The power of single molecule real-time sequencing technology in the de novo assembly of a eukaryotic genome.

Hiroaki Sakai,Eri Ogiso-Tanaka,Norihiko Tomooka,Kohtaro Iseki,Kazuhito Satou,Chiaki Muto,Takeshi Itoh,Ken Naito,Takashi Hirano,Makiko Shimoji,Akito Kaga,Kuniko Teruya,Yu Takahashi,Akino Shiroma

doi:10.1038/srep16780

Abstract

Second-generation sequencers (SGS) have been game-changing, achieving cost-effective whole genome sequencing in many non-model organisms. However, a large portion of the genomes still remains unassembled. We reconstructed azuki bean (Vigna angularis) genome using single molecule real-time (SMRT) sequencing technology and achieved the best contiguity and coverage among currently assembled legume crops. The SMRT-based assembly produced 100 times longer contigs with 100 times smaller amount of gaps compared to the SGS-based assemblies. A detailed comparison between the assemblies revealed that the SMRT-based assembly enabled a more comprehensive gene annotation than the SGS-based assemblies where thousands of genes were missing or fragmented. A chromosome-scale assembly was generated based on the high-density genetic map, covering 86% of the azuki bean genome. We demonstrated that SMRT technology, though still needed support of SGS data, achieved a near-complete assembly of a eukaryotic genome.

Highlights

Second-generation sequencers (SGS) have been game-changing, achieving cost-effective whole genome sequencing in many non-model organisms
The details of sequencing libraries are shown in Supplementary Table 1
We first carried out a hybrid de novo assembly using both Roche and Illumina data to achieve the highest coverage as possible (Supplementary Fig. 1)

Summary

Introduction

Second-generation sequencers (SGS) have been game-changing, achieving cost-effective whole genome sequencing in many non-model organisms. We reconstructed azuki bean (Vigna angularis) genome using single molecule real-time (SMRT) sequencing technology and achieved the best contiguity and coverage among currently assembled legume crops. A chromosome-scale assembly was generated based on the high-density genetic map, covering 86% of the azuki bean genome. The high-throughput sequencing capacity of these second-generation sequencers (SGS) enabled the assembly of diploid plant genomes with much less time and cost[4]. The third generation, single molecule real-time (SMRT) sequencing platform[8] successfully generates reads of 10 kb on average[9] and recently achieved an N50 of 4.3 Mb in assembling the haploid human genome[10]. If the markers are not dense www.nature.com/scientificreports/

Methods

Results

Conclusion