Evaluation of a Transposase Protocol for Rapid Generation of Shotgun High-Throughput Sequencing Libraries from Nanogram Quantities of DNA
Evaluation of a Transposase Protocol for Rapid Generation of Shotgun High-Throughput Sequencing Libraries from Nanogram Quantities of DNA
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DOI：
10.1128/aem.05610-11
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发表时间：
2011-11-01
影响因子：
4.4
通讯作者：
Wommack, K. Eric
中科院分区：
文献类型：
--
作者：
Marine, Rachel;Polson, Shawn W.;Wommack, K. Eric
构建用于下一代测序的DNA片段文库可能是具有挑战性的，特别是对于DNA产量较低的样品。为规避与低起始量DNA相关的问题而设计的方案可能会导致扩增偏差，从而扭曲元基因组数据中基因组的分布。此外，样本吞吐量可能很慢，因为当前的文库构建技术很耗时。这项研究评估了NextEra，这是一种基于转座子的新方法，旨在从少量DNA中快速产生DNA片段文库。序列读数分布在模拟病毒组合中的九个噬菌体基因组上，符合最不丰富的六个噬菌体的预测；然而，最丰富的噬菌体的排名顺序与预测略有不同。来自NextEra文库的从头开始基因组组件提供了跨越一半以上噬菌体基因组的长重叠群；在四个可用于比较的全长基因组序列的情况下，发现共同序列与超过99%的基因组匹配，具有近乎完美的同一性。对噬菌体基因组中低和高序列覆盖率区域的分析表明，GC含量可能会影响NextEra文库中序列的覆盖率。对使用NextEra和标准454Flx钛文库制备方案制备的噬菌体基因组的比较表明，根据在NextEra文库中观察到的GC含量而产生的覆盖率偏差主要是由于NextEra方案中的偏差，而不是454测序技术。然而，在适当的序列覆盖下，NextEra方案为基因组研究提供了高质量的数据。对于元基因组分析，需要考虑GC扩增偏差的影响；然而，NextEra提供的文库准备标准化应该有利于比较元基因组分析。
Construction of DNA fragment libraries for next-generation sequencing can prove challenging, especially for samples with low DNA yield. Protocols devised to circumvent the problems associated with low starting quantities of DNA can result in amplification biases that skew the distribution of genomes in metagenomic data. Moreover, sample throughput can be slow, as current library construction techniques are time-consuming. This study evaluated Nextera, a new transposon-based method that is designed for quick production of DNA fragment libraries from a small quantity of DNA. The sequence read distribution across nine phage genomes in a mock viral assemblage met predictions for six of the least-abundant phages; however, the rank order of the most abundant phages differed slightly from predictions. De novo genome assemblies from Nextera libraries provided long contigs spanning over half of the phage genome; in four cases where full-length genome sequences were available for comparison, consensus sequences were found to match over 99% of the genome with near-perfect identity. Analysis of areas of low and high sequence coverage within phage genomes indicated that GC content may influence coverage of sequences from Nextera libraries. Comparisons of phage genomes prepared using both Nextera and a standard 454 FLX Titanium library preparation protocol suggested that the coverage biases according to GC content observed within the Nextera libraries were largely attributable to bias in the Nextera protocol rather than to the 454 sequencing technology. Nevertheless, given suitable sequence coverage, the Nextera protocol produced high-quality data for genomic studies. For metagenomics analyses, effects of GC amplification bias would need to be considered; however, the library preparation standardization that Nextera provides should benefit comparative metagenomic analyses.