Virology 500 (2017) 130–138

Contents lists available at ScienceDirect

Virology
journal homepage: www.elsevier.com/locate/yviro

VirusDetect: An automated pipeline for efficient virus discovery using deep

sequencing of small RNAs
crossmark
Yi Zhenga,1, Shan Gaoa,b,1, Chellappan Padmanabhanc, Rugang Lic, Marco Galvezd,
⁎
Dina Gutierrezd, Segundo Fuentesd, Kai-Shu Lingc, Jan Kreuzed, Zhangjun Feia,e,
a
Boyce Thompson Institute, Ithaca, NY 14853, USA
b
College of Life Sciences, Nankai University, Tianjin 300071, PR China
c
U.S. Vegetable Laboratory, U.S. Department of Agriculture-Agricultural Research Service, Charleston, SC 29414, USA
d
Virology laboratory, International Potato Center (CIP), Lima, Peru
e
Robert W. Holley Center for Agriculture and Health, U.S. Department of Agriculture-Agricultural Research Service, Ithaca, NY 14853, USA

A R T I C L E I N F O A BS T RAC T

Keywords: Accurate detection of viruses in plants and animals is critical for agriculture production and human health. Deep
Virus discovery sequencing and assembly of virus-derived small interfering RNAs has proven to be a highly efficient approach
Small RNA for virus discovery. Here we present VirusDetect, a bioinformatics pipeline that can efficiently analyze large-
Next-generation sequencing scale small RNA (sRNA) datasets for both known and novel virus identification. VirusDetect performs both
VirusDetect
reference-guided assemblies through aligning sRNA sequences to a curated virus reference database and de
novo assemblies of sRNA sequences with automated parameter optimization and the option of host sRNA
subtraction. The assembled contigs are compared to a curated and classified reference virus database for known
and novel virus identification, and evaluated for their sRNA size profiles to identify novel viruses. Extensive
evaluations using plant and insect sRNA datasets suggest that VirusDetect is highly sensitive and efficient in
identifying known and novel viruses. VirusDetect is freely available at http://bioinfo.bti.cornell.edu/tool/
VirusDetect/.

1. Background chanism called RNA silencing or RNA interference (RNAi). In eukar-

Virus infections are universal and recognized as a significant threat
to agriculture production and human health. Efficient and accurate
detection of viruses in plants and animals is essential for the develop-
ment of effective strategies to manage the spread and impact of viral
diseases. Conventional virus detection methods such as enzyme-linked
immunosorbent assay (ELISA), polymerase chain reaction (PCR),
nucleic acid hybridization or microarray are useful but they require
prior knowledge or sequence information of the potential pathogens,
thus they are not highly efficient in detecting novel viruses or virus
variants. Traditional methods for the detection of unknown viruses
including electron microscopy and biological indicator hosts are
limited in their scope and only permit partial characterization of novel
agents. Recently, a new approach, virus discovery by high throughput
sequencing and assembly of total small RNAs (small RNA sequencing
and assembly; sRSA), has proven to be highly efficient in plant and
animal virus detection (Kreuze et al., 2009; Wu et al., 2010, 2015). This
approach exploits a natural and fundamental antiviral defense me-
yotes, upon viral infection an innate immunity system employs DICER
and DICER-like (DCL) enzymes to cleave viral RNAs into small
interfering RNAs (siRNAs) with sizes from 21 to 24 nucleotides (nt),
which are further amplified by RNA dependent RNA polymerases
(RdRPs). These virus-derived siRNAs direct antiviral immunity
through the RNAi mechanism that guide ARGONAUTE proteins to
silence the infected viral RNAs through cleavage or translational
inhibition (Ding, 2010). During this process, the virus-derived
siRNAs are automatically enriched in the hosts, which can be readily
detected by deep sequencing of host small RNAs (sRNAs) (Kreuze
et al., 2009; Wu et al., 2010). In contrast to most traditional methods in
virus detection, the sRSA approach does not require any prior virus
sequence information or the ability to cultivate and purify viruses. In
addition, compared to virus detection using genome or RNA sequen-
cing, the sRSA approach is very sensitive in identifying viruses and sub-
viral agents of different nucleic acid types and genome structures,
including single-stranded RNA (ssRNA) viruses of positive or negative
polarity, double-stranded RNA (dsRNA) viruses, DNA viruses, and

⁎
Corresponding author at: Boyce Thompson Institute, Ithaca, NY 14853, USA.
E-mail address: zf25@cornell.edu (Z. Fei).
1
Equal contributor.

