Reducing assembly complexity of microbial genomes with single-molecule sequencing

Abstract

BackgroundThe short reads output by first- and second-generation DNA sequencing instruments cannot completely reconstruct microbial chromosomes. Therefore, most genomes have been left unfinished due to the significant resources required to manually close gaps in draft assemblies. Third-generation, single-molecule sequencing addresses this problem by greatly increasing sequencing read length, which simplifies the assembly problem.ResultsTo measure the benefit of single-molecule sequencing on microbial genome assembly, we sequenced and assembled the genomes of six bacteria and analyzed the repeat complexity of 2,267 complete bacteria and archaea. Our results indicate that the majority of known bacterial and archaeal genomes can be assembled without gaps, at finished-grade quality, using a single PacBio RS sequencing library. These single-library assemblies are also more accurate than typical short-read assemblies and hybrid assemblies of short and long reads.ConclusionsAutomated assembly of long, single-molecule sequencing data reduces the cost of microbial finishing to $1,000 for most genomes, and future advances in this technology are expected to drive the cost lower. This is expected to increase the number of completed genomes, improve the quality of microbial genome databases, and enable high-fidelity, population-scale studies of pan-genomes and chromosomal organization.

Sequence type 1 group B Streptococcus , an emerging cause of invasive disease in adults, evolves by small genetic changes

“…As a complete genome of an ST-1 GBS strain isolated from a human had not been determined previously, we next used a combination of long reads by using a PacBio instrument and paired-end Illumina short-read data to completely assemble the genome of the ST-1 strain SGBS001 (20). The genome was 2,092,071 bp with 2,061 predicted ORFs and contains genes predicted to encode alpha-like protein (Alp) 3 (Alp3) and pilus 1 and 2a (Fig.…”

Canu: scalable and accurate long-read assembly via adaptive k-mer weighting and repeat separation

“…There are two strategies for overcoming this fundamental limitation: increasing the effective read length, and separating nonexact repeats based on copy-specific variants. Recently, single-molecule sequencing has revolutionized assembly by producing reads >10 kbp (Gordon et al 2016), which has significantly reduced the number of unresolvable repeats and enabled the complete assembly of microbial genomes (Chin et al 2013;Koren et al 2013;Koren and Phillippy 2014). These long reads also aid assembly phasing (Chin et al 2016), where the conserved alleles in a diploid, polyploid, or meta-genome can be thought of as a special kind of repeat.…”

New Advances in NGS Technologies

“…It is a complex task, since the software must compare each reads in all of the reference DNA positions Trends and Advances in Veterinary Genetics [8,10,11]. It is a computationally challenging passage and wasteful in terms of time.…”