Performance comparison of second- and third-generation sequencers using a bacterial genome with two chromosomes

被引：76

作者：

Miyamoto, Mari ^{[1
]}

Motooka, Daisuke ^{[2
]}

Gotoh, Kazuyoshi ^{[2
,6
]}

Imai, Takamasa ^{[3
]}

Yoshitake, Kazutoshi ^{[4
]}

Goto, Naohisa ^{[2
]}

Iida, Tetsuya ^{[2
]}

Yasunaga, Teruo ^{[2
]}

Horii, Toshihiro ^{[2
]}

Arakawa, Kazuharu ^{[5
]}

Kasahara, Masahiro ^{[3
]}

Nakamura, Shota ^{[2
]}

机构：

[1] CLC Bio Japan Inc, Shibuya Ku, Tokyo 1500033, Japan

[2] Osaka Univ, Microbial Dis Res Inst, Dept Infect Metagen, Suita, Osaka 5650871, Japan

[3] Univ Tokyo, Dept Computat Biol, Grad Sch Frontier Sci, Kashiwa, Chiba 2778561, Japan

[4] Natl Inst Genet, Lab DNA Data Anal, Mishima, Shizuoka 4118540, Japan

[5] Keio Univ, Inst Adv Biosci, Fujisawa, Kanagawa 2520882, Japan

[6] Okayama Univ, Grad Sch Med, Dept Bacteriol, Okayama 7008558, Japan

来源：

BMC GENOMICS | 2014年 / 15卷

关键词：

Next-generation sequencing; de novo assembly; Illumina MiSeq; Ion Torrent PGM; Roche 454 GS Junior; PacBio RS system; ASSEMBLIES; PLATFORMS; MOLECULES; REPEATS; VELVET; READS;

D O I：

10.1186/1471-2164-15-699

中图分类号：

Q81 [生物工程学（生物技术）]; Q93 [微生物学];

学科分类号：

071005 ; 0836 ; 090102 ; 100705 ;

摘要：

Background: The availability of diverse second- and third-generation sequencing technologies enables the rapid determination of the sequences of bacterial genomes. However, identifying the sequencing technology most suitable for producing a finished genome with multiple chromosomes remains a challenge. We evaluated the abilities of the following three second-generation sequencers: Roche 454 GS Junior (GS Jr), Life Technologies Ion PGM (Ion PGM), and Illumina MiSeq (MiSeq) and a third-generation sequencer, the Pacific Biosciences RS sequencer (PacBio), by sequencing and assembling the genome of Vibrio parahaemolyticus, which consists of a 5-Mb genome comprising two circular chromosomes. Results: We sequenced the genome of V. parahaemolyticus with GS Jr, Ion PGM, MiSeq, and PacBio and performed de novo assembly with several genome assemblers. Although GS Jr generated the longest mean read length of 418 bp among the second-generation sequencers, the maximum contig length of the best assembly from GS Jr was 165 kbp, and the number of contigs was 309. Single runs of Ion PGM and MiSeq produced data of considerably greater sequencing coverage, 279x and 1,927x, respectively. The optimized result for Ion PGM contained 61 contigs assembled from reads of 77x coverage, and the longest contig was 895 kbp in size. Those for MiSeq were 34 contigs, 58x coverage, and 733 kbp, respectively. These results suggest that higher coverage depth is unnecessary for a better assembly result. We observed that multiple rRNA coding regions were fragmented in the assemblies from the second-generation sequencers, whereas PacBio generated two exceptionally long contigs of 3,288,561 and 1,875,537 bps, each of which was from a single chromosome, with 73x coverage and mean read length 3,119 bp, allowing us to determine the absolute positions of all rRNA operons. Conclusions: PacBio outperformed the other sequencers in terms of the length of contigs and reconstructed the greatest portion of the genome, achieving a genome assembly of "finished grade" because of its long reads. It showed the potential to assemble more complex genomes with multiple chromosomes containing more repetitive sequences.

引用

页数：8

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