http://dx.doi.org/10.1016/j.virol.2016.10.017
Received 11 August 2016; Received in revised form 17 October 2016; Accepted 20 October 2016
0042-6822/ © 2016 Elsevier Inc. All rights reserved.
Y. Zheng et al.
viroids, even when they occur at low titers that are not readily detected
by other methods such as RNA-Seq (Kreuze, 2014; Wu et al., 2015).
Moreover, sRNA profile (e.g., size distribution) can help to identify
novel viruses in a sequence-independent manner and provide informa-
tion about virus biology (Aguiar et al., 2015; Webster et al., 2015).
Furthermore, sRNAs are short thus less expensive short-read runs can
be used, and because of the natural enrichment of viral siRNAs, only
relatively shallow sequencing is required, thus reducing the sequencing
cost significantly.
Thanks to the rapid advance of next generation sequencing (NGS)
technologies, the sRSA approach has been widely used for the
identification of known and novel DNA and RNA viruses as well as
viroids in both plants and animals (Reviewed in Wu et al. (2015)).
Despite the expanding popularity of the sRSA approach in virus
discovery, currently there is no software package available that is
specifically designed to detect viruses using large-scale sRNA sequence
data generated with NGS technologies. Several bioinformatics tools
have been developed for virus discovery using NGS data such as
PathSeq (Kostic et al., 2011), VirusFinder (Wang et al., 2013), VirusSeq
(Chen et al., 2013), VirusHunter (Zhao et al., 2013), VirFind (Ho and
Tzanetakis, 2014), PFOR2 (Zhang et al., 2014) and IVA (Hunt et al.,
2015). However, these tools are mainly designed for virus discovery
using RNA-Seq or genome sequencing data and some are specifically
designed only for human pathogen discovery. Tools designed specifi-
cally for sRNA include Paparazzi (Vodovar et al., 2011), which
reconstitutes viral genomes through alignment using a related refer-
ence sequence as scaffold, and PFOR (Wu et al., 2012), which is
specifically designed for discovery of viroids. Visitor (Antoniewski,
2011), viRome (Watson et al., 2013), SearchSmallRNA (de Andrade
and Vaslin, 2014) and MISIS (Seguin et al., 2014) are graphical
interface tools that can only provide alignments of the siRNA reads
to a chosen virus reference genome; therefore they cannot be used as
general tools for identification of novel and known viruses.
In this study, we present VirusDetect, an automated bioinformatics
pipeline that can detect both known and novel viruses efficiently and
accurately using deep sequencing of plant or animal siRNAs. We
demonstrate the high efficiency and sensitivity of VirusDetect in
detecting known and novel virus using sRNA data generated from
plants and animals. Furthermore, we demonstrate that VirusDetect can
also efficiently identify viruses using other types of NGS datasets,
suggesting VirusDetect can be used as a universal tool for virus
discovery using NGS technologies.
2. Results and discussion
2.1. Design of VirusDetect
The workflow of VirusDetect is shown in Fig. 1A. VirusDetect
accepts cleaned sRNA sequences in fasta or fastq format. The program
first maps the sRNA reads to known virus reference sequences using
BWA (Li and Durbin, 2009). The mapped sRNA reads are considered
as virus-derived and then assembled into virus contigs using the
reference-guided approach. Next VirusDetect performs de novo assem-
bly of sRNAs. De novo assembly in VirusDetect is performed using
Velvet (Zerbino and Birney, 2008). The ‘hash_length’ (the length of k-
mer) is the most important parameter that determines the assembly
quality of Velvet and the ‘cov_cutoff’ (the coverage cutoff) is another
important parameter (Zerbino, 2010). VirusDetect first determines the
best ‘hash-length’ by performing Velvet assemblies using different k-
mer lengths. It then determines the best ‘cov_cutoff’ by performing
assemblies using the determined best ‘hash_length’ and different
coverage cutoffs. Since host siRNAs are distributed sporadically across
the host genome, when de novo assembled, they normally result in very
short contigs; while virus-derived siRNAs are distributed densely
across entire virus genomes and thus can be de novo assembled into
much longer contigs. Therefore, in VirusDetect, the best de novo
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Virology 500 (2017) 130–138
assemblies are defined as those containing the longest assembled
