W16–W21 Nucleic Acids Research, 2016, Vol. 44, Web Server issue Published online 3 May 2016doi: 10.1093/nar/gkw387

PHASTER: a better, faster version of the PHAST phagesearch toolDavid Arndt1, Jason R. Grant1, Ana Marcu1, Tanvir Sajed1, Allison Pon1, Yongjie Liang1 andDavid S. Wishart1,2,3,*

1Department of Computing Science, Edmonton, AB T6G 2E8, Canada, 2Department of Biological Sciences,University of Alberta, Edmonton, AB T6G 2E9, Canada and 3National Institute for Nanotechnology, 11421Saskatchewan Drive, Edmonton, AB T6G 2M9, Canada

Received January 30, 2016; Revised April 15, 2016; Accepted April 27, 2016

ABSTRACT

PHASTER (PHAge Search Tool – Enhanced Release)is a significant upgrade to the popular PHAST webserver for the rapid identification and annotation ofprophage sequences within bacterial genomes andplasmids. Although the steps in the phage identifica-tion pipeline in PHASTER remain largely the same asin the original PHAST, numerous software improve-ments and significant hardware enhancements havenow made PHASTER faster, more efficient, more vi-sually appealing and much more user friendly. In par-ticular, PHASTER is now 4.3× faster than PHASTwhen analyzing a typical bacterial genome. Morespecifically, software optimizations have made thebackend of PHASTER 2.7X faster than PHAST, whilethe addition of 80 CPUs to the PHASTER computecluster are responsible for the remaining speed-up. PHASTER can now process a typical bacterialgenome in 3 min from the raw sequence alone, or in1.5 min when given a pre-annotated GenBank file. Anumber of other optimizations have also been imple-mented, including automated algorithms to reducethe size and redundancy of PHASTER’s databases,improvements in handling multiple (metagenomic)queries and higher user traffic, along with theability to perform automated look-ups against 14000 previously PHAST/PHASTER annotated bacte-rial genomes (which can lead to complete phageannotations in seconds as opposed to minutes).PHASTER’s web interface has also been entirelyrewritten. A new graphical genome browser has beenadded, gene/genome visualization tools have beenimproved, and the graphical interface is now moremodern, robust and user-friendly. PHASTER is avail-able online at www.phaster.ca.

INTRODUCTION

Bacterial genomes and plasmids can contain a large fraction(>20% in some cases) of functional and non-functional bac-teriophage genes (1). Indeed, these non-bacterial, prophagesequences likely account for a significant proportion of thevariation within bacterial species or clades. The fact thatphages and prophages allow bacteria to acquire antibioticresistance, to adapt to new environmental niches or to be-come pathogenic has led to renewed interest in identifyingand annotating prophage sequences in bacterial genomes.As a result, prophage finding programs and web servershave become integral to many bacterial genome annotationpipelines. One of the first web servers to be developed forphage identification and annotation was PHAST (PHAgeSearch Tool). First published in 2011 (2), PHAST has be-come one of the most popular tools for prophage identifi-cation in bacterial genomes. To date, the PHAST paper hasreceived more than 400 citations and the PHAST websitereceives up to 7300 job submissions per month with up to30% of these coming through its web API (Application Pro-gramming Interface). The popularity of the server appearsto be due to a number of factors, such as its speed, accu-racy, visually appealing output and the fact that it appearedjust as NextGen sequencing of bacterial genomes started to‘take off” (3).

The PHAST prophage analysis pipeline is relativelystraightforward. In particular, PHAST accepts both Gen-Bank and FASTA formatted genomic sequence data,and performs BLAST (4) searches against a customprophage/phage database that combines protein sequencesfrom the National Center for Biotechnology Information(NCBI) phage database and the prophage database de-veloped by Srividhya et al. (5). Phage-like genes are thenclustered into prophage regions using DBSCAN (6). Non-phage genes in these identified regions are annotated by asecond BLAST search against a non-redundant bacterialprotein database, and the prophage regions are assigned acompleteness score based on the proportion of phage genesin the identified region. All of the annotated prophage data

*To whom correspondence should be addressed. Tel: +1 780 492 0383, Fax: +1 780 492 1071; Email: david.wishart@ualberta.ca

C© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), whichpermits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contactjournals.permissions@oup.com

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is then displayed on the web through a variety of hyper-linked tables and colored chromosomal maps.

