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Nanopore sequencing reads improve assembly and gene annotation of the Parochlus steinenii genome.

ABSTRACT: Parochlus steinenii is a winged midge from King George Island. It is cold-tolerant and endures the harsh Antarctic winter. Previously, we reported the genome of this midge, but the genome assembly with short reads had limited contig contiguity, which reduced the completeness of the genome assembly and the annotated gene sets. Recently, assembly contiguity has been increased using nanopore technology. A number of methods for enhancing the low base quality of the assembly have been reported, including long-read (e.g. Nanopolish) or short-read (e.g. Pilon) based methods. Based on these advances, we used nanopore technologies to upgrade the draft genome sequence of P. steinenii. The final assembled genome was 145,366,448 bases in length. The contig number decreased from 9,132 to 162, and the N50 contig size increased from 36,946 to 1,989,550 bases. The BUSCO completeness of the assembly increased from 87.8 to 98.7%. Improved assembly statistics helped predict more genes from the draft genome of P. steinenii. The completeness of the predicted gene model increased from 79.5 to 92.1%, but the numbers and types of the predicted repeats were similar to those observed in the short read assembly, with the exception of long interspersed nuclear elements. In the present study, we markedly improved the P. steinenii genome assembly statistics using nanopore sequencing, but found that genome polishing with high-quality reads was essential for improving genome annotation. The number of genes predicted and the lengths of the genes were greater than before, and nanopore technology readily improved genome information.

SUBMITTER: Shin SC

PROVIDER: S-EPMC6434015 | biostudies-literature | 2019 Mar

REPOSITORIES: biostudies-literature

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Nanopore sequencing reads improve assembly and gene annotation of the Parochlus steinenii genome.

Shin Seung Chul SC Kim Hyun H Lee Jun Hyuck JH Kim Han-Woo HW Park Joonho J Choi Beom-Soon BS Lee Sang-Choon SC Kim Ji Hee JH Lee Hyoungseok H Kim Sanghee S

Scientific reports 20190325 1

Parochlus steinenii is a winged midge from King George Island. It is cold-tolerant and endures the harsh Antarctic winter. Previously, we reported the genome of this midge, but the genome assembly with short reads had limited contig contiguity, which reduced the completeness of the genome assembly and the annotated gene sets. Recently, assembly contiguity has been increased using nanopore technology. A number of methods for enhancing the low base quality of the assembly have been reported, inclu ...[more]

PMID: 30911035

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Project description:BackgroundLong-read sequencing technologies were launched a few years ago, and in contrast with short-read sequencing technologies, they offered a promise of solving assembly problems for large and complex genomes. Moreover by providing long-range information, it could also solve haplotype phasing. However, existing long-read technologies still have several limitations that complicate their use for most research laboratories, as well as in large and/or complex genome projects. In 2014, Oxford Nanopore released the MinION® device, a small and low-cost single-molecule nanopore sequencer, which offers the possibility of sequencing long DNA fragments.ResultsThe assembly of long reads generated using the Oxford Nanopore MinION® instrument is challenging as existing assemblers were not implemented to deal with long reads exhibiting close to 30% of errors. Here, we presented a hybrid approach developed to take advantage of data generated using MinION® device. We sequenced a well-known bacterium, Acinetobacter baylyi ADP1 and applied our method to obtain a highly contiguous (one single contig) and accurate genome assembly even in repetitive regions, in contrast to an Illumina-only assembly. Our hybrid strategy was able to generate NaS (Nanopore Synthetic-long) reads up to 60 kb that aligned entirely and with no error to the reference genome and that spanned highly conserved repetitive regions. The average accuracy of NaS reads reached 99.99% without losing the initial size of the input MinION® reads.ConclusionsWe described NaS tool, a hybrid approach allowing the sequencing of microbial genomes using the MinION® device. Our method, based ideally on 20x and 50x of NaS and Illumina reads respectively, provides an efficient and cost-effective way of sequencing microbial or small eukaryotic genomes in a very short time even in small facilities. Moreover, we demonstrated that although the Oxford Nanopore technology is a relatively new sequencing technology, currently with a high error rate, it is already useful in the generation of high-quality genome assemblies.

Dataset Information

Nanopore sequencing reads improve assembly and gene annotation of the Parochlus steinenii genome.

Publications

Nanopore sequencing reads improve assembly and gene annotation of the Parochlus steinenii genome.

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