Sequence data has been deposited in Genbank. Accession numbers for HCV sequences are MT632105-MT632194. Accession numbers for HBV sequences are MT622522-MT622525. Accession numbers for HDV sequences are MT583788-MT583813.

Introduction {#sec001}
============

Viral hepatitis represents a significant global health burden, particularly as many cases lead to cirrhosis and liver cancer, which can be fatal. Viral surveillance is essential to understand prevalence and determine appropriate public health measures. In particular, hepatitis B virus (HBV), hepatitis C virus (HCV), and hepatitis delta virus (HDV) are major burdens on human health worldwide that must be monitored. Given the large numbers of genotypes and sub-genotypes for all three of these hepatitis viruses, the wide spectrum of genetic diversity they encompass brings the inherent potential to evade detection by diagnostic tests \[[@pone.0236046.ref001]--[@pone.0236046.ref003]\]. While generation of partial sequences for a given viral genome by Sanger sequencing methods has provided the classical means of surveillance, there are several drawbacks to consider with this approach. First, sub-genomic sequences can underestimate true diversity in a population that may contain recombinant strains. Second, focused sequencing in one region may not adequately inform diagnostic assay development that targets un-sequenced regions of the virus. Third, this method requires the design of primers for amplification that may not work for all genotypes. Alternatively, these issues can be avoided by pursuing complete genome sequencing.

The application of next-generation sequencing (NGS) to obtain full genomes is an invaluable epidemiological tool for tracking where strains have traveled, identifying transmission networks, spotting an outbreak, and monitoring for mismatches in diagnostics \[[@pone.0236046.ref004]--[@pone.0236046.ref008]\]. Unbiased metagenomics using random priming permits any pathogen to be detected in patient specimens, including viruses, bacteria, parasites, and fungi \[[@pone.0236046.ref009], [@pone.0236046.ref010]\]. However, abundant host background reads can obscure the presence of many of these agents and these methods are challenged by small, diverse, low copy, and or highly structured hepatitis viruses, such as HBV and HDV. Target enrichment offers an opportunity to significantly boost sensitivity, resulting in improved coverage and higher confidence data \[[@pone.0236046.ref011]\]. Single stranded DNA probes can be hybridized to reads within mNGS libraries to selectively capture and amplify viral sequences and is particularly useful for samples with low viral loads \[[@pone.0236046.ref012], [@pone.0236046.ref013]\]. A post-library capture step (e.g. xGen) has successfully been deployed for blood borne RNA viruses in the 10 kb-range length, like HIV and HCV, although it has not yet been evaluated for small viruses like HBV (3.2 kb) and HDV (1.6 kb) \[[@pone.0236046.ref013], [@pone.0236046.ref014]\].

Molecular surveillance of hepatitis viruses is particularly important in Israel, where immigration and travel rates are high. The countrywide prevalence of chronic HBV (HBsAg+) is estimated at 1.75%, with HDV co-infection at 6.5--7.1% \[[@pone.0236046.ref015], [@pone.0236046.ref016]\]. In general, the eastern Mediterranean region has the highest levels of HCV at nearly 2.5%, but large studies specific to Israel puts country prevalence at 0.5--0.9%, largely due to eastern European and Russian immigrants from the former Soviet Union \[[@pone.0236046.ref016]--[@pone.0236046.ref019]\]. While the incidence of newly-diagnosed cases has been declining and most infections are Genotype 1b, Israel is home to numerous immigrant populations with the capacity to import new strains of HCV \[[@pone.0236046.ref019], [@pone.0236046.ref020]\]. To date, circulating strains for all three viruses have largely been determined by sub-genomic sequencing \[[@pone.0236046.ref015], [@pone.0236046.ref021]\]. Therefore, we applied metagenomic and two target enrichment NGS techniques (xGen and Pan viral probes) to study epidemiological trends of HCV-infected and HBV/HDV co-infected individuals in Israel.

Materials and methods {#sec002}
=====================

Specimens {#sec003}
---------

Patient plasma was collected from individuals seeking treatment at the Israeli National HIV and Viral Hepatitis Reference Center (NHRL). Plasma samples were remains from patients referred to the laboratory for HDV viral load measurements (HBV-HDV co-infections) or for HCV RAS analysis. HDV positivity was defined by real-time PCR \[[@pone.0236046.ref015]\]. All specimens were de-identified and IRB approval was granted for mNGS. Patients were exempt from signing a consent form by the local IRB (approval numbers for HCV and for HDV are 9329-12-SMC and 2890-15-SMC, respectively). Specimens in the HBV genotype panel were purchased from Boca Biolistics (Pompano Beach, FL) or collected from volunteer blood donors in Spain and Cameroon (HBV/HDV co-infected) to demonstrate probe efficacy on diverse strains. Spanish samples were selected from the Biobank of the Catalonia Blood Bank and de-identified. Participants were recruited from Barcelona and provided written, informed consent. IRB approval was obtained from the Vall d'Hebron Hospital Ethics Committee. Cameroonian samples were from two HBV surveillance studies conducted from 2010--2016 where participants were recruited from blood bank donors, hospitals, and chest clinics in the urban centers of Douala and Yaoundé. Written informed consent was provided and plasma was collected anonymously. Studies were approved by the Ministry of Health of Cameroon, the Cameroon National Ethical Review Board, and the Faculty of Medicine and [Biomedical Science](https://www.sciencedirect.com/topics/medicine-and-dentistry/biomedical-discipline) IRB. Israeli HDV samples were collected from October 2014-Jul 2017 and HCV samples were collected from June 2015-Jul 2017. Only DAA naïve HCV-RNA positive patients and HBV+ HDV patients with detectable HDV RNA were recruited into the study. Samples were randomly collected from these patients and can be considered representative of a larger HCV positive or HBV/HDV dually positive population. Relevant demographic details are included in [S1 Table](#pone.0236046.s003){ref-type="supplementary-material"}. Negative controls were normal human plasma (NHP) and positive controls consisted of purified stocks or infected plasma in which virus or bacteria was diluted into NHP to log 4.0 copies/ml. Parvovirus B19, HHV-5, VZV, Influenza A, Adenovirus 7, and Chlamydia stocks were from Exact Diagnostics (Dallas, TX); HIV, HBV, and HCV originated from samples sourced in Cameroon and Spain.

Viral loads {#sec004}
-----------

HDV and HBV viral loads (22 samples) were determined by quantitative PCR in Israel \[[@pone.0236046.ref015]\]. HCV and HBV viral loads were approximated at Abbott using a semi-quantitative multiplex PCR. This research-use assay simultaneously detects HBV, HCV, HIV-1, and HIV-2. Quantitation was extrapolated from relative Ct values of diluted standards.

Specimen pretreatment and extraction {#sec005}
------------------------------------

Plasma specimens were pre-treated with Ultra-pure benzonase (Sigma, St. Louis MO) for 3 hrs at 37°C and extracted on an *m*2000*sp* (Abbott Laboratories, Des Plaines IL) using the RNA/DNA protocol (500 μl input/50 μl elute).

mNGS library production {#sec006}
-----------------------

Metagenomic libraries (mNGS) were prepared and quantified essentially as described \[[@pone.0236046.ref013]\]. Briefly, total nucleic acid was concentrated to 10 μl with RNA Clean and Concentrator-5 spin columns (Zymo Research, CA) and RNA was reverse transcribed with random primers using Superscript III (SSRTIII) 1^st^ Strand reagents (Life Technologies), followed by 2^nd^ strand synthesis with Sequenase V2.0 T7 DNA pol (Affymetrix). Double stranded DNA/cDNA was recovered with DNA Clean and Concentrator-5 spin columns (Zymo Research) and -barcoded with Nextera XT indices lacking 5' biotin tags using 24 cycles of amplification (IDT, Coralville IA; Illumina, Carlsbad CA). Nextera libraries were purified with Agencourt AMPpure XP beads (Beckman Coulter) and quantified by a 2200 TapeStation (Agilent) and Qubit fluorometer (Life Technologies).

