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精确的圆形共识长读测序提高了人类基因组变异检测和组装的准确性。

Accurate circular consensus long-read sequencing improves variant detection and assembly of a human genome.

机构信息

Pacific Biosciences, Menlo Park, CA, USA.

Google Inc., Mountain View, CA, USA.

出版信息

Nat Biotechnol. 2019 Oct;37(10):1155-1162. doi: 10.1038/s41587-019-0217-9. Epub 2019 Aug 12.

Abstract

The DNA sequencing technologies in use today produce either highly accurate short reads or less-accurate long reads. We report the optimization of circular consensus sequencing (CCS) to improve the accuracy of single-molecule real-time (SMRT) sequencing (PacBio) and generate highly accurate (99.8%) long high-fidelity (HiFi) reads with an average length of 13.5 kilobases (kb). We applied our approach to sequence the well-characterized human HG002/NA24385 genome and obtained precision and recall rates of at least 99.91% for single-nucleotide variants (SNVs), 95.98% for insertions and deletions <50 bp (indels) and 95.99% for structural variants. Our CCS method matches or exceeds the ability of short-read sequencing to detect small variants and structural variants. We estimate that 2,434 discordances are correctable mistakes in the 'genome in a bottle' (GIAB) benchmark set. Nearly all (99.64%) variants can be phased into haplotypes, further improving variant detection. De novo genome assembly using CCS reads alone produced a contiguous and accurate genome with a contig N50 of >15 megabases (Mb) and concordance of 99.997%, substantially outperforming assembly with less-accurate long reads.

摘要

目前使用的 DNA 测序技术要么产生高度准确的短读长,要么产生不太准确的长读长。我们报告了对环形一致性测序(CCS)的优化,以提高单分子实时(SMRT)测序(PacBio)的准确性,并生成高度准确(99.8%)的长高保真度(HiFi)读长,平均长度为 13.5 千碱基(kb)。我们应用我们的方法对特征明确的人类 HG002/NA24385 基因组进行测序,并获得至少 99.91%的单核苷酸变异(SNV)、95.98%的<50 bp 插入和缺失(indels)和 95.99%的结构变异的精度和召回率。我们的 CCS 方法与短读长测序检测小变异和结构变异的能力相匹配或超过。我们估计在“瓶中基因组”(GIAB)基准集中,2434 个不一致是可纠正的错误。几乎所有(99.64%)的变体都可以被相位成单倍型,进一步提高了变体检测的准确性。单独使用 CCS 读取进行从头基因组组装生成了一个连续且准确的基因组,其 contig N50 大于 15 兆碱基(Mb),一致性为 99.997%,明显优于使用不太准确的长读长进行的组装。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/8d81/6776680/1cdc01f785b4/nihms-1533949-f0001.jpg

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