PHAST was introduced at a time of increasing inter-est in prophage identification, which had already seenthe development of several stand-alone phage search pro-grams, namely Prophage Finder (7), Phage Finder (8) andProphinder (9). Since PHAST’s original release, phage find-ing has continued to develop, and several other high qualityphage identification tools have since been published. Someof the newer phage and prophage identification tools in-clude PhiSpy (10) and VirSorter (11). PhiSpy, in additionto performing sequence similarity-based searches, analyzesseveral other sequence-based statistics to help identify novelphages that are not represented in existing phage databases.VirSorter is geared towards more specialized applicationssuch as finding phages in draft genomes and Single-cell Am-plified Genomes (12,13). VirSorter also handles fragmentedmetagenomic data as well as whole genomic data.

In addition to the appearance of new algorithms forphage searching and new kinds of user demands by phagescientists (i.e. handling metagenomic data), other impor-tant developments in the field have taken place. These in-clude the rapid growth in the number of sequenced bacte-rial and phage genomes. Since 2011, the size of the NCBIphage/prophage database has grown by a factor of fourwhile the number of sequenced bacteria has grown by nearlyfive fold. This has led to a massive increase in both databasesizes and search times. Likewise, the ease with which bacte-rial genomes are now sequenced has led to a 12× growth inthe number of queries being handled by PHAST (and otherphage searching programs) over the past five years. In thelast year alone, visits to PHAST have increased by ∼60%and the server now processes one job every ∼6 min.

These rapid developments in both bacterial genomics andthe phage/prophage field led us to revisit PHAST and to ac-tively explore methods to enhance and upgrade the PHASTserver. These efforts led to the creation of PHASTER(PHAge Search Tool – Enhanced Release), a faster, bet-ter, more modern version of PHAST. To improve PHAST’sprocessing speed, we made a number of algorithmic (soft-ware) and hardware improvements. We also expanded andenhanced the way PHAST’s phage and bacterial databasesare prepared, read and handled. Improvements were alsomade to the user interface to make the website more visu-ally appealing and far more convenient to use. Further, byrewriting much of the PHAST codebase, we were able tomake the website easier to maintain into the future. Theseand other improvements are described in more detail below.

WHAT’S NEW

Software upgrades and optimizations

Phage and prophage searching is a computationally inten-sive task with most of the time and computational resourcesbeing consumed with large scale sequence comparisons andalignments. The original version of PHAST used an older,legacy version of BLAST that was both slower and less ableto handle large numbers of query sequences. By upgradingto the latest version of BLAST (known as BLAST+, version2.3.0+ (14)) we were able to overcome a number of previouslimitations and improve PHAST’s overall performance. In

particular, BLAST+ is specially designed to handle longerand greater numbers of sequences more efficiently and to doits alignments more quickly than earlier versions of BLAST.Although much faster alternatives to BLAST exist, theyare generally less sensitive. Tests with one such alternative,RAPSearch2 (15), led to a ∼7% loss in sensitivity (data notshown). Upgrading to BLAST+ maintained prediction ac-curacy while achieving a 15–25% speedup to the server’sBLAST search phases.

BLAST search speeds were further improved throughvarious computing cluster optimizations. The old PHASTemployed a grid scheduler but with only minimal optimiza-tions, resulting in frequently idle CPU cores. PHASTERnow prepares input to its grid scheduler such that all CPUcores are used much more efficiently, particularly in caseswhen the cluster is handling a single user submission andmust complete it as quickly as possible. PHASTER now di-vides its bacterial database into several parts, allowing com-puting nodes to specialize in particular sub-databases. InPHASTER, each query sequence is now searched againsteach sub-database component, and the results are subse-quently combined, with appropriate adjustments made tothe BLAST expectation values (e-values) for the true to-tal size of the database searched. Each database componentcan be handled by multiple cluster nodes, providing flexibil-ity to the cluster’s job scheduler, and ensuring redundancyin case of hardware failure. In addition, the set of query se-quences is partitioned evenly into a small number of sub-groups, such that the sum of the sequence lengths in eachsub-group is approximately the same, and each sub-set isrun separately. With these optimizations, smaller individualBLAST+ jobs can be more readily distributed to availableCPU cores as they become available. The optimal numberof CPU cores per individual BLAST job and the numberof groups into which to partition the query sequences weredetermined through careful parameter optimization. It isalso important to note that the improved runtimes and en-hanced parallelism did not compromise the accuracy of theprophage identifications (see below).