Design of HBV and HDV xGen probe sets {#sec007}
-------------------------------------

Probe sets were designed essentially as described previously for HIV, with each probe 120 nt in length \[[@pone.0236046.ref013]\]. Briefly, 60 HBV complete genomes including genotypes A-I were aligned in BioEdit. A single consensus (3223 nt) was extracted, with degenerate bases replaced by specific nucleotides, and an initial 53 probes at 2X coverage (e.g. 60 nt overlap) were designed from this sequence. The alignment was surveyed in 120 nt windows to identify regions with \<80% identity and include any genotype-specific fragments each 120, 239, or 257 nt in length. An additional 25 probes with 1X coverage (e.g. 1 nt overlap) were designed for a total of 78 HBV probes. For HDV, sequence diversity is much greater, requiring probes designed from separate consensus genomes of genotypes 1--8. Genomes ranged from 1293 nt-1693 nt in length, resulting in 109 probes at approximately 1X coverage. An additional 28 HDV probes in diverse regions were included for a total of 137. HBV and HDV probe sets were combined into one reagent (215 probes) for hybridization.

xGen reagent synthesis and protocol {#sec008}
-----------------------------------

120 nt probe stocks (3 pmol/probe) modified with a 5' biotin tag, Nextera barcoding primers lacking a biotin label, and blocking oligos complementary to Nextera Set A i5 and i7 index primers (1 μl/rxn) were all synthesized at IDT. Hybridizations, capture by streptavidin beads, washes and library amplification were essentially as described \[[@pone.0236046.ref013]\]. Here, after the initial 12 cycles of amplification and elution off streptavidin beads, a repeat KAPA amplification of 10 cycles was performed and libraries were visualized on a 2200 TapeStation and quantified with a Qubit fluorometer using the dsDNA high-sensitivity kit.

Pan-viral enrichment {#sec009}
--------------------

SSRTIII-Nextera (mNGS) libraries from the HBV diversity panel were pooled together for enrichment with the commercially available Pan Viral probe set (n\>600,000 probes). Hybridization and amplification steps were followed according to manufacturer instructions (Twist Biosciences, San Francisco, CA). A complete description of the procedure is included in the [S1 Appendix](#pone.0236046.s001){ref-type="supplementary-material"}. We note that after DNA purification of library amplification on streptavidin beads, a second 15 cycle PCR 'off the beads' was performed.

Next generation sequencing and analysis {#sec010}
---------------------------------------

HCV dual barcoded libraries were multiplexed according to viral load and either sequenced on a single HiSeq run (n = 72) or batched into 4 runs of 7 (n = 28) on a MiSeq to achieve sufficient read depth. HBV and HDV mNGS libraries (n = 26) were divided over 4 MiSeq runs. xGen libraries were pooled together for a single, separate run, since these share the same barcodes as mNGS libraries. NGS data analysis was performed as described with CLC Genomics Workbench 9.0 software (CLC bio/Qiagen, Aarhus Denmark) and SURPI \[[@pone.0236046.ref022], [@pone.0236046.ref023]\]. Raw data was initially mapped to multiple reference sequences to determine the genotype with the greatest identity. An iterative approach was used to derive the final sequence, using the initial consensus as the reference to refine the consensus upon remapping. To detect possible contaminating reads from barcode hopping, raw data from each sample was individually mapped to the consensus sequences of other samples sequenced on the same run, removing any with ≥99% identity. Unmapped reads (e.g. unique to the sample of interest) were collected and realigned to generate the final consensus.

Phylogenetic analysis {#sec011}
---------------------

Multiple sequence alignments were performed in MegAlign Pro (Lasergene, DNASTAR Inc., Madison WI) using the MUSCLE algorithm and manually edited in BioEdit Sequence Alignment Editor (v 7.2.5) \[[@pone.0236046.ref024], [@pone.0236046.ref025]\]. Neighbor-Joining phylogenetic inference was performed using PHYLIP (version 3.5c; J. Felsenstein, University of Washington, Seattle, WA). Evolutionary distances were estimated with Dnadist (Kimura two-parameter method) and phylogenetic relationships were determined by Neighbor (neighbor-joining method). Branch reproducibility of trees was evaluated using Seqboot (100 replicates) and Consense. Trees were visualized using FigTree (version 1.4.2; A. Rambaut, Institute of Evolutionary Biology, University of Edinburgh, Edinburgh). Sequences basal to a genotype branch were examined for recombination breakpoints with SimPlot (version 3.5.1; S. Ray, Johns Hopkins University, Baltimore, MD).

Nucleotide sequence accession numbers {#sec012}
-------------------------------------

Full-length sequences were submitted to GenBank under the following accessions: HCV (MT632105-MT632194), HBV (MT622522-MT622525), and HDV (MT583788-MT583813).

Results {#sec013}
=======

Full genome coverage of HCV without enrichment {#sec014}
----------------------------------------------

A total of 99 HCV RNA positive specimens were collected at the NHRL. A semi-quantitative PCR of libraries indicated that the average viral load was log 5.4 copies/ml, consistent with previously observed trend of most untreated HCV patients having viral loads \>4 log IU/ml \[[@pone.0236046.ref026]\]. A median of 8.3 million reads were obtained per library ([S2 Table](#pone.0236046.s004){ref-type="supplementary-material"}). Full length HCV sequences (≥97% coverage) were obtained for 82 individuals and ≥90% of the genome was determined for 89/99 samples ([Table 1](#pone.0236046.t001){ref-type="table"}). Five of these had 62--86% coverage despite viral loads ≥ log 5, whereas the remaining 5 with \< 50% coverage had either a viral load \< log 5 or produced very few total mNGS reads. Numbers of reads and the level of coverage depth provide an indication of the confidence in the consensus sequences. HCV reads were normalized to total reads and expressed as reads/million (rpm). The majority (93%) of mNGS libraries had ≥100 HCV rpm, with most (63%) having ≥1000 HCV rpm ([Table 1](#pone.0236046.t001){ref-type="table"}). A median of 11,466 HCV reads were obtained per library. Due to the high coverage obtained for HCV specimens, no further enrichment was necessary.

10.1371/journal.pone.0236046.t001

###### HCV mNGS metrics.

![](pone.0236046.t001){#pone.0236046.t001g}

  \% Genome Coverage   Total individuals   HCV Reads/Million   Total individuals   HCV Genotype   Total individuals
  -------------------- ------------------- ------------------- ------------------- -------------- -------------------
  ≥97%                 82                  10,000--100,000     12                  1a             19
  90--97%              7                   1,000--10,000       51                  1b             68
  50--90%              5                   100--1,000          30                  2c             1
  20--50%              2                   10--100             4                   3a             8
  \<20%                3                   \<10                2                   n.d.           3

The first two columns report the breakdown of samples according to genome coverage. The second two columns report the breakdown of samples by HCV reads per million. The third two columns report the breakdown of samples by genotype.

Full-length consensus sequences were added to a complete alignment of HCV references and analyzed within neighbor joining phylogenetic trees ([Fig 1](#pone.0236046.g001){ref-type="fig"}). HCV genotypes were primarily 1b (68%) and 1a (19%), with minor representation of genotypes 2c (1%) and 3a (8%) ([Table 1](#pone.0236046.t001){ref-type="table"}). Branch lengths indicated all Israeli strains (red) were unique; nevertheless, those branching with high bootstrap values were aligned pairwise to rule out cross-contamination. These sequences shared only 91--94% identity and none shared a barcode. The basal branching pattern of Genotype 1b strain (sample 25--2000618) was evaluated in Simplot but did not show evidence of recombination. Genotype 3a sequences had highest identity (≤95%) with strains originating in Western Europe. These strains generally came from male, injection drug users with a median age of 49 ± 7 years, and not from a specific country. For the 7 partial genomes (20--90% coverage), separate trees were generated from gap-stripped alignments and there was still sufficient sequence to allow for classification.

