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Contig-Layout-Authenticator (CLA): A Combinatorial Approach to Ordering and Scaffolding of Bacterial Contigs for Comparative Genomics and Molecular Epidemiology

  • Sabiha Shaik,

    Affiliation Pathogen Biology Laboratory, Department of Biotechnology and Bioinformatics, School of Life Sciences, University of Hyderabad, Hyderabad, India

  • Narender Kumar,

    Affiliation Pathogen Biology Laboratory, Department of Biotechnology and Bioinformatics, School of Life Sciences, University of Hyderabad, Hyderabad, India

  • Aditya K. Lankapalli,

    Affiliation Pathogen Biology Laboratory, Department of Biotechnology and Bioinformatics, School of Life Sciences, University of Hyderabad, Hyderabad, India

  • Sumeet K. Tiwari,

    Affiliation Pathogen Biology Laboratory, Department of Biotechnology and Bioinformatics, School of Life Sciences, University of Hyderabad, Hyderabad, India

  • Ramani Baddam,

    Affiliation Pathogen Biology Laboratory, Department of Biotechnology and Bioinformatics, School of Life Sciences, University of Hyderabad, Hyderabad, India

  • Niyaz Ahmed

    niyaz.ahmed@uohyd.ac.in

    Affiliation Pathogen Biology Laboratory, Department of Biotechnology and Bioinformatics, School of Life Sciences, University of Hyderabad, Hyderabad, India

Contig-Layout-Authenticator (CLA): A Combinatorial Approach to Ordering and Scaffolding of Bacterial Contigs for Comparative Genomics and Molecular Epidemiology

  • Sabiha Shaik, 
  • Narender Kumar, 
  • Aditya K. Lankapalli, 
  • Sumeet K. Tiwari, 
  • Ramani Baddam, 
  • Niyaz Ahmed
PLOS
x

Abstract

A wide variety of genome sequencing platforms have emerged in the recent past. High-throughput platforms like Illumina and 454 are essentially adaptations of the shotgun approach generating millions of fragmented single or paired sequencing reads. To reconstruct whole genomes, the reads have to be assembled into contigs, which often require further downstream processing. The contigs can be directly ordered according to a reference, scaffolded based on paired read information, or assembled using a combination of the two approaches. While the reference-based approach appears to mask strain-specific information, scaffolding based on paired-end information suffers when repetitive elements longer than the size of the sequencing reads are present in the genome. Sequencing technologies that produce long reads can solve the problems associated with repetitive elements but are not necessarily easily available to researchers. The most common high-throughput technology currently used is the Illumina short read platform. To improve upon the shortcomings associated with the construction of draft genomes with Illumina paired-end sequencing, we developed Contig-Layout-Authenticator (CLA). The CLA pipeline can scaffold reference-sorted contigs based on paired reads, resulting in better assembled genomes. Moreover, CLA also hints at probable misassemblies and contaminations, for the users to cross-check before constructing the consensus draft. The CLA pipeline was designed and trained extensively on various bacterial genome datasets for the ordering and scaffolding of large repetitive contigs. The tool has been validated and compared favorably with other widely-used scaffolding and ordering tools using both simulated and real sequence datasets. CLA is a user friendly tool that requires a single command line input to generate ordered scaffolds.

Introduction

The emergence of newer platforms for whole genome sequencing has driven a revolution in the field of comparative genomics and epidemiological tracking of microorganisms [13]. Some of these platforms are essentially an adaptation of shotgun sequencing producing higher throughput and being cost-effective when compared to traditional Sanger sequencing. Though there are a variety of platforms available, such as Illumina, 454, Ion Torrent etc., their utility mainly entails reading of bases from short DNA fragments to generate read data [4]. In addition, some platforms such as Illumina also provide a paired-end sequencing option which essentially means that the generated fragments are read from both the ends providing useful information for downstream processing. Although the advent of these platforms has revolutionized the way we analyze and compare genomes, the main challenge refers to reconstructing the complete or a maximum draft genome out of millions of reads generated by the sequencers. This has underpinned development of multiple assembly tools with similar or distinct algorithms to harness and consolidate the sequence read data into larger contiguous fragments called contigs. Some examples of such assembly tools based on de novo algorithms include Velvet [5], SSAKE [6], etc. Although many of the available assembly processes reduce complexity of the data by generating contigs, the latter still require a lot of downstream analysis such as sorting based on their real biological order and resolution of repetitive elements before a high quality draft or complete genome is constructed.

