Rosani Umberto, Domeneghetti Stefania, Pallavicini Alberto, Venier Paola
Department of Biology, University of Padua, Via U. Bassi 58/b, 32121 Padua, Italy.
Department of Life Sciences, University of Trieste, Via L. Giorgeri, No. 5, 34121 Trieste, Italy.
Biomed Res Int. 2014;2014:538549. doi: 10.1155/2014/538549. Epub 2014 Jun 30.
Next generation sequencing (NGS) allows fast and massive production of both genome and transcriptome sequence datasets. As the genome of the Mediterranean mussel Mytilus galloprovincialis is not available at present, we have explored the possibility of reducing the whole genome sequencing efforts by using capture probes coupled with PCR amplification and high-throughput 454-sequencing to enrich selected genomic regions. The enrichment of DNA target sequences was validated by real-time PCR, whereas the efficacy of the applied strategy was evaluated by mapping the 454-output reads against reference transcript data already available for M. galloprovincialis and by measuring coverage, SNPs, number of de novo sequenced introns, and complete gene sequences. Focusing on a target size of nearly 1.5 Mbp, we obtained a target coverage which allowed the identification of more than 250 complete introns, 10,741 SNPs, and also complete gene sequences. This study confirms the transcriptome-based enrichment of gDNA regions as a good strategy to expand knowledge on specific subsets of genes also in nonmodel organisms.
新一代测序（NGS）能够快速大量地生成基因组和转录组序列数据集。由于目前尚无地中海贻贝（Mytilus galloprovincialis）的基因组，我们探索了通过使用捕获探针结合PCR扩增和高通量454测序来富集选定基因组区域，从而减少全基因组测序工作量的可能性。通过实时PCR验证DNA靶序列的富集情况，而应用策略的有效性则通过将454测序输出的 reads 比对到已有的地中海贻贝参考转录数据，并通过测量覆盖度、单核苷酸多态性（SNPs）、从头测序的内含子数量和完整基因序列来评估。针对近1.5兆碱基对的目标大小，我们获得了一个目标覆盖度，该覆盖度能够鉴定出超过250个完整内含子、10741个SNPs以及完整的基因序列。这项研究证实了基于转录组的基因组DNA区域富集是一种很好的策略，可用于扩展对非模式生物中特定基因子集的认识。
