Wei Hong-Ying, Huang Sheng, Wang Jiang-Yong, Gao Fang, Jiang Jing-Zhe
Key Laboratory of Aquatic Product Processing, Ministry of Agriculture, South China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Guangzhou, 510300, China.
Shanghai Ocean University, Shanghai, 201306, China.
Genes Genomics. 2018 Mar;40(3):281-288. doi: 10.1007/s13258-017-0629-1. Epub 2017 Dec 2.
The emergence and widespread use of high-throughput sequencing technologies have promoted metagenomic studies on environmental or animal samples. Library construction for metagenome sequencing and annotation of the produced sequence reads are important steps in such studies and influence the quality of metagenomic data. In this study, we collected some marine mollusk samples, such as Crassostrea hongkongensis, Chlamys farreri, and Ruditapes philippinarum, from coastal areas in South China. These samples were divided into two batches to compare two library construction methods for shellfish viral metagenome. Our analysis showed that reverse-transcribing RNA into cDNA and then amplifying it simultaneously with DNA by whole genome amplification (WGA) yielded a larger amount of DNA compared to using only WGA or WTA (whole transcriptome amplification). Moreover, higher quality libraries were obtained by agarose gel extraction rather than with AMPure bead size selection. However, the latter can also provide good results if combined with the adjustment of the filter parameters. This, together with its simplicity, makes it a viable alternative. Finally, we compared three annotation tools (BLAST, DIAMOND, and Taxonomer) and two reference databases (NCBI's NR and Uniprot's Uniref). Considering the limitations of computing resources and data transfer speed, we propose the use of DIAMOND with Uniref for annotating metagenomic short reads as its running speed can guarantee a good annotation rate. This study may serve as a useful reference for selecting methods for Shellfish viral metagenome library construction and read annotation.
高通量测序技术的出现和广泛应用推动了对环境或动物样本的宏基因组学研究。宏基因组测序的文库构建以及对所产生序列读数的注释是此类研究中的重要步骤，并会影响宏基因组数据的质量。在本研究中，我们从中国南方沿海地区采集了一些海洋软体动物样本，如香港牡蛎、栉孔扇贝和菲律宾蛤仔。这些样本被分为两批，以比较两种贝类病毒宏基因组文库构建方法。我们的分析表明，与仅使用全基因组扩增（WGA）或全转录组扩增（WTA）相比，将RNA逆转录为cDNA，然后通过全基因组扩增与DNA同时进行扩增，可产生更多的DNA。此外，通过琼脂糖凝胶提取获得的文库质量更高，而不是使用磁珠大小选择法。然而，如果结合过滤器参数的调整，后者也能提供良好结果。这一点，再加上其操作简便，使其成为一个可行的选择。最后，我们比较了三种注释工具（BLAST、DIAMOND和Taxonomer）和两个参考数据库（NCBI的NR和Uniprot的Uniref）。考虑到计算资源和数据传输速度的限制，我们建议使用DIAMOND和Uniref对宏基因组短读段进行注释，因为其运行速度能够保证较高的注释率。本研究可为选择贝类病毒宏基因组文库构建和读段注释方法提供有益参考。