The assembled contigs could be incorporated in various ways to reconstruct the genome. One important approach refers to directly ordering the contigs according to a chosen reference to build a chromosomal assembly with the help of tools such as ABACAS [7], Ragout [8], Mauve Contig Mover (MCM) [9], CONTIGuator [10] and Scaffold_builder [11] etc. These reference based approaches are known to be very efficient in analyzing specific mutations/variations among highly similar organisms since the possibilities of finding novel genomic insertions are scarce in some clonal populations. However, these methods have limitations in identifying strain-specific genes as well as handling mobile genetic elements which usually contribute to high strain to strain variation [12]. These approaches could therefore affect the resolution of an assembled genome sequence, especially in case of highly diverse and recombining organisms such as Helicobacter pylori [13].

The other approach for genome assembly is based on constructing scaffolds consisting of contigs joined together based on paired-end read information [14]. While a majority of sequence read assemblers such as Velvet [5], SOAPdenovo2 [15], SOPRA [16] and SGA [17] etc. come with an additional option of scaffolding, exclusive and stand-alone scaffolding tools such as Bambus [14], Bambus2 [18] and SSPACE [19] are also available. These tools usually generate multiple scaffolds where only intra-scaffold ordering and orientation of contigs is observed. Therefore, further ordering of scaffolds is required to obtain a draft genome. Other than this, these tools also face difficulties during the scaffolding of the repetitive contigs which sometimes result in misjoining/misassembly of the scaffolds [20]. The emergence of sequencing platforms that no longer depend on genome fragmentation and attempt to read directly from the genomic DNA such as PacBio, Oxford Nanopore etc. has shown promise to overcome these limitations by virtue of the long reads they generate. However, these platforms would take some more time to become mainstream and to displace some of the popular yet affordable platforms such as Illumina in terms of availability and affordability in some parts of the world [2123].

In order to address the limitations of both the reference and scaffolding based whole genome ordering tools mainly entailing Illumina paired-end sequencing, we developed Contig-Layout-Authenticator (CLA) which not only scaffolds the contigs but also attempts to achieve proper ordering of the scaffolds at the whole genome level. In other words, the contigs are initially ordered at the whole genome level similar to the ordering achieved by ABACAS and Ragout, based on a reference, but the order is much more refined at the later stages. The ordered contigs are further scaffolded when supported by the paired-end information; this way, if multiple scaffolds are generated, then it clearly indicates that some genome information between two consecutive scaffolds still needs to be incorporated into the assembly. Thus, CLA carries out the functions of ordering and scaffolding tools in a combinatorial and efficient manner yielding better draft genomes. The pipeline of CLA is proficient and adept in identifying probable contaminating contigs as well as misassemblies within the scaffolds. It assists the users in improving quality of the draft genome in an informed and step by step manner. The comparative analysis of CLA with other widely used ordering and scaffolding tools revealed its enhanced performance. Therefore, we believe CLA would serve as an efficient and user friendly tool for sorting and scaffolding of contigs to achieve better draft genomes of prokaryotic organisms.

Results

As CLA performs both ordering at the whole genome level followed by scaffolding, its efficiency was validated at three different levels. Firstly, it was compared with the widely used ordering tools such as ABACAS, MCM, CONTIGuator and Ragout. Secondly, the scaffolding ability of CLA was compared with Bambus2, SSPACE, SOPRA, SOAPdenovo2, SGA and the reference-based scaffolder, MeDuSa [24]. Finally, to test the hybrid approach efficiency, scaffolds from SSPACE, SOPRA, SOAPdenovo2 and SGA were further ordered with Ragout and the final results were compared against CLA. All the in-house scripts (along with their command line history) that were used for validation, are provided along with the tool.