![The majority of Israeli strains are Genotype 1b.\
Neighbor-joining phylogenetic tree of full-length HCV sequences. New Israeli sequence branches are in red and reference strains are in black labeled with accession numbers. Bootstrap values of nodes \>70 are shown.](pone.0236046.g001){#pone.0236046.g001}

mNGS output for all 99 original samples were analyzed with the SURPI metagenomics pipeline to probe for additional viruses, such as HPgV-2, a recently appreciated co-infection of HCV \[[@pone.0236046.ref023], [@pone.0236046.ref027]\]. A variety of viruses (e.g. HIV, HBV, VZV, Influenza, adenovirus, etc.) spiked into normal human plasma each at log 4.0 copies/ml served as a positive control and all were detected. Coverages and reads per million values were similar for the three positive controls included in the separate sequencing runs of HCV libraries ([S1 Fig](#pone.0236046.s002){ref-type="supplementary-material"}). Human pegivirus-1 (GBV-C) reads were enriched in 5 individuals. No additional blood borne agents or viruses besides HCV were found in any of the 99 mNGS patient libraries.

Target enrichment of HBV and HDV {#sec015}
--------------------------------

HDV antibody positive HBV/HDV specimens with detectable HDV RNA by quantitative PCR were prepared in the same manner as HCV for mNGS. Preliminary viral loads measured independently in Israel and at Abbott indicated that levels of co-infecting HBV were very low, with only a minority of the 26 samples registering \<33 Ct (1--3 log copies/ml) and most without detectable levels of DNA \[[@pone.0236046.ref015]\]. Consequently, HBV coverage by mNGS was sparse, if not completely absent ([Table 2](#pone.0236046.t002){ref-type="table"}). HDV by contrast had higher viral loads (4--7 log copies/ml) and was readily detected in 100% of samples, with coverages ranging from 20--100%.

