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Nucleic Acids Res. 2010 July 1; 38(Web Server issue): W194–W200.
Published online 2010 April 30. doi:  10.1093/nar/gkq326
PMCID: PMC2896100

mGenomeSubtractor: a web-based tool for parallel in silico subtractive hybridization analysis of multiple bacterial genomes


mGenomeSubtractor performs an mpiBLAST-based comparison of reference bacterial genomes against multiple user-selected genomes for investigation of strain variable accessory regions. With parallel computing architecture, mGenomeSubtractor is able to run rapid BLAST searches of the segmented reference genome against multiple subject genomes at the DNA or amino acid level within a minute. In addition to comparison of protein coding sequences, the highly flexible sliding window-based genome fragmentation approach offered can be used to identify short unique sequences within or between genes. mGenomeSubtractor provides powerful schematic outputs for exploration of identified core and accessory regions, including searches against databases of mobile genetic elements, virulence factors or bacterial essential genes, examination of G+C content and binucleotide distribution bias, and integrated primer design tools. mGenomeSubtractor also allows for the ready definition of species-specific gene pools based on available genomes. Pan-genomic arrays can be easily developed using the efficient oligonucleotide design tool. This simple high-throughput in silico ‘subtractive hybridization’ analytical tool will support the rapidly escalating number of comparative bacterial genomics studies aimed at defining genomic biomarkers of evolutionary lineage, phenotype, pathotype, environmental adaptation and/or disease-association of diverse bacterial species. mGenomeSubtractor is freely available to all users without any login requirement at:


Bioinformatics- and microarray-facilitated comparative analyses have revealed that some bacterial species possess extremely plastic genomes, hence giving rise at a population level to the concept of a species-associated meta- or pan-genome (1,2). The pan-genome comprises the entire set of DNA sequences that are represented invariably, frequently, sporadically or only rarely in members of the species. At an individual level the genome of each bacterium can be divided into core regions, shared by the entire species, and accessory or species-variable regions. This latter optional genomic repertoire, termed by us as the ‘mobilome’ (mobile genome) (3), includes a myriad of short strain-specific sequences and longer spans of episomal and integrative plasmids, transposons, integrons, gene cassettes, prophages, strain-specific gene clusters of unknown provenance and a growing list of genomic islands (4,5).

Many recently developed in silico comparison tools, web-servers and databases aim to exploit the expanding flow of high-throughput genome sequencing data to reveal the wider DNA blueprints of individual bacterial species and map out the extent and nature of species-associated mobilomes. The following examples are of particular note: BLAST-based webACT can be used to visualize pair-wide similarity across up to five user-supplied genome sequences (6), the CGview server can generate up to three circular genome maps with user-uploaded BLAST hits (7), MUMmer rapidly aligns a complete or partially sequenced genome against a reference template (8), Mauve functions as a genome-scale multiple sequence aligner (9) and the xBASE database offers a multi-functional comparative analysis resource for laboratory-based bacteriologists (10). GenomeSubtractor 1.0 is an in silico substractive hybridization tool, available as accessory within the tRNAcc command line package (11) and the MobilomeFINDER server (12). We first used this tool to identify protein coding sequences that were present in Salmonella enterica serovar Paratyphi A but not available non-Paratyphi A Salmonella genomes to develop a Paratyphi A-specific multiplex PCR assay (13). However, current freely available comparative genomics tools exhibit two major deficiencies: (i) an inability to analyse several genome sequences at once, especially when the genomes compared are relatively divergent and (ii) excessively long computing times and limited computational outputs as processing is typically performed on a local PC. We have now developed mGenomeSubtractor, a markedly superior version of GenomeSubtractor 1.0, to perform highly parallel genome-scale in silico ‘subtractive hybridization’ analyses of reference genomes against up to forty comparator genomes simultaneously. This mpiBLAST-based tool allows rapid alignment of tens of NCBI-archived or user-supplied genome sequences within a minute. mGenomeSubtractor is a highly intuitive cluster node architecture-based computing tool that offers efficient visualization of genomic mosaics, zoom-in options, a wide range of targeted analyses, linkages to other databases and a useful range of applications. mGenomeSubtractor will be ideal for the identification and exploration of the most dynamic regions of bacterial genomes and for the definition and preliminary characterization of wider species-associated gene pools.