CLA versus reference based sorting tools

All the tools under this category such as ABACAS (v1.3.1), MCM (Mauve-2.3.1), CONTIGuator (v2.7.3) and Ragout (v1.1) were run with default parameters and their outputs were compared with CLA using the QUAST(v3.1) [25] tool to avoid manual errors. Ragout and MeDuSa were run using multiple reference genomes (S1 and S2 Tables). The outputs generated by all the above mentioned tools using simulated and real sequence datasets were evaluated for parameters such as misassemblies within the chromosome assembly, the number of contigs (>1kb) unassigned in the final order, the size of largest unassigned contig, the genome fraction obtained and the number of large repetitive contigs (contig size greater than 500bp) correctly placed. QUAST was used to identify number of misassemblies and to calculate the genome fractions with the original genome as reference for simulated datasets. While in the case of a real dataset, due to the unavailability of the original genome, a closely related genome was used as reference for QUAST. Therefore, the statistics generated in case of real dataset could be biased towards the reference genome and may not exactly depict the efficiency of the tools as shown with the simulated dataset. In-house Perl scripts were used to calculate the number of large repetitive contigs present in the original genome, as well as number of them placed by each of these tools.

Misassemblies in the chromosome assembly

A chromosomal assembly or draft genome constructed from reference based ordering tools comprised of ordered contigs merged together after placing ‘Ns’ in between. Tools such as Ragout, CONTIGuator and ABACAS usually provide the final ordered contigs in the form of a single scaffold or chromosome assembly, whereas, MCM only provides ordered contigs in multi-FASTA format. In CLA, contigs are merged into scaffolds (by placing ‘Ns’ in between) only if supported by sufficient paired-end information. An in-house script was used to generate a chromosome assembly for the outputs of MCM and CLA. In order to identify the errors such as misassemblies encountered by each of the compared tools and judge their performance, we used QUAST. The misassemblies observed in the chromosomal assemblies were further linked to inversions, relocations and translocations by QUAST. Information about different types of misassemblies observed in case of simulated dataset(s) is given in S3 Table. As inferred from Table 1, CLA outcompeted other tools by producing least number of misassemblies in case of simulated datasets of all the test organisms except H. pylori. In case of organisms such as Bartonella quintana, and Caulobacter crescentus, CLA showed only two inversions without any relocations (S3 Table). After CLA, Ragout and CONTIGuator performed fairly well followed by ABACAS. MCM was observed to be the only tool with the highest number of misplacements in all organisms; this might be due to placement of unassigned contigs at the end of the order.

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Table 1. Comparative statistics of CLA and reference based alignment tools generated using simulated dataset.

https://doi.org/10.1371/journal.pone.0155459.t001

While dealing with real datasets, a reference genome required to act as an exact representative of the test strains was not available for some bacterial species/strains; we therefore chose a closely related genome as reference. Because this modification would definitely favor the reference based tools, the performance of CLA varied from genome to genome as revealed in the Table 2. CLA vended least number of misassemblies in all cases except Campylobacter jejuni, as inferred from Table 2. Moreover, the performance of CLA was comparable and even superior in some instances to other tools thus exhibiting the robustness of the combinatorial approach it used.

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Table 2. Comparative statistics of CLA and reference based alignment tools generated using real dataset.

https://doi.org/10.1371/journal.pone.0155459.t002

Contigs unassigned in the final chromosome assembly

Majority of the reference based ordering tools often exclude contigs (which fail to align properly with a reference genome) from the final order. These unassigned contigs are either provided as separate files or put towards the end in their output. The scenario becomes more pertinent in cases of highly recombining organisms as the reference genome architecture might differ significantly from the target genome. These unassigned contigs might encompass valuable strain specific information encoding novel elements and virulence genes etc. Since CLA utilizes paired-end information in addition to a reference genome, it is able to incorporate the maximum number of contigs in the chromosomal assembly and thereby retaining potentially novel, strain specific elements. As observed from the comparative statistics mentioned in Table 1, CLA showed consistent results by not having unassigned any contig with size greater than 1kb in all the organisms. On the other hand, CONTIGuator left more number of contigs with size greater than 1kb unassigned to the final order. It was also observed that a contig as long as 10kb in B. quintana was not considered in the final order generated by CONTIGuator. Though ABACAS performed considerably well, in some cases such as C. jejuni, Haemophilus influenzae, H. pylori and Rhizobium etli, it ended up excluding contigs of lengths 481kb, 345kb, 170kb and 798kb, respectively, in these organisms. MCM was observed to assign all the contigs in the final assembly as it places the unaligned contigs towards the end of the chromosomal assembly.

