MinION-based long-read sequencing and assembly extends the Caenorhabditis elegans reference genome

Abstract

Advances in long-read single molecule sequencing have opened new possibilities for ‘benchtop’ whole-genome sequencing. The Oxford Nanopore Technologies MinION is a portable device that uses nanopore technology that can directly sequence DNA molecules. MinION single molecule long sequence reads are well suited for de novo assembly of complex genomes as they facilitate the construction of highly contiguous physical genome maps obviating the need for labor-intensive physical genome mapping. Long sequence reads can also be used to delineate complex chromosomal rearrangements, such as those that occur in tumor cells, that can confound analysis using short reads. Here, we assessed MinION long-read-derived sequences for feasibility concerning: (1) the de novo assembly of a large complex genome, and (2) the elucidation of complex rearrangements. The genomes of two Caenorhabditis elegans strains, a wild-type strain and a strain containing two complex rearrangements, were sequenced with MinION. Up to 42-fold coverage was obtained from a single flow cell, and the best pooled data assembly produced a highly contiguous wild-type C. elegans genome containing 48 contigs (N50 contig length = 3.99 Mb) covering >99% of the 100,286,401-base reference genome. Further, the MinION-derived genome assembly expanded the C. elegans reference genome by >2 Mb due to a more accurate determination of repetitive sequence elements and assembled the complete genomes of two co-extracted bacteria. MinION long-read sequence data also facilitated the elucidation of complex rearrangements in a mutagenized strain. The sequence accuracy of the MinION long-read contigs (∼98%) was improved using Illumina-derived sequence data to polish the final genome assembly to 99.8% nucleotide accuracy when compared to the reference assembly.

Nanopore sequencing: Review of potential applications in functional genomics

“…A hybrid strategy utilizing short Illumina reads is therefore commonly adopted to polish the assembly (Figure 1a) (Giordano et al, 2017;Istace et al, 2017). Multiple rounds of polishing are often necessary, where the error correction process is validated using Benchmarking Universal Single-Copy Orthologs (BUSCO) completeness scores Simao, Waterhouse, Ioannidis, Kriventseva, & Zdobnov, 2015;Tan et al, 2018;Tyson et al, 2018). Several assemblers incorporate the hybrid error correction step prior to the assembly, in place of the initial overlap-based error correction (Bankevich et al, 2012;Zimin et al, 2013).…”

SVants – A long-read based method for structural variation detection in bacterial genomes

“…Nanopore have enabled new ways of assessing the diversity and importance of MGEs. Oxford Nanopore in particular has shown great promise with its theoretically unlimited read length and consistent sequencing accuracy across the range of sequence lengths produced [10]. The increased read length of these platforms makes it possible to not only to identify MGEs, but also to generate long enough sequences to cover the full length of the MGE structure with additional DNA sequence on both sides.…”

Real-Time Selective Sequencing with RUBRIC: Read Until with Basecall and Reference-Informed Criteria

“…Experiment C used a previously prepared frozen library and reduced the queue size from 24 to 12. Experiment D increased the queue to 16 and adjusted the mean current-based threshold with a fresh digest and library prep. Experiment E1 implemented a standard deviation-based threshold for a frozen library, and experiment E2 further adjusted that threshold.…”

“…The Oxford Nanopore Technologies (ONT) MinION sequencer represents a significant paradigm shift in the reach, applicability, and capability of nucleic acid sequencing technology 1 . Combining a portable form factor, simple library prep, long-read capability (kb to Mb) 2 , direct RNA sequencing 3 , and real-time data output, the MinION has been variously applied to forensic genotyping 4 , bacterial typing 5 , plant biology 6 , food safety 7 , environmental metagenomics 8,9 , cancer research 10,11 , antibiotic resistance studies 12,13 , and de novo genome assembly [14][15][16] . The small operational and logistical footprint of the MinION, combined with its real-time capabilities 17 , make it uniquely suited to diagnostics and surveillance in clinical and field-forward settings, where the MinION has already been applied to assay Ebola 18,19 , Zika 20 , tuberculosis 21 , and other pathogens [22][23][24][25] .…”