**6. An application of SeqAnt 2.0: Exome sequencing to discover mutations affecting neutrophil function in very-early-onset pediatric Crohn's disease**

Children with very-early-onset (VEO) pediatric Crohn's disease (CD) are found to have high levels of neutrophil dysfunction. Neutrophils are an abundant type of white blood cell that play an essential role in innate immunity. We therefore hypothesized that children with very-early-onset Crohn's disease would exhibit an increased frequency of genetic mutations affecting neutrophil function. For an initial study we selected 45 VEO CD patients (median (range) age: 8.5 (5-10) years) with CBir1 sero-reactivity and moderate-to-severe clinical disease activity at diagnosis. We used the Roche NimbleGen SeqCap EZ Human Exome Library v2.0 on genomic DNA extracted from whole blood to capture the whole exome for each patient. Barcodes were used to prepare the libraries for whole-exome capture, which allowed us to sequence two whole exomes per lane of next-generation sequencing. We performed multiplexed 100 base-pair paired-end sequencing on an Illumina HiSeq 2000 instrument. We used PEMapper (Cutler and Zwick, in revision) to map raw sequence reads and identify variants sites relative to the ~30.8 Mb human exome reference sequence (NCBI37/hg19).

We then used SeqAnt to annotate all variant sites for functional significance, frequency, presence in databases like dbSNP, and measures of evolutionary conservation. Our central hypothesis was that early-onset (pediatric) forms of IBD would be substantially influenced by deleterious mutations found in the neutrophil pathway. If true, a straightforward evolutionary model of mutation-selection balance predicts that these variants ought to be rare in the general population, found at highly evolutionarily conserved sites, and have large effects on gene function. Thus, variants found in coding regions (replacement, nonsense, exonic insertions/deletions) that putatively alter protein structure and function will be the strongest candidates as contributors to IBD in pediatric patients. A number of lines of evidence specifically implicate loci involved in neutrophil functional pathways. We therefore proposed a strategy of first discovering variation in genes known to function in the neutrophil pathway, followed by direct functional testing of alleles from specific patients.

**disease**

(NCBI37/hg19).

of alleles from specific patients.

the mutant phenotype.

We demonstrated the use of this approach to find the causative mutations induced in four novel ENU lines identified from a recent ENU screen. In all four cases, after applying our method and combining with standard mapping data used to initially localize the variant to a chromosome, we found two or fewer putative mutations (and sometimes only a single one). Confirming that the variant was in fact causative was then easily achieved via standard segregation approaches. SeqAnt gave us the ability to rapidly annotate and screen variants of lesser interest (silent, UTR, intronic, intergenic), so we could instead focus our attention on those variants (replacement) that were most likely to account for

**6. An application of SeqAnt 2.0: Exome sequencing to discover mutations**

Children with very-early-onset (VEO) pediatric Crohn's disease (CD) are found to have high levels of neutrophil dysfunction. Neutrophils are an abundant type of white blood cell that play an essential role in innate immunity. We therefore hypothesized that children with very-early-onset Crohn's disease would exhibit an increased frequency of genetic mutations affecting neutrophil function. For an initial study we selected 45 VEO CD patients (median (range) age: 8.5 (5-10) years) with CBir1 sero-reactivity and moderate-to-severe clinical disease activity at diagnosis. We used the Roche NimbleGen SeqCap EZ Human Exome Library v2.0 on genomic DNA extracted from whole blood to capture the whole exome for each patient. Barcodes were used to prepare the libraries for whole-exome capture, which allowed us to sequence two whole exomes per lane of next-generation sequencing. We performed multiplexed 100 base-pair paired-end sequencing on an Illumina HiSeq 2000 instrument. We used PEMapper (Cutler and Zwick, in revision) to map raw sequence reads and identify variants sites relative to the ~30.8 Mb human exome reference sequence