A similar pattern was observed with the real dataset (Table 2). While CLA and MCM have both incorporated in the final order all the contigs that were greater than 1kb, contigs as large as 32kb, 36kb and 93kb remained unassigned, respectively, in the final order, when Ragout, ABACAS and CONTIGuator were used to assemble genomes of C. jejuni, Escherichia coli and Salmonella Typhi. The number of contigs that were >1kb and were not considered by these tools were also observed to be very high in case of the real dataset.

Genome Fraction

The genome fraction obtained after the chromosome assembly of all the strains in simulated and real sequence datasets was calculated using QUAST. In almost all cases in simulated datasets, CLA and Ragout were observed to generate a higher genome fraction when compared to others. While the majority of tools achieved a genome fraction greater than 97%, the genome fraction from ABACAS was found to be 56.49%, 70.20%, 82.35% and 71.12% in C. jejuni, H. influenzae, H. pylori and R. etli, respectively (Table 1). In real datasets, the genome fraction obtained by all the tools was observed to be above 85% except in H. influenzae, where ABACAS was observed to generate a fraction only equal to 66.48%.

Resolving large repetitive contigs

Large repetitive contigs were defined as the contigs of length >500 bases having more than one coordinate in the original genome. To validate the placement, large repeat containing contigs of size >500 bases were considered. An in-house script was used to calculate the total number of positions for all repetitive contigs based on a BLASTn [26] alignment against a reference genome. BLAST version 2.2.29+ was used and the best hit with >90% identity and >90% query coverage was considered as a valid hit to identify the position coordinates. Then, the output of CLA and all ordering tools were scanned using another in house Perl script to evaluate the total number of positions filled by repetitive region containing contigs.

The ultimate aim of any genome sequencing project is to harness as much information as possible by formulation of better assemblies. Given this, resolution of repeats becomes an unavoidable parameter to obtain near perfect genomes. Since all the tools mentioned above are designed to aid researchers in achieving better assemblies, these tools were also compared for their performance related to the resolution of repeats. Except CLA and Ragout, other tools under this category were designed only to hint at the probable repetitive contigs and therefore required further manual intervention. As both CLA and Ragout handled repeats to some extent, they performed much better than the other tools in all the genomes. CLA and Ragout could more or less fill a similar number of true repeating positions in the majority of cases. While CLA could correctly fill 19 out of 21 positions (Table 1) in R. etli, Ragout could fill only 13. Similarly, Ragout performed better in the Treponema pallidum (T. pallidum) assembly by filling all 17 positions whereas only 9 of them could be placed by CLA. Both CLA and Ragout have shown similar performance in highly recombining organisms like B. quintana and C. jejuni, by filling 15 out of 19 positions and 10 out of 12 positions respectively. MCM and CONTIGuator were observed to be placing very few repeats in the correct order and copy number. CLA and Ragout performed in a superior way to other tools in handling the repetitive contigs by placing them in correct positions, followed by ABACAS.

In a real dataset, the number of repetitive positions was calculated with respect to the closely related genome used as reference which also served as input to these tools due to the unavailability of the original genome. Ragout was observed to perform well in all cases followed by CLA as inferred from Table 2. Therefore, it can be surmised that CLA and Ragout are comparable for this parameter in the chromosomal assembly process.

CLA versus Scaffolding tools

Scaffolding further reduces the number of sequence fragments by joining consecutive contigs and helps in the improvement of assembly. This could be achieved with the help of paired-end information or cues from the assembly graph, but was also attempted with the help of multiple reference genomes in the case of MeDuSa.

CLA, which utilizes a combination of reference alignment and paired-end information for scaffolding was compared against the recently published MeDuSa (v3) and other scaffolders such as SSPACE (v2.0), SOPRA (v1.4.6), SOAP denovo2 (v2.04-r240), SGA and BAMBUS2 (amos-3.1.0). For the simulated dataset, QUAST was used to deduce the assembly statistics from output files of each tool keeping the original genome as reference. Whereas, for the real dataset, genome of a closely related strain was used as reference genome. Parameters such as number of scaffolds formed, number of misassemblies within the scaffolds, number of misassembled scaffolds and size of genome fraction were used to compare the performance of CLA with others.

