conclusions are briefly outlined here and in Table 2. All of these experimental protocol differences can impact the number of unique receptor sequences observed in a sample, particularly whether rare sequences are observed. This can in turn result in artifacts when computing any of the common diversity metrics, such as clonality, repertoire overlap, and when constructing sequence networks. Similarly, protocol differences that affect relative abundances of starting templates, including whether gDNA or mRNA was used, primer differences, and whether UMIs were used, can result in artifacts when computing diversity and clonality measures and when analyzing gene usage. Finally, differences in the use of UMIs, sequencing platform error rates, and primers can result in artifacts for somatic hypermutation analyses.

Differences in preprocessing and sequence annotation protocols can similarly result in artifacts during re- or meta-analysis (Table 2). Differences in sequence filtering and deduplication can impact the number of unique AIRR sequences observed in a sample, as well as estimates of starting template relative abundances, in turn impacting diversity, clonality, and overlap measures, as well as sequence networks and gene usage estimates. The germline gene database and alignment algorithm used for germline gene annotations can impact germline gene assignments, thereby impacting gene usage estimates and somatic hypermutation analyses. These can also impact diversity, clonality, and other analyses when they are conducted at the annotation rather than sequence level (i.e., defining unique rearrangements according to their V gene, J gene, and CDR3 sequence rather than their full-length sequence). Finally, assigning clonal membership can depend on the clonal assignment algorithm used and could thereby impact the results of IG affinity maturation.

### Table 2 Considerations in re- and meta-analysis

(continued)

### Table 2 (continued)

Upper panel—Experimental, preprocessing, and rearrangement annotation protocol differences are shown in rows, and the impacted AIRR-seq data set features are shown in columns. X indicates when a data set feature is expected to be impacted by a particular protocol difference. Lower panel—Analysis types are shown in rows. X indicates when an analysis type is expected to be impacted by the data set feature in the corresponding column. O indicates that an analysis type is expected to be impacted when the analysis is conducted at the annotation- rather than sequence-level (e.g., V gene, J gene, and CDR3 sequence rather than full-length sequence)

> The above and Table 2 are not meant to be comprehensive, but instead serve as a guide when designing analyses that combine data sets or results across different studies. When selecting studies for such an analysis and formulating the research questions that can be reliably addressed, it is important to identify differences in experimental, preprocessing and annotation protocols and understand how these protocol differences can affect relevant data set features and analyses. It is recommended to choose research questions and analysis methods that are independent of any protocol differences where possible. Furthermore, common experimental design best practices are encouraged, such as ensuring that protocol differences do not partition with treatment groups and incorporating methods to control for and/or estimate their effects, as one would batch effects.

### Acknowledgments

We would like to thank our colleagues from the AIRR Community, who have dedicated many hours to the development of the community and the standards and initiatives on which this chapter is based. In particular, we would like to thank the authors of the other AIRR Community chapters in this volume, with a special thanks to Susanna Marquez, William Lees, and Ulrik Stervbo who assisted with content and editing of this chapter.

### References

https://doi.org/10.3389/fimmu.2017. 01418

repositories. Front Immunol 9:1877. https:// doi.org/10.3389/fimmu.2018.01877

Jason R, Cantrell Daniel K, Wheeler Alessandro, Sette Bjoern, Peters (2019) (2018) The Immune Epitope Database (IEDB): 2018 update. Nucleic Acids Research 47(D1) D339–D343. https://doi.org/10.1093/nar/ gky1006

Open Access This chapter is licensed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license and indicate if changes were made.

The images or other third party material in this chapter are included in the chapter's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the chapter's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.

# Chapter 24

# IMGT® Immunoinformatics Tools for Standardized V-DOMAIN Analysis

### Ve´ronique Giudicelli, Patrice Duroux, Mae¨l Rollin, Safa Aouinti, Ge´raldine Folch, Joumana Jabado-Michaloud, Marie-Paule Lefranc, and Sofia Kossida

### Abstract

The variable domains (V-DOMAIN) of the antigen receptors, immunoglobulins (IG) or antibodies and T cell receptors (TR), which specifically recognize the antigens show a huge diversity in their sequences. This diversity results from the complex mechanisms involved in the synthesis of these domains at the DNA level (rearrangements of the variable (V), diversity (D), and joining (J) genes; N-diversity; and, for the IG, somatic hypermutations). The recognition of V, D, and J as "genes" and their entry in databases mark the creation of IMGT by Marie-Paule Lefranc, and the origin of immunoinformatics in 1989. For 30 years, IMGT®, the international ImMunoGeneTics information system® http://www.imgt.org, has implemented databases and developed tools for IG and TR immunoinformatics, based on the IMGT Scientific chart rules and IMGT-ONTOLOGY concepts and axioms, and more particularly, the princeps ones: IMGT genes and alleles (CLASSIFICATION axiom) and the IMGT unique numbering and IMGT Collier de Perles (NUMEROTATION axiom). This chapter describes the online tools for the characterization and annotation of the expressed V-DOMAIN sequences: (a) IMGT/V-QUEST analyzes in detail IG and TR rearranged nucleotide sequences, (b) IMGT/HighV-QUEST is its high throughput version, which includes a module for the identification of IMGT clonotypes and generates immunoprofiles of expressed V, D, and J genes and alleles, (c) IMGT/StatClonotype performs the pairwise comparison of IMGT/HighV-QUEST immunoprofiles, (d) IMGT/DomainGapAlign analyzes amino acid sequences and is frequently used in antibody engineering and humanization, and (e) IMGT/Collier-de-Perles provides two-dimensional (2D) graphical representations of V-DOMAIN, bridging the gap between sequences and 3D structures. These IMGT® tools are widely used in repertoire analyses of the adaptive immune responses in normal and pathological situations and in the design of engineered IG and TR for therapeutic applications.

Key words IMGT, Immunogenetics, Immunoinformatics, Immunoglobulin, Antibody, T cell receptor, V-DOMAIN sequence analysis, Adaptive immune repertoire, IMGT Collier de Perles, IMGT-ONTOLOGY

Anton W. Langerak (ed.), Immunogenetics: Methods and Protocols, Methods in Molecular Biology, vol. 2453, https://doi.org/10.1007/978-1-0716-2115-8\_24, © The Author(s) 2022

### 1 Introduction

Immunoglobulins (IG) or antibodies [1, 2] and T cell receptors (TR) [3] are antigen receptors of the adaptive immune responses in vertebrates with jaws (gnathostomata) [4]. The huge diversity of the variable domains (V-DOMAIN) of the IG and TR chains of the immune repertoires results from several mechanisms that occur during their synthesis [1–4]. In particular, the combinatorial diversity depends on the number of variable (V), diversity (D), and joining (J) genes found in the IG and TR loci, which potentially can rearrange to form V-DOMAIN encoded by V-(D)-J regions [1–4]. It is the recognition of the V, D, and J as "genes" and their entry in databases that mark the creation of IMGT in 1989 by Marie-Paule Lefranc (Universite´ de Montpellier, CNRS) at Human Gene Mapping 10 (HGM10) and is at the origin of immunoinformatics, a new science at the interface between immunogenetics and bioinformatics [4].

Other mechanisms of diversity comprise the junctional diversity with exonuclease trimming at the ends of the V, D, and J genes and the random addition of nontemplated nucleotides, preferably "g" and "c," by the terminal deoxynucleotidyl transferase (TdT) encoded by the DNA nucleotidylexotransferase (DNTT) gene, creating the N-regions [1–3], and for IG, somatic hypermutations [1, 2]. For 30 years, IMGT®, the international ImMunoGeneTics information system® http://www.imgt.org, has implemented databases and developed tools for IG and TR immunoinformatics [5], based on the IMGT Scientific chart rules (see Subheading 2) and IMGT-ONTOLOGY concepts and axioms [6, 7], and more particularly, the princeps ones: IMGT genes and alleles (CLASSIFICA TION axiom) [8–12] and the IMGT unique numbering [13–17] and IMGT Collier de Perles [18–21] (NUMEROTATION axiom). This chapter describes the online analysis tools for the characterization and annotation of the expressed V-DOMAIN nucleotide (nt) and amino acid (AA) sequences, available from "IMGT tools" section of the IMGT® Home page. Protocols for their use and the description of main results are presented in this chapter. These concern the following: (a) IMGT/V-QUEST [22, 23] is the IMGT® online tool for the analysis of IG and TR nucleotide rearranged sequences (see Subheading 3); (b) IMGT/HighV-QUEST [24–27], the high throughput version of IMGT/V-QUEST, can analyze sets of up to one million sequences. It includes a module for the identification of IMGT clonotypes (AA) and the generation of IG and TR gene profiles for the diversity and expression of IMGT clonotypes (AA) (see Subheading 4); (c) IMGT/StatClonotype [28, 29] is a standalone package that performs statistical pairwise comparisons of IMGT clonotype (AA) diversity or expression between two IMGT/HighV-QUEST result sets (see Subheading 5); (d) IMGT/DomainGapAlign [30, 31] analyses domain AA sequences and two dimensional (2D) structures, and its results are used in antibody engineering and humanization [32, 33] (see Subheading 6); and (e) IMGT/Collier-de-Perles tool [21] generates IMGT Colliers de Perles graphical 2D representations for AA domain sequences [18–20] (see Subheading 7), it is available from the IMGT Home page and is also automatically launched by IMGT/V-QUEST and IMGT/DomainGapAlign.

### 2 IMGT Scientific Chart Rules for the Analysis of the V-DOMAIN

2.1 IMGT Gene and Allele Nomenclature and IMGT Reference Directory Sets The IMGT gene names of the IG and TR V, D, J, and C genes [1– 4] were approved by the Human Genome Organization (HUGO) Nomenclature Committee (HGNC) in 1999 [8, 9, 12] and were endorsed by the WHO-IUIS Nomenclature Subcommittee for IG and TR [10, 11]. IMGT gene and allele names are based on the concepts of classification of IMGT-ONTOLOGY "Group," "Subgroup," "Gene," and "Allele" [1–4, 10–12]. Alleles are the polymorphic variants of a gene: they are identified by their IMGT reference sequence, which corresponds to the coding V-REGION, D-REGION, J-REGION, and C-REGION sequence at the nucleotide level of V, D, J, and C gene alleles, respectively. IMGT reference directory sets include the allele IMGT reference sequences from functional (F) genes and alleles, open reading frame (ORF), and pseudogenes (P) [5]. IMGT germline V, D, and J genes and alleles, with their characteristics, their reference sequence and other sequences from the literature are managed in IMGT/ GENE-DB [34] and in IMGT Repertoire (IG and TR) Gene tables and Alignments of alleles Web resources [1–4]. The tools for V-DOMAIN analysis compare user sequences with IMGT reference directory sets for the identification of V, D, and J genes and alleles and the evaluation of mutations and AA changes. 2.2 IMGT Unique Numbering for the IG and TR V Domains An IG or TR V-DOMAIN comprises about 100 amino acids and is made of nine antiparallel beta strands (A, B, C, C0 , C00, D, E, F, and G) linked by beta turns (AB, CC0 , C00D, DE, and EF) or loops (BC,

C0 C00, and FG) [35]. At the structural level, they form a sandwich of two sheets closely packed against each other through hydrophobic interactions and joined together by a disulfide bridge between 1st-CYS at position 23 in B-STRAND (in the first sheet) and 2nd-CYS at position 104 in F-STRAND (in the second sheet) [13].

The IMGT unique numbering for IG and TR V-DOMAIN [13] delimits (1) the four framework regions: FR1-IMGT (A and B strands, from positions 1 to 26), FR2-IMGT (C and C<sup>0</sup> strands, from positions 39 to 55), FR3-IMGT (C00, D, E and F strands, from positions 66 to 104), FR4-IMGT (G strand, from positions 118 to 128), and (2) the three hypervariable or complementarity

Fig. 1 Protein displays of IG and TR V-DOMAIN based on the IMGT unique numbering for V-DOMAIN [13]. The V-DOMAIN translations were obtained from the analysis by IMGT/V-QUEST [22, 23] (see Subheading 3) of the nucleotide sequences of shown accession numbers in IMGT/LIGM-DB [36]. The identification of FR-IMGT and CDR-IMGT and of beta strands and loops was performed by IMGT/DomainGapAlign [30, 31] (see Subheading 6), which provides a standardized delimitation whatever the species, the receptor type, and the chain type. CDR-IMGT lengths are indicated between brackets, separated by dots (column on the right). 1st-CYS 23 and 2nd-CYS 104 are in pink, and W 41, hydrophobic AA 89, and W or F 118 are in blue. Taxons are in the IMGT 6 or 9-letter abbreviation: Homsap for Homo sapiens, Canlupfam for Canis lupus familiaris (dog), Musmus for Mus musculus (mouse), Turtru for Tursiops truncatus (dolphin), Macmul for Macaca mulatta (Rhesus monkey), and Felcat for Felis catus (cat)

> determining regions involved in the ligand recognition: CDR1- IMGT (BC loop, positions 27 to 38), CDR2-IMGT (C<sup>0</sup> C<sup>00</sup> loop, positions 56 to 65), and CDR3-IMGT (FG loop, positions 105 to 117, with additional positions 112.1, 111.1, 112.2 etc., if longer than 13 codons (or AA)). FR-IMGT positions, which delimit the three CDR-IMGT, are designated as anchors: they are 26 and 39, 55 and 66, and 104 and 118, respectively (Fig. 1), and shown as squares in IMGT Colliers de Perles [18–21]. According to the IMGT unique numbering [13], a V-DOMAIN is characterized by five highly conserved AA: 1st-CYS 23, tryptophan 41 (CONSERVED-TRP), hydrophobic amino acid 89, 2nd-CYS 104, and J-PHE or J-TRP 118 of the J-MOTIF (F/W-G-X-G, 118–121, where F is phenylalanine, W tryptophan, G glycine, X, any AA). The three CDR-IMGT lengths characterize a V-DOMAIN. By convention, they are indicated between brackets, separated by dots (for example [8.8.13]). The CDR1-IMGT and CDR2-IMGT are encoded by the V-REGION, whereas the CDR3-IMGT results from the V-(D)-J rearrangement. The IMGT Collier de Perles [18–20] can be generated by the IMGT/ Collier-de-Perles tool [21] (see Subheading 7).

Fig. 2 Graphical representation or prototypes of IG and TR V-DOMAIN with IMGT labels at the nucleotide level. (a) V-D-J-REGION. (b) V-J-REGION [1–4]. The JUNCTION encompasses 2nd-CYS 104, CDR3-IMGT, and J-TRP or J-PHE 118, and its length is therefore two AA longer than CDR3-IMGT. Potential palindromic nucleotides ("P") identified in case of untrimmed V, D, and/or J regions during the DNA rearrangement are not shown (With permission from M-P. Lefranc and G. Lefranc, LIGM, Founders and Authors of IMGT®, the international ImMunoGeneTics information system®, http:/www.imgt.org)

2.3 IMGT Standardized Labels and Sequence Description

The IMGT tools, which perform the analysis of sequences, provide the description of the V-DOMAIN with IMGT standardized labels (written in capital letters). The V-DOMAIN corresponds either to a V-D-J-REGION (in IG heavy (IGH)), TR beta (TRB), and TR delta (TRD) chains) (Fig. 2a) or to a V-J-REGION (in IG light lambda (IGL) and IG kappa (IGK)), TR alpha (TRA) and TR gamma (TRG) chains) (Fig. 2b), encoded by V-D-J or V-J rearrangements, respectively.