10.1371/journal.pone.0236046.t002

###### Israeli HBV/HDV mNGS and xGen metrics.

![](pone.0236046.t002){#pone.0236046.t002g}

  Specimen ID   Virus   Genotype     Viral load / *Ct*   Final sequence length (nt)   Bootstrap   Library Type   Total Reads   HBV or HDV reads   \% virus reads   Genome Coverage%   Avg coverage depth
  ------------- ------- ------------ ------------------- ---------------------------- ----------- -------------- ------------- ------------------ ---------------- ------------------ --------------------
  2000166       HBV     B2           \-                  203                          89          mNGS           526,669       211                0.04%            6%                 41
  \-            \-      \-           *not detected*      \-                           \-          xGen           8,105         2                  0.02%            2%                 1
  \-            HDV     1            \-                  654                          100         mNGS           599,442       10                 0.002%           48%                1.48
  \-            \-      \-           \-                  \-                           \-          xGen           9,056         1,594              17.60%           40%                138.03
  2000208       HBV     nd           148.45              nd                           nd          mNGS           1,221,334     0                  0%               0%                 0
  \-            \-      \-           *34*.*64*           \-                           \-          xGen           154,910       0                  0%               0%                 0
  \-            HDV     1            1150000             1541                         100         mNGS           1,221,334     6,859              0.56%            94%                406.72
  \-            \-      \-           \-                  \-                           \-          xGen           154,910       137,271            88.61%           96%                8390.27
  2000234       HBV     nd           \-                  nd                           nd          mNGS           354,688       20                 0.01%            1%                 0.17
  \-            \-      \-           \-                  \-                           \-          xGen           5,856         2                  0.03%            1%                 1.68
  \-            HDV     1            248000              894                          100         mNGS           354,688       189                0.05%            50%                21.37
  \-            \-      \-           \-                  \-                           \-          xGen           5,856         1,124              19.19%           62%                95.74
  2000236       HBV     nd           5.09                185                          nd          mNGS           2,006,742     434                0.02%            2%                 104
  \-            \-      \-           *37*.*11*           \-                           \-          xGen           970,261       226,990            23.39%           4%                 71192
  \-            HDV     1            2140000             1600                         100         mNGS           2,328,818     12,717             0.55%            94%                896.85
  \-            \-      \-           \-                  \-                           \-          xGen           1,006,436     440,930            43.81%           96%                32627.18
  2000319       HBV     nd           0.98                nd                           nd          mNGS           827,658       0                  0%               0%                 0
  \-            \-      \-           *42*.*94*           \-                           \-          xGen           11,286        0                  0%               0%                 0
  \-            HDV     1            54750               1419                         100         mNGS           827,658       43                 0.01%            79%                4.1
  \-            \-      \-           \-                  \-                           \-          xGen           11,286        77                 0.68%            79%                6.78
  2000320       HBV     nd           Indeter.            58                           nd          mNGS           61,370        2                  0.003%           1%                 1
  \-            \-      \-           \-                  \-                           \-          xGen           5,556         0                  0%               0%                 0
  \-            HDV     1            173563              1119                         100         mNGS           65,364        5                  0.01%            20%                2.18
  \-            \-      \-           \-                  \-                           \-          xGen           5,556         4,342              78.15%           30%                1240.82
  2000322       HBV     nd           410.94              138                          nd          mNGS           1,505,468     1                  0.0001%          2%                 1
  \-            \-      \-           *33*.*08*           \-                           \-          xGen           2,349,944     1                  0.0000%          2%                 0.01
  \-            HDV     1            40000               1641                         100         mNGS           1,505,468     6,048              0.40%            95%                369.02
  \-            \-      \-           \-                  \-                           \-          xGen           2,349,944     1,144,523          48.70%           98%                84759.81
  2000324       HBV     nd           \-                  85                           nd          mNGS           1,068,387     120                0.01%            2%                 62
  \-            \-      \-           *not detected*      \-                           \-          xGen           312,189       7,363              2.36%            2%                 3561
  \-            HDV     1            \-                  1210                         100         mNGS           1,154,948     467                0.04%            87%                32.5
  \-            \-      \-           \-                  \-                           \-          xGen           313,010       242,129            77.36%           85%                15973.34
  2000372       HBV     nd           \-                  139                          nd          mNGS           1,933,208     380                0.02%            2%                 26
  \-            \-      \-           *not detected*      \-                           \-          xGen           2,167,747     26                 0.001%           4%                 6
  \-            HDV     1            21800000            1642                         100         mNGS           2,215,648     62,193             2.81%            90%                4319.08
  \-            \-      \-           \-                  \-                           \-          xGen           2,236,978     1,060,712          47.42%           98%                72427.13
  2000563       HBV     nd           16.58               66                           nd          mNGS           522,433       111                0.02%            2%                 25
  \-            \-      \-           *35*.*57*           \-                           \-          xGen           79,266        0                  0%               0%                 0
  \-            HDV     1            1.4x10(8)           1471                         100         mNGS           580,988       1,747              0.30%            90%                133.92
  \-            \-      \-           \-                  \-                           \-          xGen           79,266        61,293             77.33%           90%                4422.76
  2000570       HBV     D, basal     Indeter.            449                          75          mNGS           120,604       0                  0%               0%                 0
  \-            \-      \-           \-                  \-                           \-          xGen           165,848       904                0.55%            14%                96
  \-            HDV     1            1.24x10(9)          1676                         100         mNGS           120,604       11,661             9.67%            90%                1039.97
  \-            \-      \-           \-                  \-                           \-          xGen           165,436       96,813             58.52%           98%                7365.02
  2000741       HBV     nd           \-                  nd                           nd          mNGS           376,532       0                  0%               0%                 0
  \-            \-      \-           *41*.*06*           \-                           \-          xGen           2,400         0                  0%               0%                 0
  \-            HDV     1            \-                  1103                         100         mNGS           376,532       32                 0.01%            55%                4.1
  \-            \-      \-           \-                  \-                           \-          xGen           2,400         15                 0.63%            40%                2.24
  2000742       HBV     A1           218.75              1810                         79          mNGS           2,171,531     38                 0.002%           13%                1
  \-            \-      \-           *35*.*07*           \-                           \-          xGen           33,656        1,361              4.04%            54%                26
  \-            HDV     1            44800               1510                         100         mNGS           4,415,132     1,180              0.03%            93%                43.65
  \-            \-      \-           \-                  \-                           \-          xGen           37,374        28,799             77.06%           90%                1380.97
  2000744       HBV     D            984.64              1779                         100         mNGS           2,342,415     179                0.01%            5%                 1
  \-            \-      \-           *32*.*80*           \-                           \-          xGen           4,680,211     1,697              0.04%            55%                58
  \-            HDV     1            87400               1634                         100         mNGS           2,835,334     17,587             0.62%            94%                1286.69
  \-            \-      \-           \-                  \-                           \-          xGen           4,767,854     1,734,301          36.37%           98%                123135.8
  2000999       HBV     B                                514                          98          mNGS           4,026,934     52                 0.001%           28%                2.02
  \-            \-      \-           *not detected*      \-                           \-          xGen           2,155,897     169                0.01%            15%                26
  \-            HDV     1            1150000             1675                         100         mNGS           4,026,934     22,098             0.55%            99%                1426.64
  \-            \-      \-           \-                  \-                           \-          xGen           2,218,134     1,279,533          57.69%           99%                84602.53
  2001063       HBV     nd           \-                  173                          nd          mNGS           49,274        0                  0%               0%                 0
  \-            \-      \-           *not detected*      \-                           \-          xGen           31,248        3                  0.01%            5%                 1
  \-            HDV     1            3.03x10(7)          1531                         100         mNGS           49,274        3,141              6.37%            91%                267.68
  \-            \-      \-           \-                  \-                           \-          xGen           32,590        11,334             34.78%           91%                920.13
  2001073       HBV     A2           \-                  2038                         100         mNGS           2,371,838     127                0.01%            10%                1
  \-            \-      \-           *25*.*09*           \-                           \-          xGen           426,656       6,000              1.41%            63%                174
  \-            HDV     1            1.26x10(6)          1675                         100         mNGS           2,918,854     11,643             0.40%            94%                790.47
  \-            \-      \-           \-                  \-                           \-          xGen           464,694       203,456            43.78%           100%               12967.16
  2001149       HBV     (B/D dual)   \-                  2346                         100         mNGS           2,167,544     110                0.01%            8%                 1
  \-            \-      \-           *29*.*18*           \-                           \-          xGen           3,467,598     756                0.02%            69%                20
  \-            HDV     1            \-                  1677                         100         mNGS           2,523,132     151,554            6.01%            99%                10179.87
  \-            \-      \-           *16*.*8*            \-                           \-          xGen           3,775,760     1,236,439          32.75%           99%                84803.89
  2001167       HBV     nd           \-                  nd                           nd          mNGS           3,613,434     47                 0.001%           22%                2.23
  \-            \-      \-           *not detected*      \-                           \-          xGen           761,172       856                0.11%            2%                 568.18
  \-            HDV     1            \-                  1677                         100         mNGS           3,613,434     30,647             0.85%            96%                2109.39
  \-            \-      \-           *22*.*2*            \-                           \-          xGen           761,172       340,999            44.80%           98%                23133.83
  2001178       HBV     A            \-                  530                          76          mNGS           2,601,041     515                0.02%            1%                 1
  \-            \-      \-           *not detected*      \-                           \-          xGen           21,182        568                2.68%            16%                75
  \-            HDV     1            \-                  1526                         100         mNGS           3,387,460     669                0.02%            95%                45.54
  \-            \-      \-           \-                  \-                           \-          xGen           23,480        12,194             51.93%           91%                841.31
  2001190       HBV     D basal      \-                  486                          75          mNGS           3,579,444     129                0.004%           18%                7.38
  \-            \-      \-           *not detected*      \-                           \-          xGen           2,662,835     38                 0.001%           15%                4
  \-            HDV     1            \-                  1683                         100         mNGS           3,579,444     130144             3.64%            100%               8261.17
  \-            \-      \-           *17*.*17*           \-                           \-          xGen           2,913,414     1382215            47.44%           100%               88874.32
  2001210       HBV     nd           \-                  nd                           nd          mNGS           4,217,318     56                 0.001%           34%                1.73
  \-            \-      \-           *not detected*      \-                           \-          xGen           99,342        0                  0%               0%                 0
  \-            HDV     1            \-                  1538                         100         mNGS           4,217,318     1232               0.03%            92%                85.4
  \-            \-      \-           *24*.*7*            \-                           \-          xGen           99,342        59937              60.33%           93%                4347.25
  2001212       HBV     nd           39 IU/ml            239                          nd          mNGS           1,866,015     65                 0.003%           7%                 2
  \-            \-      \-           \-                  295                          40          xGen           851,225       681                0.08%            9%                 147
  \-            HDV     1            5.3x10(6)           1521                         100         mNGS           2,377,234     2694               0.11%            89%                198.87
  \-            \-      \-           \-                  \-                           \-          xGen           866,568       509708             58.82%           92%                34959.53
  2001222       HBV     nd           \-                  110                          nd          mNGS           1,610,506     16                 0.001%           8%                 2.11
  \-            \-      \-           *not detected*      \-                           \-          xGen           522,338       1079               0.21%            3%                 266
  \-            HDV     1            9.11x10(6)          1423                         100         mNGS           1,610,506     1929               0.12%            89%                157.42
  \-            \-      \-           \-                  \-                           \-          xGen           532,072       343668             64.59%           90%                26124.36
  2001228       HBV     nd           \-                  244                          nd          mNGS           5,186,758     56                 0.001%           32%                1.91
  \-            \-      \-           *not detected*      \-                           \-          xGen           3,975,042     4                  0.0001%          8%                 0.0127
  \-            HDV     1            7.52x10(7)          1683                         100         mNGS           5,186,758     73594              1.42%            100%               4697.18
  \-            \-      \-           \-                  \-                           \-          xGen           3,975,042     1393290            35.05%           100%               91278.85
  2001234       HBV     D            \-                  494                          86          mNGS           2,821,662     20                 0.001%           13%                1.7
  \-            \-      \-           *not detected*      \-                           \-          xGen           14,849        44                 0.30%            15%                9
  \-            HDV     1            1.66x10(5)          1509                         100         mNGS           2,821,662     230                0.01%            86%                15.5
  \-            \-      \-           \-                  \-                           \-          xGen           16,784        7154               42.62%           90%                499.78

Summary of results for HBV and HDV sequencing of Israeli specimens.