mGenomeSubtractor web-interface: a mpiBLAST-based tool for parallel in silico subtractive hybridization of bacterial genomes

Using an mpiBLAST-based procedure, mGenomeSubtractor performs a simple and intuitive in silico ‘subtractive hybridization’ by comparing selected closely related genomes to generate a list of conserved/strain-specific protein coding sequences (CDS) of the query genome and subsequently obtain the core/accessory regions. To examine the degree of sequence similarity at a nucleotide or amino acid level between each query CDS and the set of subject genomes, mGenomeSubtractor employs standard BLAST-derived identity and ratio of matching length to query length cut-offs or the simple H-value homology score (3,14). For each query, the H-value was calculated as follows: H = i × (lm/lq), where i was the level of identity of the region with the highest Bit score expressed as a frequency of between 0 and 1, lm the length of the highest scoring matching sequence (including gaps) and lq the query length. In this study, the H-value was used to measure BLASTN-derived sequence similarity at nucleotide level. By analogy, we defined an equivalent Ha-value that reflected BLASTP-based similarity at an amino acid level.

mGenomeSubtractor provides a flexible and biologist-friendly web-interface, consisting of an input page, a retrieve and Results pages. Users select a reference (or query) genome and a set of subject genomes for comparison from a full listing of complete bacterial genomes, regularly updated by NCBI Microbial Genome Resources. In the example shown in Figure 1, the Streptomyces coelicolor A3(2) genome was selected as the reference genome, with the subject set comprising two genomes: the S. avermitilis MA-4680 genome selected from the NCBI genome listing and the complete S. lividans TK24 genome downloaded from the Broad Institute and user-uploaded to the mGenomeSubtractor website. Parallel implementation of mpiBLASTP was used to measure amino acid sequence similarity with default NCBI BLASTP parameters, with the exception that ‘F’ was set to ‘F (no filter for repeated sequences)’. mGenomeSubtractor allows users to select or upload complete sequence and annotation details of reference (and comparator) genomes. Users can also simultaneously upload thousands of annotated protein sequences as Multi-Fasta formatted files to run high-throughput BLAST searches against subject genomes. Similarly, the subject set also accepts newly sequenced genomes rapidly annotated via the xBASE Annotation server (10), and Multi-Fasta CDS and protein sets. A key innovation offered by mGenomeSubtractor is the ability to fragment genomes into thousands of short segments using a flexible sliding window algorithm, thus allowing for the identification of much shorter intergenic or intragenic unique sequences.

Figure 1.
In silico ‘subtractive hybridization’ based comparative genomics using mGenomeSubtractor with chromosomal sequences of Streptomyces coelicolor A3(2) (reference genome), S. avermitilis MA-4680 and S. lividans TK24 as the inputs. (A) Histogram ...

Typically alignment is computed ‘on the fly’ in a matter of tens of seconds. Users are then directed to the retrieve page, which displays a histogram of H-values (Figure 1A) that helps users to set an appropriate cutoff to discriminate between conserved and strain-specific CDS. With the example shown, S. coelicolor A3(2) CDS were defined as conserved if the two H-values resulting from individual alignments versus S. lividans TK24 and S. avermitilis MA-4680 were >0.42. Advanced options such as the pre-definition of certain genomic regions as core or accessory and the option of matching to whole or partial subject set genomes allow users greater flexibility.