It was observed that CLA performed better under this category owing to its combinatorial approach. The comparative statistics for simulated and real datasets are mentioned in Tables 3 and 4, respectively. The lesser number of scaffolds with minimum number of misassemblies clearly depict the efficiency and accuracy of CLA over other scaffolding tools. For example, in the R. etli genome of the simulated dataset, CLA could combine all 30 contigs into just two scaffolds whereas MeDuSa, Bambus2, SSPACE with extension, SSPACE without extension, SOPRA, SOAPdenovo2 and SGA generated 10, 23, 24, 28, 30, 24 and 30 scaffolds, respectively. CLA’s performance was consistent for all the genomes under comparison except in H. pylori where the number of misassemblies slightly increased to 7. In C. jejuni, 33 contigs were combined by CLA into 6 scaffolds without any misassemblies. In B. quintana, C. crescentus and S. Typhi, only 2 inversions were found within scaffolds from CLA (S4 Table). Numbers of misassemblies performed by CLA were relatively fewer when compared with MeDuSa even in the real dataset (Table 4).

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Table 3. Comparative statistics of CLA and Scaffolding tools generated using simulated dataset.

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Table 4. Comparative statistics of CLA and Scaffolding tools generated using real dataset.

https://doi.org/10.1371/journal.pone.0155459.t004

Though MeDuSa generated a lesser number of scaffolds in a few cases of both the real and simulated datasets, the number of misassemblies was significantly higher in all the organisms. While misassemblies in case of SSPACE, SOPRA and SOAPdenovo2 were more or less similar to that of CLA, these tools gave a higher number of scaffolds. Considering the trade-off between number of scaffolds generated and the number of misassemblies, CLA showed the best performance with least number of scaffolds having the minimum misassemblies and the highest genome fraction recovered.

CLA versus combination of reference based and scaffolding tools

CLA being able to perform ordering followed by scaffolding, its performance was also compared by ordering already generated scaffolds. Ragout was used to order scaffolds from SSPACE with extension, SOPRA, SOAPdenovo2 and SGA and the final results were compared to that of CLA. The comparative statistics are listed out in Table 5.

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Table 5. Comparative statistics of CLA and Ragout ordering of scaffolds using simulated dataset.

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CLA performed better with the least number of misassemblies in the majority of cases like B. quintana, C. crescentus, R. etli, S. Typhi and T. pallidum. Ragout and SGA together generated the least number of misassemblies in C. jejuni, H. influenzae and H. pylori followed by CLA. Amongst the combinations used, Ragout and SGA gave good results similar to that of CLA while Ragout and SSPACE with extension did not seem to be an ideal and consistent combination.

Additional features of CLA

Handling intra-contig repeating segments.

Small repetitive sequences which form only part of a contig also create problems during assembly. Some of these elements consist of insertion sequences (IS) and transposable elements. CLA could split and precisely place certain segments of contigs which comprised of IS and transposable elements and displayed connections at different places, given sufficient availability of paired-end information. Such segments within the contigs are extracted based on the read alignment positions and are placed according to their connections in the map-file ([b] in S1 Fig). CLA in simulated data of S. Typhi placed a 500-600bp intra-contig repeat at about 24 places. This segment was found to be a transposable element that was present at about 26 different positions in the original genome of S. Typhi TY2. The positioning of these small repeats according to CLA and their original position in the reference are mentioned in S5 Table. The performance of CLA in terms of placing small repeats appeared to vary and was dependent on the sequencing quality and coverage depth. Therefore, CLA appears to be an efficient tool in handling even small repetitive elements in comparison to other tools which could not address these issues.

Contigs unrelated to the chromosomal assembly.

Possible contamination of DNA samples during sequencing and inaccurate de-multiplexing of read data may lead to contamination of the reads and potentially resulting in un-related contigs. Such contigs might cause significant problems during downstream analysis. In other cases, plasmids which are not a part of chromosomal sequences also pose problems in achieving accurate genome assembly. Given this, CLA was observed to filter out such contigs and separate them from the main chromosomal assembly. Contigs with BLASTn identity (against reference) of less than 5% and with no paired-end link information are tagged as unrelated contigs. Such contigs were effectively excluded out by CLA in real datasets of E. coli, H. influenzae, H. pylori, Mycobacterium tuberculosis and S. Typhi. In S. Typhi, one of the contigs tagged as unrelated was found to be a plasmid contig thus preventing false scaffolding with the chromosomal contigs. To examine the performance of CLA in the detection of possible contamination, a few random contigs from other genomes were introduced into simulated data. CLA could detect these contigs as contamination from a different source and correctly labelled them as possibly unrelated contigs. Contigs tagged as unrelated could be cross-checked by the user for its use in the final assembly.