contigs. The de novo assembled contigs are pooled together with those
generated from reference-guided assemblies, and then processed to
remove redundant sequences using the same method described in
Zheng et al. (2011). VirusDetect then employs a homology-dependent
strategy to identify known and novel virus sequences from the
assembled contigs. The program first compares the contigs against
reference virus nucleotide sequences using the BLASTN program
(Altschul et al., 1990), and those having hits to virus nucleotide
sequences are identified as virus contigs. The remaining contigs are
further compared against the reference virus protein sequences using
the BLASTX program (Altschul et al., 1990), and those having hits to
the virus protein sequences are also identified as virus contigs. Contigs
matched to the same reference sequence are merged to form the final
output, and used to derive the coverage of the reference by virus
contigs. In addition, the depth of each virus contig covered by sRNA
reads is calculated and used to derive the average sRNA sequencing
depth of the matched virus contigs. To make it comparable among
different samples, raw depth is normalized to reads per million (RPM).
VirusDetect derives the coverage and the depth information since they
are the major parameters indicating whether the identified viruses are
true or due to alignment artifacts as true viruses generally have higher
coverage and depth. Finally, VirusDetect generates user-friendly hy-
perlinked html tables to display the list of viruses identified from
BLASTN and BLASTX searches, respectively (Fig. 1B). Each reference
virus sequence in the table is linked to a page, which shows the detailed
sequence alignment information of the virus contigs to the reference
(Fig. 1C). In addition, VirusDetect also provides the sequences of
detected virus contigs and their corresponding reference viruses in
fasta format. The alignment information between the contigs and the
reference viruses is also provided in files of Excel and SAM (Li et al.,
2009) format.
It has been reported that subtraction of host derived sRNAs
enriches virus-derived siRNAs and reduces noise introduced from host
sRNAs, thus improving the assembly of virus siRNAs and increasing
the efficiency of virus detection (Isakov et al., 2011; Li et al., 2012).
Therefore, prior to de novo assembly, VirusDetect can optionally map
the sRNA reads to host reference sequences to subtract host-derived
sRNAs. In addition, the contigs generated from de novo assemblies are
also aligned to the host sequences and those having high nucleotide
identity (e.g., > 90%) to the host sequences are discarded. Some host
genomes contain integrated viral sequences that are related to extant
replicating viruses, but are mostly inactive fragments and could be
falsely identified as an infecting virus if the genome subtraction is not
employed. On the other hand, host genomes used for subtraction
should be carefully examined to ensure they do not contain any
inadvertent viral sequence contamination.
It is worth to point out that homology-dependent strategies
employed by VirusDetect would not be able to identify novel viruses
whose genomes do not have similarity to any known virus sequences at
both nucleotide and protein levels. It has been reported that virus-
derived siRNAs are predominantly 21 or 22 nt in length (Ding, 2010;
Webster et al., 2015). Using this type of siRNA profile to identify novel
viral contigs in a sequence independent manner has been demon-
strated in insects and plants (Aguiar et al., 2015; Webster et al., 2015),
however its general applicability is not yet clear. Nonetheless, for
assembled contigs showing no viral sequence homology, VirusDetect
generates an html table listing their siRNA size profiles and high-
lighting those with viral-like siRNA signatures (i.e., enrichment of 21-
nt and 22-nt siRNAs) (Supplementary Fig. 1). However, it is suggested
that although the majority of VirusDetect-reported viral-like contigs
based on siRNA size profiles are virus derived, they should be further
evaluated in more detail to confirm their authenticity.
For virus detection using VirusDetect, the quality and size of the
reference virus database are important factors affecting the efficiency of
sRNA alignments and homology searches of virus contigs. For this
Y. Zheng et al. Virology 500 (2017) 130–138

