Direct Squencing from the Minimal Number of DNA Molecules Needed to Fill a 454 Picotiterplate
Direct Squencing from the Minimal Number of DNA Molecules Needed to Fill a 454 Picotiterplate
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DOI：
10.1371/journal.pone.0097379
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发表时间：
2014-06-02
期刊：
影响因子：
3.7
通讯作者：
Moya, Andres
中科院分区：
文献类型：
--
作者：
Dzunkova, Maria;Garcia-Garcera, Marc;Moya, Andres
在下一代测序方案中制备文库所需的大量 DNA 阻碍了小 DNA 样品的直接测序。这种限制通常可以通过全基因组扩增 (WGA) 富集此类样品来克服，主要是通过基于 phi 29 聚合酶的多重置换扩增 (MDA)。然而，该技术可能会受到样品的 GC 含量的影响，并且容易产生嵌合体以及富集过程中的污染，这会在序列数据分析过程中产生不需要的噪音，并且还会妨碍正确的功能和/或分类学分配。 MDA 的替代方案是直接 DNA 测序 (DS)，它代表了基因组测序的理论黄金标准。在这项工作中，我们根据一珠一分子的概念，探索从焦磷酸测序所需的最小数量的 DNA 分子中对大肠杆菌 fs 24 基因组进行测序的可能性。使用 DS 的优化方案，我们构建了一个鸟枪库，其中包含填充 picotiterplate 选定区域所需的最少数量的 DNA 分子。我们收集了大部分具有统一覆盖范围的参考基因组延伸。我们将 DS 方法与应用于相同量起始 DNA 的 MDA 方法进行了比较。正如预期的那样，MDA 产生了稀疏且有偏差的读取分布，并且具有大量未分配和非特异性 DNA 扩增。优化的 DS 方案允许对含有极少量 DNA 的样品进行无偏测序。
The large amount of DNA needed to prepare a library in next generation sequencing protocols hinders direct sequencing of small DNA samples. This limitation is usually overcome by the enrichment of such samples with whole genome amplification (WGA), mostly by multiple displacement amplification (MDA) based on phi 29 polymerase. However, this technique can be biased by the GC content of the sample and is prone to the development of chimeras as well as contamination during enrichment, which contributes to undesired noise during sequence data analysis, and also hampers the proper functional and/or taxonomic assignments. An alternative to MDA is direct DNA sequencing (DS), which represents the theoretical gold standard in genome sequencing. In this work, we explore the possibility of sequencing the genome of Escherichia coli fs 24 from the minimum number of DNA molecules required for pyrosequencing, according to the notion of one-bead-one-molecule. Using an optimized protocol for DS, we constructed a shotgun library containing the minimum number of DNA molecules needed to fill a selected region of a picotiterplate. We gathered most of the reference genome extension with uniform coverage. We compared the DS method with MDA applied to the same amount of starting DNA. As expected, MDA yielded a sparse and biased read distribution, with a very high amount of unassigned and unspecific DNA amplifications. The optimized DS protocol allows unbiased sequencing to be performed from samples with a very small amount of DNA.