To improve target identification, 5' biotin-tagged xGen probes each 120 nt in length were designed to include the entire spectrum of HBV (genotypes A-I) and HDV (genotypes 1--8) genetic diversity. Our present samples only required enrichment for HBV, but given that probes to other strains or viruses do not interfere with each other, we combined these into one probe set (n = 215) for future use ([Fig 2A](#pone.0236046.g002){ref-type="fig"}). The HBV-HDV xGen probe methodology was first validated on 12 unrelated mono- and co-infected samples from a variety of countries that are known to include a diverse array of genotypes. Since xGen libraries derive from mNGS libraries and thus have the same barcodes, they were sequenced on different runs to be able to discriminate the source of reads. Coverage plots of an HBV genotype F1 (1007-HBV-0036) from Peru with a viral load of 3.6 log copies/ml demonstrate the dramatic improvement with probe enrichment ([Fig 2B](#pone.0236046.g002){ref-type="fig"}, left). Only 6% coverage at 1X depth was obtained by mNGS, which increased to 90% at 27X depth with xGen. A modest improvement was observed for an HDV genotype 1 with a 4.59 log cp/ml viral load (U160953A), increasing from 90% to 94% coverage, but primarily with an increase in depth ([Fig 2B](#pone.0236046.g002){ref-type="fig"}, right). Note that in this sample, HBV (genotype B2) present at 3.24 log IU/ml increased from 8% coverage with mNGS to 84% with xGen ([Fig 2C](#pone.0236046.g002){ref-type="fig"}, [Table 3](#pone.0236046.t003){ref-type="table"}).

![HBV/HDV-xGen greatly enhances sensitivity.\
(A) HBV/HDV xGen probes tile HBV genotypes A-I with 2X coverage and HDV genotypes 1--8 with 1X coverage. (B) Representative genome coverage plots of NGS data for HBV (left) and HDV (right) strains from Cameroon. For HBV, mNGS reads are shown in orange and xGen reads in blue. For HDV, mNGS reads are shown in green and xGen reads in purple. (C) Histograms of coverage (top) and reads/million (bottom) on co-infections or HBV-only infections. Country, genotype, and viral load are listed beneath each plot with the same color scheme as in Fig 2B.](pone.0236046.g002){#pone.0236046.g002}

10.1371/journal.pone.0236046.t003

###### HBV and HDV diversity panel results.

![](pone.0236046.t003){#pone.0236046.t003g}

  Specimen ID     Country             Virus   Genotype   Final length (nt)   Boot strap   VL (log IU/mL)   Library Type   Total Reads   Viral reads   Reads per million   Percent Genome Coverage   Avg coverage depth
  --------------- ------------------- ------- ---------- ------------------- ------------ ---------------- -------------- ------------- ------------- ------------------- ------------------------- --------------------
  23--23          Cameroon            HBV     E          440                 99           1.66             mNGS           3,963,160     594           150                 3%                        1
  \-              \-                  \-      \-         \-                  \-           \-               xGen           2,284,203     95            42                  14%                       16
  \-              \-                  HDV     6          1685                100          4.89             mNGS           4,583,420     73,543        16045               100%                      4863
  \-              \-                  \-      \-         \-                  \-           \-               xGen           2,322,206     2,270,392     977688              100%                      137586
  U160953A        Cameroon            HBV     B2         2731                98           3.24             mNGS           1,624,386     10            6                   8%                        1
  \-              \-                  \-      \-         \-                  \-           \-               xGen           3,160,577     191           60                  84%                       3
  \-              \-                  HDV     1          1554                100          4.59             mNGS           1,624,386     3,074         1892                90%                       212
  \-              \-                  \-      \-         \-                  \-           \-               xGen           3,205,212     2,926,340     912994              94%                       204100
  \-              \-                  \-      \-         \-                  \-           \-               Pan viral      1,162,542     390,959       336297              90%                       369999
  U22343          Cameroon            HBV     A3         3221                100          nd               mNGS           1,583,088     859           543                 100%                      19
  \-              \-                  \-      \-         \-                  \-           \-               xGen           3,548,047     879,717       247944              100%                      19624
  \-              \-                  HDV     8          1676                100          6.98             mNGS           1,904,026     2,136         1122                100%                      119
  \-              \-                  \-      \-         \-                  \-           \-               xGen           3,628,496     2,666,770     734952              100%                      169534
                                                                                                                                                                                                    
  0111A838        USA                 HBV     B2         3215                100          5.92             mNGS           1,531,143     173           113                 92%                       3
  \-              \-                  \-      \-         \-                  \-           \-               xGen           55,902        44,229        791188              100%                      923
  \-              \-                  \-      \-         \-                  \-           \-               Pan viral      690,304       40,003        57950               100%                      1768
  1010A324        USA                 HBV     C1         2814                100          4.18             mNGS           1,354,611     14            10                  3%                        1
  \-              \-                  \-      \-         \-                  \-           \-               xGen           9,880         1,034         104656              87%                       26
  \-              \-                  \-      \-         \-                  \-           \-               Pan viral      405,484       321           792                 78%                       18
  1007-HBV-0036   Peru                HBV     F1         2901                100          3.6              mNGS           2,781,836     282           101                 6%                        1
  \-              \-                  \-      \-         \-                  \-           \-               xGen           12,905        931           72143               90%                       27
  \-              \-                  \-      \-         \-                  \-           \-               Pan viral      401,432       255           635                 63%                       18
  1019-HBV-0001   Republic of Congo   HBV     E          2479                100          3.53             mNGS           1,554,926     123           79                  15%                       1
  \-              \-                  \-      \-         \-                  \-           \-               xGen           54,413        6,495         119365              76%                       193
                                                                                                           Pan viral      22,393,622    4,162         186                 70%                       264
  0211A172        USA                 HBV     H          3178                100          3.52             mNGS           478,506       35            73                  21%                       1
  \-              \-                  \-      \-         \-                  \-           \-               xGen           16,763        9,313         555569              98%                       215
  \-              \-                  \-      \-         \-                  \-           \-               Pan viral      147,056       1,746         11873               81%                       96
  0610A006        Egypt               HBV     D1         1496                91           3.48             mNGS           2,582,798     11            4                   6%                        2
  \-              \-                  \-      \-         \-                  \-           \-               xGen           56,962        406           7128                47%                       12
  \-              \-                  \-      \-         \-                  \-           \-               Pan viral      424,558       291           685                 58%                       22
  U160953A        Cameroon            HBV     B2         2731                98           3.24             mNGS           1,624,386     10            6                   8%                        1
  \-              \-                  \-      \-         \-                  \-           \-               xGen           3,160,577     191           60                  84%                       3
  \-              \-                  \-      \-         \-                  \-           \-               Pan viral      1,162,542     0             0                   0%                        0
  HBV0073         Spain               HBV     D          56                  nd           1.34             mNGS           76,111        22            289                 1%                        2
  \-              \-                  \-      \-         \-                  \-           \-               xGen           2,044         4             1957                10%                       1
  \-              \-                  \-      \-         \-                  \-           \-               Pan viral      21,630        0             0                   0%                        0
  HBV0285         Spain               HBV     A2         43                  nd           \<1.18           mNGS           1,969,686     6             3                   6%                        1
  \-              \-                  \-      \-         \-                  \-           \-               xGen           22,230        36            1619                1%                        29
  \-              \-                  \-      \-         \-                  \-           \-               Pan viral      237,712       0             0                   0%                        0

Summary of results for HBV and HDV sequencing (mNGS, xGen, and Pan viral) for diversity panel specimens.

The boost in sensitivity over a range of viral loads and genetic diversity is summarized for each virus by comparing genome coverage (top) and reads per million (bottom) ± xGen enrichment ([Fig 2C](#pone.0236046.g002){ref-type="fig"}, [Table 3](#pone.0236046.t003){ref-type="table"}). While samples with very low viral loads did see some increase for both metrics, those with titers ≥3.5 log saw the most improvement. The median increase in HBV genome coverage was 61% (range 8--84%). The same Nextera mNGS libraries obtained from mono- and co-infected samples from a variety of countries were similarly captured and amplified with a "Pan-viral" probe set which contains \>600,000 probes against 1000 human viruses, but only includes probes tiling a single HBV (NC_003977.2: genotype D) and HDV (NC_001653.2: genotype 1) reference sequence. Genome coverage was comparable when titers were high, however at lower levels, sequence diversity impacted HBV detection with the Pan-viral method, stressing the importance of including probes to multiple genotypes ([Table 3](#pone.0236046.t003){ref-type="table"}). For example, both methods obtained 100% coverage of an HBV B2 with a titer of log 5.92 copies/ml, whereas for the B2 strain (log 3.24 copies/ml) that increased from 8% to 84% with xGen, no coverage (0%) was obtained with the Pan viral method.