Information about probable misassemblies.

Another advantage of using CLA is that the user is provided with additional files containing information on the efficiency of read-pair mapping and the extent of possible misassembling. The same can be useful for advanced users in order to further improve their assembly. CLA flags this information by vending a log file to the user.

Discussion

Genome assembly could be a challenging task especially for prokaryotes. This is mainly due to the plasticity of prokaryotic genomes as dictated by discrete evolutionary events and bottlenecks that shape adaptation dynamics and lifestyles of bacterial organisms in different ecosystems [1]. Consequently, prokaryotic genomes are often replete with signatures reminiscent of various genetic rearrangements occurring due to frequent insertion, deletion and substitution events as well as enriched with multiple homopolymeric tracts (arising out of replication errors), repeat motifs of different composition and lengths, insertion sequences, prophages and genomic islands etc. [27]. All these plastic regions pose serious difficulties in assembling a genome mainly because of sequence redundancy that they bring in the form of multiple alleles, palindromes, inverted repeats and tandem duplications. Most of the available tools either rely on reference to order contigs at the whole genome level, or scaffold them based on the read data [7, 9, 10, 14, 16, 18]. While using a reference genome could result in omission/exclusion of certain strain specific elements [10], the scaffolding methods struggle to resolve repetitive regions and limit ordering within the scaffolds [20]. Given these practical difficulties, we developed CLA, which combines the benefits of individual approaches to minimize errors while generating a draft genome. CLA uses a reference at the beginning to create a preliminary sort order which then undergoes extensive validation based on paired-end read information to resolve repetitive elements and re-sorting of the contigs. The sorted contigs are only scaffolded based on the available read-pair information. Although ordering is tried to be attained at the whole genome level, contigs are linked into scaffolds only when supported by paired-end information indicating their connectivity. Hence, it is easier for the users to fill in the information between the scaffolds using further downstream processing in order to achieve a complete genome.

The existing reference based tools though efficient in sorting the contigs at the whole genome level, were observed to remove those contigs from the final chromosome assembly which failed to properly align with the reference (Tables 1 and 2). These excluded contigs may lead to loss of significant genome information. For example, BLAST analysis of such excluded contigs from the H. influenzae genome identified several genes encoding metabolic functions, which were otherwise discarded. On the other hand, the scaffolding of contigs is performed only based on paired-end information [14, 18, 19] or in the case of MeDuSa using multiple reference genomes [24]. In the case of final assembly/genome obtained with multiple scaffolds, ordering is limited within the scaffolds and also repetitive regions with their misleading connections sometimes lead to intra-scaffold misassemblies. From our validation study, it was observed that CLA could address all these issues better than the compared tools (Tables 15). Since CLA utilizes both reference and paired-end information, it performed better in retaining maximum number of contigs in the final output without compromising on accuracy. It also utilizes read-pair information to place some of the repetitive elements. The overall performance of CLA was found to be much better than the existing reference based ordering tools as well as scaffolding tools with a minimum number of misassemblies. In all our case studies and comparative analyses, CLA was seen to be misplacing contigs in just two cases: 1) firstly, when there was insufficient paired-end information, 2) secondly, when two contigs had same flanking contig connections at both ends leading to their misjoining within the scaffold. To avoid such scenarios, CLA lists out contigs with probable swapping, in a log file to alert the users of probable misassemblies within scaffolds.

The performance with the real and simulated datasets pointed out the capability of CLA in not only handling data from monomorphic bacteria such as S. Typhi but also highly diverse ones as H. pylori. The higher abundance of transposases in bacteria and important biological roles proposed for them in previous studies underlines the need to handle them carefully during genome assembly [28]. CLA was observed to be efficient in this aspect and was able to resolve 24 out of 26 transposable elements in S. Typhi.

