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Volume 6(2)
ISSN 0973-2063 (online) 0973-8894 (print)
Bioinformation 6(2): 91-94 (2011) 91 © 2011 Biomedical Informatics
ProViDE: A software tool for accurate estimation
of viral diversity in metagenomic samples
Tarini Shankar Ghosh, Monzoorul Haque Mohammed, Dinakar Komanduri, Sharmila
Shekhar Mande*
Bio-Sciences Division, Innovation Labs, Tata Consultancy Services, 1 Software Units Layout, Hyderabad 500 081, Andhra Pradesh, India; Sharmila Shekhar
Mande - Email: sharmila@atc.tcs.com; *Corresponding author
Received March 04, 2011; Accepted March 07, 2011; Published March 26, 2011
Selected publications from Asia Pacific Bioinformatics Network (APBioNet) Ninth International Conference on Bioinformatics (InCoB 2010), Tokyo, Japan 26-
28 September 2010.
Abstract:
Given the absence of universal marker genes in the viral kingdom, researchers typically use BLAST (with stringent E-values) for taxonomic classification of viral
metagenomic sequences. Since majority of metagenomic sequences originate from hitherto unknown viral groups, using stringent e-values results in most
sequences remaining unclassified. Furthermore, using less stringent e-values results in a high number of incorrect taxonomic assignments. The SOrt-ITEMS
algorithm provides an approach to address the above issues. Based on alignment parameters, SOrt-ITEMS follows an elaborate work-flow for assigning reads
originating from hitherto unknown archaeal/bacterial genomes. In SOrt-ITEMS, alignment parameter thresholds were generated by observing patterns of sequence
divergence within and across various taxonomic groups belonging to bacterial and archaeal kingdoms. However, many taxonomic groups within the viral kingdom
lack a typical Linnean-like taxonomic hierarchy. In this paper, we present ProViDE (Program for Viral Diversity Estimation), an algorithm that uses a customized
set of alignment parameter thresholds, specifically suited for viral metagenomic sequences. These thresholds capture the pattern of sequence divergence and the
non-uniform taxonomic hierarchy observed within/across various taxonomic groups of the viral kingdom. Validation results indicate that the percentage of 'correct'
assignments by ProViDE is around 1.7 to 3 times higher than that by the widely used similarity based method MEGAN. The misclassification rate of ProViDE is
around 3 to 19% (as compared to 5 to 42% by MEGAN) indicating significantly better assignment accuracy. ProViDE software and a supplementary file
(containing supplementary figures and tables referred to in this article) is available for download from http://metagenomics.atc.tcs.com/binning/ProViDE/
Background:
A number of metagenomic studies have been initiated in the past 3-4 years to
explore, characterize and compare the taxonomic diversity of viruses present in
various environments [1, 2]. Besides cataloguing viral diversity, these studies
have identified several hitherto unknown groups of viruses that play a critical
role in transferring genes involved in a variety of metabolic functions [1, 3].
Given the absence of universal marker genes (such as 16S rRNA in bacteria /
archaea) in the viral kingdom, researchers typically use similarity-based
approaches like BLAST (with stringent E-values) for taxonomic classification
of viral metagenomic sequences. However, since a majority of sequences in
typical metagenomes originate from hitherto unknown viral groups, the use of
such stringent thresholds will result in a large fraction of sequences remaining
unclassified. Furthermore, using less stringent E-values (observed for BLAST
hits with poor alignment quality) will result in a high number of incorrect
taxonomic assignments. The recently published SOrt-ITEMS algorithm
provides an approach to address the above issues [4]. Based on alignment
parameters, an elaborate work-flow is followed by SOrt-ITEMS for assigning
reads originating from genomes of hitherto unknown archaeal/bacterial
organisms. Alignment parameter thresholds used by SOrt-ITEMS are generated
by observing the pattern of sequence divergence within and across various
taxonomic groups belonging to bacterial and archaeal kingdoms. However,
majority of taxonomic groups within the viral kingdom are characterized by the
absence of a typical Linnaean-like taxonomic hierarchy (phylum, class, order,
family, genus and species). This motivated us to develop ProViDE (Program
for Viral Diversity Estimation), a novel algorithm that uses a customized set of
alignment parameter thresholds/ranges, specifically suited for the accurate
taxonomic labelling of viral metagenomic sequences. These thresholds take
into the account the pattern of sequence divergence and the non-uniform
taxonomic hierarchy observed within/across various taxonomic groups of the
viral kingdom.
Methodology:
Determination of alignment parameter thresholds:
Using MetaSim [5], simulated data sets were generated from 50 diverse viral
genomes (Supplementary Table 1). Subsequently the alignment parameter
thresholds were determined (Supplementary Figures 1-4, Supplementary
Tables 2-5) using a methodology similar to that adopted in SOrt-ITEMS [4].
Based on these, flow charts (Figure 1) were devised (for various query lengths)
in order to identify an appropriate taxonomic level of assignment for a given
query sequence.