HBV and HDV cohort of patients from Israel {#sec016}
------------------------------------------

The HBV/HDV mNGS libraries from Israel were revisited with xGen selection. Given the particularly low titers for HBV, most samples (n = 15) only obtained 2--20% genome coverage (60--600 nt/3.2kb), but this was still an increase from zero coverage with mNGS and provided an indication that HBV was indeed present ([Table 2](#pone.0236046.t002){ref-type="table"}). When HBV sequences were originally obtained by mNGS, xGen enrichment significantly (28--73%) improved the overall coverage. As an example, sample 2001149 originally had 8% HBV coverage which was extended to 69% with enrichment ([Fig 3A](#pone.0236046.g003){ref-type="fig"}). Interestingly, this individual was dually-infected with genotypes B2 and D1. Incomplete coverage and reliance on a consensus sequence created the appearance of a recombinant, however, overlapping sequences from both strains were detected whereas contiguous reads spanning putative 'recombination breakpoints' were not. ([Fig 3A](#pone.0236046.g003){ref-type="fig"}). Phylogenetics from the other patients with \>50% of the HBV genome revealed infections with genotypes A1, A2, and D ([Table 2](#pone.0236046.t002){ref-type="table"}).

![Dual BD infection and clustering of HDV genotype 1 strains in Israel.\
(A) Coverage plot of HBV specimen, 2001149. mNGS reads are shown in orange and xGen reads in dark blue (upper panel). Genotype D1 reads are in gold and genotype B2 reads are in brown (lower panel). (B) Coverage plot of HDV specimen, 2001234. (C) Neighbor-joining phylogenetic tree of near full-length HDV sequences. New Israeli sequence branches are in red and reference strains are in black labeled with accession numbers. Bootstrap values of nodes \>70 are shown.](pone.0236046.g003){#pone.0236046.g003}

HDV titers were much higher and thus the abundance of viral reads in mNGS libraries were often sufficient to already provide near full-length sequences ([Table 2](#pone.0236046.t002){ref-type="table"}) \[[@pone.0236046.ref015]\]. Sample 20001234 was representative of most cases, with HDV-xGen providing significant boosts in sequence depth, but with genome coverage only minimally extended (3--11%) ([Fig 3B](#pone.0236046.g003){ref-type="fig"}). We obtained 12 full genomes (\>95%) and 14 partial (33--92%) sequences. HDV consensus genomes generated ± enrichment were compared to one another and all agreed with \>96% identity unless the mNGS initially yielded partial sequences with low coverage ([S3 Table](#pone.0236046.s005){ref-type="supplementary-material"}). This indicates the additional rounds of amplification required for xGen did not bias the final sequence \[[@pone.0236046.ref013], [@pone.0236046.ref028]\]. Twenty-one strains with \>84% genome coverage were included in the phylogenetic analysis. Consistent with previous Sanger sequencing classifications off the HDAg coding region, all strains were HDV genotype 1 ([Fig 3C](#pone.0236046.g003){ref-type="fig"}) \[[@pone.0236046.ref015]\]. Sample 2000742 from Ethiopia branched with HDV strains from Somalia (U81988.1) and Ethiopia (U81989.1). Notably, the HBV sequence from this same individual is genotype A1, indicating both infections likely originated in Africa. Sample 2001222, the only patient from Romania, branches with Gen 1 strains from a variety of geographies, but the rest of the Israeli samples cluster together on a Gen 1 branch absent reference strains from other countries. The 2001149 and 2001063 strains with short branch lengths are from the same patient (blue) and share 99.28% identity.

Discussion {#sec017}
==========

Metagenomics is an extremely powerful approach that allows one to query for the presence of any pathogen in patient samples without any prior knowledge of the sequence. Yet, due to its unbiased nature, mNGS often only scratches the surface for virus detection, especially for low viral load specimens. Here, the high viral loads typical of HCV, and also with HDV, allowed us to readily obtain full genomes using standard methodology, whereas the low titers of HBV required enrichment \[[@pone.0236046.ref026]\]. This cohort included 53 males and 43 females with an overall median age of 58. Most were likely exposed to HCV through IDU or blood transfusion, while others had no identifiable risk factor. Sensitivity for numerous viruses in our controls suggested we should have detected other blood borne pathogens if they were there ([S1 Fig](#pone.0236046.s002){ref-type="supplementary-material"}), but no additional co-infections besides GBV-C were present among the HCV positive individuals.

In Israel, the age-adjusted prevalence of HCV infection was recently estimated to be 5 per thousand, with immigrants from Eastern Europe making up most patients \[[@pone.0236046.ref019]\]. Indeed, many immigrants to Israel are from countries with high HCV prevalence, such as Georgia, Turkmenistan, Moldova, Uzbekistan, Ukraine, Morocco, Romania, and Kazakhstan. The disparity in HCV infection rates for native-born Israelis (0.1%) versus immigrants (5.7%) is significant and further borne out in studies focusing on IDU populations \[[@pone.0236046.ref018], [@pone.0236046.ref029]\]. While the prevailing trend reported is that HCV Genotype 1b predominates in Israel, the presence of other genotypes besides type 1 (70%) has been noted, including type 2 (8%), type 3 (20%) and type 4 (2%) \[[@pone.0236046.ref016], [@pone.0236046.ref019]--[@pone.0236046.ref021], [@pone.0236046.ref030]\]. The majority of HCV strains characterized here were genotype 1b and found in individuals immigrating from the former Soviet Union and neighboring Eastern European countries. Nearly half (10/22) of the native born in Israelis in the cohort were genotype 1a, with the remaining 9 genotype 1a strains coming from Western Europe and the Middle East/North Africa. Genotype 3a strains were primarily from middle aged (49 yr old), IDU males from any country.

Sequence-independent (nuclease treatment, filtration, ultracentrifugation) and -dependent (rRNA depletion, CRISPR cas9-mediated DASH) methods represent enrichment strategies intended to concentrate viral nucleic acid or lower host background \[[@pone.0236046.ref031], [@pone.0236046.ref032]\]. Target enrichment represents an alternate approach based on positive selection which greatly enhances sensitivity for low viral load infections in clinical samples and consequently reduces the overall depth of sequencing required \[[@pone.0236046.ref012], [@pone.0236046.ref028], [@pone.0236046.ref033]\]. Comprehensive approaches like ViroCap which tile \>185,000 sequences totaling \~200 Mb, include probes against RefSeqs, near-neighbors, and other viral databases, just as VirCapSeq covers 207 viral taxa with nearly 2 million probes, even after clustering highly identical regions \[[@pone.0236046.ref034], [@pone.0236046.ref035]\]. These methods provide amazing boosts in coverage and depth, but these probe sets are prohibitively expensive to synthesize and use on a routine basis. A commercialized, and somewhat leaner probe set (\~600,000 probes) covering \~1000 human viruses from Twist Biosciences now puts this technology within reach of more labs. The trade-off we have observed is that while sensitivity for many viruses is substantially improved, performance is compromised for highly diverse viruses where probes are designed against only one or a few representative strains ([Table 3](#pone.0236046.t003){ref-type="table"}). When public health measures require full genome sequencing to halt outbreaks, observe transmission clusters, or track diagnostically relevant mismatches, mere identification of specific viruses like HIV and HCV is insufficient. We and others have shown that probes to all subtypes and groups (HIV) and genotypes (HCV) are required to reliably obtain full genomes \[[@pone.0236046.ref013], [@pone.0236046.ref014], [@pone.0236046.ref036]\].

