key: cord-0999864-gbguv4r2
authors: Briese, Thomas; Kapoor, Amit; Mishra, Nischay; Jain, Komal; Kumar, Arvind; Jabado, Omar J.; Lipkin, W. Ian
title: Virome Capture Sequencing Enables Sensitive Viral Diagnosis and Comprehensive Virome Analysis
date: 2015-09-22
journal: mBio
DOI: 10.1128/mbio.01491-15
sha: d48ae48fa33c489b576489afc16c00f5b1532178
doc_id: 999864
cord_uid: gbguv4r2

Insensitivity and technical complexity have impeded the implementation of high-throughput nucleic acid sequencing in differential diagnosis of viral infections in clinical laboratories. Here, we describe the development of a virome capture sequencing platform for vertebrate viruses (VirCapSeq-VERT) that increases the sensitivity of sequence-based virus detection and characterization. The system uses ~2 million probes that cover the genomes of members of the 207 viral taxa known to infect vertebrates, including humans. A biotinylated oligonucleotide library was synthesized on the NimbleGen cleavable array platform and used for solution-based capture of viral nucleic acids present in complex samples containing variable proportions of viral and host nucleic acids. The use of VirCapSeq-VERT resulted in a 100- to 10,000-fold increase in viral reads from blood and tissue homogenates compared to conventional Illumina sequencing using established virus enrichment procedures, including filtration, nuclease treatments, and RiboZero rRNA subtraction. VirCapSeq-VERT had a limit of detection comparable to that of agent-specific real-time PCR in serum, blood, and tissue extracts. Furthermore, the method identified novel viruses whose genomes were approximately 40% different from the known virus genomes used for designing the probe library. The VirCapSeq-VERT platform is ideally suited for analyses of virome composition and dynamics. Importance VirCapSeq-VERT enables detection of viral sequences in complex sample backgrounds, including those found in clinical specimens, such as serum, blood, and tissue. The highly multiplexed nature of the system allows both the simultaneous identification and the comprehensive genetic characterization of all known vertebrate viruses, their genetic variants, and novel viruses. The operational simplicity and efficiency of the VirCapSeq-VERT platform may facilitate transition of high-throughput sequencing to clinical diagnostic as well as research applications.

to select 100-mer oligonucleotides spaced by approximately 25 to 50 nucleotides (nt) along each sequence. To address sequence variation, oligonucleotide mutant or variant sequences were retained if sequences diverged by more than 90%. Where technical complexity in oligonucleotide synthesis was challenging due to melting temperature (T m ) or homopolymer repeats, probe sequences were refined by shortening and adjusting their start/stop positions. The final library comprised 1,993,176 oligonucleotides ranging in length from 50 to 100 nt and in T m from 58.7°C to 101°C (see Table S2 in the supplemental material).

We evaluated in silico whether the selected probe library provides uniform coverage of the targeted virus sequences. Our analysis indicated that probe numbers were proportional to the amount of available sequence information, resulting in an 88 to 98% estimated coverage of target sequences when an "outreach" for each probe of approximately 100 nt to either side is assumed (see Table S3 in the supplemental material). We mapped the probe library against a database of 100 reference virus genome sequences representing double-and single-stranded DNA and RNA, positive and negative RNA, and circular, linear, and segmented viruses, using a minimum nucleotide identity of 90%. The probe library covered targeted genome sequences with probes spaced at Ͻ150-nt intervals ( Fig. 1 ) but provided no coverage of noncoding regions (e.g., poliovirus 5= untranslated region [UTR]) (Fig. 1A) . The highest probe coverage was evident in divergent genome regions (e.g., yellow fever virus E gene region; approximately position 1000 to 2500) (Fig. 1B) . In silico analysis indicated that the VirCapSeq-VERT probe library included oligonucleotides that selectively hybridize to genomes of vertebrate viruses but not to those of bacteriophages or plant or fungal viruses.