The V-DOMAIN labels according to the chain type or locus are: VH, V-KAPPA, V-LAMBDA for the IGH, IGK, and IGL, respectively, and V-ALPHA, V-BETA, V-DELTA, V-GAMMA for the TRA, TRB, TRD, and TRG, respectively [1–4].

the same reading frame)) or "unproductive" (stop codons and/or

2.4 IMGT Functionality of IG and TR Genes and Alleles and of Rearranged Sequences The "Functionality" concept identifies the functionality based on the configuration of the IG and TR genes. The functionality of the germline (V, D and J) and undefined (C) IG and TR genes and alleles, defined on the same criteria as conventional genes and alleles, is either functional (F), open reading frame (ORF), or pseudogene (P). The functionality of the IG and TR V-(D)-J rearranged sequences is either "productive" (no stop codon and in-frame JUNCTION (2nd-CYS 104 and J-TRP/J-PHE 118 in

out-of-frame JUNCTION) [2].

### 3 IMGT/V-QUEST

IMGT/V-QUEST [22, 23] identifies the V, D, and J genes and alleles in IG and TR V domains. It characterizes the nucleotide (nt) mutations and amino acid (AA) changes resulting from somatic hypermutations in IG V-REGION. It provides a detailed characterization of the V-D-J or V-J junctions by the integrated IMGT/ JunctionAnalysis tool [37, 38] and the full annotation of the V-DOMAIN with IMGT labels by IMGT/Automat [39, 40]. 3.1 IMGT/V-QUEST Sequence Submission The top of the IMGT/V-QUEST Welcome page (Fig. 3) provides two links: the first one gives access to the list of the IMGT/V-QUEST reference directory sets to which the users' own sequences can be compared (see Note 1), and the second one provides examples of human rearranged sequences to test the tool. The page then includes five sections to configure the analysis: 3.1.1 Your Selection 1. Select the species or taxon first and then the receptor type or locus (see Note 2) in the lists. 3.1.2 Sequence Submission IMGT/V-QUEST analyses up to 50 FASTA formatted IG or TR rearranged nucleotide sequences per run, from genomic DNA or cDNA indifferently. 1. Enter the sequences in the text area "Type (or copy/paste) your nucleotide sequence(s) in FASTA format".

Fig. 3 IMGT/V-QUEST Welcome page with the five sections: "Your selection," "Sequence submission," "Display results," "Advanced parameters," and "Advanced functionalities" [22, 23]

	- 1. Select "A. Detailed view" to get the results for each sequence individually. Results consist in a "Result summary" with the main results of the analysis and 14 detailed result sections that can be selectively checked or unchecked by the user (see Note 4). In sequence alignments, the number of IMGT reference sequences aligned with the user sequence (five by default) can be modified from 1 to 20.
	- 2. Select "B. Synthesis view" to display the sequences that express the same V gene and allele aligned together. Results include a "Summary table" with the main results of the analysis which can be ordered by "V-GENE and allele name" (default) or by the sequence "input" order. There are eight detailed result sections that can be checked or unchecked (see Note 5).
	- 3. Select "C. Excel file" to download the results, either in a spreadsheet (default) or as a zip archive. The results may include 11 sheets (or text files in the zip archive (see Note 6)), which can be checked or unchecked. The 12th sheet (or text file) is available if the option "Analysis of single chain Fragment variable (scFv)" is selected in "Advanced functionalities" (see Subheading 3.1.5) [41]. An alternative is to display the content of one given sheet in your browser ("Display 1 CSV file in your browser") or to "Download AIRR formatted results" as a zip archive (see Note 7) [42, 43].

3.1.4 Advanced Parameters The default values of the advanced parameters are used by IMGT/ V-QUEST for classical analyses [22, 23]. They may be modified for specific studies and/or unusual sequences. The user may:

insertions and/or deletions. Selecting "Yes" allows to identify the somatic hypermutations by nucleotide insertions and deletions in the V-REGION that may occur in normal and malignant cells [44] and/or potential sequencing errors.

	- (a) "Nb of accepted D-GENE" (number of D genes searched by the tool in IGH, TRB or TRD junctions).
	- (b) "Nb of accepted mutations" in 30 V-REGION, D-REGION, and 50 J-REGION: by default, 2, 4 and 2 mutations are accepted in the 30 V-REGION, D-REGION, and 5<sup>0</sup> J-REGION, respectively, for IGH, 7 in the 3<sup>0</sup> V-REGION and 5<sup>0</sup> J-REGION for IGK and IGL junctions (see Note 9). By default, no mutation is accepted for the TR junctions.
	- (a) "Nb of nucleotides to exclude in 5<sup>0</sup> of the V-REGION for the evaluation of the number of mutations" (useful in case of primer specific nucleotides).
	- (b) "Nb of nucleotides to add (or exclude) in 3<sup>0</sup> of the V-REGION for the evaluation of the alignment score" (useful in case of low (or high) exonuclease activity).

3.1.5 Advanced Functionalities "Advanced functionalities" [22, 23] corresponds to specific analyses, with additional dedicated results, for engineered/artificial sequences, and for the search of specific sequences for clinical applications. The user may:

3.2 IMGT/V-QUEST Results for A. Detailed View The page "A. Detailed results for the IMGT/V-QUEST analyzed sequences" [22, 23] indicates at the top the number of analyzed sequences and the list of sequences identifiers with links allowing to browse directly the corresponding individual results. Individual results include the FASTA submitted sequence and the "Result summary" of the analysis, followed by the detailed result sections selected in the Welcome page. Importantly, the result sections allow to explore in depth the results of the analysis regarding the identification of V, (D), J genes and alleles, the description of the V-DOMAIN with the delimitation of FR-IMGT and CDR-IMGT, and the characterization of the mutations.

3.2.1 Sequence and Result Summary The numbers of 50 trimmed-n and 30 trimmed-n from the submitted sequence before the analysis if any (see Note 3), the sequence length, the sequence analysis category (see Note 12), and the IMGT reference directory set with which the sequence was compared (e.g., Homo sapiens (human) IG set) are indicated above the submitted sequence provided in FASTA format [22, 23] (Fig. 4). The part of the sequence corresponding to the V-DOMAIN is underlined in green. If a sequence was submitted in antisense orientation, it is complementary reversed and displayed, as well as the results, in the V gene sense orientation.

> The "Result summary" provides the main characteristics of the analyzed sequence [22, 23]:

IMGT/V-QUEST provides warnings (not shown) that appear as notes in red to alert the user, if potential insertions or deletions are suspected in the V-REGION (see Note 15), or if other possibilities for the J-GENE and allele names are identified. Users are encouraged to check alignments in related detailed result sections.

Below the "Result summary," notes in black (not shown) may appear to indicate:

Fig. 4 IMGT/V-QUEST "Detailed results" [22, 23]. The parameters of the analysis are recalled on the top of the page. The first part the "Detailed results" for "seq\_1" (IMGT/LIGM-DB [36] accession number X81732) includes the sequence in FASTA format (the first 57 nt not underlined in green are not part of the V-DOMAIN) and the "Result summary." Seq\_1 functionality is "productive." This human IGHV sequence expresses the IGHV3-9\*01, IGHD3-3\*01, and IGHJ4\*02 genes and alleles. The lengths of the four FR-IMGT are 25, 17, 38, and 11. The lengths of the three CDR-IMGT are 8, 8, and 13. The JUNCTION length is 45 nt, and the decryption [45] shows that it is composed of 8 nt for the 3<sup>0</sup> V-REGION (5 nt were trimmed from the germline V during DNA rearrangement), 3 nt for N1-REGION, and 17 nt for the D-REGION (7 nt in 5<sup>0</sup> and 7 in 3<sup>0</sup> were trimmed from the germline D, 6 nt for the N2-REGION, and 11 for the 5<sup>0</sup> J-REGION (6 nt were trimmed from the germline J))

3.2.2 Detailed Result Sections

If selected in the Welcome page, the 14 detailed result sections are displayed [22, 23]. They allow to verify, detail, and complete the "Result summary."

	- (a) The "Analysis of the JUNCTION" (Fig. 5) [22, 23] shows the details of the junction at the nucleotide level with delimitation of the IMGT labels (Fig. 2 in Subheading 2.3). Dots indicate the number of nucleotides trimmed at the germline V, D, and J gene ends. Vmut, Dmut, and Jmut indicate the number of mutations in the 30 V-REGION, D-REGION, and 5<sup>0</sup> J-REGION, respectively, and the corresponding mutated nucleotides are underlined in the sequence. "Ngc" corresponds to the ratio of the number of g+c nucleotides to the total number of N nucleotides. The JUNCTION decryption is also provided [45] (see Note 14). If selected "Eligible D genes" (not shown), all D genes, which match the junction with their corresponding score, are displayed below.
	- (b) The "Translation of the JUNCTION" displays the AA JUNCTION with AA colored according to the eleven IMGT physicochemical classes [46] (see Note 18), the JUNCTION frame ('+' for in-frame, and '-' for out-offrame), the CDR3-IMGT length, the molecular mass, the isoelectric point (pI), and a link to detailed physicochemical descriptor (not shown). Gaps (represented by dots) are inserted in "out-of-frame" JUNCTION to maintain the J-REGION frame, and the corresponding codon, which cannot be translated, is represented by "#" in AA translation (not shown).

Fig. 5 IMGT/V-QUEST "Detailed results" [22, 23]. Results of IMGT/JunctionAnalysis [37, 38] for AB063867 IMGT/LIGM-DB accession number. This human IGH sequence results from the rearrangement of IGHV3- 11\*05 F, IGHD6-13\*01 F, and IGHJ4\*02 F. The JUNCTION is in-frame. The length of the CDR3-IMGT is 14 AA (42 nt). The JUNCTION length is of 48 nt, and the decryption [45] shows that it is composed of 9 nt for the 3<sup>0</sup> V-REGION (2 nt were trimmed from the germline V), 6 nt for N1-REGION, and 17 nt for the D-REGION (1 nt in 5<sup>0</sup> and 3 in 3<sup>0</sup> were trimmed from the germline D, 3 nt for the N2-REGION, and 13 for the 5<sup>0</sup> J-REGION (4 nt was trimmed from the germline J)

> FASTA format with the formatted header required as input by IMGT/JunctionAnalysis online [37, 38]. These results are provided even if IMGT/JunctionAnalysis gives no results.

	- (a) "6. V-REGION alignment according to the IMGT unique numbering" for the nt sequences with the FR-IMGT and CDR-IMGT delimitations according to the IMGT unique numbering [13].
	- (b) "7. V-REGION translation" for the nt sequence and its AA translation, aligned with the closest germline V-REGION.
	- (a) "9. V-REGION mutation and AA change table" lists the nt mutations and, if nonsilent, the corresponding AA changes. They are described for each FR-IMGT and CDR-IMGT with their nt and codon positions according to the IMGT unique numbering [13]. In parentheses, the "AA class Change Type" indicates if, between germline AA and replaced AA, the hydropathy, volume, and physicochemical properties have been conserved (+) or not (-) according to the IMGT physicochemical classes [46].
	- (b) "10. V-REGION mutation and AA change statistics" comprises two tables for the detailed and complete characterization of nt mutations and AA changes: "Nucleotide (nt) mutations" table quantifies nt positions with or without gaps, the identical nt, the total number of mutations, and the silent and nonsilent ones for the V-REGION and per FR-IMGT and CDR-IMGT. It then details the same evaluation for the four types of transitions and of the eight types of transversions. "Amino acid (AA) changes" table quantifies the codons or amino acid positions, with or without gaps, the unchanged AA, and AA changes for the V-REGION and per FR-IMGT and CDR-IMGT (see Note 19). It then evaluates the number of changes in 4 "AA class Similarity Degree": "Very similar" (the three properties hydropathy, volume, and physicochemical properties are conserved), "Similar" (one of the three properties is changed), "Dissimilar" (two of the three properties are changed), and "Very dissimilar" (the three properties are changed).
	- (c) "11. V-REGION mutation hot spots" shows the localization of the hot spot patterns (a/t)a (or wa) and (a/g) g(c/t)(a/t) (or rgyw) and their complementary reverse motifs t(a/t) (or tw) and (a/t)(a/g)c(c/t) (or wrcy) in the closest germline V gene and allele. Finally, this section includes a table for the "Correlation between V-REGION mutations, AA changes, codons changes, and hotspot motifs." It provides a synthesis for each mutation: the position in nt, the AA change and its position according

to the AA numbering [13, 16, 17], the AA class Change Type, the germline and mutated codon, and the corresponding hotspot if any. An illustration is provided in Fig. 6.

	- (a) "12. V-REGION and V-(D)-J-REGION" provides nt and AA FASTA sequences with gaps according to the IMGT unique numbering [13, 16, 17] of the V-REGION (nt sequence with access to the IMGT/PhyloGene tool [47]) and of V-J or V-D-J-REGION. In case of out-of-frame junctions V-J or V-D-J-REGION, a note is added, and the V-J or V-D-J-REGION is shown in red.
	- (b) "13. Annotation by IMGT/Automat" provides a full automatic annotation for the V-J-REGION or V-D-J-REGION by IMGT/Automat [39, 40] with IMGT labels (see Subheading 2.3).

The insertions and/or deletions that are detected by using the "Advanced parameters" and "Search for insertions" are described in the "Result summary" row [22, 23] (Fig. 7) with their localization in FR-IMGT or CDR-IMGT, the number of inserted or deleted nt, and, for insertions, the inserted nucleotides, the presence or absence of frameshift, the V-REGION codon from which the insertion or deletion starts, and the nt position in the user sequence.

The Advanced functionality "Analysis of single chain Fragment variable (scFv)" [41] allows the analysis of scFv sequences from phagedisplay combinatorial libraries [48, 49]. IMGT/V-QUEST [22, 23] identifies, localizes, and characterizes the two V-DOMAIN of a scFv (Fig. 8). At the top of the result page, the number of analyzed sequences and the number of identified V-DOMAIN are indicated. V-DOMAIN identifiers are automatically generated by adding to the sequence identifier a suffix composed of an underscore plus a letter for the locus (H, K, L for IGH, IGK, IGL or A, B, D, G for TRA, TRB, TRD, TRG, respectively). Below the list of V-DOMAIN identifiers is a table that indicates the positions of each V-DOMAIN and of the linker in the identified scFv. The detailed analysis of each individual V-DOMAIN is then provided classically.