The Results page offers the following functional tabs for further analysis of the identified conserved or strain-specific CDS: ‘List’ [H-value, CDS annotation, multiple sequence alignment with MUSCLE (15) and Jalview (16), and/or primer design with Primer3Plus (17)], ‘Download’ (download sequence and annotation data in bulk), ‘Blast’ [BLAST search against the DEG (18), VFDB (19) and/or ACLAME (20) databases], ‘Core/Accessory Region’ (investigation of core and/or accessory genomic regions), ‘Design Probes’ [link to an integrated YODA (21) page for genome-wide CDS-specific probe selection for high-density oligonucleotide array construction]. mGenomeSubtractor also generates schematic outputs with zoom in utility if the reference genome is from the complete genome list or uploaded as a complete sequence with an associated annotation file in NCBI .ptt format. A colour-coded comparative genomic map of S. coelicolor A3(2) is shown in Figure 1C; CDS judged as shared using a Ha-value cutoff of 0.42 by none, either or both comparator genomes (S. lividans TK24 and S. avermitilis MA-4680) are as indicated. The 4776 S. coelicolor A3(2) CDS conserved across all three genomes shown in black with the legend label ‘2’ indicating conservation across both comparator genomes. Subsequent selection of the region of interest opens up a second and third graphical output comprising the G+C content and COG assignment of each CDS (Figures 1B and and22D).

Figure 2.
In silico ‘subtractive hybridization’ of Streptomyces lividans TK24 against its close relative S. coelicolor A3(2) using mGenomeSubtractor. (A) Circular map of S. lividans TK24 linear chromosome showing inferred contigs (ICs) and inferred ...

The Results page ‘Core/Accessory Region’ tab outputs mGenomeSubtractor-generated ‘core’ and/or ‘accessory’ reference genome regions by merging adjacent genes classified as ‘conserved’ or ‘strain-specific’. Accessory regions are tabulated to detail boundary coordinates and classical features of horizontally acquired genomic islands such as immediate flanking tRNA genes, internal integrase and/or transposase genes, and genome-atypical G+C content and/or dinucleotide distribution bias. The mGenomeSubtractor web-server also generates a graphical output comprising a circular map of the reference genome that highlights the locations and sizes of identified core and accessory regions within this reference template (Figure 2A).

mGenomeSubtractor::Gene Pool for estimate of species-specific gene pool

The size of a species gene pool, comprising the complete set of unique genes in a particular species, is probably a function of the extent of genetic diversity within and close to the evolutionary branch bearing the aforementioned species. The mpiBLAST-based mGenome-Subtractor::Gene Pool tool allows for estimation and classification of the known species-specific gene pool as represented within the set of sequenced genomes analysed. Supplementary Figure S3 shows the mGenome-Subtractor::Gene Pool Input and Results pages corresponding to the example analysis of seven completely sequenced Pseudomonas aeruginosa chromosomes as the input, four NCBI complete genomes (LESB58, PA7, UCBPP-PA14 and PAO1) and other three sequences (PACS2, C3719 and 2192) annotated by xBASE server (10). Firstly, all 5925 annotated CDS present on the P. aeruginosa LESB58 genome were included in the gene pool. Then each annotated CDS on the P. aeruginosa PA7 genome was used as a query in an mpiBLASTN search against the LESB58 genome sequence. The 1003 PA7-specific CDS identified by mGenomeSubtractor::Gene Pool (H-value <0.42) based on this two genome comparison were added to the gene pool. Next, a similar reiterative process was used to analyse each of the remaining five P. aeruginosa genomes leading to the sequential inclusion of a further 460 genes into the growing gene pool. Finally, single representative unique CDSs were selected from the gene pool by removing redundant ‘duplicated’ genes as determined using the H-value criteria (H-value >0.81). mGenome-Subtractor::Gene Pool analysis of the seven complete P. aeruginosa genomes using the criteria stated above defined a known P. aeruginosa gene pool comprising 7285 unique CDS, a number that exceeded the total number of non-duplicated CDS in any one chromosome by 16–39%.

The Results page displays summary data of the stored gene sequences, associated annotation information and COG classification categories as interactive tables, bar charts and pie charts (Supplementary Figure S3C–H). Users can also select ‘Design oligonucleotide probes with YODA’ to aid production of high-density oligonucleotide arrays representing entire defined gene pools or particular sub-pools of interest. YODA (21) can be run with default parameters or with user-defined constraints.


