Amplification-free Illumina sequencing-library preparation facilitates improved mapping and assembly of (G+C)-biased genomes.
Amplification-free Illumina sequencing-library preparation facilitates improved mapping and assembly of (G+C)-biased genomes.
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DOI：
10.1038/nmeth.1311
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发表时间：
2009-04
期刊：
影响因子：
48
通讯作者：
Turner, Daniel J.
中科院分区：
文献类型：
--
作者：
Kozarewa, Iwanka;Ning, Zemin;Quail, Michael A.;Sanders, Mandy J.;Berriman, Matthew;Turner, Daniel J.
在Illumina基因组分析仪的文库制备期间引入的扩增伪影增加了这些序列的可观比例将是重复的可能性，并导致跨靶向测序区域的读段覆盖率的不均匀分布。因此，这些不利的特征导致基因组组装和来自短读段的变异分析的困难，特别是当序列来自具有高或低GC含量的极端碱基组成的基因组时。在这里，我们提出了一种无扩增的文库制备方法，其中簇扩增步骤，而不是聚合酶链反应，富集完全连接的模板链，减少重复序列的发生率，提高读取映射和SNP调用，并帮助从头组装。我们说明了这一点，通过生成和分析DNA序列，从极GC-穷人（恶性疟原虫），GC-中性（大肠杆菌）和高GC（百日咳杆菌）基因组。
Amplification artifacts introduced during library preparation for the Illumina Genome Analyzer increase the likelihood that an appreciable proportion of these sequences will be duplicates, and cause an uneven distribution of read coverage across the targeted sequencing regions. As a consequence, these unfavorable features result in difficulties in genome assembly and variation analysis from the short reads, particularly when the sequences are from genomes with base compositions at the extremes of high or low GC content. Here we present an amplification-free method of library preparation, in which the cluster amplification step, rather than the polymerase chain reaction, enriches for fully ligated template strands, reducing the incidence of duplicate sequences, improving read mapping and SNP calling and aiding de novo assembly. We illustrate this by generating and analysing DNA sequences from extremely GC-poor (Plasmodium falciparum), GC-neutral (Escherichia coli) and high GC (Bordetella pertussis) genomes.
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