3.2.3 Sequence and Result Summary with the Search for Insertions and Deletions in V-REGION

3.2.4 Top of Detailed Results for the Analysis of single chain Fragment variable (scFv)

Fig. 6 IMGT/V-QUEST "Detailed results" [22, 23]. Correlation between V-REGION mutations, AA changes, codons changes, and hotspots motifs in FR1-IMGT of seq\_3 (accession number AJ006165 of IMGT/LIGM DB [36]). (a) "7. V-REGION translation" and (b) "11. V-REGION mutation hotspots." Only the FR1-IMGT parts of the results are displayed: the two silent mutations g48>a (G16) and t54>c (S18) are shown in green and light blue rectangles, respectively. The nonsilent mutation g74>c (shown in the red rectangle) leads to the AA dissimilar change G25>A (hydropathy and physicochemical properties are not conserved). The codon "ggt" in position 73-75 is changed in "gct." The nt mutation occurs in the hotspot "ggtt" in position 73–76

Fig. 7 IMGT/V-QUEST "Detailed results" [22, 23]. Sequence and Result summary with "Search for insertions and deletions." An insertion of 18 nt is identified in seq\_4 (IMGT/LIGM-DB [36] accession number MG950400) from CDR2-IMGT position 56 (from nt 151 in the submitted sequence). The insertion is shown in capital letters

### 3.3 IMGT/V-QUEST Results for B. Synthesis View

At the top of the page, the parameters used for the analysis are recalled, and the number of analyzed sequences is indicated. The results include a summary table and potentially eight detailed result sections if selected by the user.

3.3.1 Summary Table The "Summary table" (Fig. 9) displays one row for each input sequence with the corresponding results, including 22 columns [22, 23]: (1) the sequence order in the submission; (2) the sequence identifier (Sequence ID); (3) the name of the closest V-GENE and allele; (4) the functionality of the sequence (when found, the presence of stop codons is indicated); (5) the V-REGION score; (6) the V-REGION percentage of identity with, between parentheses, the ratio of number of identical nucleotides (nt)/number of aligned nt; (7) the name of the closest J-GENE and allele; (8) the J-REGION score; (9) the J-REGION percentage of identity and the ratio of number of identical nucleotides (nt)/number of aligned nt; and provided according to the IMGT/JunctionAnalysis results [37, 38] (10) the D-GENE and allele name; (11) the D reading frame; (12) the CDR-IMGT lengths; (13) the AA JUNCTION; and (14) the JUNCTION frame (in the absence of results of IMGT/JunctionAnalysis, only the AA JUNCTION defined by IMGT/V-QUEST [22, 23] is displayed); (15) the JUNCTION nt length and decryption [45]; (16) the number of missing in 5<sup>0</sup> partial V-REGION; (17) the number of uncertain nt; (18) the number of missing nt in 3<sup>0</sup> partial J-REGION; (19) and (20) the numbers of 5<sup>0</sup> and 3<sup>0</sup> trimmed 'n' nucleotides; (21) the length of the sequence; and (22) the sequence analysis category (see Note 12). Clicking on the sequence ID provides the corresponding Detailed View in a separate tab (depending on your browser). Warnings in red may be indicated to highlight specific features of the sequence (Fig. 9). 3.3.2 Detailed Analysis of the JUNCTION A link to access the IMGT/JunctionAnalysis [37, 38] results is provided for sequences of the same locus. AA translations are aligned on the longest CDR3 length according to the IMGT unique numbering [13] (Fig. 10). 3.3.3 Detailed Result Sections for Alignment of Sequences Expressing the In "Alignment with the closest alleles" below the summary table, the V genes and alleles are listed with the number of assigned sequences in parentheses [22, 23]. Click on the associated link to reach the corresponding detailed result sections. They provide six

Same V Gene and Allele different displays (if all were selected) of alignment of sequences

Fig. 7 (continued) in the FASTA sequence. IMGT/V-QUEST then performs a classical analysis (for gene and allele identification, analysis of the JUNCTION, evaluation of nt mutation, and AA changes) after removal of the insertion(s) and addition of gaps to replace the deletions. The evaluation of the identity percentage added in square brackets includes each insertion or deletion as an additional mutation

Fig. 8 IMGT/V-QUEST "Detailed results" [22, 23]. Top of Detailed results for the "Advanced functionality" "Analysis of single chain fragment variable (scFv)". scFv\_1 and scFv\_2 sequences correspond to the accession numbers AJ006120 and AF117956 in the IMGT/LIGM-DB database [36]. The 5<sup>0</sup> V-DOMAIN are VH and 3<sup>0</sup> V-DOMAIN are V-KAPPA for both scFv. The detailed results are then provided for each domain

Fig. 9 IMGT/V-QUEST Synthesis view [22, 23]. (a) The 12 first columns of the "Summary table": the four analyzed sequences are shown in the "V-GENE and allele name" order in the Summary table. The sequences ID are accession numbers of IMGT/LIGM-DB [36]. The hyperlinks allow to get the corresponding results in "A Detailed view." The two sequences assigned to IGHV3–9 and the two sequences assigned to IGHV5-51 will be, respectively, aligned together in the detailed result sections 1 to 6. In the column "J-GENE and allele," a warning "(a)" in red indicates that other IGHJ genes and alleles may be solutions for the sequence 3 (not shown). (b) The last 12 columns of the "Summary table": the V-DOMAIN of sequence 1 is partial: 5 nt are missing in the 3<sup>0</sup> part of the J-REGION

> that express the same V gene and alleles: "1. Alignment for V-GENE," "2. V-REGION alignment according to the IMGT unique numbering" [13], "3. V-REGION translation," and three different formats for the "V-REGION protein display." Section "7. V-REGION most frequently occurring AA per position and per FR-IMGT and CDR-IMGT" shows the most frequent AA in sequences expressing the same V genes and alleles per FR-IMGT and CDR-IMGT and per position according to the IMGT unique numbering [13].

3.4 IMGT/V-QUEST Output for Excel File "Excel file" allows the users to open and save a spreadsheet including the results of the IMGT/V-QUEST analysis [22, 23]. The file contains 11 sheets or 12 for the Advanced Functionality "Analysis of single chain Fragment variable (scFv)" [41] (see Subheading 4.3 for the detail of their content in IMGT/HighV-QUEST sequence analysis results).

Fig. 10 IMGT/V-QUEST Synthesis view [22, 23]. Results of IMGT/JunctionAnalysis for four IGH junctions [37, 38]: "Analysis of the JUNCTIONs" displays for the four junctions, the sequences of the IMGT labels 3<sup>0</sup> V-REGION, N1, D-REGION, N2, and 5<sup>0</sup> J-REGION. The mutated nt are underlined. "Translation of the JUNCTIONs" displays the four junctions aligned per position according to the IMGT unique numbering [13]

### 4 IMGT/HighV-QUEST

IMGT/HighV-QUEST [24–27] is the high throughput version of IMGT/V-QUEST [22, 23]. It is freely available for academics, but it requires the user's registration. This allows the tool to automatically notify the users on the availability of the results. A link to the "New user" form is provided in the IMGT/HighV-QUEST welcome page. When the user logs in, the tool uses reCAPTCHA (https://developers.google.com/recaptcha) to protect the site from spam and abuse.

IMGT/HighV-QUEST provides two main functionalities [24–27]:

Subheading 4.4), and use "Statistics history" page for the download of IMGT clonotypes results (see Subheading 4.5)).

Links to the four pages are displayed on the top of the IMGT/ HighV-QUEST web interface.

### 4.1 IMGT/HighV-QUEST Sequence Set Submission IMGT/HighV-QUEST Search page (Fig. 11) is provided when the user logs in. It includes the four following sections [24–27]: 4.1.1 The Sequence Submission Form 1. Provide an analysis title, select the species (see Note 1), and the receptor type or locus (see Note 2) as for IMGT/V-QUEST. 2. Upload a simple text-formatted file containing your FASTA sequences (up to 1,000,000 of IG or TR rearranged sequences can be submitted in a single run). 3. When an analysis is launched ("Start" button), it is firstly dispatched and queued on the IMGT servers and is then performed depending on the available resources. Choose to be notified by e-mail "when analysis is queued" and/or "when analysis is completed" (selected by default). 4.1.2 Display Results 1. Select Result format: the default "CSV" result format includes 11 (or 12 with the Advanced Functionality "Analysis of single chain Fragment variable (scFv)") CSV files equivalent to those provided by the "Excel file" of IMGT/V-QUEST. Result format "AIRR" [42, 43] (see Note 7) or "Both formats" can be selected. 2. The individual result files (equivalent to IMGT/V-QUEST "Detailed view" in text format) can be included in the results for submissions of maximally 200,000 sequences only. 4.1.3 Advanced Parameters The analysis can be customized with exactly the same advanced parameters as proposed by IMGT/V-QUEST (see Subheading 3.1.4). 4.1.4 Advanced Functionalities "Analysis of single-chain Fragment variable (scFv)" [41] can be included in the analysis (default is "no") (see Subheading 3.1.5). 4.2 IMGT/HighV-QUEST Analysis History Page: Follow-Up and Download of Results The "Analysis history" page allows the user to check the status of the submitted analyses [24–27]. A table displays for each of them its title, its status (queued, running, or completed), the submission date, the number of submitted sequences, the species and the receptor type or locus (as selected by the user), and the actions that can be performed. When the analysis is completed, the user can download the results as a single archive file in TXZ format (commonly supported by archive tools for windows and other operating

systems). The availability of the results is guaranteed for two weeks

Fig. 11 The IMGT/HighV-QUEST Search page [24–27]

4.3 IMGT/HighV-QUEST Sequence Analysis Results

after the analysis is completed. After that, the files can be removed by the system. In that case, "File removed" is indicated in red instead of the archive logo.

A user may delete an analysis at any time except if it is used by the second module "Statistics" of IMGT/HighV-QUEST. In such cases, "Used by Statistics" is indicated in place of the "delete" button.

The content of the TXZ file depends on the selected "Result format" ("CSV," "AIRR," or "Both formats") [24–27]:

1. "CSV" format contains a tar folder (which needs to be extracted by an archive tool) with 11 (or 12) files (equivalent to the results of the excel file provided by the classical IMGT/ V-QUEST) in CSV format, and, if selected in the IMGT/ HighV-QUEST Search page, one subfolder with individual result files, in text format for each sequence (equivalent to the classical IMGT/V-QUEST "Detailed view" results (see Subheading 3.2)).

The content of each CSV file is indicated in Table 1 [27].

4.4 IMGT/HighV-QUEST Launch Statistics Page for the Evaluation of IMGT Clonotypes

An IMGT clonotype (AA) is defined by a unique V-(D)-J-rearrangement (V and J genes and alleles), with a unique CDR3- IMGT amino acid sequence and the presence of the conserved anchors C 104 and W/F 118 [26]. An IMGT clonotype (AA) is linked to one or more IMGT clonotype (nt): they are defined by a unique V-(D)-J-rearrangement with a unique CDR3-IMGT nucleotide sequence, whose translation corresponds to the CDR3- IMGT of the IMGT clonotype (AA) [26]. When IMGT/HighV-QUEST "Statistics" is launched, the tool evaluates IMGT clonotypes in batches of analyzed sequence sets per locus and provides immunoprofiles for IMGT clonotypes (AA) diversity (number of different IMGT clonotypes per V, D, and J gene and allele) and expression (number of sequences assigned to IMGT clonotypes (AA) per V, D, and J gene and allele) [26]. Moreover, IMGT/ HighV-QUEST can perform the comparison of multiple batches and provide the list of the IMGT clonotype (AA), which are common to two or more batches [26].

1theIMGT/HighV-QUESTCSVfileswiththenumberofcolumnsandresultcontent[27]

Table (continued)

Table 1 (continued)

IMGT® Immunoinformatics for Standardized V-DOMAIN Analysis 503

Table 1 (continued) File number Result type File name Number of columns Results content (see Note 20) #10 "V-REGION-mutationhotspots" 8 Hotspot motifs (a/t)a, t(a/t), (a/g)g(c/t)(a/t), and (a/t)(a/g)c(c/t) detected in the closest germline V-REGION with their localization in FR-IMGT and CDR-IMGT #11 "Parameters" "Parameters" ∙ Date of the analysis ∙ IMGT/V-QUEST program version, IMGT/V-QUEST reference directory release ∙ Parameters used for the analysis: species, receptor type or locus, IMGT reference directory set, advanced parameters, advanced functionalities #12 Sequence description scFv 40 Available only for the advanced functionality "Analysis of single chain Fragment variable (scFv)," one line per scFv: Positions and length of the V-(D)-J-REGION, CDR\_lengths, JUNCTION for the 2 V-DOMAIN of the scFv, positionsandlengthofthelinker[41]

For launching statistics, the following steps should be followed:

It allows to follow the status of the submitted statistics analysis and to download the results once completed [24–27]. The IMGT/ HighV-QUEST statistical output is provided as a zip file.

The IMGT/HighV-QUEST statistics output [26] is organized in the sections listed in Table 2 (see also http://www.imgt.org/ HighV-QUEST/doc.action#statistical-outputs-results).

The illustration of the content of file 4.2.1 is shown in Fig. 12: it shows the first seven most expressed IMGT clonotypes (AA) of a list of 27,080.

4.5.2 "Data" Directory Importantly, the archive includes a "data" directory: it contains text files named 'stats\_xxx' where 'xxx' is composed of 'batch name'\_' locus'. They include the list of all the IMGT clonotypes (AA) (that are displayed through html sections of Table 2) and their characteristics separated by tabulations. These files include the fields needed by the external IMGT/StatClonotype [28, 29] tool (see Subheading 5). Their content is described in the IMGT/HighV-QUEST Documentation at http://www.imgt.org/HighV-QUEST/doc. action#datastatsxxx.

