Novel degenerate PCR method for whole-genome amplification applied to Peru Margin (ODP Leg 201) subsurface samples
Novel degenerate PCR method for whole-genome amplification applied to Peru Margin (ODP Leg 201) subsurface samples
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DOI：
10.3389/fmicb.2012.00017
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发表时间：
2012-01-01
影响因子：
5.2
通讯作者：
House, Christopher H.
中科院分区：
文献类型：
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作者：
Martino, Amanda J.;Rhodes, Matthew E.;House, Christopher H.
开发并测试了一种基于简并聚合酶链反应（PCR）的全基因组扩增方法，该方法旨在与454测序技术配合使用。虽然在此针对Roche 454技术进行了优化，但所提出的一般框架也可适用于其他下一代测序系统（例如，Illumina，Ion Torrent）。我们称之为随机扩增宏基因组PCR（RAMP）的方法涉及使用来自Roche 454扩增子测序的特异性引物，通过在3'端添加简并区域进行修改。它利用PCR反应，即使在50个PCR循环后，也不会从空白中扩增。通过对实验条件的优化，用从培养的大肠杆菌中提取的DNA对该方法进行了验证。大肠杆菌细胞，并在三个不同的场合测序后估计基因组覆盖率。覆盖率在测试的不同实验条件下变化不大，并且在测序工作量相当于理论基因组覆盖率14.10x的情况下约为62%。测序扩增产物的GC含量在该菌株预测值的2%以内。杆菌该方法也适用于从海洋地下样品提取的DNA从ODP腿201站点1229（秘鲁边缘），和分类分析的结果显示微生物群落占主导地位的变形菌，Chloroflexi，厚壁菌门，Euryarchaeota和Crenarchaeota，等等。这些结果与以前获得的那些样品相似，然而，确定的分类群比例的变化很好地说明了普遍接受的观点，即群落分析对所使用的扩增技术和将序列分配给分类群的方法都很敏感。总的来说，我们发现RAMP代表了从低生物量样品扩增宏基因组的有效方法。
A degenerate polymerase chain reaction (PCR)-based method of whole-genome amplification, designed to work fluidly with 454 sequencing technology, was developed and tested for use on deep marine subsurface DNA samples. While optimized here for use with Roche 454 technology, the general framework presented may be applicable to other next generation sequencing systems as well (e.g., Illumina, Ion Torrent). The method, which we have called random amplification metagenomic PCR (RAMP), involves the use of specific primers from Roche 454 amplicon sequencing, modified by the addition of a degenerate region at the 3' end. It utilizes a PCR reaction, which resulted in no amplification from blanks, even after 50 cycles of PCR. After efforts to optimize experimental conditions, the method was tested with DNA extracted from cultured E. coli cells, and genome coverage was estimated after sequencing on three different occasions. Coverage did not vary greatly with the different experimental conditions tested, and was around 62% with a sequencing effort equivalent to a theoretical genome coverage of 14.10x. The GC content of the sequenced amplification product was within 2% of the predicted values for this strain of E. coli. The method was also applied to DNA extracted from marine subsurface samples from ODP Leg 201 site 1229 (Peru Margin), and results of a taxonomic analysis revealed microbial communities dominated by Proteobacteria, Chloroflexi, Firmicutes, Euryarchaeota, and Crenarchaeota, among others. These results were similar to those obtained previously for those samples; however, variations in the proportions of taxa identified illustrates well the generally accepted view that community analysis is sensitive to both the amplification technique used and the method of assigning sequences to taxonomic groups. Overall, we find that RAMP represents a valid methodology for amplifying metagenomes from low-biomass samples.