Experimental assessment of efficiency. Nucleic acid (NA) extracts of human lung tissue or whole blood were spiked with various amounts of NAs representing large and small, positive-and negative-strand, segmented and nonsegmented, and DNA and RNA viruses (Table 1) . Spiked lung (pool 1) and blood (pool 2) NA preparations were divided and processed in parallel using a standard Illumina HTS protocol or the VirCapSeq-VERT system, whereby viral sequences are enriched by positive selection. Each of the preparations was sequenced on an Illumina HiSeq 2500 sequencer, loading 2 lanes per pool. VirCapSeq-VERT resulted in a 100-to 1,000-fold increase in on-target (viral) reads and a reduction of host background reads from 99.7% to 68.2% in lung and from 99.4% to 38.5% in blood ( Fig. 2 ; also, see Table S4 in the supplemental material). The average coverage also increased dramatically, with nearly full-length sequences (Ͼ95%) being obtained for all viruses (Table 2 ). Figure 3 shows selected examples of To determine the threshold for detection of viral sequence, we used NA from lung tissue homogenate and EDTA-blood that contained different amounts of WNV and herpes simplex virus 1 (HHV-1) NA. Nearly complete genome recovery (Ͼ90%) was achieved for both viruses at input levels of 100 viral copies in 50 ng of blood NA or 1,000 viral copies in 100 ng lung NA (Fig. 4) . Extrapolated to clinical samples, these values correspond to a blood specimen containing approximately 1,200 copies/ml or a tissue specimen containing approximately 16,000 copies/mg, assuming 100% extraction yield (see Table S5 in the supplemental material). Even at the lowest level of virus input tested, 10 viral copies per 50 ng background NA, corresponding to approximately 100 copies/ml blood, VirCapSeq-VERT enabled recovery of 45 kb (29%) of HHV-1 and 0.75 kb (7%) of WNV genome sequence. We then tested human blood and serum samples (1 ml) spiked with live enterovirus D68 (EV-D68) virus stock. VirCapSeq-VERT enabled detection in both sample types at a concentration of 10 copies/ml (Fig. 5) , comparable to the sensitivity of real-time PCR (see Table S6 in the supplemental material).

Comparison with other enrichment regimens. Analysis of samples of human blood spiked with live EV-D68, HHV-1, and influenza A virus (FLUAV) stock indicated that VirCapSeq-VERT yielded up to a 10,000-fold increase in mapped read counts over samples treated after extraction with DNase and RiboZero rRNA depletion, individually or in combination, and then processed by standard HTS. VirCapSeq-VERT resulted in nearly full genome recovery for most viruses even with less than 1,000 copies of target input (Table 3 ; also, see Table S7 in the supplemental material).

Clinical specimens included a human nasal swab sample containing EV-D68 that was divided into three aliquots (i) treated with filtration and nuclease digestion prior to extraction and standard HTS, (ii) treated with filtration and nuclease digestion prior to extraction and VirCapSeq-VERT, or (iii) not treated prior to extraction and VirCapSeq-VERT. VirCapSeq-VERT with no prior treatment enabled the highest sequence recovery and depth (see Table S8 in the supplemental material).

Since fecal material is frequently challenging for viromic analyses, we tested a sample of fecal pellets from bats known to contain rotavirus sequences. The sample was divided into four aliquots and (i) treated with filtration and nuclease digestion prior to extraction, followed by standard HTS, (ii) treated with filtration and nuclease digest prior to extraction, followed by DNase digestion after extraction and standard HTS, (iii) treated with filtration and nuclease digestion prior to extraction and VirCapSeq-VERT, or (iv) not treated prior to extraction and VirCapSeq-VERT. VirCapSeq-VERT again yielded the highest mapped read count (see Table S9 in the supplemental material).

The specificity of VirCapSeq-VERT for relevant targets was readily apparent in comparison with results obtained by conventional HTS. Whereas up to 36% of the viral reads found by conventional HTS represented insect-infecting dicistroviruses (Fig. 6 ), these reads were reduced to 15% by VirCapSeq-VERT. Vertebrate rotavirus, coronavirus, astrovirus, and circovirus sequences detected only at low levels in conventional HTS were increased 4-100 fold by VirCapSeq-VERT.

