Evaluation of strategies for the assembly of diverse bacterial genomes using MinION long-read sequencing

Abstract

BackgroundShort-read sequencing technologies have made microbial genome sequencing cheap and accessible. However, closing genomes is often costly and assembling short reads from genomes that are repetitive and/or have extreme %GC content remains challenging. Long-read, single-molecule sequencing technologies such as the Oxford Nanopore MinION have the potential to overcome these difficulties, although the best approach for harnessing their potential remains poorly evaluated.ResultsWe sequenced nine bacterial genomes spanning a wide range of GC contents using Illumina MiSeq and Oxford Nanopore MinION sequencing technologies to determine the advantages of each approach, both individually and combined. Assemblies using only MiSeq reads were highly accurate but lacked contiguity, a deficiency that was partially overcome by adding MinION reads to these assemblies. Even more contiguous genome assemblies were generated by using MinION reads for initial assembly, but these were more error-prone and required further polishing. This was especially pronounced when Illumina libraries were biased, as was the case for our strains with both high and low GC content. Increased genome contiguity dramatically improved the annotation of insertion sequences and secondary metabolite biosynthetic gene clusters, likely because long-reads can disambiguate these highly repetitive but biologically important genomic regions.ConclusionsGenome assembly using short-reads is challenged by repetitive sequences and extreme GC contents. Our results indicate that these difficulties can be largely overcome by using single-molecule, long-read sequencing technologies such as the Oxford Nanopore MinION. Using MinION reads for assembly followed by polishing with Illumina reads generated the most contiguous genomes and enabled the accurate annotation of important but difficult to sequence genomic features such as insertion sequences and secondary metabolite biosynthetic gene clusters. The combination of Oxford Nanopore and Illumina sequencing is cost effective and dramatically advances studies of microbial evolution and genome-driven drug discovery.

Assembly methods for nanopore-based metagenomic sequencing: a comparative study

“…The mock communities were composed by the same ten microorganisms, but in different proportions (Table 1). With the aim of reducing the computational resources needed for the first screening of the selected assemblers, the GridION and Prome-thION datasets were subsampled to obtain an output comparable with recent genomic or metagenomic studies based on MinION (approximately 3 Gbp and 6 Gbp) 9,[34][35][36][37][38][39] . In general, mean read length remained the same in the subsampled datasets in comparison to the original sequencing data 15 .…”

“…In the present work, however, ZymoBIOMICS genomes were used as a reference for carrying out the comparative analyses, due to their higher level of completeness. Although these reference genomes cannot be considered as "gold standards", Goldstein et al 9 demonstrated that the error profile obtained through hybrid assembly (ONT + Illumina MiSeq) was similar to the one obtained with MiSeq-only assembly, but the former resulted in higher contiguity. Reference genomes were gathered in a single multi-FASTA file to create a single-reference metagenome.…”

“…This tool was employed to align the draft assemblies to the reference metagenome. Then, the script 'count_SNPS_indels.pl' from Goldstein et al 9 was used to calculate the final number of SNPs and indels. In both strategies, the number of variants was normalized to the total assembly size of each metagenome.…”

“…Biosynthetic gene clusters (BGCs) are usually composed of repetitive genetic structures that are hard to assemble with short reads, and with long-read technologies being therefore more suitable to overcome this issue. However, BGCs are also very sensitive to frameshift errors, which have been reported to frequently occur in nanopore data 9 . For that reason, AntiSMASH web service (v. 5.0) 44 was used to compare the performance on BGC prediction BGCs number and profile among the different assembly tools.…”

Antimicrobial resistance profiling and phylogenetic analysis of Neisseria gonorrhoeae clinical isolates from Kenya in a resource-limited setting

“…Whole-genome sequencing (WGS) allows for timely detection and elucidation of AMR determinants in bacteria 7 . Third-generation sequencing has many advantages, like long read lengths, short time, and reduction of bias introduces through amplification by PCR-steps 8 . WGS has been used to investigate quinoloneresistant gonorrhoea outbreaks 9,10 in Kenya.…”