Fig. 1. Design of VirusDetect. (A) Flowchart of VirusDetect for virus identification using sRNA sequencing data. (B, C) Screenshots of VirusDetect output html pages showing the
list of identified viruses (B) and alignments of virus contigs to the reference virus genome (C).

Table 1 reason, we classified the GenBank virus entries into eight different host
Comparison of potato viruses detected by standard virus indexing procedure (indicator kingdoms: vertebrate, invertebrate, plant, protozoa, algae, fungus,
plant, ELISA, NASH) and small RNA sequencing and assembly (sRSA) through bacteria, and archaea. sRNA samples generated from a specific host
VirusDetect.
would only need to be compared to the reference virus sequences from
Sample (CIP Standard Indexing sRSAa (from in PCR confirmation the kingdom the host belongs to, thus substantially reducing the
germplasm (from potato and/or vitro potato (from in vitro running time and the potential noise introduced from virus sequences
accession indicator plants plant potato plant of other unrelated host kingdoms. Nonetheless, reference virus se-
number) grown in extractions) extractions)
quences from multiple host kingdoms can be combined and used to
greenhouse)
identify viruses that may have hosts from different kingdoms. Currently
706735 PVXb,c,d PVX, PVA PVX, PVA GenBank (release 211) contains approximately two million virus
396009.258 – − − entries, among which ~94.8% were classified into the vertebrate
703471 PVSb PVS PVS
kingdom, ~4.4% into the plant kingdom, ~2.6% into the invertebrate
705268 PLRVb, PVXb,c,d PLRV, PVX PLRV, PVX
700744 PVSb,c,d, PVTb PVS, PVT PVS, PVT kingdom, and very few ( < 0.5%) into each of the other five kingdoms. It
706851 PVXc,d, PVSb PVX, PVS, PVT PVX, PVS, PVT is worth to note that ~2% of the virus entries could be classified into
703518 PVSb PVS PVS more than one kingdom. To further improve the efficiency of
704832 PLRVd, APMMVb,d, PLRV, APMMV, PLRV, APMMV, VirusDetect, the virus databases from different host kingdoms were
PVXd PVT PVT
processed to remove redundant sequences. The number of virus
703573 − − −
308328.32 − − − sequences in the resulting non-redundant databases (95% redundancy
398098.203 − − − level) is approximately 11.6%, 13%, and 14.3% of that in the original
396272.22 PVSb,d PVS PVS GenBank virus database for vertebrate, invertebrate, and plant viruses,
396063.16 PLRVd PLRV PLRV
respectively (Supplementary Table 1).
598198.4 − − −
304413.45 − − −
393046.7 PVXb,c,d, PVSb,c PVX, PVS PVX, PVS 2.2. Identification of known viruses using VirusDetect
a
By sRSA using VirusDetect. We first evaluated the performance of VirusDetect in detection of
b
Potato plant.
c
Indicator plant (mechanical inoculation).
known viruses using sRNA sequences generated from a set of 16 potato
d
Datura stramonium (graft-inoculation). accessions. These accessions were evaluated using the ISO17025
certified standard virus indexing procedure implemented at the
International Potato Center (CIP) for the infection of known potato