BIOINFORMATION open access
ISSN 0973-2063 (online) 0973-8894 (print)
Bioinformation 6(2): 91-94 (2011) 92 © 2011 Biomedical Informatics
Figure 1: Flow-charts showing various steps followed to arrive at an appropriate taxonomic level, where the assignment of each read (A) Sanger (~800 bp length)
(B) 454 – Titanium (~400 bp length) (C) 454- Standard (~ 250 bp) and (D) 454-GS20 (~ 100 bp). (I: Identity; P: Positives) is to be restricted. Hit taxon denotes the
taxon/organism corresponding to the hit sequence.
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Steps followed for taxonomic classification of viral metagenomic
sequences:
Supplementary Figure 5 depicts the various steps followed by ProViDE
algorithm. The output of a BLASTx search against the nr database is taken as
input for ProViDE. For each hit, ProViDE first parses the values of various
alignment parameters. For each read, based on its length, an appropriate
taxonomic level of assignment (TL) is subsequently identified (Figure 1). The
taxonomic assignment of the read is done using the orthology approach as used
in SOrt-ITEMS [4]. The final taxonomic assignment of the read is thus
restricted to taxonomic level that lies at or above the TL.
Data-sets and Database variants used for evaluating binning accuracy and
specificity:
1,40,000 sequences were generated from 35 viral genomes (Supplementary
Table 6). These genomes were different from the ones taken for obtaining
alignment parameter thresholds. Based on their length, these sequences were
divided into four test data-sets, namely Sanger, 454-400, 454-250 and 454-100.
To evaluate the performance of ProViDE with respect to sequences originating
from unknown viral genomes, sequences in each data-set were queried (using
BLASTx) against 2 variants of the nr database, namely, (a) nr database
excluding sequences belonging to the query genome ('MINUS SPECIES') and
(b) nr database excluding all sequences which fall under the immediate higher
level taxonomic group to which the query species belongs ('MINUS ONE
LEVEL UP'). The BLASTx outputs obtained were given as input to ProViDE.
The results of ProViDE were also compared with corresponding results
generated with a similarity based binning method, MEGAN [6]. Both the
programs were run using a min-support value of 2 and a bit score threshold
value of 35.
Categorization of taxonomic assignments:
The assignments of a read to a taxon that lies in the path between the root and
the taxon corresponding to the source organism of the read was categorized as
'correct'. To quantify the specificity, these 'Correct assignments' were sub-
grouped into two categories. All correct assignments at the level of root or
cellular organisms or super-kingdom (Viruses) were considered as 'non -
specific'. Assignments below the level of super-kingdom were considered as
'specific assignments'. The assignment of a read to a taxon that does not lie in
the path between the root and the taxon corresponding to the source organism
of the read was categorized as 'Wrong'. Reads having hits having a bit-score
less than 35 and/or an alignment length of less than 25 were categorized as
'Unassigned'. All reads with no BLAST hits were categorized as 'No hits'.
Discussion:
Table 1 shows evaluation results with respect to the total number of correct
assignments, wrong assignments, and the number of sequences categorized as
unassigned. As expected, the percentage of total correct assignments is seen to
increase with increasing read length. However, it is observed that (for all four
test data-sets), the percentage of 'correct' assignments by ProViDE is around
1.7 to 3 times higher than that by MEGAN. Since for both methods, most (if
not all) correct assignments are at specific levels, the relative specificity
obtained with ProViDE is around 1.7 to 3 times higher than that with MEGAN.
Furthermore, the percentage of sequences misclassified by ProViDE is in the
range of 3-19% (as compared to 5 - 42% by MEGAN) indicating significantly
better assignment accuracy. A similar number of sequences are categorized as
'unassigned' by both programs indicating that the relatively high levels of
accuracy obtained using ProViDE are not at the cost of decreased number of
assignments. One of the important aspects of metagenomic sequence analysis is
to assign metagenomic sequences to correct taxonomic groups. Given that
metagenomic sequence data sets typically contain millions of sequences,
majority of which originate from new/hitherto unknown organisms, accurate
and specific taxonomic assignment of metagenomic sequences still remains a
major computational challenge.
In the current study, we have presented an algorithm (ProViDE) that is
specifically customized for taxonomic analysis of viral metagenome data sets.
Majority of reads in viral metagenomic data-sets originate from hitherto
unknown viral groups, the sequences of which are absent in existing reference
databases. Consequently, a majority of these sequences generate poor quality
alignments with sequences in reference databases. Assignment of these
sequences directly to the taxon corresponding to the best hit (irrespective of
alignment quality) is expected to generate a large number of incorrect
assignments. Besides, validation results generated in the present study also
indicate that the popular binning algorithm, namely MEGAN, which is based
on the principle of least common ancestor approach, also has an extremely high
misclassification rate (which is as high as 40% for some of the data sets). This
high misclassification rate of MEGAN is expected since it uses a single
alignment parameter (bit-score) for judging alignment quality (prior to
assignment). Consequently, MEGAN ends up misclassifying a majority of
reads, especially those having poor quality alignments (with identities as low as
20%). Furthermore, as demonstrated by earlier studies [4], the least common
ancestor (LCA) approach used by MEGAN is generally associated with poor
binning specificity (especially in metagenomic scenarios wherein majority of
reads originate from unknown organisms).