PHASTER now provides the option to search for phageregions in contigs assembled from metagenomic data. Inthis mode, complete and partial genes are predicted us-ing FragGeneScan (16), and the predicted prophages arearranged by contig in the presented results. We assessedcontigs of various lengths derived from a test genome (Es-cherichia coli O157:H7, GenBank accession NC 002655)to determine both the minimum and the optimal contiglengths for detecting prophages in metagenomic data. Wefound that a small number of phage regions (∼15%) couldbe identified in contigs as short as 2000 bp, while ∼90%of prophages could be identified in contigs >20 000 bplong. Based on these data we set a threshold minimum con-tig length of 2000 bp (although we obviously recommendlonger contig lengths, if available). The full set of contig per-formance data are available on the PHASTER website.

Database preparation

As noted earlier, the rapidly growing size of both bacte-rial and phage/prophage sequence databases resulted insubstantial increases in the runtimes for PHAST’s BLAST

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Figure 1. A screenshot montage of the upgraded PHASTER user interface.

searches. For instance, the number of bacterial prophage se-quences has increased from around 45 000 as of January1, 2011 to over 187 000 today. In addition, the bacterialsequence database, which is used in PHAST’s annotationstep, has grown by a factor of nearly 5X since PHAST wasfirst released. The latest version of the bacterial sequencedatabase consists of >16 million protein sequences fromRefSeq (17). However, the bacterial sequence database hasa high degree of sequence redundancy with many of the in-dividual gene sequences being very similar to one another.This sequence redundancy adds little value to the anno-tation process while needlessly slowing down the BLASTsearches. For PHASTER, we reduced the size of the bacte-rial sequence database by removing sequences with >70%sequence identity to any other sequence in the database, us-ing CD-HIT (18). The resulting database now contains justunder nine million bacterial protein sequences, or 55% ofthe pre-filtered database size. This database reduction ledto a two-fold reduction in the search time for this phase ofthe PHASTER annotation pipeline.

A surprisingly high percentage of queries submitted toPHAST consist of genomes that had been previously an-notated by PHAST. Rather than treating each query as

a completely novel sequence and spending several min-utes repeating the same annotation process that had beencompleted previously, we have implemented a ‘quick querycheck”. With this new checking routine in place, sequencesor sequence files that are submitted to PHASTER arerapidly compared against a local database of >14 000 non-redundant, previously annotated (via PHAST) bacterial orplasmid genomes. In the quick query check the query se-quence’s nucleotide frequencies and total sequence lengthare computed and rapidly matched against a database ofthese same statistics for all cached sequences. Potential se-quence matches are isolated (usually just one, if any) andthen aligned against the query sequence to ensure that onlyexact sequence matches are used. Having identified a querythat is identical to an entry in PHASTER’s database, theannotations are transferred and the result is returned to theuser in a few seconds. As a result, while the average de novoquery to PHASTER may take 2–3 min, a significant num-ber of queries can be returned in 2–3 s.

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Table 1. Detail of PHASTER’s performance upgrades, and a comparison of their impact on runtimes for a 5.5 Mbp test genome (Escherichia coli O157:H7,GenBank accession NC 002655)

Cumulative set of performance enhancementsBLAST versus viral

DB runtime (s)BLAST versus bacterial DB

runtime (s)Total runtime on GenBank

annotated genome (s)Total runtime on unannotated

genome (s)

PHAST (baseline) current DBs, no other upgrades 191 576 270 899PHASTER (upgrade 1) – filter bacterial DB 191 309 270 632PHASTER (upgrade 2) – cluster upgrade 88 166 167 386PHASTER (upgrade 3) – BLAST+ 77 132 156 341PHASTER (upgrade 4) – partition query sequences evenly 47 103 126 282PHASTER (upgrade 5) – bacterial DB, optimize parameters 47 48 126 227PHASTER (upgrade 6) – faster front-end server 47 48 100 205