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Y. Zheng et al.
706735 (PVX) 700744 (PVS)
706735 (PVA) 700744 (PVT)
703471 (PVS)
706851 (PVX)
705268 (PLRV)
706851 (PVS)
705268 (PVX)
706851 (PVT)
703518 (PVS)
396272.22 (PVS)
Fig. 2. Viruses identified from the potato samples by VirusDetect. Alignments of identified virus contigs to the reference virus genomes. Blue tracks represent reference virus
genomes, and red tracks represent assembled virus contigs. PVA, Potato virus A; PVS, Potato virus S; PVX, Potato virus X; PLRV, Potato leafroll virus; PVT, Potato virus T; and APMMV,
Andean potato mild mosaic virus.
viruses including Potato virus S (PVS), Potato virus X (PVX), Potato
leafroll virus (PLRV), Potato virus T (PVT) and Andean potato mild
mosaic virus (APMMV) (Table 1). A total of 0.5–1.3 million high-
quality sRNA reads were generated for each of these samples, among
which 60–70% could be mapped to the potato genome while 0.6–5.6%
mapped to the plant virus database (Supplementary Table 2).
Consistent with the results from the virus indexing analysis,
VirusDetect did not detect any viruses in six accessions (396009.258,
703573, 308328.32, 398098.203, 598198.4, and 304413.45) while it
identified the corresponding viruses in the remaining accessions except
PVX in accession 704832 (Table 1). For the majority of the identified
viruses (15 out of 18), the assembled contigs from sRNAs covered more
than 50% of their genomes, and genomes of approximately half of the
identified viruses were covered by more than 95% (Fig. 2 and
Supplementary Table 3). In addition, Potato virus A (PVA) was
detected in accession 706735 by VirusDetect with assembled contigs
covering nearly the entire PVA genome (Fig. 2 and Supplementary
Table 3), but not identified by the current indexing procedure since an
ELISA test for this virus was not included in the standard procedure
and symptoms corresponding to PVA infection may have been masked
by those of PVX in the indicator hosts. Furthermore, PVT was detected
in accessions 706851 and 704832 by VirusDetect, but not by nucleic
acid spot hybridization (NASH) or indicator host ranges, likely because
the concentration of the virus was below the threshold of those tests.
All viruses identified by VirusDetect were confirmed by RT-PCR tests
from the original in vitro plants (Table 1). Sequencing of PCR
fragments corresponding to the viruses detected by VirusDetect also
confirmed accurate sequence assembly over the amplified regions.
Conversely, PVX was detected in accession 704832 by ELISA only
from grafted Datura stramonium but not by VirusDetect, nor PCR
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Virology 500 (2017) 130–138
704832 (PLRV)
704832 (PVT)
704832 (APMMV)
396063.16 (PLRV)
393046.7 (PVX)
393046.7 (PVS)
100% 75%
Identity
from the original plant, suggesting a possible greenhouse
contamination during the indexing process. Indeed, PVX is highly
contagious and other indicator plants inoculated with this accession
should have shown symptoms of infection; however, this was not the
case.
For this analysis, VirusDetect was run on a laptop with i5-3320M
CPU using one thread. It took 4–13 min for VirusDetect to analyze
each of these 16 samples for virus detection (Supplementary Table 2).
The main workflow of VirusDetect does not support multi-threads, but
several time-consuming steps include read mapping using BWA,
removing redundant virus contigs and comparing assembled contigs
against the virus reference database and the host sequences using
BLAST can be run using multiple CPUs to increase the speed.
Nonetheless, given the fact that VirusDetect can process large datasets
on desktop computers with reasonable time, coupled with its high
sensitivity in virus discovery, VirusDetect can serve as a tool for
efficient identification of viruses from deep siRNA sequencing datasets.
2.3. Detection of a novel plant virus using VirusDetect based on
sequence homology
We further evaluated the efficiency of VirusDetect using a sRNA
dataset we generated from an unspecified weed species from Brazil,
showing virus symptoms. The dataset contained a total of 710,854
high-quality cleaned sRNA reads. The sizes of these sRNA reads were
predominantly ranging from 21 to 24 nt (Fig. 3A). VirusDetect mapped
a total of 16,175 reads (2.28%) to non-redundant plant virus database.
However, the reference-guided assembly did not yield any contigs from
these mapped reads as these sRNA reads were sporadically aligned to
the reference sequences. De novo assembly of these sRNAs without
Y. Zheng et al.
Fig. 3. VirusDetect identified a novel potyvirus, Brazilian weed virus Y (BWVY), from a weed sRNA dataset. (A) Size distribution of total sRNAs and siRNAs derived
from BWVY. (B) siRNA distribution across the genome of BWVY in both positive (+) and negative (−) strands. (C) Alignments of virus contigs (blue lines) assembled from different
depths of sRNAs to the genome (black line) of BWVY identified in the weed sample.
subtraction of host sRNAs (since no host references were available)
generated a total of 73 non-redundant contigs. Homology search of
these contigs against the plant virus database identified one virus
contig, which was the longest one of 9837 nt and took up 66.1% of total
assembled bases (14,891 nt). The open reading frame (ORF) of this
contig had a length of 9237 nt, which could be translated into a single
polyprotein with 3078 amino acids. It was identified as a novel
potyvirus by VirusDetect since the translated protein sequence of its
ORF had significant and complete alignments (59.2% amino acid
sequence identity) to the genome of Verbena virus Y (GenBank
accession no. ACB69755), which belongs to the potyvirus genus.
Mapping sRNA reads back to the complete genome of the newly
identified virus (tentatively named Brazilian weed virus Y or BWVY)
indicated that these virus-derived siRNAs were predominantly of 21
and 22 nt (Fig. 3A) and they covered the entire genome of BWVY at
both positive and negative strands (Fig. 3B).
To validate the authenticity of BWVY, we performed Sanger
sequencing through genome walking using overlapping RT-PCR pro-
ducts that covered the entire virus genome. A complete genome
sequence of BWVY (9837 nt) was assembled from the Sanger
sequences. Alignment of the genome sequence assembled from
Sanger sequences and that assembled from sRNA sequences indicated
that the two genomes are largely similar, with 99% sequence identity. A
small percentage of sequence variations (~1%) between the two
genomes generated by two different methods were due to the presence
of single nucleotide polymorphisms scattered throughout the genome.
The virus genome sequence generated by Sanger sequencing was
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Virology 500 (2017) 130–138
assembled from a limited number of individual cloned molecules. On
the other hand, the virus genome sequence generated by deep sRNA
sequencing was likely resulted from millions of individual virus
molecules. Due to the existence of different virus haplotypes in a field
population, such low level of sequence variation (~1%) between the
consensus sequences generated from the two different sequencing
technologies was not unexpected, as reported in other studies (e.g.,
Di Giallonardo et al., 2014).
We further evaluated the sensitivity of VirusDetect in detecting the
novel BWVY using the randomly selected 15 K, 30 K, 60 K, 100 K, 150
K, 300 K, 450 K and 600 K sRNA reads from the dataset. VirusDetect
could confidently detect the novel virus, BWVY, even with only 60 K
sRNA reads, with 96.6% of the BWVY genome covered by the
assembled contigs (Supplementary Table 4), although most of the
assembled virus contigs were relatively short (Fig. 3C). However,
increasing the depth improved the coverage of the BWVY genome by
assembled contigs at low depths ( < 20 X). Further increase of the
sRNA sequencing depth would not improve the coverage very much but
would generate much longer virus contigs (Fig. 3C and Supplementary
Table 4).
2.4. Identification of novel viruses using VirusDetect based on siRNA
profile
The efficiency of VirusDetect in identifying novel viruses based on
siRNA profiles was evaluated using an sRNA dataset from the D.
melanogaster ovary somatic sheet (OSS) cell line described in Robine
Y. Zheng et al. Virology 500 (2017) 130–138

