Towards quantitative metagenomics of wild viruses and other ultra-low concentration DNA samples: a rigorous assessment and optimization of the linker amplification method.
Towards quantitative metagenomics of wild viruses and other ultra-low concentration DNA samples: a rigorous assessment and optimization of the linker amplification method.
复制标题
DOI：
10.1111/j.1462-2920.2012.02791.x
复制
发表时间：
2012-09
影响因子：
5.1
通讯作者：
Sullivan MB
中科院分区：
文献类型：
--
作者：
Duhaime MB;Deng L;Poulos BT;Sullivan MB
宏基因组学生成并测试有关野生种群动态和机制驱动因素的假设，但通常会遇到用于测序的起始基因组材料不足（< 1 ng）的问题。目前为宏基因组分析扩增足够 DNA 的解决方案包括深度测序线性扩增 (LADS)（需要比正常情况下更多的 DNA）、链接扩增鸟枪文库 (LASL)（通量极低）和全基因组扩增（有明显偏差，因此是非定量的）。在这里，我们将 LASL 方法应用于下一代测序，为具有挑战性的样品提供替代聚合酶，开发更有效的大小步骤，整合“修复 PCR”步骤以提高产量并最大限度地减少后期 PCR 伪影，并根据实验室分离株和野生群落病毒 DNA 经验记录优化方法的定量能力。我们优化的接头扩增方法只需要 1 pg 的 DNA，是目前最精确和准确的方法，G + C 含量扩增偏差小于 1.5 倍，即使对于野生病毒群落等多样化的复杂样品也是如此。虽然此处针对 454 测序进行了优化，但这种接头扩增方法可用于准备宏基因组文库，以便使用下一代平台进行测序，包括 Illumina 和 Ion Torrent，我们在此处测试了第一个平台并提供了数据。
Metagenomics generates and tests hypotheses about dynamics and mechanistic drivers in wild populations, yet commonly suffers from insufficient (< 1 ng) starting genomic material for sequencing. Current solutions for amplifying sufficient DNA for metagenomics analyses include linear amplification for deep sequencing (LADS), which requires more DNA than is normally available, linker-amplified shotgun libraries (LASLs), which is prohibitively low throughput, and whole-genome amplification, which is significantly biased and thus non-quantitative. Here, we adapt the LASL approach to next generation sequencing by offering an alternate polymerase for challenging samples, developing a more efficient sizing step, integrating a ‘reconditioning PCR’ step to increase yield and minimize late-cycle PCR artefacts, and empirically documenting the quantitative capability of the optimized method with both laboratory isolate and wild community viral DNA. Our optimized linker amplification method requires as little as 1 pg of DNA and is the most precise and accurate available, with G + C content amplification biases less than 1.5-fold, even for complex samples as diverse as a wild virus community. While optimized here for 454 sequencing, this linker amplification method can be used to prepare metagenomics libraries for sequencing with next-generation platforms, including Illumina and Ion Torrent, the first of which we tested and present data for here.