In contrast, multiple alignment parameters like bit-score, identities, positives
(thresholds of which were specially identified for viral metagenomic
sequences) are used by ProViDE for ascertaining the quality of the alignment.
This ensures that assignment of reads at specific levels is done only for those
reads that generate high quality alignments with database sequences. As the
alignment quality decreases, ProViDE assigns these reads at progressively
higher taxonomic levels. Validation results have indicated that employing this
approach helps in significantly reducing the number of incorrectly assigned
sequences. Validation results also indicate that ProViDE correctly assigns a
greater number of sequences at specific levels (as compared to MEGAN). This
indicates the overall utility of the ProViDE algorithm for accurate and specific
taxonomic assignment of viral metagenomic sequences. A comparative
evaluation of binning time indicates that the ProViDE algorithm takes
approximately an hour to process the blastx output obtained for a data-set
having 100,000 reads. This is marginally higher than the time taken by
MEGAN for analysing the same number of reads. Supplementary Figure 6
gives a time comparison analysis plot of this analysis.
Conclusion:
Performance evaluation with data-sets or database variants simulating typical
metagenomic scenarios indicates that ProViDE has significantly high
specificity and accuracy. To the best of our knowledge, ProViDE is the first
ever similarity-based binning algorithm that provides an accurate and specific
taxonomic label to most of the reads constituting viral metagenomic data sets.
References:
[1] Williamson SJ et al. PLoS ONE. 2008 3(1): e1456 [PMID: 18213365]
[2] Lindell D et al. Nature 2005 438: 86 [PMID: 16222247]
[3] Willner D et al. PLoS ONE. 2009 4(10): e7370 [PMID: 19816605]
[4] Monzoorul Haque M et al. Bioinformatics 2009 25:1722 [PMID:
19439565]
[5] Richter DC et al. PLoS ONE. 2008 3(10): e3373 [PMID: 18841204]
[6] Huson DH et al. Genome Res. 2007 17: 377 [PMID: 17255551]
Edited by TW Tan
Citation: Ghosh et al. Bioinformation 6(2): 91-94 (2011)
License statement: This is an open-access article, which permits unrestricted use, distribution, and reproduction in any medium, for non-commercial purposes,
provided the original author and source are credited.
BIOINFORMATION open access
ISSN 0973-2063 (online) 0973-8894 (print)
Bioinformation 6(2): 91-94 (2011) 94 © 2011 Biomedical Informatics
Supplementary material:
Table 1: Comparison of the percentage of reads assigned under various bin categories by ProViDE and MEGAN for the (A) 454-100 data sets (B) 454-250 data
sets (C) 454-400 data sets, and (D) Sanger data sets. In this table the terms 'MINUS SPECIES', and 'MINUS ONE LEVEL UP' refer to the database variants used.
A detailed description of the database variants is given in the Methodology section of the manuscript. Note that the subtotals may vary by a value of 0.1, since the
individual values were rounded off to single decimals.
(A) 454_100
ASSIGNMENT CATEGORIES MINUS SPECIES MINUS ONE LEVEL UP
ProViDE MEGAN ProViDE MEGAN
NON SPECIFIC LEVELS 0 1.2 0 0
SPECIFIC LEVELS 25.4 13.5 5 2.4
TOTAL CORRECT ASSIGNMENTS 25.4 14.7 5 2.4
WRONG 5.2 12.3 2.7 5.2
UNASSIGNED + NO HITS 69.4 73.1 92.4 92.4
(B) 454_250
ASSIGNMENT CATEGORIES MINUS SPECIES MINUS ONE LEVEL UP
ProViDE MEGAN ProViDE MEGAN
NON SPECIFIC LEVELS 0 1.7 0 0
SPECIFIC LEVELS 44.2 23.0 18.8 6.7
TOTAL CORRECT ASSIGNMENTS 44.2 24.7 18.8 6.7
WRONG 5.2 24.7 3.5 15.4
UNASSIGNED + NO HITS 50.6 50.7 77.7 77.8
(C) 454_400
ASSIGNMENT CATEGORIES MINUS SPECIES MINUS ONE LEVEL UP
ProViDE MEGAN ProViDE MEGAN
NON SPECIFIC LEVELS 0 1.7 0 0.1
SPECIFIC LEVELS 52.5 26.2 27.2 8.7
TOTAL CORRECT ASSIGNMENTS 52.5 27.9 27.2 8.8
WRONG 4.7 29.4 3.4 21.8
UNASSIGNED + NO HITS 42.7 42.8 69.4 69.4
(D) SANGER
ASSIGNMENT CATEGORIES MINUS SPECIES MINUS ONE LEVEL UP
ProViDE MEGAN ProViDE MEGAN
NON SPECIFIC LEVELS 0 2.3 0 0.5
SPECIFIC LEVELS 60.2 32.0 35.3 14.2
TOTAL CORRECT ASSIGNMENTS 60.2 34.3 35.3 14.7
WRONG 14.2 41.1 19.7 41.9
UNASSIGNED + NO HITS 25.7 24.7 45.0 43.4
