Shotgun metagenomic sequencing is performed to determine diversity and abundance, with the addition of functional analysis. This complex technique allows for parallel sequencing of all organisms within the community with high coverage and tons of data, with special consideration from samples of low biomass.
DNA extraction and quality control
DNA is extracted from samples using the Qiagen MagAttract PowerSoil DNA KF Kit (Formerly MO BIO PowerSoil DNA Kit) optimized for the Thermofisher KingFisher robot. This kit uses magnetic beads to specifically capture DNA while excluding organic inhibitors. Inhouse testing has shown this kit to have a good balance of DNA yield and quality as demonstrated on a variety of environmental sample types.
Whole DNA is prepared for sequencing using the Illumina Nextera XT DNA library prep kit. Samples are barcoded and mixed together for sequencing. The mix is then sequenced using Illumina HiSeq technologies using the high-throughput mode producing 2 x 150 bp reads (100-120 Gbases per sequencing run).
The number of samples multiplexed depends on the sample type (ie. samples that contain more host DNA or complex communities requiring more sequencing depth need consideration). For fecal samples, we recommend at least 16 bases per sample to achieve shallow sequencing which provides basic taxonomic functional profiling. For applications that require genome reconstruction more sequencing is required.