et al. (2009) (GenBank GEO accession no. GSE15378). First, using the possible to be of viral origin, had the proportion > 40% (41.7–68.9%)
homology-based approaches, VirusDetect identified all six known (Supplementary Table 5).
viruses that were reported in Wu et al. (2010), including the In summary, our results suggest that combined with homology-
Drosophila X virus (DXV), Drosophila birnavirus (DBV), Drosophila dependent and homology-independent strategies, VirusDetect is highly
C virus (DCV), and Nora virus, as well as the Flock house virus (FHV) sensitive in identifying novel viruses.
and Drosophila A virus (DAV) which were highly similar to the
American nodavirus (ANV; 93% and 95% nt identity for RNA2 and
2.5. Testing VirusDetect on other types of NGS data
RNA1, respectively) and Drosophila tetravirus (DTrV; 98% nt identity)
reported in Wu et al. (2010), respectively. VirusDetect was able to
Besides sRNA sequencing, several other strategies involving NGS
detect additional viruses in the cell line, including Bloomfield virus,
have been used for virus detection such as those employing sequencing
Autographa californica nucleopolyhedrovirus (AcMNPV), and
of polyA-enriched or rRNA-depleted RNAs and dsRNAs (Wu et al.,
Spodoptera frugiperda rhabdovirus (Sf-rhabdovirus) (Supplementary
2015). Therefore, we tested the performance of VirusDetect on one of
Fig. 2).
these types of NGS datasets.
Bloomfield virus is a new virus that was discovered recently
We applied VirusDetect to a polyA enriched RNA-Seq dataset from
(Webster et al., 2015). To evaluate the ability of VirusDetect to identify
Prunus domestica that included two samples infected with Plum pox
new viruses with no sequence similarity to known viruses, we run
virus (PPV) and two healthy samples (Rodamilans et al., 2014). The
VirusDetect on the OSS sRNA dataset using the reference virus
genome of P. mume (Zhang et al., 2012) was used for host read
database in which all sequences homologous to the segments of the
subtraction and HISAT (Kim et al., 2015) was included in VirusDetect
Bloomfield virus were removed. As expected, homology-based ap-
to replace BWA for aligning RNA-Seq reads to the P. mume genome.
proaches (BLASTN and BLASTX) failed to identify any segments of
Consistent with findings in Rodamilans et al. (2014), VirusDetect
this virus. However, based on the siRNA profile (proportion of 21-nt
successfully assembled contigs covering 98% of PPV-Rec and PPV-D
and 22-nt siRNAs of at least 40%), VirusDetect was able to identify
genomes from the two PPV-infected samples (Supplementary Fig. 3),
viral-like contigs corresponding to all the reported nine segments of the
while no virus was identified from the two healthy samples.
Bloomfield virus, with at least 80% of each segment covered by the
Furthermore, VirusDetect was able to identify Cherry virus A in the
identified viral contigs except the segment 5, which was covered by
infected samples that was not described in Rodamilans et al. (2014).
57.5% (Fig. 4). For all the assembled contigs derived from the
The assembled contigs of Cherry virus A has high sequence identity to
Bloomfield virus, only seven short contigs (53–79 nt) of segment 5
the reference virus genome (Supplementary Fig. 3), indicating that it is
and one (41 bp) of segment 9 were not identified as viral-like contigs
very likely that the identified virus is real. Our analysis supports that
(Supplementary Table 5). In addition, VirusDetect identified several
VirusDetect is also highly efficient and sensitive in virus discovery
novel viral-like contigs that were not reported before, including one of
using RNA-Seq datasets.
1034 nt. However, further confirmation of these newly identified viral-
like contigs is required. It is worth to note that here we chose
proportion of 21-nt and 22-nt siRNAs of 40% as the cutoff because 3. Conclusion
all the assembled long contigs (e.g., > 500 nt), which were highly
We have developed and described an automated bioinformatics