QUEST Statistics History Page: Follow-Up and Download of Statistics

4.5 IMGT/HighV-

4.5.1 Results Sections to be Displayed in the User Web Browser

(continued)

(continued)

### Table 2 (continued)

Fig. 12 Top of the file 4.2.1 'IMGT clonotypes (AA) per Nb<sup>0</sup> [26]. (a) List of IMGT clonotypes (AA) ordered by decreasing number of assigned sequences. The first seven of IMGT clonotypes (AA) are shown. The table provides the Exp. ID (IMGT clonotype (AA) identifier in the set), the numbers of "1 copy," "More than one," and the total. The IMGT clonotype (AA) definition includes the names of the V, D, and J genes and alleles, the CDR3-IMGT length, the AA CDR3-IMGT, and the anchors. The IMGT clonotype (AA) representative sequence is characterized by the identity percentage with the closest V gene and allele, the sequence length, the sequence functionality, and a link to the FASTA sequence. An additional link allows to display all "1 copy" assigned to the IMGT clonotype (AA). The batch S3 results from the analysis of the run SRR1168790 available on Sequence Read Archive (SRA) (https:/www.ncbi.nlm.nih.gov/sra). (b) Example of IMGT clonotypes (nt) linked to the IMGT clonotypes (AA) #7 (extracted from file 4.2.2) to which 96 "1 copy" sequences were assigned. Ninety-four of them are assigned to the same IMGT clonotype (nt) with a CDR3-IMGT of 42 nucleotide "gcgtgtgacgtccagacgtcacaatatgtagcttttgactac". Two other IMGT clonotypes (nt) (with one sequence each) are also linked to #7. One shows a mutation (t>c) on the nt 6 of the CDR3 and the second a mutation (t>c) on the nt 33 of the CDR3 (shown in red in the figure)

### 5 IMGT/StatClonotype

Fig. 13 IMGT/StatClonotype Welcome page [28, 29]. The files stats\_S3\_IGH.txt (IMGT/HighV-QUEST set 1) and stats\_S4\_IGH.txt (IMGT/HighV-QUEST set 2) were uploaded from the "data" directory of IMGT/HighV-QUEST statistical output (obtained from the runs SRR1168790 and SRR1168789, respectively, available on Sequence Read Archive (SRA) (https:/www.ncbi.nlm.nih.gov/sra)). The range for CDR3-IMGT lengths is 4 and 36. IMGT/HighV-QUEST set 1 includes 27,075 IMGT clonotypes (AA) to which 44,446 sequences were assigned

statistical procedures (not shown).

Fig. 14 IMGT/StatClonotype Multiple testing procedures plots for genes [28, 29]. In the left panel, "IMGT clonotype (AA) diversity," "Single gene," and "Hide null or smallest gene occurrences" were selected (not shown). "Multiple testing procedures plots" displays an interactive line graph on the left and a scatter plot on the right for genes. Hovering the mouse on the interactive the line graph on the left allows the display of the exact number of significant differences in proportions, that is 21 for a Type I error <sup>α</sup> <sup>¼</sup> 0.05 with the multiple testing procedure BH. On the right, the scatter plot shows the coordinates of z-score and log\_10 (SidakSS) for IGHV1-69, for which the difference in proportion is significant whatever the multitesting procedure as indicated in the table. The graphs can be saved in PNG, JPG, or PDF and the tables in CSV format

> confidence intervals (CI), for genes (on the top) (Fig. 15) and for alleles (at the bottom) (see Note 27). In synthesis graphs for genes, IMGT gene names are ordered by their positions in the locus with their known functionalities. Below the normalized bar graph are listed the not ordered genes (not shown). They are grouped and shown at the bottom of the gene list in the synthesis graph. The values for the normalized proportions of genes (or alleles) in set 1 and set 2, the differences in proportions, the lower and upper bound of the confidence indices for differences in proportions, and the Test interpretation are recorded in "Statistical test results" tab Tables.

5. "CDR-IMGT lengths" tab [28, 29] displays, in the right panel, interactive bar graphs for set 1 and set 2 showing the distribution of the number of IMGT clonotypes (AA) (for IMGT clonotype (AA) diversity) or of the number of sequences assigned to IMGT clonotypes (AA) (for IMGT clonotype (AA) expression), per CDR-IMGT length (see Note 28). The left panel allows to choose the CDR-IMGT (CDR1-IMGT, CDR2-IMGT, or CDR3-IMGT) and to select the length of

Fig. 15 IMGT/StatClonotype synthesis graph for IMGT clonotype (AA) diversity per V gene [28, 29]. It displays visual comparison of the normalized proportions of IMGT clonotype (AA) diversity of the IGHV genes between sets 1 and 2. For example, the diversity of IMGT Clonotypes (AA) expressing the IGHV1-2, IGHV4- 39, and IGHV1-69 genes is significantly higher in set 1 than in set 2 whatever the multiple testing procedure. In the left panel, "Single gene" and "Hide null or smallest gene occurrences" were selected. Synthesis graphs are downloadable in PNG, JPG, or PDF format

the CDR-IMGT for the "List of IMGT clonotypes (AA) with selected CDR3-IMGT length" displayed below the bar graphs for sets 1 and 2.

6. "CDR-IMGT AA Properties" tab displays the distribution of the IMGT classes [46] of the 20 amino acids at CDR-IMGT (CDR1-IMGT, CDR2-IMGT, or CDR3-IMGT) positions in sets 1 and 2 for a given CDR-IMGT length. The left panel allows (1) to select the IMGT classes to be displayed for the amino acids: "20 amino acids," "Physicochemical" (Fig. 16), "Hydropathy," "Volume," "Chemical," "Charge," "Hydrogen donor or acceptor atoms," and "Polarity"; (2) to show

Fig. 16 IMGT/StatClonotype CDR-IMGT AA properties distribution [28, 29]. Examples for the CDR3-IMGT of length 15 in set 1: (a) "20 amino acids" and (b) "Physicochemical"

results by absolutes values (number of occurrences of an amino acid (or IMGT amino acid class) at a given position, for a given CDR length) or percentages; and (3) to modify the length and width of the graphs. The major right panel includes, for each set, a table with numbers (or percentages) of the amino acids (or IMGT amino acid classes) (in rows), at a given position (in columns). The table includes a row for undefined amino acids ("X") and for stop codons. The tables can be downloaded as CSV files. The corresponding graphical representation is shown as an interactive bar graph to visualize the amino acid distribution per position. At the bottom of the page, the variability plots based on the indexes according to "Shannon entropy," 'Wu-Kabat variability," or "Simpson index" with tables for numerical values are displayed. Comparisons of two sets are useful in detecting the characteristics of amino acids at positions important for the V domain antibody diversity or, by contrast, for maintaining its structure.

7. "V-D-J gene associations" tab [28, 29] displays interactive heat maps to represent V-J, V-D, or D-J gene associations in set 1 and set 2. The left panel allows (1) to display the Dendrogram for V-J, V-D, or D-J gene association, (2) to get the results with clustering or not, (3) to get the results in normalized values, and (4) to select the color palettes. The major right panel includes interactive heat maps to represent V-J, V-D, or D-J gene associations in set 1 and set 2. If the "Results with clustering" is selected, a double Ward hierarchical clustering with Euclidean distance is performed (this classification operates simultaneously on the lines and columns of a matrix intersecting two different types of genes), otherwise heat maps are shown without dendrograms and ordering. Such an analysis permits to detect genes with similar diversity or expression profiles, which can be further explored for given and/or related specificities in immune repertoire comparative analysis. Under heat maps, tables crossing the V-J, V-D, or D-J gene occurrences in set 1 and set 2 are given.

### 6 IMGT/DomainGapAlign

IMGT/DomainGapAlign [30, 31] analyzes the amino acid sequences of the IG and TR V-DOMAIN (see Note 29). IMGT/ DomainGapAlign identifies the closest V and genes and alleles of the user's amino acid domain sequences by comparison with the IMGT reference directory sets composed of the translations of the germline V and J regions of the genes managed in IMGT/GENE-DB [34]. The reference amino acid sequences are available by querying IMGT/DomainDisplay (IMGT® Home page, http:// www.imgt.org). Importantly, IMGT/DomainGapAlign can analyze V-DOMAIN from different species and different locus in a single run. The tool gaps the sequences, numbers the AA of each V-DOMAIN, and provides the delimitations of the FR-IMGT and CDR-IMGT and those of the beta strands and loops by applying the IMGT unique numbering [13]. It also characterizes the amino acid changes (see Note 30).

### 6.1 IMGT/ DomainGapAlign Query and Customization of the Analysis

6.1.1 Standard Parameters and Sequence (s)

Select the number of alignments displayed for each V-DOMAIN in the results (default is 5).

5. Check "IMGT Colliers de Perles" [21] to include the IMGT Collier de Perles [18–20] in the results (see Subheading 7)

Fig. 17 IMGT/DomainGapAlign Welcome page [30, 31]

Fig. 18 IMGT/DomainGapAlign Results [30, 31]. Top of the result page: the VH domain of daclizumab 3nfp\_H chain (PDB code 3nfp of IMGT/3Dstructure-DB [30, 50, 51]) is compared with the Homo sapiens reference directory. It is aligned with the human IGHV1-46\*01 and IGHJ4\*01 genes alleles

A second table is displayed for J genes and alleles with the species, the IMGT J gene and allele name, the number of the domain, the Smith-Waterman alignment score, the identity percentage, and the overlap.

Below are displayed two additional parallel tables: on the left the "AA changes in strands and loops" and on the right the "AA changes in FR-IMGT and CDR-IMGT" with the number of different AA, the description of the AA change with the "AA class Change Type'" (+) or not (-) (for hydropathy, volume and physicochemical characteristics [46] according to the AA IMGT classes), and "AA class Similarity Degree" (very similar, similar, dissimilar, and very dissimilar).

5. IMGT Colliers de Perles [18–20] (See Subheading 7) are shown, if selected, on one or two layers, without or with AA change positions shown in pink circles (or squares for CDR-IMGT anchors).

### 7 IMGT/Collier-de-Perles

The IMGT/Collier-de-Perles tool [21] generates "'IMGT Colliers de Perles" [18–20]. For V-DOMAIN, IMGT Colliers de Perles are obtained on one or two layers, provided that the V-DOMAIN

Fig. 19 IMGT/DomainGapAlign Results [30, 31]. Bottom of the result page for VH domain of daclizumab 3nfp\_H chain (PDB code 3nfp of IMGT/3Dstructure-DB [30, 50, 51]): the CDR-IMGT lengths are [8.8.9] with a total of three AA changes. The FR-IMGT lengths are [<IP_ADDRESS>] with a total of 14 AA changes

(AA) sequence is gapped according to the IMGT unique numbering [13] (see Note 32). Resulting IMGT Colliers de Perles show the standardized delimitation of FR-IMGT and CDR-IMGT, and of beta strands with their orientation in the IG and TR V-DOMAIN, allowing the visualization of the amino acids, which are important for a 3D structural configuration and bridging the gap between sequences and structures.

7.1 IMGT/Collier-de-Perles Launched from IMGT Sequence Analysis Tools

	- 2. Starting from a V-DOMAIN amino acid sequence, use IMGT/ DomainGapAlign [30, 31] (see Subheading 6) to generate the IMGT Colliers de Perles and select "IMGT Colliers de Perles" in the submission form (see Note 33).

7.2 IMGT/Collier-de-Perles Submission Interface

	- 1. Select the "Domain type" ("Variable (V)"), the number of layers for the IMGT Collier de Perles representation (1 or 2) (see Note 34).
	- 2. Select the "CDR-IMGT color type" [46] according to the locus of the sequence (1 for IGH, TRB, or TRD sequences and 2 for IGK, IGL, TRA or TRG sequences) and the "Background color," which will be applied to the FR-IMGT positions (see Note 35).
	- 3. Enter the CDR3-IMGT length.
	- 4. Enter the gapped AA sequence without any header.
	- 5. In case of detected amino acid insertions compared with the IMGT unique numbering for V domain [13], provide in "Amino acid insertions" the position that precedes the insertion, its length in AA, and the numbering label for each inserted position.
	- 6. A title for the resulting IMGT Collier de Perles can be optionally provided.
	- 7. Click on "Show" to launch the tool.

### 7.3 IMGT/Collier-de-Perles Results The IMGT Collier de Perles for a V-DOMAIN [18–21] displays the graphical representation of a V-DOMAIN with one position (1 AA) per bead (circle or square). Numbers allow an easy delimitation of the FR-IMGT, of the CDR-IMGT, and of the beta strands

Fig. 20 The IMGT/Collier-de-Perles Welcome page [21]

and the localization of the conserved amino acids. The anchor positions of CDR-IMGT are in square (see Subheading 2.2). The hatched positions represent gaps according to the IMGT unique numbering for V domain [13]. AA written in red letters indicate the five conserved positions in V-DOMAIN (1st-CYS 23, CON-SERVED-TRP 41, hydrophobic 89, 2nd-CYS 104 and J-TRP or J-PHE 118). CDR-IMGT are colored according to "IMGT CDR-IMGT color type" [46] of the corresponding locus and

Fig. 21 IMGT Colliers de Perles for V-DOMAIN [18–21]. (a–d) Background color is "50% Hydrophobic positions," and Proline (P) is in yellow [46]. (a) IMGT Collier de Perles on one layer generated from the

FR-IMGT according to the "Background color" (see Note 35) selected by the user. The orientations of the nine beta-strands are indicated at the bottom of the IMGT/Collier-de-Perles. Illustrations of IMGT/Collier-de-Perles output are shown in Fig. 21.

### 8 Notes

Fig. 21 (continued) IMGT/V-QUEST [22, 23] analysis of a VH (nt) (accession number X81732 of IMGT/LIGM-DB [36]). (b) IMGT Collier de Perles on two layers of a VH with hydrogen bonds between the amino acids of the C, C0 , C<sup>00</sup>, F, and G strands and those of the CDR-IMGT (daclizumab 3nfp\_H, PDB code 3nfp of IMGT/3Dstructure-DB [30, 50, 51]). (c, <sup>d</sup>) IMGT Colliers de Perles with AA changes of a V-LAMBDA domain generated from the IMGT/DomainGapAlign [30, 31] analysis (translation of the AF063723 IMGT/LIGM-DB accession number), (c) on one layer, and (d) on two layers. (e, <sup>f</sup>) IMGT/Collier-de-Perles [21] results on one layer for the entry code p00149 from IMGT/2Dstructure-DB (e) with background color "IGH 80% hydrophopathy classes [46] and (f) with background color "IGH 80% physicochemical classes" [46]

deletions in V-REGION," and (4) analysis on complementary reverse sequence with "Search for insertions and deletions in V-REGION" and corrections if any.

in 5' of the V-REGION for the evaluation of the number of mutations") before launching the analysis.

### Acknowledgements

We are very grateful to Ge´rard Lefranc, founder of the Laboratoire d'ImmunoGe´ne´tique Mole´culaire LIGM (Universite´ de Montpellier and CNRS), for his unique contribution in the creation of IMGT® in 1989 and his unwavering support for these 30 years. We thank all members of the IMGT® team for their expertise and constant motivation. IMGT® was funded in part by the BIOMED1 (BIOCT930038), Biotechnology BIOTECH2 (BIO4CT960037), 5th PCRDT Quality of Life and Management of Living Resources (QLG2-2000-01287), and 6th PCRDT Information Science and Technology (ImmunoGrid, FP6 IST-028069) programs of the European Union (EU). IMGT® received financial support from the GIS IBiSA, the Agence Nationale de la Recherche (ANR) Labex MabImprove (ANR-10-LABX-53-01), the Re´gion Occitanie Languedoc-Roussillon (Grand Plateau Technique pour la Recherche (GPTR), and BioCampus Montpellier. IMGT® is currently supported by the Centre National de la Recherche Scientifique (CNRS), the Ministe`re de l'Enseignement Supe´rieur, de la Recherche et de l'Innovation (MESRI), the University of Montpellier, and the French Infrastructure Institut Franc¸ais de Bioinformatique (IFB) ANR-11-INBS-0013. IMGT® is a registered trademark of CNRS. IMGT® is member of the International Medical Informatics Association (IMIA) and a member of the Global Alliance for Genomics and Health (GA4GH). This work was granted access to the High Performance Computing (HPC) resources of Meso@LR and of Centre Informatique National de l'Enseignement Supe´rieur (CINES), to Tre`s Grand Centre de Calcul (TGCC) of the Commissariat a` l'Energie Atomique et aux Energies Alternatives (CEA) and to Institut du de´veloppement et des ressources en informatique scientifique (IDRIS) under the allocation 036029 (2010-2022) made by GENCI (Grand Equipement National de Calcul Intensif).

