Institut des Sciences de l'Evolution, ISEM, Université de Montellier, CNRS, IRD, EPHE, France.
Genome Biol Evol. 2018 Feb 1;10(2):616-622. doi: 10.1093/gbe/evy022.
Standard Illumina libraries are biased toward sequences of intermediate GC-content. This results in an underrepresentation of GC-rich regions in sequencing projects of genomes with heterogeneous base composition, such as mammals and birds. We developed a simple, cost-effective protocol to enrich sheared genomic DNA in its GC-rich fraction by subtracting AT-rich DNA. This was achieved by heating DNA up to 90 °C before applying Illumina library preparation. We tested the new approach on chicken DNA and found that heated DNA increased average coverage in the GC-richest chromosomes by a factor up to six. Using a Taq polymerase supposedly appropriate for PCR amplification of GC-rich sequences had a much weaker effect. Our protocol should greatly facilitate sequencing and resequencing of the GC-richest regions of heterogeneous genomes, in combination with standard short-read and long-read technologies.
标准的 Illumina 文库偏向于中等 GC 含量的序列。这导致在具有不均匀碱基组成的基因组（如哺乳动物和鸟类）的测序项目中，GC 丰富区域的代表性不足。我们开发了一种简单、经济有效的方法，通过减去富含 AT 的 DNA 来富集剪切基因组 DNA 中的 GC 丰富部分。这是通过在进行 Illumina 文库制备之前将 DNA 加热至 90°C 来实现的。我们在鸡 DNA 上测试了新方法，发现加热的 DNA 使 GC 最丰富的染色体的平均覆盖率提高了多达六倍。使用据称适用于 GC 丰富序列 PCR 扩增的 Taq 聚合酶的效果要弱得多。我们的方案应与标准的短读长和长读长技术结合使用，极大地促进了异质基因组中 GC 最丰富区域的测序和重测序。
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