We therefore tailored a specific probe set to ensure capture of all HBV (A-I) and HDV (1--8) genotypes. Boosts in coverage and sequence depth on a variety of HBV specimens with a range of viral loads validated this approach ([Fig 2C](#pone.0236046.g002){ref-type="fig"}). xGen did extend coverage for HBV in some co-infected Israeli samples, however at titers this low, there was only so much improvement to be expected. This suppression of HBV titers by HDV that we observed is consistent with numerous reports \[[@pone.0236046.ref037], [@pone.0236046.ref038]\]. Where HBV sequences were obtained, these were genotype A1, A2, D, and a B/D dual infection. HDV viral loads on the other hand were considerably higher than HBV and enrichment was often not necessary. Our full genome sequencing confirmed previous sub-genomic HDAg protein sequencing wherein genotype 1 was the only strain detected \[[@pone.0236046.ref015]\]. Levels of co-infection in most countries are simply not known, and as we have seen in Cameroon, they can be far higher than expected \[[@pone.0236046.ref039]\]. The significant prevalence of HDV (6.5%) in Israel mandates HDV RNA testing for all coinfected patients.

mNGS has yielded a wealth of information and at times, actionable results for patients \[[@pone.0236046.ref040]--[@pone.0236046.ref042]\]. However, due to costs, turn-around time, validation, and reimbursement among many issues, the arrival of mNGS in the clinic as a test to replace all other infectious disease diagnostics does not appear imminent \[[@pone.0236046.ref043]\]. Nevertheless, as we demonstrate here, it can play an important role as a research tool and a means of insight into epidemiologic trends. While levels of hepatitis are going down, Israel is home to many immigrants and surveillance is needed. The numerous applications for these and other viruses ensure that mNGS will play an even greater role in dictating public health policy in Israel and elsewhere.

Supporting information {#sec018}
======================

###### Pan-viral enrichment protocol.

(PDF)

###### 

Click here for additional data file.

###### Viral detection in mNGS libraries.

A positive control consisting of 8 viruses and chlamydia trachomatis spiked into normal human plasma each at log 4.0 copies/ml was included with samples in three separate extractions, library preps and sequencing runs of HCV positive samples. Reads were taxonomically assigned by the SURPI pipeline. The top histogram represents the genome coverages and the bottom histograms represent reads per million for each pathogen.

(PDF)

###### 

Click here for additional data file.

###### Patient demographic data.

Demographic information for HCV and HBV/HDV positive patients enrolled in study at the Israeli National HIV and Viral Hepatitis Reference Center (NHRL).

(PDF)

###### 

Click here for additional data file.

###### HCV mNGS library metrics.

HCV positive library results are listed and include total reads, HCV reads, percent genome coverage, genotype classification, and HCV reads per million.

(PDF)

###### 

Click here for additional data file.

###### HDV sequence agreement.

Pairwise nucleotide identity values comparing HDV consensus sequences from mNGS versus xGen is expressed as a percent.

(PDF)

###### 

Click here for additional data file.

We thank Guixia Yu, Scot Federman, and Dr. Charles Chiu at University of California San Francisco for sequencing and data processing of HCV samples. We thank Dr. Matthew Frankel for the HBV genotype panel specimens. We thank Dave Campbell and Dr. Nicholas Downey at IDT for assistance with xGen probe design, and John Robichaud and Mark Consugar at Twist Biosciences for assistance with the Pan-viral protocol.

10.1371/journal.pone.0236046.r001

Decision Letter 0

Khudyakov

Yury E

Academic Editor

© 2020 Yury E Khudyakov

2020

Yury E Khudyakov

This is an open access article distributed under the terms of the

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1 May 2020

PONE-D-20-06995

Advanced Molecular Surveillance Approaches for Characterization of Blood Borne Hepatitis Viruses

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4\. Is the manuscript presented in an intelligible fashion and written in standard English?

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Reviewer \#1: Yes

Reviewer \#2: Yes

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5\. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer \#1: The present manuscript reports on HCV, HBV, and HDV molecular epidemiology in infected patients from Israel by using the NGS MiSeq system to obtain full genome sequences. HCV sequencing was successfully performed directly on 99 samples carrying relatively high viral RNA loads as expected. Phylogenetic analysis indicated that genotype 1 was dominant in these patients (clinical status?) with subgenotype differences between patients born in Israel (HCV-1a) and patients originating from Russia and Eastern Europe. Diversity was illustrated further by the presence of genotypes 2-4. It would be interesting to know if there was difference in genome coverage and HCV reads/million according to genotypes. It is unexpected that the authors did not report the detection of any pegivirus which are quite prevalent in humans.

In parallel, 26 samples previously documented HBV/HDV co-infected samples were investigated. Due to the extremely low level of HBV DNA in these samples, two methods of viral targets enrichment were evaluated. xGen probe sets (2x coverage) for the main HBV and HDV genotypes were designed and combined in the same assay. The method was first evaluated on a panel of characterized HBV mono-infected and HBV/HDV dual-infected samples. The results obtained with both panel and patient samples showed the efficiency of the method to recover HBV sequences. The use of a commercial pan viral probe set for HBV was less efficient showing the importance to use probes covering the genetic diversity of all HBV genotypes. Target enrichment showed poor added value for HDV sequencing. The HBV and HDV genotypes observed were consistent with previous data from the region. An interesting feature is the identification of a rare HBV-BD recombinant strain. However, the Simplot profile was derived from a consensus sequence generated by NGS. How the authors excluded the possibility of dual infection? Did they check that there was no overlapping reads characteristic of the two genotypes?

Reviewer \#2: In this manuscript, the authors presented a metagenomic next generation sequencing approach (mNGS) for the molecular characterization of blood borne viral hepatitis viruses (HCV, HBV and HDV).

Overall, the methods employed by the authors to accomplish their objectives were adequate and well-presented. The viral enrichment method, crucial for mNGS and a topic of great interest for virological studies, presented solid results when applied in the international HBV panel but not so consistent regarding Israeli samples. Nonetheless, the technique allowed to detect a previously unreported recombinant B/D strain which was a great achievement. The tables presented in the article should be clearer. It lacks proper legend to understand what hyphen means in many lines (not done, not available?).

The employment of new methodologies to provide tools for virological surveillance is relevant for scientific community and this paper surely contributes to a remarkable bottleneck of viral metagenomics which is the low sensibility of detection of low-titer viral DNA/RNA. However, as highlighted by the authors, its use in routine diagnosis does not appear imminent, especially in low income countries.

The quality of the written English is good thorough out the article.

Minor revision

1\. Table 1 is not self-explanatory and should be clearer. Only in the text it is possible to understand that the values shown are either absolute numbers or percentages. This must be clearly indicated in the table.

2\. The description of the cohort in line 381 repeats the male gender twice. Authors must correct the typing error.

3\. A typo in the word "Kazakhstan" (line 391) must be corrected.

\*\*\*\*\*\*\*\*\*\*

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Reviewer \#1: No

Reviewer \#2: No

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10.1371/journal.pone.0236046.r002

Author response to Decision Letter 0

22 Jun 2020

Reviewer \#1: The present manuscript reports on HCV, HBV, and HDV molecular epidemiology in infected patients from Israel by using the NGS MiSeq system to obtain full genome sequences. HCV sequencing was successfully performed directly on 99 samples carrying relatively high viral RNA loads as expected. Phylogenetic analysis indicated that genotype 1 was dominant in these patients (clinical status?) with subgenotype differences between patients born in Israel (HCV-1a) and patients originating from Russia and Eastern Europe. Diversity was illustrated further by the presence of genotypes 2-4. It would be interesting to know if there was difference in genome coverage and HCV reads/million according to genotypes.