Bloomfield virus segment 1 (81.2%) Bloomfield virus segment 6 (98.5%)

Bloomfield virus segment 7 (79.9%)

Bloomfield virus segment 2 (96.2%) Bloomfield virus segment 8 (96.1%)

Bloomfield virus segment 4 (85.5%) Bloomfield virus segment 9 (97.1%)

Bloomfield virus segment 5 (57.5%)

Bloomfield virus segment 10 (95.3%)

Fig. 4. Identification of the Bloomfield virus by VirusDetect using siRNA size profiles. Blue tracks represent reference virus genomes, and red tracks represent assembled
virus contigs.

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Y. Zheng et al.
pipeline, VirusDetect, for virus discovery from sRNA sequencing
datasets. Extensive validation revealed that VirusDetect is a sensitive
and efficient tool for detection of known and novel viruses from plants,
animals and other kingdoms. The high sensitivity and efficiency of
VirusDetect in virus discovery are achieved by employing a combina-
tion of the following strategies: applying both reference-guided and de
novo assemblies of virus-derived siRNAs, using a curated and classified
virus reference database, and performing host sRNA subtraction and
automated parameter optimization for de novo assemblies. In some
cases, VirusDetect can assemble virus contigs that cover nearly the
entire genomes of most identified viruses from relatively small-scale
sRNA datasets. This combined with the increasing throughput and
decreasing cost of NGS technologies represents a cost-effective ap-
proach for virus detection and discovery through sequencing sRNAs
from a large number of multiplexed samples, and thus may facilitate
metagenomics researches of viral communities (viromes) on different
species across geographic ranges. Furthermore, although VirusDetect is
mainly designed for virus discovery using sRNA sequences, we
demonstrate that VirusDetect can be used as a universal bioinformatics
tool for virus discovery using different types of NGS datasets.
4. Materials and methods
4.1. Implementation of VirusDetect
The VirusDetect package is implemented in Perl. BWA (Li and
Durbin, 2009) is employed to align siRNA reads to the reference virus
sequences, host sequences or assembled virus contigs. For reference-
guided assembly, SAMtools (Li et al., 2009) is used to process BWA
alignments and generate per-position alignment information in pileup
format, which is used to guide the construction of virus contigs. De
novo assembly of viral siRNAs is performed using Velvet (Zerbino and
Birney, 2008). VirusDetect uses the BLAST program (Altschul et al.,
1990) to compare assembled contigs against virus reference nucleotide
and protein sequence databases. Perl modular Bio::Graphics (Stajich
et al., 2002) is used to generate the track images according to the
BLAST result of virus contigs.
4.2. Curation and classification of GenBank virus sequence database
Virus sequences downloaded from GenBank were classified into
eight different host kingdoms: vertebrate, invertebrate, plant, protozoa,
algae, fungus, bacteria, and archaea, based on the virus taxonomy
information provided by the International Committee on Taxonomy of
Viruses (ICTV; http://www.ictvonline.org/) using a perl script, which is
included in the VirusDetect package. Virus sequences that were not
classified in ICTV were then manually classified according to the
descriptions of the virus sequences and their high sequence similarity
to those classified viruses. Virus sequences classified into each of the
eight host kingdoms were further processed to remove redundant
sequences with sequence identity cutoff of 95%, 97% and 100%,
respectively, using cd-hit (Li and Godzik, 2006). The corresponding
protein sequences of each virus were also extracted. All the classified
virus nucleotide and protein sequences are available at the VirusDetect
website.
4.3. Plant material, RNA extraction and sRNA library construction
and sequencing
For potato sRNA sequencing and standard virus indexing, two
copies of 16 in vitro accessions were selected from the germplasm
collection at the CIP (Table 1). One copy of in vitro plants was
processed for standard virus indexing and the other set was processed
for sRNA sequencing. Total RNA was extracted from 1 g of fresh leaf
tissue using the CTAB method. The quantity of the total RNA was
determined using a NanoDrop analyzer (Thermo Fisher Scientific,
136
Virology 500 (2017) 130–138
USA) and the quality was checked by agarose gel electrophoresis.
sRNAs of 20–30 nt were purified after excising the band from a 3.5%
agarose gel. To demonstrate the efficiency of VirusDetect in identifying
and assembling genomes of novel viruses from sRNA sequences, a leaf
sample of an unspecified weed species with yellowing and mosaic-like
symptoms was collected from a tomato field in Barbacena, Brazil in
February 2013. Total RNA from this sample was purified using TRIzol
reagents following the manufacturer's instructions (Invitrogen, USA).
After quantification in a NanoDrop (Thermo Fisher Scientific, USA),
RNA molecules, ranging from 18 to 28 nt, were excised from a
polyacrylamide gel and extracted. sRNA libraries of the weed and
potato samples were constructed following the protocol described in
Chen et al. (2012) and sequenced on an Illumina HiSeq 2500 system
with the 50-bp single-end mode.
4.4. sRNA read processing
Raw sRNA reads were first processed to identify the 3′ adaptor
sequences using an in-house perl script, which is included in the
VirusDetect package. Briefly, the adaptor sequences were identified in
the sRNA reads if the first eleven nucleotides of the adaptor could
