Construction of high coverage whole-genome sequencing libraries from single colon crypts without DNA extraction or whole-genome amplification.
Construction of high coverage whole-genome sequencing libraries from single colon crypts without DNA extraction or whole-genome amplification.
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DOI：
10.1186/s13104-023-06333-y
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发表时间：
2023-04-27
影响因子：
1.8
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中科院分区：
文献类型：
--
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由于基因组覆盖偏倚、PCR过度循环和昂贵技术的要求，对少量细胞进行全面可靠的全基因组变异分析一直具有挑战性。为了全面鉴定反映干细胞基因组异质性的单个结肠隐窝中的基因组改变，我们开发了一种从单个结肠隐窝构建全基因组测序文库的方法，而无需DNA提取、全基因组扩增或增加PCR富集循环。我们提供了81个单隐窝（每个包含的DNA比传统方法的要求少4 - 8倍）和16个散装组织文库的比对后统计数据，以证明在获得人类基因组的可靠覆盖方面的一致成功，无论是在深度（≥ 30 X）和广度（≥ 92%的基因组覆盖在≥ 10 X深度）。这些单穴文库具有与使用高质量和大量的纯化DNA的常规方法产生的文库相当的质量。可以想象，我们的方法可以应用于许多组织的小活检样本，并可以与单细胞靶向测序相结合，以全面分析癌症基因组及其演变。该方法的广泛潜在应用为以高分辨率在少量细胞中经济有效地检查基因组异质性提供了扩展的可能性。在线版本包含补充材料，可通过10.1186/s13104-023-06333-y获得。
Comprehensive and reliable genome-wide variant analysis of a small number of cells has been challenging due to genome coverage bias, PCR over-cycling, and the requirement of expensive technologies. To comprehensively identify genome alterations in single colon crypts that reflect genome heterogeneity of stem cells, we developed a method to construct whole-genome sequencing libraries from single colon crypts without DNA extraction, whole-genome amplification, or increased PCR enrichment cycles. We present post-alignment statistics of 81 single-crypts (each contains four- to eight-fold less DNA than the requirement of conventional methods) and 16 bulk-tissue libraries to demonstrate the consistent success in obtaining reliable coverage, both in depth (≥ 30X) and breadth (≥ 92% of the genome covered at ≥ 10X depth), of the human genome. These single-crypt libraries are of comparable quality as libraries generated with the conventional method using high quality and quantities of purified DNA. Conceivably, our method can be applied to small biopsy samples from many tissues and can be combined with single cell targeted sequencing to comprehensively profile cancer genomes and their evolution. The broad potential application of this method offers expanded possibilities in cost-effectively examining genome heterogeneity in small numbers of cells at high resolution. The online version contains supplementary material available at 10.1186/s13104-023-06333-y.
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影响因子：
13.6
作者：
Ruan Q;Ruan W;Lin X;Wang Y;Zou F;Zhou L;Zhu Z;Yang C
通讯作者：
Yang C
影响因子：
12.3
作者：
Fan X;Yang C;Li W;Bai X;Zhou X;Xie H;Wen L;Tang F
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Tang F
影响因子：
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Chang CC;Chow CC;Tellier LC;Vattikuti S;Purcell SM;Lee JJ
通讯作者：
Lee JJ
影响因子：
64.8
作者：
Lee-Six, Henry;Olafsson, Sigurgeir;Stratton, Michael R.
通讯作者：
Stratton, Michael R.
影响因子：
4.3
作者：
Nicolas, Pierre;Kim, Kyoung-Mee;Tavare, Simon
通讯作者：
Tavare, Simon