Hirai Miho, Nishi Shinro, Tsuda Miwako, Sunamura Michinari, Takaki Yoshihiro, Nunoura Takuro
Research and Development (R&D) Center for Marine Biosciences, Japan Agency for Marine-Earth Science and Technology (JAMSTEC).
Ecosystem Observation and Evaluation Methodology Research Unit, Project Team for Development of New-generation Research Protocol for Submarine Resources, Japan Agency for Marine-Earth Science and Technology (JAMSTEC).
Microbes Environ. 2017 Dec 27;32(4):336-343. doi: 10.1264/jsme2.ME17132. Epub 2017 Nov 28.
Shotgun metagenomics is a low biased technology for assessing environmental microbial diversity and function. However, the requirement for a sufficient amount of DNA and the contamination of inhibitors in environmental DNA leads to difficulties in constructing a shotgun metagenomic library. We herein examined metagenomic library construction from subnanogram amounts of input environmental DNA from subarctic surface water and deep-sea sediments using two library construction kits: the KAPA Hyper Prep Kit and Nextera XT DNA Library Preparation Kit, with several modifications. The influence of chemical contaminants associated with these environmental DNA samples on library construction was also investigated. Overall, shotgun metagenomic libraries were constructed from 1 pg to 1 ng of input DNA using both kits without harsh library microbial contamination. However, the libraries constructed from 1 pg of input DNA exhibited larger biases in GC contents, k-mers, or small subunit (SSU) rRNA gene compositions than those constructed from 10 pg to 1 ng DNA. The lower limit of input DNA for low biased library construction in this study was 10 pg. Moreover, we revealed that technology-dependent biases (physical fragmentation and linker ligation vs. tagmentation) were larger than those due to the amount of input DNA.
鸟枪法宏基因组学是一种用于评估环境微生物多样性和功能的低偏差技术。然而，对足够量DNA的要求以及环境DNA中抑制剂的污染导致构建鸟枪法宏基因组文库存在困难。我们在此使用两种文库构建试剂盒（KAPA Hyper Prep试剂盒和Nextera XT DNA文库制备试剂盒）并进行了一些修改，研究了从亚北极地表水和深海沉积物中纳克以下量的输入环境DNA构建宏基因组文库的情况。还研究了与这些环境DNA样本相关的化学污染物对文库构建的影响。总体而言，使用这两种试剂盒从1 pg到1 ng的输入DNA构建了鸟枪法宏基因组文库，且文库没有受到严重的微生物污染。然而，与从10 pg到1 ng DNA构建的文库相比，从1 pg输入DNA构建的文库在GC含量、k-mer或小亚基（SSU）rRNA基因组成方面表现出更大的偏差。本研究中低偏差文库构建的输入DNA下限为10 pg。此外，我们发现技术依赖性偏差（物理片段化和接头连接与转座酶标签法）比输入DNA量导致的偏差更大。