Though manual curation is inevitable for completing a genome, CLA leaves less scope for manual intervention and also provides all the required blueprints to complete further re-construction of the genome. Therefore, we believe that in the light of existing difficulties regarding the genome assembly, CLA would be a significant step forward in improving the genome assembly pipeline with a user friendly approach and efficient data usage.

Materials and Methods

Real and simulated datasets

Eight complete bacterial genomes with varied genome characteristics were downloaded from NCBI. The paired reads were simulated for each of the genomes with the help of GemSIM (v.1.6) [29] using its Illumina error model. The genome characteristics of these genomes along with their accession numbers are provided in S1 Table. The 100 bases long read-pairs were simulated with an insert length of 400(±20) along with genome coverage of 100X. The reads were filtered using NGS QC Toolkit (v.2.3) [30] to remove bad quality reads.

These filtered reads were then assembled into contigs using Velvet de novo assembler (1.2.08) with an optimal k-mer chosen with the help of VelvetOptimiser (2.2.5) (http://bioinformatics.net.au/software.velvetoptimiser.shtml). The assembled contigs were used as input for validating CLA with other tools using QUAST. For the real dataset, five paired-read datasets with different organismal background from SRA data were considered. Information about these datasets is provided in the S2 Table. Filtering followed by the assembly of reads was carried out in a similar manner as described above.

Pipeline of CLA

The tool was designed and developed to run in the following stages:

Sort order creation.

The detailed schematic of CLA is explained in Fig 1. Contigs, paired-end sequence reads and a suitable reference genome sequence (preferably of the same species as the genome being assembled) serve as input for the tool. The process starts with the exclusion of contigs that are less than 200bp. A preliminary sort order is created after alignment of the contigs with the reference genome by using BLASTn. Contigs with a BLASTn best hit of less than a threshold identity value (for contigs of size <1kb: 50% identity; size>1-10kb: 25% identity; size >10kb: 10% identity) are placed at the end of the order. The individual contigs are arranged according to the sort order with consequent reverse complementation of contigs wherever required in accordance with BLASTn output.

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Fig 1. Schematic overview of CLA pipeline.

The schema of CLA pipeline is divided into three stages. In the first stage, a reference based order is derived followed by the second stage where connections between the individual contigs are extracted based on alignment information. The final stage makes use of the information from the first two stages to decide the final order followed by scaffolding and gap filling.

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Extracting the connections.

The paired reads are then mapped to the sorted contigs (obtained as above) using BWA [31] and their information regarding the alignment position and orientation is extracted. Each of the contigs is theoretically divided into start, mid and end regions as depicted in Fig 2 wherein the sequence read having its pair in any other contig constitutes a potential link. The information of all possible links (formed by the read pairs) at the start, mid and end regions of each of the contigs is then tabulated in a map file. Since the read pairs are derived from a single insert, these links strongly suggest the proximity or contiguity of these two contigs in the real genome. For example, as depicted in Fig 2, if two contigs are in proximity then the reads aligned at the end of one contig would have their linking pairs aligned at the start of the following contig. The map file contains four columns representing the contig ID, start, end and mid region of the contigs, respectively. To avoid any erroneous assembly at the extreme ends of the contigs, the first and last ten bases from the start and the end of each of the contigs are not considered for calculating the links. A minimum of 10 read pair links are considered to calculate the proximity of two contigs in the genome (valid connection between contigs). Therefore, the map file generation is instrumental in validation of the reference sorted contigs and also helps in resolving some of the issues such as repeats, duplications and inversions. Some examples regarding the resolutions of repetitive regions and possible duplications are detailed in S1 Fig.

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Fig 2. Defining the start and end of a contig based on the insert size.

Connectivity between two consecutive contigs is decided by the paired reads from a single insert. To find such connections between contigs, each contig is theoretically divided into start, mid and end regions based on the insert size.

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Sort-order validation.