Since the precise sequence of the rotavirus present in bat samples was unknown, contigs obtained by de novo assembly were used to identify the closest GenBank match for each segment. Table 4 shows that nearly a full sequence was obtained for sequences differing by up to 25% from the known sequences used for VirCapSeq-VERT probe design. Partial sequence in conserved regions was obtained even for sequences differing by as much as 50% from known sequences (NSP1 and NSP4) ( Table 4) .

Detection of novel sequences. To further test the capacity of VirCapSeq-VERT to detect novel viral sequences, we used an extract of a liver homogenate from a deer mouse experimentally infected with the rodent hepacivirus isolate RHVpl-01. The complete genome sequence of this isolate has a Ͻ65% global nucleotide sequence identity with the sequences used to design the VirCapSeq-VERT probes. Nonetheless, VirCapSeq-VERT selectively enriched RHVpl-01 sequence in conserved regions encoding the helicase and polymerase genes, for which bioinformatics analysis showed the presence of probes with up to 90% nucleotide identity in the VirCapSeq-VERT probe pool. We conclude therefore, from rotavirus and hepacivirus experiments, that while not an ideal platform for viral discovery, VirCapSeq-VERT can detect novel viruses through hybridization to short conserved sequence motifs within larger genome fragments. Sample multiplexing. During the estimation of the limit of detection of VirCapSeq-VERT, we processed samples individually or together during hybridization capture. The results obtained with the samples processed individually were superior (see Table S5 in the supplemental material). This finding suggested that competition for probe populations may compromise application in diagnostic settings where patient samples may have widely divergent virus loads. We investigated the practical impact of this potential confounding factor in assays using 21 barcoded libraries representing samples containing seven different viruses at genome loads that varied from 10 2 to 10 8 . One set represented the seven different viruses each at a concentration of approximately 10 4 genome copies/library. To mimic competition anticipated in some clinical samples, the second set contained the same seven libraries at 10 4 copies, combined with an additional 14 libraries prepared with the seven viruses at 10 2 and at 10 5 to 10 8 copies. Virus detection was not impaired in multiplex assays even with samples that varied up to 10 4 in target concentration (Table 5) ; however, genome coverage was typically higher in 7-plex than in 21-plex assays.

To determine the utility of VirCapSeq-VERT in characterization of virome diversity and dynamics, we analyzed a set of 23 serum samples collected from multiply transfused hemophilia patients known to contain hepatitis C virus (HCV), GB virus C (GBV-C), human immunodeficiency virus (HIV), and torque teno virus (TTV). Samples were amplified using unique bar codes, and two pools were generated for VirCapSeq-VERT. Pool 1 contained 9 samples. Pool 2 contained the same 9 samples mixed with the remaining 14 samples. All viruses in the 9-plex as well as in the 23-plex sample pool were efficiently characterized (see Fig. S1 in the supplemental material).

Sequencing approaches have enabled metagenomics, including virome studies, and are of increasing interest in the field of diagnostics. However, concerns regarding sensitivity, especially in high-host-background settings, cumbersome and timeconsuming sample processing, and cost pose hurdles that would need to be overcome in order to realize the potential of HTS. Unlike 16S rRNA of bacteria, viruses lack universally conserved markers and have plastic genomes that easily generate mutants, strains, and variants. Virus variants differing in sequence, even by a single point mutation, can vary in host range, transmissibility and pathogenicity (23, 24) . Accordingly, an ideal viral diagnostic platform should enable sensitive multiplexed detection of all vi-ruses and their variants. Nucleic acid capture with oligonucleotides has been used to enhance the efficiency of HTS for characterizing host (25, 26) or selected microbial (27, 28) targets at low scale. However, to our knowledge, VirCapSeq-VERT is the first example wherein a positive selection method for HTS has been employed for comprehensive, sensitive application in microbial diagnostics and whole virome analysis.