match the sRNA reads with at most one mismatch. sRNA reads with no
adaptor sequences identified were discarded and the remaining reads
were then processed to trim the 3′ adaptor sequences. The processed
reads that were in low quality (containing ambiguous bases) or shorter
than 15 nt were excluded from downstream analysis. The cleaned
sRNA reads were used for virus discovery using VirusDetect. The non-
redundant (95%) plant virus database was used as the reference. The
potato genome (Xu et al., 2011) was used as the reference for host
sRNA subtraction for the potato sRNA datasets.
4.5. Standard virus indexing of potato
Indexing of the 16 potato accessions was performed by virology
service unit at the CIP following an ISO/IEC 17025 accredited
procedure which includes the following tests: i) NASH for Potato
spindle tuber viroid (PSTVd) and PVT from in vitro and greenhouse-
grown plants, respectively, ii) DAS-ELISA from three replicates of
greenhouse grown plants with antibodies for PVX, PLRV, PVS,
APMMV, Potato virus Y (PVY), Potato yellowing virus (PYV),
Arracacha virus B-Oca strain (AVB-O), and Andean potato mottle
virus (APMoV), and iii) mechanical inoculation and symptom evalua-
tion of eleven biological indicator plants: Nicotiana benthamiana, N.
clevelandii×N. bigelovii, N. debneyii, N. glutinosa, N. tabacum "White
Burley", Chenopodium murale, C. quinoa, Datura stramonium,
Gomphrena globosa, Solanum lycopersicum "Rutgers" and Physalis
floridana. In addition graft inoculation of D. stramonium was also
performed.
4.6. Validation of known and novel plant viruses detected by
VirusDetect
Potato viruses were confirmed by RT-PCR using virus specific
primers for PVX, PVS, PVT, PLRV, and APMMV (Supplementary
Table 6). Same total RNA used for sRNA library construction was
used for PCR. For cDNA synthesis 1 μg of total RNA and 250 ng/μl of
random hexamer primers were used in a total reaction mix of 20 μl
using 200 U of M-MLV reverse transcriptase (Invitrogen). After
incubation at 37 °C for 50 min, the reaction was diluted 1:10 with
nuclease free water and 5 μl was used for PCR. The PCR was performed
using GoTaq® DNA Polymerase (Promega) in a volume of 20 μl with a
final primer concentration of 0.5 µM each. The PCR protocol consisted
of 5 min initial denaturation at 94 °C, followed by 35 cycles of 94 °C for
30 s, 50–60 °C for 30 s and 72 °C between 30s to 1 min, with final
extension at 72 °C for 10 min. PCR products were visualized on 1%
agarose gels stained with GelRed (Biotium). PCR fragments amplified
Y. Zheng et al.
for each isolate were purified using the High Pure PCR product
purification kit (Roche), then cloned into pGEM-T easy vector
(Promega) following standard procedures and transformed into
Escherichia coli (DH 5α). Samples were sent to Macrogen (Korea)
for Sanger sequencing.
To validate the genome sequence of the novel virus (BWVY)
assembled by VirusDetect, a series of 16 primer pairs
(Supplementary Table 7) were designed to amplify overlapping frag-
ments covering the entire virus genome. Amplicons generated using the
above primer pairs in reverse transcription-PCR (RT-PCR) were
sequenced directly or cloned using a TOPO TA cloning kit
(Invitrogen) and sequenced using the Sanger technology by the
Functional Biosciences, Inc. (Madison, WI).
4.7. RNA-Seq data processing
Raw RNA-Seq dataset of P. domestica described in Rodamilans
et al. (2014) was downloaded from NCBI SRA database under
accession SRP041925. Adaptor and low quality sequences were re-
moved using Trimmomatic (Bolger et al., 2014). The remaining high
quality reads were aligned to the rRNA database (Quast et al., 2013)
using bowtie (Langmead et al., 2009) allowing up to 3 mismatches. The
unaligned reads were used for virus discovery using VirusDetect.
Competing interests
The authors declare that they have no competing interests.
Author contributions
Z.F., Y.Z., and S.G. designed the project. Y.Z. and S.G. implemented
the program. Y.Z. performed data analyses. C.P., R.L. and K.L.
constructed the weed sRNA library and performed experiments to
validate the novel weed virus. D.G., S.F., M.G. and J.K. contributed to
the design and evaluation of the program. J.K. and S.F. designed, M.G.
and D.G. constructed sRNA libraries, and S.F. and M.G. performed the
validation experiment with potato samples. Z.F., Y.Z., K.L. and J.K.
wrote the manuscript. All authors read and approved the manuscript.
Availability of supporting data
The genome sequence of BWVY is available in NCBI GenBank
under accession no. KU685505. The nearly complete genome
sequences of viruses assembled from the potato samples are available
in NCBI GenBank under the following accession nos.: KU58645,
KU586452, KU586454, KU586455, KU586456, KU586451, and
KU586453. The sRNA read data from the Brazil weed and the 16
potato samples are available in NCBI Sequence Read Archive under the
BioProject ID PRJNA311127.
Acknowledgements
The collection of the weed sample in Brazil by HM.CLAUSE is
greatly appreciated. We thank Andrea Gilliard and Alan Wilder for
their excellent technical assistance. This work was supported by grants
from National Science Foundation (IOS-1110080) to Z.F. and J.K., the
USDA SCRI (2012-01507-229756) to Z.F. and K.L., the USDA SCRI
(2010-600-25320) to K.L., and the CGIAR research program on roots,
tubers and bananas to J.K.
Appendix A. Supplementary material
Supplementary data associated with this article can be found in the
online version at doi:10.1016/j.virol.2016.10.017.
137
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