### References

Harb Protoc 2011:726–736. https://doi.org/ 10.1101/pdb.prot5635

Recognit 17:17–32. https://doi.org/10. 1002/jmr.647

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# IMGT/3Dstructure-DB: T-Cell Receptor TR Paratope and Peptide/Major Histocompatibility pMH Contact Sites and Epitope

### Marie-Paule Lefranc and Ge´rard Lefranc

### Abstract

T-cell receptors (TR), the antigen receptors of T cells, specifically recognize peptides presented by the major histocompatibility (MH) proteins, as peptide/MH (pMH), on the cell surface. The structure characterization of the trimolecular TR/pMH complexes is crucial to the fields of immunology, vaccination, and immunotherapy. IMGT/3Dstructure-DB is the three-dimensional (3-D) structure database of IMGT®, the international ImMunoGenetics information system®. By its creation, IMGT® marks the advent of immunoinformatics, which emerged at the interface between immunogenetics and bioinformatics. The IMGT® immunoglobulin (IG) and TR gene and allele nomenclature (CLASSIFICATION axiom) and the IMGT unique numbering and IMGT/Collier-de-Perles (NUMEROTATION axiom) are the two founding breakthroughs of immunoinformatics. IMGT-ONTOLOGY concepts and IMGT Scientific chart rules generated from these axioms allowed IMGT® bridging genes, structures, and functions. IMGT/3Dstructure-DB contains 3-D structures of IG or antibodies, TR and MH proteins of the adaptive immune responses of jawed vertebrates (gnathostomata), IG or TR complexes with antigens (IG/Ag, TR/pMH), related proteins of the immune system of any species belonging to the IG and MH superfamilies, and fusion proteins for immune applications. The focus of this chapter is on the TR V domains and MH G domains and the contact analysis comparison in TR/pMH interactions. Standardized molecular characterization includes "IMGT pMH contact sites" for peptide and MH groove interactions and "IMGT paratopes and epitopes" for TR/pMH complexes. Data are available in the IMGT/3Dstructure database, at the IMGT Home page http://www.imgt.org.

Key words IMGT, T-cell receptor, CDR-IMGT, Major histocompatibility, Paratope, Epitope, TR/ pMH, IMGT-ONTOLOGY, Immunoinformatics, IMGT/3Dstructure-DB

### 1 Introduction

The adaptive immune responses were acquired by jawed vertebrates (or gnathostomata) more than 450 million years ago and are found in all extant jawed vertebrate species from fishes to humans [1]. The adaptive immune responses are characterized by a remarkable specificity and memory, which are the properties of the B and T cells owing to an extreme diversity of their antigen receptors [1]. The

Anton W. Langerak (ed.), Immunogenetics: Methods and Protocols, Methods in Molecular Biology, vol. 2453, https://doi.org/10.1007/978-1-0716-2115-8\_25, © The Author(s) 2022

specific antigen receptors comprise the immunoglobulins (IG) or antibodies of the B cells and plasma cells [2–5] and the T-cell receptors (TR) [6]. Whereas the IG recognize antigens in their native (unprocessed) form, the TR recognize processed antigens, which are presented as peptides by the highly polymorphic major histocompatibility (MH) proteins (in humans HLA for human leukocyte antigens, encoded by genes in the MHC locus) (Fig. 1). T cells are involved in cell-mediated immune response, against a stress of viral, bacterial, fungal, or tumoral origin and identify antigenic peptides presented by the MH proteins as

Fig. 1 A T-cell receptor (TR)/peptide-major histocompatibility 1 (pMH1) complex. A TR (here, TR-alpha\_beta) is shown (on top, upside down) in complex with an MH (here, MH1) presenting a peptide in its groove [1]. In vivo, a TR is anchored in the membrane of a T cell as part of the signaling T-cell receptor (TcR <sup>¼</sup> TR +CD3). A TR is made of two chains, each comprising a variable domain (V-DOMAIN) at the N-terminal end and a constant domain (C-DOMAIN) at the C-terminal end. The domains are V-ALPHA and C-ALPHA for the TR-ALPHA chain and V-BETA and C-BETA for the TR-BETA chain. An MH1 is made of the I-ALPHA chain with two G-DOMAIN (G-ALPHA1 and G-ALPHA2) and a C-LIKE-DOMAIN (C-LIKE), noncovalently associated with the B2M (a C-LIKE-DOMAIN). In this representation (with G-ALPHA1 on the left, G-ALPHA2 and B2M on the right), the peptide is oriented in the groove from front of the figure to back. The TR/pMH1 complex structure is 3qfj from IMGT/3Dstructure-DB (http:/www.imgt.org). (With permission from M-P. Lefranc and G. Lefranc, LIGM, Founders and Authors of IMGT®, the international ImMunoGeneTics information system®, http:/www.imgt.org)

peptide/MH (pMH) on cell surface [1]. The recognition and signal transduction are carried out by the multiprotein bifunctional T-cell receptor (TcR) assembly that comprises the TR responsible of the specific pMH recognition plus the associated transmembrane signaling CD3 proteins [6]. The TcR is itself associated, in the immunological synapse, with the CD4 or CD8 coreceptors, to the activating CD28 and inhibitory CTLA4 costimulatory proteins, to the CD2 adhesion molecule and to intracellular kinases. The CD8 expressed on most cytotoxic T cells binds the MH class I (MH1) that is expressed ubiquitously on cells of the organism [7]. The CD4 expressed on most helper T cells binds the MH class II (MH2) that is expressed by professional antigen presenting cells (dendritic cells, macrophages, monocytes, and B cells) [7].

IMGT®, the international ImMunoGeneTics information system®, http://www.imgt.org, is the global reference in immunogenetics and immunoinformatics [1], founded in 1989 by Marie-Paule Lefranc at Montpellier (Universite´ de Montpellier and CNRS). It is a high quality integrated knowledge resource comprising 7 databases, 17 online tools, and more than 25,000 pages of web resources [8–11]. IMGT® is specialized in the sequences, structures, and genetic data of the IG, TR, and MH of human and other vertebrate species, in the immunoglobulin superfamily (IgSF) and the MH superfamily (MhSF) of vertebrates and invertebrates, and in related proteins of the immune system (RPI), fusion protein for immune applications, and composite proteins for clinical applications [1, 8–11]. IMGT/3Dstructure-DB [12–14] is the three-dimensional (3-D) structure database of IMGT®. This database provides the standardized IMGT annotation and analysis of the 3-D structures of the TR, pMH, and TR/pMH complexes and comprises detailed molecular characterization and description of their interactions [15–17]. The standardized analysis is based on the concepts of IMGT-ONTOLOGY, the first ontology in immunogenetics and immunoinformatics [18–24]. The IMGT-ONTOLOGY concepts are generated from seven axioms [25–31], of which the CLASSIFICATION axiom (IG and TR gene and allele nomenclature) (see Note 1) at the birth of IMGT® and immunoinformatics [1, 25] and the NUMEROTATION axiom (IMGT unique numbering [7, 26–27, 32–35] and IMGT Colliers de Perles [28, 29, 36–39]) allow bridging sequences, structures, and functions. The IMGT unique numbering for variable (V) domain includes the IG and TR V-DOMAIN and the V-like domains of IgSF other than IG and TR [32–34]. The IMGT unique numbering for constant (C) domain includes the IG and TR C-DOMAIN and the C-like domains of IgSF other than IG and TR [35]. The IMGT unique numbering for G domain includes the groove (G) domains of the MH G-DOMAIN and the G-like domains of MhSF other than MH (or RPI-MH1Like) [7]. The IMGT/ DomainGapAlign tool [13, 40, 41] analyzes the amino acid

### sequences of the V, C, and G domains using the IMGT unique numbering [7, 34, 35] and provides a direct link to the IMGT/ Collier-de-Perles tool [39]. The IMGT Scientific chart rules provide a standardized description of the contact analysis [15–17] and comparison of TR/pMH complexes and their interactions, irrespective of the TR chains and domains, the MH class (MH1 or MH2), or the species (Homo sapiens, Mus musculus, etc.). Eleven "IMGT pMH contact sites" were defined for the comparison of pMH interactions, regardless of the peptide lengths [15–17]. The "IMGT pMH contact sites" visualize the interactions between the amino acids (AA) (see Note 2) of the peptide and those of the MH groove based on the contact analysis. They are a useful asset in peptide vaccine design and epitope prediction, and they precisely identify and visualize AA of the peptide located in the MH2 groove. The standardized "IMGT paratope and epitope" for TR/pMH complexes comprises the TR paratope and the pMH epitope, determined from contact analysis, in IMGT/3Dstructure-DB, at the IMGT Home page http://www.imgt.org.

### 2 TR and MH Standardized Description in IMGT/3Dstructure-DB

2.1 TR and MH Chains and Domains 2.1.1 TR Chains and Domains The TR is made of two chains, an alpha chain (TR-ALPHA) and a beta chain (TR-BETA) for the TR-ALPHA\_BETA receptor and a gamma chain (TR-GAMMA) and a delta chain (TR-DELTA) for the TR-GAMMA\_DELTA receptor [6] (Table 1). Each complete TR chain comprises an extracellular region made up of a V-DOMAIN) (for instance, V-ALPHA for the alpha chain) and a C-DOMAIN) (for instance, C-ALPHA for the alpha chain), a

### Table 1

IMGT standardized labels for the DESCRIPTION of the T-cell receptors (TR) and of their chains and domains. IMGT® labels (concepts of description) are written in capital letters [1]

a The TR chain C-REGION also includes the CONNECTING-REGION (CO), the TRANSMEMBRANE-REGION (TM), and the CYTOPLASMIC-REGION (CY), which are not present in the 3-D structures (IMGT® http://www.imgt. org, IMGT Scientific chart >1. Sequence and 3D structure identification and description > Correspondence between labels for IG and TR domains in IMGT/3Dstructure-DB and IMGT/LIGM-DB)

connecting region ( CONNECTING -REGION (CO)), a transmembrane region (TRANSMEMBRANE-REGION (TM)), and a short cytoplasmic region (CYTOPLASMIC-REGION (CY)) [6, 7] (Fig. 2, Table 1). The TR V domains that are directly involved in the TR/pMH interactions are described in Subheading 2.2.

2.1.2 MH Chains and Domains The MH1 is formed by the association of a heavy chain (I-ALPHA) and a light chain (beta-2-microglobulin or B2M). The MH2 is an heterodimer formed by the association of an alpha chain (II-ALPHA) and a beta chain (II-BETA) [7] (Table 2) The I-ALPHA chain of the MH1 and the II-ALPHA and II-BETA chains of the MH2 comprise an extracellular region, made of three domains for the MH1 chains and of two domains for the MH2 chains, and CO, TM, and CY regions [7] (Fig. 2, Table 2). The I-ALPHA chain comprises two groove domains (G-DOMAIN), G-ALPHA1 [D1] and G-ALPHA2 [D2], and one C-LIKE domain [D3] [7]. The B2M corresponds to a single C-LIKE domain. The II-ALPHA chain and the II-BETA chain each comprises two domains, G-ALPHA [D1] and C-LIKE [D2], and G-BETA [D1] and C-LIKE [D2] [7] (Fig. 2). Only the extracellular region that corresponds to these domains has been crystallized. The MH G domains that are directly involved in the TR/ pMH interactions are described in Subheading 2.3.

2.2 TR V Domains 2.2.1 Definition A V domain [32–34] comprises about 100 AA and is made of nine antiparallel beta strands (A, B, C, C<sup>0</sup> , C00, D, E, F, and G) linked by beta turns (AB, CC<sup>0</sup> , C00D, DE, and EF) or loops (BC, C<sup>0</sup> C00, and FG) and forming a sandwich of two sheets (Table 3). The sheets are closely packed against each other through hydrophobic interactions giving a hydrophobic core and joined together by a disulfide bridge between first-CYS at position 23 in the B-STRAND in the first sheet and the second-CYS 104 in the F-STRAND in the second sheet [34]. The V domain type includes the V-DOMAIN of the TR (and IG), which corresponds to the V-J-REGION or V-D-J-REGION encoded by V-(D)-J rearrangements [1–6, 36], and the V-LIKE-DOMAIN of the IgSF other than IG and TR [37–44]. In a V-DOMAIN, the three hypervariable loops BC, C0 C00, and FG involved in the ligand (antigen for IG or pMH for TR) recognition are designated as complementarity determining regions (CDR-IMGT) [1–6].