Current virus diagnostic assays that are commonly based on PCR assays targeting one or a few specific agents may fail to detect virus variants and provide only limited genotypic information. VirCapSeq-VERT addresses many of the current challenges of PCR and HTS for diagnostics and virome analysis. The sensitivity and specificity of the VirCapSeq-VERT are comparable to those of agent-specific real-time PCR (see Table S6 in the supplemental material). Additionally, the 100-to 10,000-fold increase achieved in on-target reads enables leveraging of sequencing depth against , particularly given its advantages in sensitivity, genome coverage, and ease of use. The capacity for highly multiplexed sample processing and simplified sample handling is cost-effective and reduces the risk of cross contamination. The VirCapSeq-VERT system is not specifically designed for viral discovery; nonetheless, it enables sequencing of genomes with as little as 75% overall sequence identity. Results of our rotavirus and hepacivirus analyses indicate that where the goal is de- (WNV) nucleic acid. The two preparations were serially diluted to generate six samples containing both viruses at 5,000, 1,000, 300, 100, 30, or 10 copies in 100 ng lung tissue or 50 ng whole-blood nucleic acid. Samples were processed with VirCapSeq-VERT. tection rather than comprehensive genome sequencing, VirCapSeq-VERT has the potential, through hybridization to conserved regions, to detect novel viruses with an overall nucleotide divergence in the range of 40%.

In summary, VirCapSeq-VERT has promise as a tool for diagnostic and research applications. It has sensitivity similar to that obtained with targeted real-time PCR, with the advantage of detecting viral variants that would not be captured with specific PCR assays as well as the potential to provide the complete genome sequence needed for assessment of viral diversity and evolution for epidemiological and public health applications.

Facsimiles of clinical specimens were generated in a background of NA extracted from normal human lung tissue, EDTAblood, or serum. The samples were spiked with viral NA and quantitated by virus-specific TaqMan real-time (reverse transcription) PCR (qPCR). NA from cell culture or blood, serum, or tissue samples was extracted using the easyMAG system (bioMérieux, Marcy l'Etoile, France) or All-Prep DNA/RNA kits (Qiagen, Hilden, Germany). Background NA was quantitated by NanoDrop (Wilmington, DE, USA) or Bioanalyzer 2100 (Agilent, Santa Clara, CA, USA) and mixed with variable quantities of viral NA derived from enterovirus D68 (EV-D68) ( (42) , representing nonsegmented and segmented negative-strand RNA viruses; and herpes simplex virus 1 (HHV-1, ATCC VR-733), as a large double-strand DNA virus. Spiking was performed using NA stocks banked at the Center of Infection and Immunity originally derived from virus cultures or positive diagnostic specimens, with the exception of EBOV, which was provided as noninfectious nucleic acid extract by Peter Jahrling at the National Institute of Allergy and Infectious Diseases, National Institutes of Health. TaqMan PCR primer and probes for the various viruses are cited or available on request.

To determine the limit of detection and to assess VirCapSeq-VERT in comparison to conventional target enrichment procedures, normal human lung tissue homogenate, EDTA-blood, or serum samples were spiked with different amounts of live EV-D68, HHV-1, and FLUAV stock quantitated by qPCR.

Clinical samples included a human nasal swab sample known to be positive for EV-D68 (30), liver specimens from deer mice infected with deer mouse hepacivirus (31), a sample of bat feces pellets in which rotaviral sequences had been identified (unpublished), and serum samples from hemophilia patients coinfected with hepatitis C virus (HCV), GB virus C (GBV-C), human immunodeficiency virus (HIV), and torque teno virus (TTV).

