Mind the gap: upgrading genomes with Pacific Biosciences RS long-read sequencing technology.
Mind the gap: upgrading genomes with Pacific Biosciences RS long-read sequencing technology.
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DOI：
10.1371/journal.pone.0047768
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发表时间：
2012
期刊：
影响因子：
3.7
通讯作者：
Gibbs RA
中科院分区：
文献类型：
--
作者：
English AC;Richards S;Han Y;Wang M;Vee V;Qu J;Qin X;Muzny DM;Reid JG;Worley KC;Gibbs RA
利用桑格毛细管电泳和/或较新的短读序列数据和全基因组组装技术，许多基因组已经测序到高质量的草稿状态。然而，即使是最好的基因组草图，由于输入数据和用于构建草图组装的技术的限制，也存在空白和其他不完善之处。测序偏差、重复的基因组特征、基因组多态性和其他复杂因素共同使某些区域难以或不可能组装。传统上，草图基因组通过耗时且昂贵的基于sanger的人工精加工过程升级到“第三阶段完成”状态。为了更方便地组装和自动整理草图基因组，我们在这里提出了一种使用太平洋生物科学RS （PacBio）平台的长读段自动整理方法。我们的算法和相关的软件工具PBJelly（可在https://sourceforge.net/projects/pb-jelly/公开获得）在参考指导的组装过程中使用长序列读取自动化精加工过程。PBJelly还提供了“提升”坐标表，可以轻松地将现有注释移植到升级后的程序集。利用PBJelly和长PacBio reads，我们升级了模拟黑腹果蝇（Drosophila melanogaster）、伪果蝇（Drosophila pseudo - oobscura） 2版草图、assembly athon 2.0版虎鹦鹉数据集的汇编和black mangabey的初步汇编。在PacBio长读段的24倍覆盖范围内，我们解决了99%的缺口，并能够关闭69%的缺口，改善了D. pseudoobscura所有缺口的12%。通过4倍的PacBio长读取覆盖，我们看到读取解决了我们的虎皮鹦鹉组装中63%的差距，其中32%已经关闭，63%得到改善。通过6.8倍的白眉猴PacBio长读图谱覆盖率，我们解决了97%的空白，关闭了66%的空白，改善了19%。缺口闭合的准确性通过与Sanger测序对原始D. pseudoobscura草稿组装的缺口进行比较验证，并显示依赖于初始参考质量。
Many genomes have been sequenced to high-quality draft status using Sanger capillary electrophoresis and/or newer short-read sequence data and whole genome assembly techniques. However, even the best draft genomes contain gaps and other imperfections due to limitations in the input data and the techniques used to build draft assemblies. Sequencing biases, repetitive genomic features, genomic polymorphism, and other complicating factors all come together to make some regions difficult or impossible to assemble. Traditionally, draft genomes were upgraded to “phase 3 finished” status using time-consuming and expensive Sanger-based manual finishing processes. For more facile assembly and automated finishing of draft genomes, we present here an automated approach to finishing using long-reads from the Pacific Biosciences RS (PacBio) platform. Our algorithm and associated software tool, PBJelly, (publicly available at https://sourceforge.net/projects/pb-jelly/) automates the finishing process using long sequence reads in a reference-guided assembly process. PBJelly also provides “lift-over” co-ordinate tables to easily port existing annotations to the upgraded assembly. Using PBJelly and long PacBio reads, we upgraded the draft genome sequences of a simulated Drosophila melanogaster, the version 2 draft Drosophila pseudoobscura, an assembly of the Assemblathon 2.0 budgerigar dataset, and a preliminary assembly of the Sooty mangabey. With 24× mapped coverage of PacBio long-reads, we addressed 99% of gaps and were able to close 69% and improve 12% of all gaps in D. pseudoobscura. With 4× mapped coverage of PacBio long-reads we saw reads address 63% of gaps in our budgerigar assembly, of which 32% were closed and 63% improved. With 6.8× mapped coverage of mangabey PacBio long-reads we addressed 97% of gaps and closed 66% of addressed gaps and improved 19%. The accuracy of gap closure was validated by comparison to Sanger sequencing on gaps from the original D. pseudoobscura draft assembly and shown to be dependent on initial reference quality.
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