Performance comparison of second- and third-generation sequencers using a bacterial genome with two chromosomes.
Performance comparison of second- and third-generation sequencers using a bacterial genome with two chromosomes.
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DOI：
10.1186/1471-2164-15-699
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发表时间：
2014-08-21
期刊：
影响因子：
4.4
通讯作者：
Nakamura S
中科院分区：
文献类型：
--
作者：
Miyamoto M;Motooka D;Gotoh K;Imai T;Yoshitake K;Goto N;Iida T;Yasunaga T;Horii T;Arakawa K;Kasahara M;Nakamura S
不同的第二代和第三代测序技术的可用性使得能够快速确定细菌基因组的序列。然而，确定最适合于产生具有多个染色体的成品基因组的测序技术仍然是一个挑战。我们通过对副溶血性弧菌的基因组进行测序和组装来评估以下三种第二代测序仪的能力：Roche 454 GS Junior（GS Jr）、Life Technologies Ion PGM（Ion PGM）和Illumina MiSeq（MiSeq）以及第三代测序仪Pacific Biosciences RS测序仪（PacBio），所述副溶血性弧菌的基因组由包含两条环状染色体的5-Mb基因组组成。我们用GS Jr、Ion PGM、MiSeq和PacBio对副溶血性弧菌的基因组进行测序，并用几种基因组组装器进行从头组装。虽然GS Jr在第二代测序仪中产生最长的平均读取长度418 bp，但来自GS Jr的最佳组装的最大重叠群长度为165 kbp，重叠群的数量为309。Ion PGM和MiSeq的单次运行产生的数据具有相当大的测序覆盖率，分别为279×和1，927 ×。Ion PGM的优化结果包含由77×覆盖度的读段组装的61个重叠群，最长的重叠群大小为895 kbp。MiSeq的结果分别为34个重叠群、58×覆盖度和733 kbp。这些结果表明，更高的覆盖深度对于更好的组装结果是不必要的。我们观察到多个rRNA编码区在第二代测序仪的组装中被片段化，而PacBio生成了两个非常长的重叠群，分别为3，288，561和1，875，537 bp，每个重叠群都来自一条染色体，覆盖率为73×，平均读取长度为3，119 bp，使我们能够确定所有rRNA操纵子的绝对位置。PacBio在重叠群的长度方面优于其他测序仪，并重建了基因组的最大部分，由于其长读段，实现了“成品级”的基因组组装。它显示了用含有更多重复序列的多条染色体组装更复杂基因组的潜力。本文的在线版本（doi：10.1186/1471-2164-15-699）包含补充材料，可供授权用户使用。
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. 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, 279× and 1,927×, respectively. The optimized result for Ion PGM contained 61 contigs assembled from reads of 77× coverage, and the longest contig was 895 kbp in size. Those for MiSeq were 34 contigs, 58× 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 73× coverage and mean read length 3,119 bp, allowing us to determine the absolute positions of all rRNA operons. 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. The online version of this article (doi:10.1186/1471-2164-15-699) contains supplementary material, which is available to authorized users.
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影响因子：
12.3
作者：
Koren S;Harhay GP;Smith TP;Bono JL;Harhay DM;Mcvey SD;Radune D;Bergman NH;Phillippy AM
通讯作者：
Phillippy AM
影响因子：
14.9
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作者：
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影响因子：
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作者：
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Kasarskis, Andrew