Selection of probe sequences. The EMBL Coding Domain Sequence database (release 122, December 2014; ftp://ftp.ebi.ac.uk/pub/databases/ embl/cds/release/std/), containing 2,199,467 records was clustered at 96% sequence identity by CD-Hit (32), yielding a database of 401,716 representative sequences spanning all virus sequence records, excluding bacteriophages. A list of all virus genera known to infect vertebrates was generated from the master species list of the International Committee on Taxonomy of Viruses (ICTV; http://talk.ictvonline.org/files/ictv_documents/m/msl/5208.aspx). Through cross-referencing of protein IDs with NCBI taxonomy IDs, a set of 342,438 coding sequence records was identified for the selected virus genera. The sequences were broken into fragments, clustered at 90% sequence identity, and used to generate 100-nt probe sequences that were tiled across the genes at approximately 25-to 50-nt intervals. A library of 1,993,200 oligonucleotide probes was selected. The NimbleGen cleavable array platform was employed for synthesis of the biotinylated, soluble probe library (SeqCap EZ Choice; Roche/ NimbleGen, Basel, Switzerland), and probe sequences were refined by a Human blood was spiked with live virus stocks derived from tissue culture to result in approximately 10 4 copies of herpes simplex virus 1 (HHV-1), influenza A virus (FLUAV), and enterovirus D68 (EV-D68) per 250 ng extracted blood NA. The sample was divided into equivalent aliquots to be processed with the indicated treatment prior to RT reaction and subjected to either conventional sequence library preparation or VirCapSeq-VERT. b Determined by qPCR of double-stranded cDNA/DNA used for sequence library construction. c HHV-1 detection was impaired due to DNase. d Normalized to 10,000,000 total reads. e Prepared with additional dilution of the sample in a blood background.

adjusting their lengths to conform to NimbleGen synthesis parameters, such as maximum T m or homopolymer repeat length. Conventional target sequence enrichment procedures. Conventional virus enrichment methods commonly used in metagenomic sequencing-based virus discovery include filtration and pre-extraction nuclease treatments, often combined with postextraction DNase I and/or depletion of ribosomal rRNA sequences. Briefly, samples (100 to 300 l) were filtered through 0.45-m-pore-size sterile disk filters (Merck/Millipore, Billerica, MA, USA) to enrich for viruses over cells or bacteria. The flowthrough was treated with 1 l RNase A (10 mg/ml; Thermo, Fisher, Waltham, MA, USA) for 15 min at room temperature, followed by a cocktail of 8 U Turbo DNase (Thermo, Fisher), 250 U Benzonase (Merck/ Millipore), and 10 mM MgCl 2 for 45 min at room temperature to digest non-particle-protected NAs. Protected NAs, such as those in viral particles, were extracted by easyMAG (bioMérieux) or AllPrep kits (Qiagen). Postextraction digestion by DNase I (2 U/g DNA for 15 min at 37 o C; Thermo, Fisher) was added in some instances to digest chromosomal DNA (cellular and bacterial), but it will also digest viral DNA (e.g., HHV-1 DNA), whereas mRNA transcripts generated from actively replicating cellular virus would be maintained. Depletion of nondesired host mRNA sequences was achieved using RiboZero magnetic kits (Illumina, San Diego, CA, USA). Enriched preparations were subjected to reverse transcription and sequence library preparation.

Conventional HTS. Total NA extracts were reverse transcribed using SuperScript III (Thermo, Fisher) with random hexamers. The cDNA was RNase H treated prior to second-strand synthesis with Klenow fragment (New England Biolabs, Ipswich, MA, USA). The generated doublestranded cDNA was sheared to an average fragment size of 200 bp using the manufacturer's standard settings (E210 focused ultrasonicator; Covaris, Woburn, MA, USA). Sheared product was purified (AxyPrep Mag Virome capture sequencing. Libraries were prepared by essentially following the standard KAPA protocol but including viral sequence capture, following mainly the SeqCap RNA enrichment system protocol (Roche/NimbleGen). Briefly, total NA extract was reverse transcribed using SuperScript III (Thermo, Fisher) with random hexamers. The cDNA was RNase H treated prior to second-strand synthesis with Klenow fragment (New England Biolabs). The resulting double-stranded cDNA/DNA mix was sheared to an average fragment size of 200 bp using the manufacturer's standard settings (Covaris E210 focused ultrasonicator). Sheared product was purified (AxyPrep), and libraries were constructed using KAPA library preparation kits (KAPA) with Roche/NimbleGen adapter kits. The quality and quantity of libraries were checked using a Bioanalyzer (Agilent). The libraries were then mixed with a SeqCap HE universal oligonucleotide, SeqCap HE index oligonucleotides, and COT DNA and vacuum evaporated at 60°C for approximately 40 min. Dried samples were mixed with 2ϫ hybridization buffer and hybridization component A (Roche/NimbleGen) prior to denaturation at 95°C for 10 min. The VirCap probe library (4.5 l) was added and hybridized at 47°C for 12 h in a standard PCR thermocycler. SeqCap Pure capture beads (Roche/ NimbleGen) were washed twice, mixed with the hybridization mix, and kept at 47°C for 45 min with vortexing for 10 s every 10 to 15 min. The streptavidin capture beads complexed with biotinylated VirCapSeq-VERT probes were trapped (DynaMag-2 magnet; Thermo, Fisher) and washed once at 47°C and then twice more at room temperature with wash buffers of increasing stringency. Finally, beads were suspended in 50 l water and directly subjected to posthybridization PCR (SeqCap EZ accessory kit V2; Roche/NimbleGen). The PCR products were purified (Agencourt Ampure DNA purification beads; Beckman Coulter, Brea, CA, USA) and quantitated by Bioanalyzer (Agilent) for Illumina sequencing.