We then used SeqAnt to annotate all variant sites for functional significance, frequency, presence in databases like dbSNP, and measures of evolutionary conservation. Our central hypothesis was that early-onset (pediatric) forms of IBD would be substantially influenced by deleterious mutations found in the neutrophil pathway. If true, a straightforward evolutionary model of mutation-selection balance predicts that these variants ought to be rare in the general population, found at highly evolutionarily conserved sites, and have large effects on gene function. Thus, variants found in coding regions (replacement, nonsense, exonic insertions/deletions) that putatively alter protein structure and function will be the strongest candidates as contributors to IBD in pediatric patients. A number of lines of evidence specifically implicate loci involved in neutrophil functional pathways. We therefore proposed a strategy of first discovering variation in genes known to function in the neutrophil pathway, followed by direct functional testing

**affecting neutrophil function in very-early-onset pediatric Crohn's**

SeqAnt 2012: Recent Developments in Next-Generation Sequencing Annotation 99

We have shown many useful features of SeqAnt and how it can be applied in a variety of experiments, yet we continue to develop SeqANt and plan to expand its functionalities going forward. Our goal is to create a one-stop online tool that readily accepts raw sequencing data and generates output through the annotation and functional characterization stages. Moreover, because our software and libraries are open source, they can be downloaded and optimized locally as part of a next-generation sequencing pipeline. SeqAnt is a truly dynamic application that is updated regularly to keep up with the constant flow of new sequencing data, genome assemblies, and improved annotation information

Genomic sequence annotation requires an up-to-date and comprehensive database of DNA sequence information for a given organism. Our first aim is to continue adding to our database organisms whose genomic information could be annotated. We plan on including several other mammals, vertebrates, invertebrates, and ultimately bacteria strains in the near future. This will give researchers a web application they can use to speed their genetic studies of such organisms. We are also in the process of updating the dbSNP information

Another area of future focus is to broaden the types of input and output files that SeqAnt could work with, while embracing standards in broad use in the bioinformatics community. We intend to include the capability to directly annotate .vcf files as a standard input file format. Presently, all our output files are either text files or BED files. We also plan to provide the option of having the annotation output in .vcf format. Furthermore, we intend to modify SeqAnt to make the .map and .ped files (PLINK formats) from the snp variant file, which will be beneficial for substructure analysis and several other analyses that can be

The inclusion of additional custom tracks from the UCSC browser to annotate for conserved and putatively functional sites will also be a future area of SeqAnt development. Our hope is that this will improve the effectiveness of downstream functional analysis. We also plan to have the application hosted in a cloud computing environment, side by side with other bioinformatics tools. This is relevant not only because of the wider accessibility it guarantees, but there is often the added ease of using other tools in the same environment to

SeqAnt was set up to be a dynamic application, and our improvements to this software make it possible to apply SeqAnt to different genomic variant analysis situations. Inevitable advances in sequencing technologies will spur continued demand for tools that can make sense out of the enormous raw sequence data generated, and we will work continually to make SeqAnt adaptable to these improvements and even more accessible to the wider public.

Great advances in targeted enrichment methods and DNA sequencing are beginning to allow individual investigators to sequence significant portions of many genomes; the

generate and modify input and output files from SeqAnt for further analysis.

available from public databases like those found at the UCSC Genome Browser.

**7. Future directions**

contained in the SeqAnt database.

done using PLINK.

**8. Conclusion**

**Table 7.** Genetic variants found in genes that regulate neutrophil function.

We used SeqAnt to annotate all the sequence variations from the 45 exomes and identified a total of 60,682 variant sites of interest in coding regions (54,313 replacement SNPs, 2953 indels covering 6369 bases). For our exploratory genome-wide analysis of SNPs, we restricted our analysis to those variants with phyloP scores greater than 2.0, which corresponds to the top 1% of conserved sites in the human genome. Remaining were 12,575, of which 51% (6490) were not cataloged in dbSNP 132 and might constitute novel mutations contributing to early-onset IBD. We then restricted our analysis to 33 neutrophil genes. Table 6 contains a list of these 33 neutrophil genes with the number of rare putative functional variants (replacement SNPs or exonic indels). These variants are to be followed up using direct functional assays to assess function. Again, SeqAnt enabled us to rapidly annotate all variants, ignore those variants of lesser interest, and focus our attention on those most likely to contribute to the VEO CD in our sequenced patients.