Contigs that have less than 5% BLASTn identity against reference (as inferred from the earlier BLASTn output) and lacking suitable connections in the map-file are flagged as un-related and are removed from the downstream analysis. These might represent contigs formed due to contaminating reads or sometimes due to plasmid contigs as observed from the results of the real datasets. A network file is then created from the map file, where start/end of a contig is represented as vertex/node and their respective connections as edges. Contigs that are less than 1kb in size are initially excluded from the sort order. The tool then scans the map file to validate the reference based sort order while looking for connections between the neighboring contigs (contigs >1kb). In cases where there is no direct connection (no links connecting the end of the former and start of the later contig) between two consecutive contigs from the reference based sort order, the tool then looks for any intermediary contigs linking these two contigs based on the link information from the map file. Each path is a representation of one or more contigs and a shortest path (defined by one or more contigs connecting to other contigs based on the link information gleaned from the map file) was found using the Floyd Warshall algorithm [32]. Only paths that include contigs greater than 1kb in size are considered at this stage. The connecting contig is either copied or moved from its position based on the connections in the map file. All the contigs at the end of this stage are referred to as anchors.

Connecting the anchors.

The link information of the excluded contigs of size less than 1kb were then used to connect the defined anchors using the Floyd Warshall algorithm as discussed in the previous section. All such connected anchors were then merged into scaffolds after placing ‘Ns’ between them. To prevent any false positive placements, the sort-order was scanned at every step based on the number of connections from the map file.

Ordering the scaffolds.

An iterative process of scaffold merging and extension is performed by the CLA based on the unused connections and leftover contigs from the network file. After a search for inter scaffold connections, if two scaffolds are found to be connected, the one smaller in size is moved and oriented in accordance with the larger scaffold. Once merged, the map-file is scanned for any further unused connections at the ends of the newly extended scaffolds. Merging is again performed and the process is repeated until either all the connections are exhausted or no proper connections could be found. A final sort order is then created and the contigs are merged into scaffolds followed by their ordering, all in accordance with the new sort order. The gaps between the scaffolds are then closed by using GapFiller [33]. Even though the order is defined, all the contigs are not merged into a single pseudogenome and are left at the stage of scaffolds because of lack of valid connections available from the map file. Thus, inter-scaffold gaps indicate the existence of gaps at the level of the sequencing data itself. CLA uses the FASTX toolkit (http://hannonlab.cshl.edu/fastx_toolkit/) for easy handling and manipulation of the intermediate files.

Supporting Information

S1 Fig. Pictorial representation of various scenarios where map-file can be used to resolve repetitive contigs.

(a) A normal case scenario where a repetitive contig 38 is placed at two different positions based on its connections from the map-file. (b) An example of an intra-contig repeating segment, where mid-region of contig 20 is connecting two contigs—contig 9 and contig 35. (c) Example of a tandem repeat, where the whole contig 95 has connections at both start and end pointing to another contig 82.

https://doi.org/10.1371/journal.pone.0155459.s001

(TIF)

S1 Table. Genome characteristics and information of the strains utilized for simulation.

Table with the accession numbers of the strains and their genomic characteristics that were used for the simulated dataset

https://doi.org/10.1371/journal.pone.0155459.s002

(PDF)

S2 Table. Information about strains under real dataset.

Table with the accession numbers of the strains and reference genomes used for the study

https://doi.org/10.1371/journal.pone.0155459.s003

(PDF)

S3 Table. Misassembly details of CLA and reference based ordering tools in simulated dataset.

Table listing out number of relocations, translocations and inversions which amounted to the total number of misassemblies

https://doi.org/10.1371/journal.pone.0155459.s004

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S4 Table. Misassembly details of CLA and Scaffolding tools in simulated dataset.

Table listing out number of relocations, translocations and inversions which amounted to the total number of misassemblies

https://doi.org/10.1371/journal.pone.0155459.s005

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S5 Table. BLAST output of intra-contig repeat from CLA result in simulated S. Typhi Ty2 data against the original Ty2 genome.

The positions tabulated depict that a ~600bp intra-contig repeat is present at 26 different locations in the original genome

https://doi.org/10.1371/journal.pone.0155459.s006

(PDF)

Acknowledgments

We would like to thank all the members of Pathogen Biology Laboratory for fruitful discussions and suggestions. NA is an Adjunct Professor of the Academy of Scientific and Innovative Research, India.

Author Contributions

Conceived and designed the experiments: NA SS NK. Performed the experiments: SS AKL NK SKT RB. Analyzed the data: SS AKL SKT RB. Contributed reagents/materials/analysis tools: NA. Wrote the paper: NA SS.

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