Hardware upgrades

There is only so much software optimization that can beperformed before one reaches a point of diminishing re-turns. As a result, a number of hardware upgrades hadto be made to the PHASTER server. In particular, wehave expanded the number of CPU cores in the comput-ing cluster available from 32 (in the original PHAST) to112 (in PHASTER). The PHASTER cluster now has 32 In-tel Xeon 5150 @ 2.66 GHz, 32 AMD Opteron 6320, and48 AMD Opteron 6348 processing cores. To further en-hance PHASTER’s performance, we have removed a num-ber of other web servers from the PHASTER cluster. Thishas freed up more CPU cycles to accommodate the server’sheavy daytime use. All of PHASTER’s gene prediction andBLAST+ search analyses are now run on this 112 core clus-ter. In addition, the front-end for the PHASTER websitehas been installed on a much quicker virtual server whichis leased through the Google Compute Engine. This front-end server features 2 Intel Xeon CPUs @ 2.30GHz anda local solid-state drive. The front-end performs many ofPHASTER’s other computations, and now does so ∼50%faster. Moreover, because PHASTER now has a dedicatedfront-end server, it is able to accommodate two jobs simul-taneously for the most memory-intensive portions of thepipeline. This provides faster results during periods of heav-ier use.

User interface enhancements

The original PHAST web interface, while very functionaland utilitarian, was actually quite rudimentary and re-lied on a number of web technologies that are no longercurrent. PHASTER has a much more modern and user-friendly interface. PHAST’s front-end, which was imple-mented using Perl and CGI, has been completely re-writtenfor PHASTER using Ruby on Rails. PHASTER’s graphi-cal genome viewer has also been completely rewritten andmodernized. The old genome viewer in PHAST providedcircular and linear genome browsers implemented in AdobeFlash. Flash is a technology that is slowing being phasedout by many websites and is not available on most mo-bile devices. As a result, PHASTER’s interactive phageviewer has now been re-done using JavaScript, using An-gularPlasmid (http://angularplasmid.vixis.com) for the cir-cular genome view and D3 (http://d3js.org) for the lineargenome view. As a result, PHASTER’s graphical user in-terface is faster, more robust and better-looking (Figure 1).A more robust, threaded queuing system has also been im-plemented using Sidekiq (http://sidekiq.org). Another in-terface upgrade involved the reformatting of PHAST’s vast

collection of pre-computed prophage predictions for over14 000 different bacterial genomes. PHAST presented thesedata in a single static table that was becoming increasinglyslow and unwieldy to view or search. In PHASTER thisdatabase is now fast-loading, paginated and dynamicallysearchable.

PHASTER also allows visitors to optionally save theirsearches using a cookie-based storage mechanism by click-ing on the appropriate check box. This will work for anyonereturning to the PHASTER website using the same browseron the same computer, provided that the browser has cook-ies enabled. Previously submitted jobs saved in this way willbe available under the new ‘My Searches” section, withoutany need to log in. This feature is optional, and users canstill bookmark their results pages instead.

PERFORMANCE AND FEATURE EVALUATIONComparisons of typical runtimes between PHAST andPHASTER are provided in Table 1. Note that PHAST(which is still operational) runs on 32 CPU cores whilePHASTER runs on 112 CPU cores. Table 1 highlights thespeed-ups obtained for each of the six different optimiza-tions made, from the redundancy reduction of the bacterialgenome database to the implementation of BLAST+ to theaddition of another 80 CPU cores. In particular, reducingthe bacterial database’s redundancy nearly halved searchtimes for that database, increasing the number of comput-ing cluster cores to from 32 to 112 improves BLAST run-times to ∼6 min for an unannotated genome, upgrading toBLAST+ further improves the total time by ∼20%, parti-tioning the query sequences more evenly via total aminoacid count offers a ∼40% overall BLAST speed improve-ment, and the division of the bacterial database into sepa-rate parts to be searched separately more than halved thebacterial database search time, reducing it to just 48 sec-onds on average. Using a faster, dedicated front-end server,allowed processing times to be reduced from approximately72 to 45 s. With all these enhancements, PHASTER’s totalruntime is now ∼210 s for a typical raw genome sequenceand ∼100 s for pre-annotated GenBank input. All run timesquoted in Table 1 were measured using the same test genome(E. coli O157:H7, with GenBank accession NC 002655).