2.2.2 IMGT Unique Numbering for V Domain The V domain strands and loops and their delimitations and lengths are based on the IMGT unique numbering for V domain (V-DOMAIN and V-LIKE-DOMAIN) [33, 34] (Table 3). In the IG and TR V-DOMAIN, the G-STRAND is the C-terminal part of the J-REGION, with J-PHE or J-TRP 118 and the canonical motif F/W-G-X-G at positions 118–121 [1]. The loop length (number of AA (or codons), which is the number of occupied positions, is a

Fig. 2 T-cell receptor/peptide/MH complexes with MH class I (TR/pMH1) and MH class II (TR/pMH2). (a) 3-D structures of TR/pMH1 and TR/pMH2. (b) Schematic representation of TR/pMH1 and TR/pMH2. The TR (TR-ALPHA and TR-BETA chains), the MH1 (I-ALPHA and B2M chains), and the MH2 (II-ALPHA and II-BETA chains) are shown with the extracellular domains (V-ALPHA and C-ALPHA for the TR-ALPHA chain; V-BETA and C-BETA for the TR-BETA chain; G-ALPHA1, G-ALPHA2, and C-LIKE for the I-ALPHA chain; C-LIKE for B2M; G-ALPHA and C-LIKE for the II-ALPHA chain; II-BETA and C-LIKE for the II-BETA chain), and the connecting, transmembrane, and cytoplasmic regions. [D1], [D2], and [D3] indicate the domains. Arrows indicate the peptide localization in the MH groove made of two G-DOMAIN [7]. In these representations (with G-ALPHA1 on the right, G-ALPHA2 and B2M on the left), the peptide is oriented in the groove from back of the figures to front. (With permission from M-P. Lefranc and G. Lefranc, LIGM, Founders and Authors of IMGT®, the international ImMunoGeneTics information system®, http:/www.imgt.org)

### Table 2

IMGT-standardized labels for the DESCRIPTION of the major histocompatibility (MH) and of their chains and domains. IMGT® labels (concepts of description) are written in capital letters [1]

a The I-ALPHA, II-ALPHA and II-BETA chains includes at the C-terminal end of the C-LIKE-DOMAIN, the CON NECTING-REGION (CO), the TRANSMEMBRANE-REGION (TM), and the CYTOPLASMIC-REGION (CY), which are not present in the 3-D structures

### Table 3

V domain strands and loops, IMGT positions and lengths, based on the IMGT unique numbering for V domains (V-DOMAIN and V-LIKE-DOMAIN) [33, 34]

a IMGT® labels (concepts of description) are written in capital letters <sup>b</sup>

In number of AA (or codons)

c See Subheading 2.4 <sup>d</sup>

In the IG and TR V-DOMAIN, the G-STRAND (or FR4-IMGT) is the C-terminal part of the J-REGION, with J-PHE or J-TRP 118 and the canonical motif F/W-G-X-G at positions 118–121. The JUNCTION refers to the CDR3-IMGT plus the two anchors second-CYS 104 and J-PHE or J-TRP 118 [1]

crucial and original concept of IMGT-ONTOLOGY. The lengths of the loops BC (or CDR1-IMGT), C0 C00, (or CDR2-IMGT) and FG (or CDR3-IMGT) characterize the V-DOMAIN (Table 3). They are delimited by anchor positions (see Note 3). The BC loop (or CDR1-IMGT) comprises positions 27–38, the C0 C00 (or CDR2-IMGT) positions 56–65, and the FG (or CDR3- IMGT) positions 105–117. In a V-DOMAIN, the CDR3-IMGT that encompasses the V-(D)-J junction resulting from V-J or V-D-J rearrangements [1] is more variable in sequence and length than the CDR1-IMGT and CDR2-IMGT that are encoded by the V-REGION only. The lengths of the three loops BC, C0 C00, and FG are shown in number of AA (or codons), into brackets and separated by dots. For example, [9.6.9] means that the BC, C<sup>0</sup> C00, and FG loops (or CDR1-IMGT, CDR2-IMGT, and CDR3-IMGT for a V-DOMAIN) have a length of 9, 6, and 9 AA (or codons), respectively.

2.2.3 IMGT Colliers de Perles for V Domain The V domain nine strands are indicated, with their orientation, in the IMGT Colliers de Perles [28, 29, 32–34, 36–39], which are IMGT 2D graphical representations based on the IMGT unique numbering. IMGT Colliers de Perles of the TR V-ALPHA and V-BETA domains from 1ao7 (see Note 4) a TR/pMH1 3-D structure complex are shown as examples (Fig. 3). The V-ALPHA and V-BETA domains share the main conserved characteristics of the V-DOMAIN, which are the disulfide bridge between cysteine 23 (first-CYS) and cysteine 104 (second-CYS), and the three other hydrophobic core residues tryptophan 41 (CONSERVED-TRP), leucine (or hydrophobic) 89, and phenylalanine 118 (J-PHE) (see Note 5). In Fig. 3, the V-ALPHA (1ao7\_D chain; [6.6.11]) has a CDR1-IMGT and a.

CDR2-IMGT of 6 AA and a CDR3-IMGT of 11 AA, whereas the V-BETA (1ao7\_E chain [5.6.14]) has a CDR1-IMGT, CDR2- IMGT, and CDR3-IMGT of 5, 6, and 14 AA, respectively (Subheading 2.2.2) (see Note 6). In IMGT/3Dstructure-DB, the IMGT genes and alleles that contribute to the V-DOMAIN are determined automatically by IMGT/DomainGapAlign [13, 40, 41], based on the standardized IMGT nomenclature [1, 2, 6] and IMGT unique numbering [34]. Thus, the V-ALPHA of 1ao7\_D corresponds to Homo sapiens TRAV12-2\*02-TRAJ24\*02 and the V-BETA of 1ao7\_E corresponds to Homo sapiens TRBV6-5\*01- (TRBD2)-TRBJ2-7\*01 [16, 17].

2.3 MH G Domains 2.3.1 Definition A G domain [7] comprises about 90 AA and is made of a sheet of four antiparallel beta strands linked by turns and of a helix (Table 4); the helix sits on the beta strands, its axis forming an angle of about 40 degrees with the strands [16, 17]. Two G domains are needed to form the MhSF groove made of a "floor" and two "walls" [7]. Each G domain contributes by its four strands

Fig. 3 IMGT/Collier-de-Perles for TR V domain (V-DOMAIN). (a) IMGT/Collier-de-Perles for TR V-ALPHA (chain 1ao7\_D). The CDR-IMGT lengths are [6.6.11]. (b) IMGT/Collier-de-Perles for TR V-BETA (chain 1ao7\_E). The CDR-IMGT lengths are [5.6.14]. AA ais shown in the one-letter abbreviation (see Note 2). Position at which hydrophobic AA (hydropathy index with positive value: I, V, L, F, C, M, A) and tryptophan (W) are found in more than 50% of analyzed sequences are shown in blue, online. All proline (P) are shown in yellow, online. Anchor positions are shown in squares (see Note 3). Arrows indicate the direction of the beta strands [28, 29]. Hatched circles correspond to missing positions according to the IMGT unique numbering for V domain [33, 34]. IMGT color menu for CDR1-IMGT, CDR2-IMGT, and CDR3-IMGT is blue, green, and greenblue, for V-ALPHA, and red, orange and purple, for V-BETA (see Note 6). IMGT/Collier-de-Perles are shown on one layer (on the left hand side) and two layers (on the right hand side). The IMGT Colliers de Perles on two layers show, in the forefront, the GFCC<sup>0</sup> C<sup>00</sup> strands and, in the back, the ABED strands. Hydrogen bonds (from the IMGT/3Dstructure-DB entry) are show in green, online. Only those between the AA of the C, C<sup>0</sup> , C<sup>00</sup>, F, and G strands (in the forefront) and those of the CDR-IMGT are shown here. IMGT/Collier-de-Perles are from IMGT/3Dstructure-DB, http:/ www.imgt.org. (With permission from M-P. Lefranc and G. Lefranc, LIGM, Founders and Authors of IMGT®, the international ImMunoGeneTics information system®, http:/www.imgt.org)

and turns to half of the groove floor and by its helix to one wall of the groove [7, 16, 17]. The G domain type includes the G-DOMAIN of the MH [7] and the G-LIKE-DOMAIN of the MhSF other than MH or RPI-MH1Like [7, 45, 46] (see Note 7).

### Table 4

G domain strands, turns, and helix, IMGT positions and lengths, based on the IMGT unique numbering for G domains (G-DOMAIN and G-LIKE-DOMAIN) [7]

a IMGT® labels (concepts of description) are written in capital letters <sup>b</sup>

In number of AA (or codons)

c See Subheading 2.4 <sup>d</sup>

For details on the characteristic Residue@Position and additional positions, see Ref. [7] <sup>e</sup>

Or 9 in some G-BETA

f Or 0 in some G-ALPHA2-LIKE [7]

the G-DOMAIN are determined automatically by IMGT/

Fig. 4 IMGT/Collier-de-Perles of MH G domains (G-DOMAIN). (a) MH1 G-ALPHA1 and G-ALPHA2 domains from 1ao7 (I-ALPHA chain 1ao7\_A). (b) MH2 G-ALPHA and G-BETA domains from 1j8h (II-ALPHA chain 1j8h\_A and II-BETA 1j8h\_B, respectively). AA positions and gaps (hatched positions) are according to the IMGT unique numbering for G domain [7]. Positions 61A, 61B, and 72A are characteristic of the G-ALPHA2 and G-BETA domains (and are not reported in the G-ALPHA1 and G-ALPHA IMGT/Collier-de-Perles) [7]. IMGT/Collier-de-Perles are from IMGT/3Dstructure-DB, http:/www.imgt.org. G-domain terminal hatched positions (MH1 G-ALPHA1 91 and 92 and MH2 G-BETA 90, 91 and 92) are not reported in online IMGT/Collier-de-Perles. The IMGT Colliers de Perles can also be obtained, with the sequences gapped by IMGT/DomainGapAlign [40, 41], using the IMGT/Collier-de-Perles tool [39]. (With permission from M-P. Lefranc and G. Lefranc, LIGM, Founders and Authors of IMGT®, the international ImMunoGeneTics information system®, http:/www.imgt.org)

DomainGapAlign [13, 40, 41], based on the standardized IMGT nomenclature and numbering [1, 7]. Thus the G-ALPHA1 and G-ALPHA2 of 1ao7\_A are encoded by HLA-A\*0201 [15–17].

2.4 Residue@ Position and Atom Pair Contacts "Residue@Position" is an IMGT® concept of numerotation that numbers the position of a given residue (or by extension that of a conserved property AA class [47]), based on the IMGT unique numbering (see Note 8). A "Residue@Position" (R@P) is defined by the position numbering according to the IMGT unique numbering [7, 34, 35], the residue name (in the 3-letter abbreviation and/or in the one-letter abbreviation) (see Note 2), the IMGT domain label (Tables 1 and 2), and either the gene and allele name for AA sequences (see Note 1), or the "IMGT chain ID" for 3-D structures. In IMGT/3Dstructure-DB, a 'Residue@Position is described in a "Residue@Position card" (Fig. 5) that provides information on its characteristics (see Note 9) and the list of the other R@P with which it interacts [16, 17]. Each interaction is characterized by the total number of "atom pair contacts" (see Note 10) and, as selected by the user for display, the number of atom pair contacts per type ("noncovalent," "polar," "hydrogen bond," "non polar," "covalent," or "disulfide") and/or per category ("(BB) Backbone/backbone," "(SS) Side chain/side chain," "(BS) Backbone/side chain," and "(SB) Side chain/backbone") [16, 17].

### 3 IMGT pMH Contact Analysis

3.1 IMGT pMH Contact Sites Definition and Determination

"IMGT pMH contact sites" [15–17] highlight the contacts between the amino acids of a presented peptide and those of the floor and helix walls of the MH groove, in 3-D structures of pMH and TR/pMH complexes [12–14]. The "IMGT pMH contact sites" are visualized in IMGT Colliers de Perles for G-DOMAIN [7]. The "IMGT pMH contact sites" provide a standardized comparison of the interactions between a presented peptide and the MH, regardless of the MH class (MH1 or MH2), the G domain (G-ALPHA1, G-ALPHA2, G-ALPHA, and G-BETA), and the peptide length. The "IMGT pMH contact sites" also allow one to precisely identify the AA that is effectively bound in the MH groove. This is particularly informative for the peptides bound to MH2 as these peptides can be much longer than the actual groove length with the N-terminal and C-terminal ends extending outside the groove [7]. In order to deal with different peptide lengths in the groove, 11 standard "IMGT pMH contact sites" were defined (C1–C11) [15–17] (Fig. 6). They correspond to a theoretical maximum length of 11 AA in the groove. This means that, in 3-D structures, some (usually two or three) "IMGT pMH contact sites" are absent as peptides are shorter than 11 AA (usually nine or eight AA long).

Fig. 5 IMGT Residue@Position card. The "Residue@Position: 61A—ALA (A)—G-ALPHA2—1ao7\_A" is defined by the position numbering ("61A") according to the IMGT unique numbering for G domain [7], the residue name in the three-letter abbreviation and in the one-letter abbreviation for AA ("ALA (A)") (see Note 2), the IMGT domain label (G-ALPHA2) (Table 2) and the IMGT chain ID (1ao7\_A) (see Note 4). The list of atom pair contacts shows that this R@P interacts with 5 R@P of the same domain (G-ALPHA2) and, of interest for the TR/ pMH interactions, with 4 R@P of the V-BETA and one of the peptide (Ligand). The "Residue@Position" card is from IMGT/3Dstructure-DB, http:/www.imgt.org. (With permission from M-P. Lefranc and G. Lefranc, LIGM, Founders and Authors of IMGT®, the international ImMunoGeneTics information system®, http:/www.imgt.org)

> The peptide binding mode to MH1 is characterized by the N-terminal and C-terminal peptide ends docked deeply with the C1 and C11 contact sites (red and pink, respectively, in the IMGT/ Collier-de-Perles) and by the peptide length that mechanically constrains the peptide conformation in the groove. Thus, for a peptide of 10 AA, one "IMGT pMH contact sites" is absent (C2), and for a peptide of 9 AA, two "IMGT pMH contact sites" are absent (C2 and C7), whereas for a peptide of 8 AA, three pMH contact sites are absent (C2, C7, and C8) [15–17] (see Note 11).

Fig. 6 Standard "IMGT pMH contact sites'. Eleven standard 'IMGT pMH contact sites' (C1 to C11) were defined for the standardized analysis and comparison of pMH interactions [16, 17]. (With permission from M-P. Lefranc and G. Lefranc, LIGM, Founders and Authors of IMGT®, the international ImMunoGeneTics information system®, http:/www.imgt.org)

The peptide binding mode to MH2 is different with the peptide lying in the groove. Thus, for nine amino acids lying in an MH2 groove, C2 is present but there are no C7 and C8. For a given 3-D structure in IMGT/3Dstructure-DB, the determination of the "IMGT pMH contact sites" combines contact analysis between the peptide and the MH (in a pMH or in a TR/pMH complex), with an interaction scoring function (see Note 12). The MH AA automatically selects the highest score that is listed and displayed in "IMGT/Collier-de-Perles with pMH contact sites." The characterization of the "IMGT pMH contact sites" based on contact analysis has superseded the previous identification of "pockets" in the MH groove (see Note 13).

