Assessing the benefits of using mate-pairs to resolve repeats in de novo short-read prokaryotic assemblies

Abstract

BackgroundNext-generation sequencing technologies allow genomes to be sequenced more quickly and less expensively than ever before. However, as sequencing technology has improved, the difficulty of de novo genome assembly has increased, due in large part to the shorter reads generated by the new technologies. The use of mated sequences (referred to as mate-pairs) is a standard means of disambiguating assemblies to obtain a more complete picture of the genome without resorting to manual finishing. Here, we examine the effectiveness of mate-pair information in resolving repeated sequences in the DNA (a paramount issue to overcome). While it has been empirically accepted that mate-pairs improve assemblies, and a variety of assemblers use mate-pairs in the context of repeat resolution, the effectiveness of mate-pairs in this context has not been systematically evaluated in previous literature.ResultsWe show that, in high-coverage prokaryotic assemblies, libraries of short mate-pairs (about 4-6 times the read-length) more effectively disambiguate repeat regions than the libraries that are commonly constructed in current genome projects. We also demonstrate that the best assemblies can be obtained by 'tuning' mate-pair libraries to accommodate the specific repeat structure of the genome being assembled - information that can be obtained through an initial assembly using unpaired reads. These results are shown across 360 simulations on 'ideal' prokaryotic data as well as assembly of 8 bacterial genomes using SOAPdenovo. The simulation results provide an upper-bound on the potential value of mate-pairs for resolving repeated sequences in real prokaryotic data sets. The assembly results show that our method of tuning mate-pairs exploits fundamental properties of these genomes, leading to better assemblies even when using an off -the-shelf assembler in the presence of base-call errors.ConclusionsOur results demonstrate that dramatic improvements in prokaryotic genome assembly quality can be achieved by tuning mate-pair sizes to the actual repeat structure of a genome, suggesting the possible need to change the way sequencing projects are designed. We propose that a two-tiered approach - first generate an assembly of the genome with unpaired reads in order to evaluate the repeat structure of the genome; then generate the mate-pair libraries that provide most information towards the resolution of repeats in the genome being assembled - is not only possible, but likely also more cost-effective as it will significantly reduce downstream manual finishing costs. In future work we intend to address the question of whether this result can be extended to larger eukaryotic genomes, where repeat structure can be quite different.

Identification of antimicrobial resistance genes in multidrug-resistant clinical Bacteroides fragilis isolates by whole genome shotgun sequencing

“…If WGS is to be used for rapid clinical diagnostics and antiobiogram generation, genome finishing requiring multitudes of Sanger sequencing experiments is too laborious and costly to be applicable. Sequencing mate-pair libraries on the Illumina platform and utilizing these with the paired-end sequences in the de novo assembly would result in a "higher quality" assemblies with significant reduction in the contig numbers [33]. This would possibly result in upstream regions encompassing the IS elements in the final assemblies.…”

The limitations of draft assemblies for understanding prokaryotic adaptation and evolution

“…The computational issues that repetitive genomes pose to NGS assembly has been discussed in other recent papers [2][3][4]13], but there has been remarkably little emphasis on the relative value of the portion of the genome that remains fragmented in these draft assemblies. To this end, we performed an in silico experiment using simulated long and short read data for the fully sequenced genome of Cupriavidus metallidurans CH34 (hereafter simply referred to as CH34).…”

“…These methods rely on newer sequencing chemistries [1] and highly parallel operations that result in high yields at low costs per read but so far produce considerably shorter reads (in the range of 35-500 nucleotides) than Sanger sequencing (600 to 1500 nucleotides). Shorter reads increase the required complexity of the assembly algorithms [2], although the ability to sequence to very high coverage can overcome many of the original issues in genome assembly including read errors and coverage gaps [3]. The utility of next generation sequencing has been demonstrated in examining new variants, or very close relatives, of previously sequenced strains (reviewed in [4]).…”

“…Whole genome shotgun assemblies using traditional Sanger sequencing have been utilized for many years for this purpose but the cost and effort required to do this type of intense sequencing has been prohibitive for all but the largest laboratories [4]. The advent of NGS platforms promises to alleviate the financial and technical demands of obtaining high quality sequence data however the issue of repetitive elements in genomic sequence remains a confounding issue in genome assembly that is difficult to resolve through coverage alone [3].…”

“…To this end, we performed an in silico experiment using simulated long and short read data for the fully sequenced genome of Cupriavidus metallidurans CH34 (hereafter simply referred to as CH34). This organism was sequenced by the Joint Genome Institute (JGI) using whole genome shotgun cloning (WGS) with a combination of three randomly sheared libraries (3,8 and 40 kb insert sizes) and an additional 3752 individual Sanger reads for finishing [14]. It was chosen for this study because of the high quality finishing and annotation that has been performed [14,15] as well as the nature of the genome, which contains two large chromosomes and many types of mobile elements.…”