The mGenomeSubtractor server consists of two basic components: the web interface and the computational pipeline. The computational component is written in Perl/Bioperl and uses mpiBLAST as the core module for checking nucleotide/amino acid sequence similarity. mpiBLAST (, a parallel implementation of NCBI BLAST using the message passing interface (MPI) library, allows time-consuming local alignment processes to be done in parallel on computing cluster architecture. The following freely available components were integrated by the mGenomeSubtractor server: (i) oligo/primer design tools, YODA (21) and Primer3Plus (17); (ii) multiple sequence alignment and visualization tools (MUSCLE) (15) and Jalview (16); (iii) genome visualization tool CGview (22); (iv) database of essential genes (DEG) (18); (v) a classification of genetic mobile elements (ACLAME) (20); (vi) virulence factors database (VFDB) (19). mGenomeSubtractor is now run on a high-performance 23-node cluster (21 computing nodes, one management node and one storage node) interconnected with a 1000 Mb ethernet network. Each computing node is equipped with two quad-core Xeon 2.33 GHz processors and 8 GB of RAM.

Compared to our previously reported stand-alone tool GenomeSubtractor 1.0 (11,12), mpiBLAST-facilitated mGenomeSubtractor offers the following major enhancements: (i) several orders of magnitude accelerated sequence alignment by up to 168 cores; (ii) query genome fragmentation using a flexible sliding window algorithm; (iii) facilitation of preliminary functional analyses of core/accessory genomic regions supported by powerful interactive schematic outputs and tabulated data of sequence/CDS features; (iv) automated extraction, assembly and summary analyses of species-specific gene pools based on input genome sets. Presently, mGenome-Subtractor takes ~1 min to compare all 5312 CDS in the 5.5 Mb Escherichia coli O157:H7 EDL933 chromosome against 23 completely sequenced divergent E. coli chromosomes. In addition, mGenomeSubtractor displays a URL for subsequent retrieval of results for jobs demanding exceedingly large amounts of computing and hence requiring greater than a minute for completion. Alternatively, results can be emailed automatically upon completion of the job. Each run is assigned a job-id and associated output files are stored on the server for 7 days.


See Figures 1 and and2,2, and Supplementary Figure S3, and more details in Supplementary Data for examples of applications of mGenomeSubstractor.


The mGenomeSubtractor web server, which integrates a wide variety of useful analytical and functional tools, has been developed to perform mpiBLAST-based single-process comparison of a reference bacterial genome against up to 30 user-selected genomes and/or 10 user-supplied genomes. With parallel computing PC cluster architecture, ease of navigation and flexible input options, we present it as a quick and comprehensive genome-mining tool dedicated to biologists. In a near future version of mGenomeSubtractor, we will improve its computing power markedly with enhanced inter-node communication over a 20 Gb Infiniband network. The upgraded version will be able to directly align mass short-read sequence data against selected bacterial genomes or even the full catalogue of finished microbial genomes, thus providing an invaluable analytical tool to help exploit the next generation sequencing data ‘tsunami’ in our midst. We propose that a tool such as mGenomeSubtractor will support the rapidly escalating number of comparative bacterial genomics studies aimed at defining genomic biomarkers of evolutionary lineage, phenotype, pathotype, environmental adaptation and/or disease-association of diverse bacterial species.


Supplementary Data are available at NAR Online.


National Natural Science Foundation of China (30871345, 30700013, 30970080 and 30821005); a Royal Society—National Natural Science Foundation of China International Joint Project grant (2007/R3 to to K.R. and Z.D.); Chen Xing young scholars programme, Shanghai Jiaotong University (to H.Y.O.). Funding for open access charge: National Natural Science Foundation of China.

Conflict of interest statement. None declared.

Supplementary Material

[Supplementary Data]


We are grateful to Prof. Chun-Ting Zhang and Dr Ren Zhang for their supporting work. We thank the Broad Institute of MIT and Harvard and the Genome Center at Washington University for their policy of making preliminary sequence data publicly available and acknowledge the use in this study of unpublished genome data of S. lividans strain TK24, P. aeruginosa strain C3719, 2192 and PACS2.


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