### 3.2 Access to IMGT pMH Contact Sites

Fig. 7 "IMGT pMH contact sites" between MH1 and a 9-AA peptide. (a) "IMGT pMH contact sites" for MH1 (human HLA-A\*0201, 1ao7\_A) and peptide 1ao7\_C. The numbers 1–9 refer to the peptide AA numbering ( LLFGYPVYV). C1–C11 refer to the "IMGT pMH contact sites" (there are no C2 and C7 in agreement with MH1 binding a 9-AA peptide). In that 3-D structure, there is no C5 because the glycine G4 score is too low. The G-ALPHA1 and G-ALPHA2 AA positions assigned automatically to the "IMGT pMH contact sites" are listed. (b) "IMGT/Collier-de-Perles with pMH contact sites." View is from above the cleft, with G-ALPHA1 on top and G-ALPHA2 on bottom. (c) Groove 3-D structure. The groove is shown with and without the peptide (on the left and right hand side, respectively). The IMGT Color menu for "IMGT pMH contact sites" is used in (a), (b), and (c). (a) and (b) are from IMGT/3Dstructure-DB, http:/www.imgt.org. (With permission from M-P. Lefranc and G. Lefranc, LIGM, Founders and Authors of IMGT®, the international ImMunoGeneTics information system®, http:/www.imgt.org)

3.2.1 IMGT pMH Contact Sites for pMH1 An example of "IMGT pMH contact sites" for pMH1 is shown in Fig. 7. In that 3-D structure of a TR/pMH1 complex (1ao7), the groove made by the G-ALPHA1 and G-ALPHA2 of the I-ALPHA chain (1ao7\_A) binds a 9-AA peptide (1ao7\_C). "IMGT pMH contact sites" results provide first a table, which shows the positions 1–9 of the peptide AA (each AA is clickable, giving access to its Residue@Position card). Nine of the 11 C1–C11 contact sites are displayed, C2 and C7 being absent, in agreement with a 9-AA peptide bound in a MH1 groove (see Subheading 3.1). The G-ALPHA1 and G-ALPHA2 AA positions that contribute to each "IMGT pMH contact site" are listed. For example, G-ALPHA1 59 and G-ALPHA2 73, 77, and 81 contribute to the "IMGT pMH contact site" C1 that predominantly interacts with leucine (L) 1 of the peptide (N-terminal end) (Fig. 7a). The "IMGT pMH contact sites" are displayed in "IMGT/Collier-de-Perles with pMH contact sites" (Fig. 7b). Clicking on one residue in the IMGT/Collier-de-Perles gives access to its "IMGT Residue@Position card" (see Subheading 2.4). The 3-D structure, with or without peptide, is shown in Fig. 7c.

3.2.2 IMGT pMH Contact Sites for pMH2 An example of "IMGT pMH contact sites" for pMH2 is shown in Fig. 8. In that 3-D structure of a TR/pMH2 complex (1j8h), the groove made by the G-ALPHA (of the II-ALPHA chain) (1j8h\_A) and the G-BETA (of the II-BETA chain) binds a 13-AA peptide (1j8h\_C). "IMGT pMH contact sites" results provide first a table, which shows the AA 1–9 in the groove (each AA is clickable, giving access to its Residue@Position card). However, in contrast to MH1 (see Subheading 3.2.1), the nine AA shown in Fig. 8a only correspond to the central part of the peptide. Indeed, the peptide bound to MH2 is longer than the length of the groove and extends outside its N-terminal and C-terminal ends, as the MH2 groove is "open" at both ends [7]. One major breakthrough of the "IMGT pMH contact sites" is the identification of the AA that is located in the MH2 groove [15–17]. Whereas the peptide (1j8h\_C) is 13 AA long ( PKYVKQNTLKLAT ), the "IMGT pMH contact sites" results allow one to determine that the 9 AA in the MH2 groove are YVKQNTLKL (Fig. 8a). Nine of the 11 C1–C11 contact sites are displayed, C7 and C8 being absent, in agreement with 9 AA inside a MH2 groove (see Subheading 3.1). The G-ALPHA and G-BETA AA positions that contribute to each 'IMGT pMH contact sites' are listed. They are visualized in the "IMGT Collier de Perles with pMH contact sites" (Fig. 8b). Clicking on one residue in the IMGT Colliers de Perles gives access to its "IMGT Residue@Position card" (see Subheading 2.4). The 3-D structure, with or without peptide, is shown in Fig. 8c.

### 4 IMGT/3Dstructure-DB Domain Pair Contacts

4.1 IMGT/ 3DStructure-DB Domain Pair Contacts (Overview)

"IMGT/3Dstructure-DB Domain pair contacts (overview)" (Fig. 9) is accessed by clicking on "Domain contacts (overview)" of "Contact analysis" in an IMGT/3Dstructure-DB card. The example shown in Fig. 9 is that of the TR/pMH1 structure 1ao7. Eight "Domain pair contacts" are of interest for TR/pMH interactions, two for pMH1 (see Subheading 4.1.1) and six for TR/ pMH1 (see Subheading 4.1.2). Similar results are obtained for the

Fig. 8 "IMGT pMH contact sites" between MH2 and 9 AA in the groove. (a) "IMGT pMH contact sites" for MH2 (human HLA-DRA\*0101\_HLA-DRB1\*0401) (1j8h\_A-1j8h\_B) and a 13 AA long peptide (1j8\_C). The numbers 1–9 refer to the AA numbering in the groove (YVKQNTLKL) as determined by the "IMGT pMH contact sites." C1–C11 refer to the "IMGT pMH contact sites" (there are no C7 and C8 in agreement with MH2 binding 9 AA in the groove). In that 3-D structure, there is no C5 because the asparagine N5 score is too low. The G-ALPHA and G-BETA AA positions assigned automatically to the "IMGT pMH contact sites" are listed. (b) "IMGT/Collierde-Perles with pMH contact sites." View is from above the cleft, with G-ALPHA on top and G-BETA on bottom. (c) Groove 3-D structure. The groove is shown with and without the peptide (on the left and right hand side, respectively). The IMGT Color menu for "IMGT pMH contact sites" is used in (a), (b), and (c). (a) and (b) are from IMGT/3Dstructure-DB, http:/www.imgt.org. (With permission from M-P. Lefranc and G. Lefranc, LIGM, Founders and Authors of IMGT®, the international ImMunoGeneTics information system®, http:/www.imgt.org)

> TR/pMH2 structure, e.g., 1j8h (the only difference being the names of the G-DOMAIN, G-ALPHA, and G-BETA, instead of G-ALPHA1 and G-ALPHA2) (not further detailed here).

4.1.1 Domain Pair Contacts for pMH1 Interactions

The two domain pair contacts for pMH1 interactions are "(Ligand)/G-ALPHA1" and "(Ligand)/G-ALPHA2" (Fig. 9). Thus, for the pMH1 interactions in 1ao7, the domain pair "(Ligand)/G-ALPHA1" shows that 26 residues are involved,

Fig. 9 IMGT/3Dstructure-DB Domain pair contacts (overview). The IMGT/3Dstructure-DB entry is the TR/pMH1 3-D structure 1ao7. The domain partners considered are designated as "Unit 1" and "Unit 2." The number of residue pair contacts, the number of residues involved (total, from Unit 1 and from Unit 2), the number of total atom pair contacts, and, as selected by the user for the display, the number of contacts per type and/or by category are provided. "(Ligand)" refers to the peptide. Two red frames highlight the domain pair contacts for pMH interactions. Two blue rectangles highlight the domain pair contacts for TR/pMH interactions, three for V-ALPHA and three for V-BETA. (With permission from M-P. Lefranc and G. Lefranc, LIGM, Founders and Authors of IMGT®, the international ImMunoGeneTics information system®, http:/www.imgt.org)

> 8 AA of the peptide (Ligand) interacting with 18 AA of G-ALPHA1 (creating 29 residue pair contacts with a total of 305 atom pair contacts). Similarly, the domain pair "(Ligand)/G-ALPHA2" shows that 24 residues are involved, 8 AA of the peptide interacting with 16 AA of G-ALPHA2 (creating 26 residue pair contacts with a total of 281 atom pair contacts).

4.1.2 Domain Pair Contacts for TR/pMH1 Interactions The six domain pair contacts for TR/pMH1 interactions include three domain pairs involving V-ALPHA and three domain pairs involving V-BETA (Fig. 9). The TR/pMH1 interactions in 1ao7 are the following:

Fig. 10 pMH1 interactions. (a) Interactions "G-ALPHA1/(Ligand)" of 1ao7. (b) Interactions "G-ALPHA2/ (Ligand)" of 1ao7. Clicking on a R@P link gives access to the corresponding IMGT Residue@Position card. "(Ligand)" refers to the peptide. The contact analysis of the TR/pMH 3-D structure 1ao7 is from IMGT/ 3Dstructure-DB, http:/www.imgt.org. The "IMGT pMH contact sites" for G-ALPHA1 (a) and G-ALPHA2 (b) were added on the right hand side of the figure, for a comparison with Fig. 7. (With permission from M-P. Lefranc and G. Lefranc, LIGM, Founders and Authors of IMGT®, the international ImMunoGeneTics information system®, http:/www.imgt.org)

4.3 IMGT Paratope and Epitope IMGT paratope and epitope are concepts of the "SpecificityType" in IMGT-ONTOLOGY [48–50]. Paratope, or "antigen-binding site," identifies the part of the V-DOMAIN of an IG or antibody ("IG paratope") or of a TR ("TR paratope") that, respectively, recognizes (binds to) the antigen (Ag) or the peptide/major histocompatibility (pMH) ("epitope" or "antigenic determinant") [49]. Epitope, or "antigenic determinant," identifies the part of the antigen (Ag) or of the peptide/major histocompatibility

Fig. 11 TR V-ALPHA/pMH1 interactions. (a) Interactions "V-ALPHA/G-ALPHA1" of 1ao7. (b) Interactions "V-ALPHA/G-ALPHA2" of 1ao7. (c) Interactions "V-ALPHA/(Ligand)" of 1ao7. Clicking on a R@P link gives access to the corresponding IMGT Residue@Position card. "(Ligand)" refers to the peptide. The contact analysis of the TR/pMH 3-D structure 1ao7 is from IMGT/3Dstructure-DB, http:/www.imgt.org. The IMGT color menu is blue, green, and greenblue for CDR1-IMGT, CDR2-IMGT, and CDR3-IMGT, respectively (see Note 6). (With permission from M-P. Lefranc and G. Lefranc, LIGM, Founders and Authors of IMGT®, the international ImMunoGeneTics information system®, http:/www.imgt.org)

Fig. 11 (continued)

(pMH) that is recognized by the paratope of the V-DOMAIN of an IG or antibody or of a TR, respectively [50].

The amino acids that constitute the TR paratope belong to the paired V domains of a TR (V-alpha and V-beta for a TR-alpha\_beta, V-gamma, and V-delta for a TR-gamma\_delta), and more precisely to the CDR-IMGT [49]. Among the CDR-IMGT, the CDR3- IMGT that results from the V-J and V-D-J junction play the major role in TR/pMH interactions [15–17]. T-cell epitopes are usually identified as "linear" when referring to the processed peptide (p) presented in the groove of the MH proteins. However, in IMGT-ONTOLOGY, the "T-cell epitope" concept is identified as "discontinuous" as it comprises amino acids of the MH that bind to the TR V domains [50]. Thus, in a TR/pMH complex, the AA in contact at the interface between the TR and the pMH constitute the paratope on the TR surface and the epitope on the pMH surface (Fig. 13). In IMGT/3Dstructure-DB, the "IMGT paratope and epitope" for TR/pMH complexes are determined by combining contact analysis (Table 5) with an interaction scoring function, which roughly complies with the true mean energy ratio [15–17]. A standardized description of the "IMGT paratope and

Fig. 12 TR V-BETA/pMH1 interactions. (a) Interactions "V-BETA/G-ALPHA1" of 1ao7. (b) Interactions "V-BETA/ G-ALPHA2" of 1ao7. (c) Interactions "V-BETA/(Ligand)." Clicking on a R@P link gives access to the corresponding IMGT Residue@Position card. "(Ligand)" refers to the peptide. The contact analysis of the TR/pMH 3-D structure (1ao7) is from IMGT/3Dstructure-DB, http:/www.imgt.org. R@P belonging to CDR1- IMGT is in red and those belonging to the CDR3-IMGT are in purple according to the IMGT color menu (see Note 6). (With permission from M-P. Lefranc and G. Lefranc, LIGM, Founders and Authors of IMGT®, the international ImMunoGeneTics information system®, http:/www.imgt.org)

Fig. 12 (continued)

epitope" is provided. Thus, the pMH1 epitope of 1ao7 (Fig. 13) comprises AA of G-ALPHA1 and G-ALPHA2 (1ao7\_A) (HLA-A\*0201) and of the peptide (1ao7\_C, Tax peptide 11–19). Twenty-four AA form the pMH1 epitope: sixteen from the MH1 (six from G-ALPHA1 and ten from G-ALPHA2) and eight from the peptide. Each AA that belongs to the epitope is characterized by its position according to the IMGT unique numbering for G domain [7] and by its position in the peptide.

The TR paratope of 1ao7 (T-cell receptor A6) (Fig. 13) comprises AA of V-ALPHA (1ao7\_D chain) and of V-BETA (1ao7\_E chain). Sixteen AA of the TR (11 from V-ALPHA and 5 from V-BETA) form the paratope. The IMGT/Collier-de-Perles (Fig. 3) show that nine out of the 11 AA of the V-ALPHA paratope belong to the CDR-IMGT (D27, R28 and G29, Q37 to the CDR1-IMGT, Y57 to the CDR2-IMGT, T108, D109, and W113 and G114 to the CDR3-IMGT) and that five AA of the V-BETA paratope belong to the CDR3-IMGT and are localized at the top of loop. Clicking on "Epitope IMGT Residue@Position cards" and "Paratope IMGT Residue@Position cards" (Fig. 13) provide detailed contacts for each AA belonging to the epitope and paratope, respectively. IMGT paratope and epitope are

Fig. 13 "IMGT paratope and epitope" of an IMGT TR/pMH complex. Each AA that belongs to the pMH epitope is characterized by its position in the peptide or in the G domains according to the IMGT unique numbering [7]. For examples, "E (58G1\_A)" means that the glutamate (E) is at position 58 of the G-ALPHA1 domain (1ao7\_A), "AAH (61G2-62G2\_A)" means that the alanine (A), alanine (A), and histidine (H) are at positions 61, 61A, and 62 of the G-ALPHA2 domain (1ao7\_A) (see also Fig. 4a). Each AA that belongs to the TR paratope is characterized by its position in the V domains according to the IMGT unique numbering [32–34]. Thus, "DRG (27 V1-29V1\_D1)" means that the aspartate (D), arginine (R), and glycine (G) are at positions 27, 28, and 29 of the V domain 1 of 1ao7\_D (V-ALPHA) (see also Fig. 3a). In the same way, "GGRP (112.1V1-114V1\_E)" means that the glycine (G), glycine (G), arginine (R), and proline (P) are at positions 112.1, 112, 113, and 114 of the V domain 1 of 1ao7\_E (V-BETA) (see also Fig. 3b). The "IMGT paratope and epitope" analysis of the TR/pMH1 3-D structure (1ao7) is from IMGT/3Dstructure-DB, http:/www.imgt.org. (With permission from M-P. Lefranc and G. Lefranc, LIGM, Founders and Authors of IMGT®, the international ImMunoGeneTics information system®, http:/www.imgt.org)

> determined automatically for the TR/pMH 3D structures in IMGT/3Dstructure-DB (see Note 15). Clicking on the "References and links" tag in the IMGT/3Dstructure-DB card gives access to external links. Links to the Immune Epitope Database (IEDB) [51, 52] are provided. Clicking on the "IMGT numbering comparison" displays, per chain, a table providing the correspondence between the IMGT unique numbering per domain and the PDB numbering of the chain entry.