Previous comparisons of PHAST to other prophage iden-tification software have shown that PHAST’s accuracy is ei-ther superior or comparable to other leading tools when an-alyzing complete genomic sequence data (2,11). Tests on aset of 54 bacterial genomes in 2011 showed that PHASTachieved a sensitivity of 79.4% and positive predictive value(PPV) of 86.5% on unannotated DNA sequences, and asensitivity of 85.4% and PPV of 94.2% on GenBank an-

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Table 2. A feature comparison between PHAST and PHASTER

Feature PHAST (as of January 2011) PHASTER

Viral sequence database ∼45 000 sequences ∼187 000 sequencesBacterial sequence database ∼4 million sequences ∼9 million sequences, streamlined through CD-HIT filteringComputing cluster 32 CPU cores 112 CPU coresBLAST Legacy version 2.2.16 BLAST+ version 2.3.0+Cluster use optimization Rudimentary Smart partitioning of query sequences and target bacterial

DB; optimized execution parametersFront-end server Shared, single CPU 50% faster, dedicatedFront-end website Perl and CGI Ruby on RailsGenome viewer Adobe Flash JavaScript, AngularPlasmid and D3Queuing system Flat file Uses Sidekiq for threading submissionsRecall previous user submissions Bookmark page ‘My Searches’ feature or bookmarkPre-computed genome results for quickquery searching

0 >14 000

Retrieve previously annotated genomeresults

GenBank accession or GI number only GenBank accession, GI number, or full sequence

Metagenomic data handling NA Supported for contigs >2000 bp

notated genomes (2). Since PHAST’s original publication,larger phage databases and small refinements to PHAST’sphage identification routines have led to improved accu-racy. In response to user feedback, slight parameter adjust-ments have also been made to increase the algorithm’s sen-sitivity so that more prophages can be identified. Tests onthe same set of 54 bacterial genomes (1) used for evalu-ation in 2011 show that PHASTER now achieves a >5%higher sensitivity of 85.0% (versus 79.4% for PHAST) onunannotated genomes, with a slightly improved PPV of87.3% (versus 86.5% for PHAST). On GenBank annotatedgenomes, PHASTER has a superior sensitivity of 86.9%(versus 85.4% for PHAST), and a PPV of 91.0% (versus94.2% for PHAST). Because of the parameter adjustmentsmade to increase PHASTER’s sensitivity, the latest scoresshow a slight increase in the number of false positives.However, given that the ‘gold standard’ annotations usedfor evaluation are now 13 years old (1), many prophagesidentified as ‘false positives’ relative to this standard arelikely to be true prophages. Indeed, manual inspection ofPHASTER’s predictions reveals many with highly signif-icant hits to telltale phage genes or clusters of predictedphage genes from the same species, suggesting that 40–50% of these ‘false positive’ predictions are in fact trueprophages. If these corrections to the gold standard anno-tations were included in our calculations, PHASTER’s per-formance would be even better. Further evaluation detailsare posted on the PHASTER website.

Table 2 highlights some of the main feature differencesbetween PHAST and PHASTER. In this table we see thatPHAST takes 899 s for a typical genome/phage annotationwhile PHASTER takes 205 s for the same genome. PHASTonly uses 32 cores, while PHASTER uses 112 cores, PHASTuses BLAST while PHASTER uses BLAST+, PHASTdoes not handle metagenomic data while PHASTER eas-ily handles assembled contigs from metagenomic data,PHAST lacks a ‘quick query check’ look-up feature whilePHASTER has quick query check capabilities, PHAST wascoded in Perl and CGI while PHASTER was coded in Rubyon Rails, PHAST uses Adobe Flash for visualization whilePHASTER uses more modern JavaScript tools for visual-ization and finally both PHAST an PHASTER achieve thesame accuracy in prophage identification.

CONCLUSIONS

To handle the rapidly expanding databases sizes and in-creasing popularity of PHAST we decided to undertake anear complete back-end re-write and a near complete front-end overhaul of the PHAST web server. This work led tothe creation of a new, enhanced release of PHAST calledPHASTER. In many respects, PHASTER is faster, better,easier to use and more robust than PHAST. These per-formance enhancements were achieved through code op-timization, improved database preparation, and hardwareupgrades. We also made PHASTER’s web interface moreconvenient, user-friendly, and substantially more robust interms of its ability to handle higher user traffic loads. Over-all, PHASTER is 4–5× faster than PHAST and, in somecases, it can be up to 400 times faster (if a previously anno-tated genome is submitted). PHASTER is now capable ofhandling assembled contig sets from metagenomic data andit offers number of useful interface enhancements for view-ing its output, testing its performance, and exploring previ-ously annotated genomes. These improvements should notonly make PHASTER more appealing for users, they willalso make the server far easier to maintain and sustain intothe future.

FUNDING

Canadian Institutes of Health Research (CIHR); GenomeAlberta, a division of Genome Canada. Funding for openaccess charge: CIHR.Conflict of interest statement. None declared.

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