Data analysis and bioinformatics pipeline. Sequencing on the Illumina HiSeq 2500 platform (Illumina) resulted in an average of 210 million reads per lane. Samples were demultiplexed using Illumina software, and FastQ files were generated. Demultiplexed and Q30-filtered FastQ files were mapped against reference genomes from GenBank with Bowtie2 mapper 2.0.6 (http://bowtie-bio.sourceforge.net). SAMtools (v 0.1.19) (33) were used to generate the consensus genomes and coverage statistics. Integrative Genomics Viewer (v 2/3/55) (34) was used to generate coverage plots. Host background levels were determined from Bowtie2 mappings against the host genomes downloaded from the NCBI. Sequencing data obtained from the unknown samples was preprocessed using PRIN-SEQ (v 0.20.2) (35) software, and filtered reads were aligned against the host reference databases to remove the host background. The resulting reads were de novo assembled using MIRA (v 4.0) (36) or SOAPdenovo2 (v 2.04) (37) assemblers, and contigs and unique singletons were subjected to homology search using MegaBlast against the GenBank nucleotide database; sequences that showed poor or no homology at the nucleotide level were screened by BLASTX against the viral GenBank protein database. Viral sequences from BLASTX analysis were subjected to another round of BLASTX homology search against the entire GenBank protein database to correct for biased E values and taxonomic misassignments. Based on the contigs identified for different viral strains, GenBank sequences were downloaded and used for mapping the whole data set to recover partial or complete genomes. Viral read numbers were obtained from counting the number of reads mapping to contig sequences and unassembled singletons, and percentages were calculated in relation to the total read number obtained. Percentages were converted into heatmaps using MultiExperiment Viewer (MeV v4.9) (38).

Supplemental material for this article may be found at http://mbio.asm.org/ lookup/suppl/doi:10.1128/mBio.01491-15/-/DCSupplemental. Figure S1 , DOCX file, 0.3 MB. a qPCR quantitated nucleic acid extracts representing seven different viruses were used to spike a background of human blood nucleic acid at levels of approximately 10 4 copies/ 100 ng, 10 2 copies/100 ng, and 10 5 to 10 8 copies/100 ng. Individual sequence libraries were prepared using 21 different indexes for bar coding. Libraries were mixed for capture hybridization into a 7-plex mix (libraries prepared from 10 4 loads) and the complete 21-plex mix. 

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We thank Alexandra Petrosov for expert sequence library preparation, Parisa Zolfaghari for virus culture and generation of PCR standards, James Ng for executing PCR assays, Lokendra Singh Chauhan for sample preparation, Rafal Tokarz for EV-D68 qPCR, and Peter Jahrling for Ebola virus nucleic acid extract. We thank Bohyun Lee and Adrian Caciula for excellent bioinformatics support and Ellie Kahn for assistance with the manuscript.The study was supported by the National Institutes of Health (U19 AI109761 to W.I.L., T.B., A.K., N.M., K.J., and A.K. and R01 HL119485 to A. Kapoor) and the Department of Homeland Security (KSUNI S11045.01 to T.B.).