### 4.4 Bridging IMGT Clonotype (AA), TR-Mimic Antibody and Paratope

4.4.1 IMGT Clonotype (AA) Repertoire and TR Paratope

Next-generation sequencing (NGS) data, analyzed by IMGT/ HighV-QUEST, provides a standardized characterization of the TR repertoire diversity and expression in normal (e.g., before and after vaccination) and pathological situations. The results include the IMGT variable (V), diversity (D), and joining (J) gene and allele names (identified at the nucleotide level) and the identification of the JUNCTION lengths and amino sequences, which, together, characterize the IMGT clonotypes (AA) [53]. TR V domain analysis, using IMGT nomenclature [1, 6] and IMGT unique numbering [34] for both NGS and 3-D structures of TR/pMH complexes

Table 5 Amino acids of the TR paratope (V-ALPHA, V-BETA) and of the pMH1 epitope (G-ALPHA1, peptide, and G-ALPHA2) of 1ao7 based on Contact analysis from IMGT/3Dstructure-DB, http://www.imgt.org [12 –14]. (A) TR V-ALPHA paratope – pMH1 epitope. (B) TR V-BETA paratope—pMH1 epitope. Amino acid positions of the TR V-ALPHA and V-BETA are according to the IMGT unique numbering for V-DOMAIN [33, 34]. Amino acid positions of the TR V-ALPHA and V-BETA are according to the IMGT unique numbering for G-DOMAIN [7]. The list of contact analysis below is complete. Differences observed in visual displays are due to filters, based on contact types or scores

<sup>(</sup>continued)

Table 5 (continued)

(A)

TR V-ALPHA paratope – pMH1 epitope of 1ao7 EPITOPE G-ALPHA1 (Ligand) Peptide

 epitope)

cAtom [7, 12–14], provides a paradigm for bridging IMGT clonotypes (AA) of NGS repertoires and TR paratope CDR-IMGT (particularly CDR3-IMGT) delimitations.

### 4.4.2 TR-Mimic Antibody Paratope The 3-D structures of an engineered TR-mimic antibody and that of a TR targeting peptide-HLA were recently compared: the IG Fab 3M4E5 and the TR 1G4\_a58b61 are receptors targeting the NY-ESO-1 peptide presented by HLA-A\*02:01 [54]. The pMH contacts of the NY-ESO-1 peptide SLLMWITQC with the MH1 HLA-A\*02:01 groove are similar in the two peptide-HLA complexes, as expected [55]. The paratope of the IG Fab (TR-mimic antibody) includes amino acids of VH [8.8.12] and V-LAMBDA [9.3.9], whereas the paratope of the TR, classically, includes amino acids of V-BETA [5.6.12] and V-ALPHA [6.7.13]. The IMGT unique numbering for V-DOMAIN [34] was used for the four domains in the description of the paratopes. Similarly, in both 3-D structures, the IMGT unique numbering for G-DOMAIN [7] was used for the description of the pMH epitope, which comprises the G-ALPHA1 helix, the peptide, and the G-ALPHA2 helix [55].

### 5 Availability and Citation

Authors who use IMGT® databases and tools are encouraged to cite this article and to quote the IMGT® Home page, http://www. imgt.org. Online access to IMGT® databases and tools is freely available for academics and under licenses and contracts for companies.

### 6 Notes

1. Since the creation of IMGT® in 1989, at New Haven during the tenth Human Genome Mapping Workshop (HGM10), the standardized classification and nomenclature of the IG and TR of human and other vertebrate species have been under the responsibility of the IMGT Nomenclature Committee (IMGT-NC). In 1995, following the first demonstration online of the nucleotide database IMGT/LIGM-DB at the ninth International Congress of Immunology in San Francisco, IMGT-NC has become the World Health Organization-International Union of Immunological Societies (WHO-IUIS)/IMGT Nomenclature SubCommittee for IG and TR. IMGT® gene and allele names are based on the concepts of classification of "Group," "Subgroup," "Gene," and "Allele," generated from the IMGT-ONTOLOGY CLASSIFICATION axiom. The IMGT® gene nomenclature for IG and TR genes was approved at the international level by the Human Genome Organisation (HUGO) Nomenclature Committee (HGNC) in 1999 and by the WHO-IUIS [56, 57]. The IMGT® IG and TR gene names [2, 6, 58, 59] are the official reference for the vertebrate genome projects and, as such, have been entered in IMGT/ GENE-DB, the IMGT® gene database [60], in National Center for Biotechnology Information (NCBI) Gene [61], in European Bioinformatics Institute (EBI) Ensembl, and in the Vega Genome Browser (Wellcome Trust Sanger Institute).

15. In January 2021, IMGT/3Dstructure-DB [12–14] contained 239 TR/pMH complexes (of which 186 are TR/pMH1 and 53 are TR/pMH2) [15–17].

### Acknowledgements

We thank Patrice Duroux for the IMGT/3Dstructure-DB database and associated tools computing management, Anjana Kushwaha for the IMGT/3Dstructure-DB entries biocuration, and Franc¸ois Ehrenmann for help with the 3-D structure figures. We are grateful to the IMGT® team for its expertise and constant motivation. We thank Cold Spring Harbor Protocol Press for the pdf of the IMGT Booklet available in IMGT references. IMGT® is a registered trademark of CNRS. IMGT® is member of the International Medical Informatics Association (IMIA) and a member of the Global Alliance for Genomics and Health (GA4GH). All figures are used with permission from M-P. Lefranc and G. Lefranc, LIGM, Founders and Authors of IMGT®, the international ImMunoGeneTics information system®, http://www.imgt.org).

Funding: IMGT® was funded in part by the BIOMED1 (BIOCT930038), Biotechnology BIOTECH2 (BIO4CT960037), fifth PCRDT Quality of Life and Management of Living Resources (QLG2-2000-01287), and sixth PCRDT Information Science and Technology (ImmunoGrid, FP6 IST-028069) programs of the European Union (EU). IMGT® received financial support from the GIS IBiSA, the Agence Nationale de la Recherche (ANR) Labex MabImprove (ANR-10-LABX-53-01), the Re´gion Occitanie Languedoc-Roussillon (Grand Plateau Technique pour la Recherche (GPTR), and BioCampus Montpellier. IMGT® is currently supported by the Centre National de la Recherche Scientifique (CNRS), the Ministe`re de l'Enseignement Supe´rieur, de la Recherche et de l'Innovation (MESRI), and the University of Montpellier.

### References

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Mol Biol 882:605–633. https://doi.org/10. 1007/978-1-61779-842-9\_33

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# Chapter 26

# ARResT/Interrogate Immunoprofiling Platform: Concepts, Workflows, and Insights

### Nikos Darzentas

### Abstract

ARResT/Interrogate was built within the EuroClonality-NGS working group to meet the challenge of developing and applying assays for the high-throughput sequence-based profiling of immunoglobulin (IG) and T-cell receptor (TR) repertoires. We herein present basic concepts, outline the main workflow, delve into EuroClonality-NGS-specific aspects, and share insights from our experiences with the platform.

Key words Immunoglobulin, Antigen receptors, IG, TR, Bioinformatics, Pipeline, Sequence analysis

### 1 Introduction

Immunoglobulins (IG) and T-cell receptors (TR) are highly adaptive molecular receptors involved in antigen recognition and enormously variable immunological responses. The advent of sequence-based profiling of IG and TR repertoires has been instrumental for understanding such responses, both normal and pathologic, the latter encompassing a wide range of diseases with an underlying immune cause. This unprecedented capability has also brought along novel and unique challenges [1]; this chapter will cover the bioinformatic one, from the perspective of the ARResT/ Interrogate immunoprofiling platform.

ARResT (abbreviation of Antigen Receptors Research Tool, http://bat.infspire.org/arrest) comprises a handful of tools developed over the years within focused groups. It originated in the days of Sanger sequence analysis toward delineating subsets of stereotyped antigen receptor sequences in chronic lymphocytic leukemia (CLL) [2, 3].

ARResT/Interrogate [http://arrest.tools/interrogate] was built from the grounds up within the EuroClonality-NGS working group [http://euroclonality.org] to initially support the development of the group's NGS assays and eventually to apply them in

Anton W. Langerak (ed.), Immunogenetics: Methods and Protocols, Methods in Molecular Biology, vol. 2453, https://doi.org/10.1007/978-1-0716-2115-8\_26, © The Author(s) 2022

research and clinical applications [4, 5]. ARResT/Interrogate is able to: automatically paired-end-join and concatenate input files; use spreadsheet sample sheets to make data and metadata available to itself and the user; identify, tag, trim, and report on primer sequences (and primer dimers); annotate and identify all rearrangement types (or 'junction classes') of all IG/TR loci; offer powerful interactive tools to the user for mining results; identify, filter, and use the EuroClonality-NGS central in-tube quality/quantification control (cIT-QC, or spike-ins) for abundance normalization; generally support EuroClonality-NGS assays, also with bespoke analytical and visual functionalities; and provide detailed logs and feedback to the user.

ARResT/Interrogate will be continuously updated, and therefore bioinformatic and user interface details included herein may not stay the same over time. We advise readers/users to seek the latest information on the ARResT/Interrogate browser [http:// arrest.tools/interrogate] and on the EuroClonality-NGS website [http://euroclonality.org]. For the same reason, we chose not to focus on application-specific methods, also because they are covered in other chapters in this book. Still, the general concepts and workflows included in this chapter should be considered safe, as should the Notes below from our years of experience both developing and using ARResT/Interrogate.

1.1 Design ARResT/Interrogate consists of the pipeline and the browser (its user interface). The browser features four main "panels" for logically organized and ordered steps (Fig. 1):

There is also the "HQ" panel that offers introductory text and specific notes and advice (in separate "tabs"). There are more panels to serve special applications, e.g., clonality assessment, but they are by default hidden and may be accessed by switching "user modes" with the widget on the top left (set at "Interrogate.simple" in Fig. 1).

Fig. 1 ARResT/Interrogate browser (user interface), with "panels," "tabs," and the "user mode" selection widget

1.2 Primers ARResT/Interrogate is able to identify, tag, trim, and report on primer sequences (and primer dimers), including making the results available for a fully interactive analysis. The trimming allows for less artificial sequence data to be processed more accurately and more efficiently, while the reporting allows for the primer-based results to be directly used for quality control and development. See also Notes 2 and 3 about trimming.

1.3 Rearrangements and Junctions ARResT/Interrogate is able to annotate and identify all rearrangement types of all IG/TR loci. We call these rearrangement types "junction classes." They include "complete," e.g., IG's VJ:Vh- (Dh)-Jh; "incomplete," e.g., TR's DJ:Db-Jb; and "other," e.g. IG's Vk-Kde or intron-Kde (Table 1). For junction classes with no biologically relevant junctional anchors (i.e., residues that define the CDR3 region, as per IMGT), we decided to introduce virtual ones—this enables consistent and informative results across all junction classes, assisting the user to focus on the most variable part of the rearrangement. For the D genes in DJ, VD, and DD incomplete junction classes, we use recombination signal sequence (RSS) heptamers: the last triplet of the heptamer in 5<sup>0</sup> and the first triplet of the heptamer in 3<sup>0</sup> . For the intron RSS in the IGK locus, we use a CCC triplet between the EuroClonality-NGS primer and the RSS heptamer, while for Kde in the IGK locus the final triplet after the RSS heptamer and before the EuroClonality-NGS primer is used. In the majority of cases, these anchors are far enough from the junctional point to allow for nucleotide trimming without affecting their presence, but ARResT/Interrogate is anyway able to report rearrangements even with the anchors trimmed or mutated. This is also true for normal anchors in complete rearrangements.

> Anchors overview: 5<sup>0</sup> side of junction.

V genes: C aa ¼ TG[CT] nt.

D genes: V aa ¼ GT[any] nt, the last triplet of the 5<sup>0</sup> heptamer.

intron: P aa ¼ CCC nt, a triplet between primer and heptamer.

30 side of junction.

J genes: W aa ¼ TGG nt or F aa ¼ TT[CT] nt.

D genes: H aa ¼ CA[CT] nt, the first triplet of the 3<sup>0</sup> heptamer.

Kde: R aa ¼ CGA nt, final triplet after heptamer and before primer.

### Table 1

Junction classes supported by ARResT/Interrogate and the EuroClonality-NGS amplicon and capture assays

### 2 Materials

	- 2. Sample sequences should be uploaded in FASTQ (preferably) or FASTA format, preferably also compressed in "gunzip" format (extension ".gz"). Also see Note 4.
	- 2. The ARResT/Interrogate sample sheet offers a number of predefined columns (i.e., ARResT/Interrogate expects these column names for the information to be used properly) and the possibility to add many others with flexible column names.
	- 3. The most important predefined columns (again, do not change the column names or use them for other purposes) are:
		- (a) Sample: required—unique for every sample and part or whole of the sample's sequence filenames.
		- (b) Cells: number of cells, based on amount of DNA of, e.g., patient, to be used for quantification.

Fig. 2 Sample sheet example. Red are predefined columns, and blue are flexible columns

### 3 Methods

3.1 A Basic Workflow

	- (a) Create a new analysis or select an existing one, otherwise the "default" will be used, which is OK. Also see Note 6.
	- (b) Upload sample sequences in compressed FASTQ/A format (see Subheading 2).
	- (c) The default scenario ("ARResT.profile") should work fine in any case. One may select a different user mode or pipeline scenario, especially when deploying EuroClonality-NGS assays (see Subheading 3.2).
	- (d) One may use own primer sequences by uploading them in uncompressed FASTA format and selecting them under "scenario options" (there are instructions on the user interface). In general, please study primers (e.g., see Notes 3, 7, and 8 as to why). Also see Note 9.
	- (a) Select results in the drop-down widget, select filtering level (see Note 11), click "load results".
	- (b) One may browse the run and sample reports, paying attention to quality control (QC) information, alarms (and our hints and tips for possible causes and solutions), basic numbers like percentage of reads with junction (see Note 7) that are also color-coded to provide visual feedback. Alarms include:
		- <sup>l</sup> Low number of reads "5<sup>0</sup> primed in R1" or "3<sup>0</sup> primed in R2", indicating wrong or missing primers, noisy reads, i.e., compromised primer alignment, etc.
		- <sup>l</sup> Low number of reads "3<sup>0</sup> primed in R1" or "5<sup>0</sup> primed in R2", indicating long or trimmed amplicons (with FR1 or FR2 primers for example) not covered by the sequenced read length, or wrong or missing primers.
		- <sup>l</sup> High number of reads "short"—sequence artifacts are generally an explanation, and if primers were used with the pipeline, one may also see an alarm about primer dimers.
	- (a) The main series of widgets are split into "select" on the left and "filter" on the right (Fig. 3).
	- (b) Note that if samples are "QC-failed" (see Note 12), they will not be available here by default; uncheck appropriate widget in "samples options" to include them back in.