The answer can be found in Supplemental Table S1. All the 2c and 3a sequences had \>98% genome coverage and \>1200 rpm. A few had \<97% genome coverage, and these were genotype 1A or 1B, which made up the majority of samples.

It is unexpected that the authors did not report the detection of any pegivirus which are quite prevalent in humans.

We regret the confusion here; an initial version of the text attempted to make the following distinction, which we ought to have kept in. HPgV-2 is a novel pegivirus that our lab discovered in 2015: it is primarily seen in HCV infections, so we were looking specifically for this virus, but we did not detect it in these HCV+ individuals. We did however observe human pegivirus-1, also known as GBV-C, in 5 individuals. As the reviewer notes, it is quite common. We have included the following sentence on lines 264-265, "Human pegivirus-1 (GBV-C) reads were enriched in 5 individuals." We also further clarify this in the Discussion on lines 389-91,

"Sensitivity for numerous viruses in our controls suggested we should have detected other blood borne pathogens if they were there (S1 Fig), but no additional co-infections besides GBV-C were present among the HCV positive individuals."

In parallel, 26 samples previously documented HBV/HDV co-infected samples were investigated. Due to the extremely low level of HBV DNA in these samples, two methods of viral targets enrichment were evaluated. xGen probe sets (2x coverage) for the main HBV and HDV genotypes were designed and combined in the same assay. The method was first evaluated on a panel of characterized HBV mono-infected and HBV/HDV dual-infected samples. The results obtained with both panel and patient samples showed the efficiency of the method to recover HBV sequences. The use of a commercial pan viral probe set for HBV was less efficient showing the importance to use probes covering the genetic diversity of all HBV genotypes. Target enrichment showed poor added value for HDV sequencing. The HBV and HDV genotypes observed were consistent with previous data from the region. An interesting feature is the identification of a rare HBV-BD recombinant strain. However, the Simplot profile was derived from a consensus sequence generated by NGS. How the authors excluded the possibility of dual infection? Did they check that there was no overlapping reads characteristic of the two genotypes?

We thank the reviewer for their very astute inquiry. After careful scrutiny of the data, we have reached the conclusion that indeed it is not a recombinant, but rather a dual infection. As they noted, there were overlapping reads indicative of both genotypes, but because a consensus sequence was used for phylogenetics and SIMPLOT analysis, these less abundant reads were obscured by the majority sequence. As the plot in Fig 3A indicated and we noted in the text, coverage was incomplete at 69%. Upon closer inspections, the transition from one genotype to the other occurred at these gaps. In addition, there were no contiguous reads that spanned the putative 'breakpoints'. Rather, one could see where reads from one genotype ended and a new set of reads for the other genotype began. We regret this oversight and are grateful to the reviewer for raising this possibility and allowing us to catch this mistake.

We have modified the abstract (lines 51-54) to read as follows:

When HBV-xGen was applied to Israeli samples, coverage was improved by 28-73% in 4 samples and identified HBV genotype A1, A2, D1 specimens and a previously never described BD recombinant dual B/D infection.

We have modified the results (lines 339-343):

Before

Interestingly, this strain was classified as a rare BD recombinant, which we confirmed by bootscanning in SIMPLOT and individual trees of fragments (Fig 3B; S2 Fig). A separate bleed from this individual did not yield HBV sequence, but prior PCR confirmed D1 sequence in the RT region.

After

Interestingly, this individual was dually-infected with genotypes B2 and D1. Incomplete coverage and reliance on a consensus sequence created the appearance of a recombinant, however, overlapping sequences from both strains were detected whereas contiguous reads spanning putative 'recombination breakpoints' were not. (Fig 3A).

Note that the original Fig 3B has been removed, as well the accompanying phylogenetic trees of sub-fragments in Fig S2. In its place, Fig 3A now includes a lower panel that illustrates which HBV genotype reads are coming from.

The legend for Fig 3A now reads as follows:

\(A\) Coverage plot of HBV specimen, 2001149. mNGS reads are shown in orange and xGen reads in dark blue (upper panel). Genotype D1 reads are in gold and genotype B2 reads are in brown (lower panel).

We have modified the Discussion now to remove two sentences (lines 437-441) and it reads as follows:

Where HBV sequences were obtained, these were genotype A1, A2, D, and a BD recombinant B/D dual infection. Recombinants of HBV are themselves rare, and a BD mosaic genome has never been described until here (Fig 3B, S2 Fig) \[39\]. Consistent with reports that recombination sites are not distributed randomly in HBV, we observed breakpoints within in the 1700-2000 region.

Reviewer \#2: In this manuscript, the authors presented a metagenomic next generation sequencing approach (mNGS) for the molecular characterization of blood borne viral hepatitis viruses (HCV, HBV and HDV).

Overall, the methods employed by the authors to accomplish their objectives were adequate and well-presented. The viral enrichment method, crucial for mNGS and a topic of great interest for virological studies, presented solid results when applied in the international HBV panel but not so consistent regarding Israeli samples. Nonetheless, the technique allowed to detect a previously unreported recombinant B/D strain which was a great achievement. The tables presented in the article should be clearer. It lacks proper legend to understand what hyphen means in many lines (not done, not available?).

The employment of new methodologies to provide tools for virological surveillance is relevant for scientific community and this paper surely contributes to a remarkable bottleneck of viral metagenomics which is the low sensibility of detection of low-titer viral DNA/RNA. However, as highlighted by the authors, its use in routine diagnosis does not appear imminent, especially in low income countries.

The quality of the written English is good thorough out the article.

Minor revision

1\. Table 1 is not self-explanatory and should be clearer. Only in the text it is possible to understand that the values shown are either absolute numbers or percentages. This must be clearly indicated in the table.

The columns headers in Table 1 have been modified to indicate the absolute number of total individuals.

2\. The description of the cohort in line 381 repeats the male gender twice. Authors must correct the typing error.

It now reads "...53 males and 43 females..."

3\. A typo in the word "Kazakhstan" (line 391) must be corrected.

Corrected.

###### 

Submitted filename: Response to Reviewers.docx

###### 

Click here for additional data file.

10.1371/journal.pone.0236046.r003

Decision Letter 1

Khudyakov

Yury E

Academic Editor

© 2020 Yury E Khudyakov

2020

Yury E Khudyakov

This is an open access article distributed under the terms of the

Creative Commons Attribution License

, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

29 Jun 2020

Advanced Molecular Surveillance Approaches for Characterization of Blood Borne Hepatitis Viruses

PONE-D-20-06995R1

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Additional Editor Comments (optional):

Reviewers\' comments:

10.1371/journal.pone.0236046.r004

Acceptance letter

Khudyakov

Yury E

Academic Editor

© 2020 Yury E Khudyakov

2020

Yury E Khudyakov

This is an open access article distributed under the terms of the

Creative Commons Attribution License

, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

6 Jul 2020

PONE-D-20-06995R1

Advanced Molecular Surveillance Approaches for Characterization of Blood Borne Hepatitis Viruses

Dear Dr. Berg:

I\'m pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they\'ll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact <onepress@plos.org>.

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[^1]: **Competing Interests:**MGB, AO, KF, BJH, JY, MAR, GAC are all employees and shareholders of Abbott Laboratories. This commercial affiliation does not alter our adherence to all PLOS ONE policies on sharing data and materials. Sequences generated in this study have been deposited in GenBank. No patents have been applied for and no products are in development related to this research.
