Metagenomic Profiling (shotgun metagenomics)
This TechNote addresses key quality parameters of three commonly used sequencing library protocols for shotgun metagenomics. Protocol A: Illumina Nextera DNA Flex (input: 100 ng), Protocol B: Illumina Nextera XT DNA (input: 1 ng), Protocol C: Down-scaled Illumina Nextera XT DNA (input: 0.1 ng). In a recent publication, Hillmann et al. (2018) recommended the down-scaled Nextera XT DNA approach as a cost-efficient protocol for shotgun metagenomics.
Sample and analysis details
To test how well the protocols can detect the theoretical composition of a microbial community standard, we performed three library preparations for each protocol using the same standard. We used a commercially available microbial community standard that consists of DNA from ten microbial species (five gram-positive bacteria, three gram-negative bacteria, two fungi) at a known composition. The standard was designed to explicitly cover a wide variety of genome sizes and GC contents and thus mimics the conditions in more complex communities. Also, we investigated the repeatability and detectable diversity of all protocols using a complex microbial DNA pool, generated from a fecal sample containing stool from ten individuals.