Comparison of methods for library construction and short read annotation of shellfish viral metagenomes
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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.
KeywordsShellfish Viral metagenome Whole genome amplification Size selection Library construction Read annotation
We express our thanks to Dr. Wang Rui-Xuan, Dr. Ye Ling-Tong and Dr. Yao Tuo (South China Sea Fisheries Research Institute, Guangzhou, China) for the collection of samples. This work was supported by the “Central Public-interest Scientific Institution Basal Research Fund,CAFS” (2016RC-LX05), the “Earmarked Fund for Modern Agro-industry Technology Research System” (CARS-49), the “Guangdong Province Marine Fishery Development Projects” and the “Guangdong Special Support Program” (00-201620641).
Compliance with ethical standards
Conflict of interest
Hong-Ying Wei has declared that no conflict of interest exists, Sheng Huang has declared that no conflict of interest exists, Jiang-Yong Wang has declared that no conflict of interest exists, Fang Gao has declared that no conflict of interest exists, Jing-Zhe Jiang has declared that no conflict of interest exists.
All animal work have been conducted according to relevant national and international guidelines. South China Sea Fisheries Research Institute Academic Committee approved this research.
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