What is Shotgun Metagenomic Sequencing?
Shotgun metagenome sequencing is performed for taxonomic profiling (diversity and abundance), as well as functional analysis. This complex technique allows for parallel sequencing of DNA from all organisms within the community, with high coverage for species-level detection. The data generation allows for more advanced reporting and genome assemblies.
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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. In-house testing has shown that this kit has a good balance of DNA yield and quality, as demonstrated on a variety of environmental sample types.
Library preparation
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 technologies for paired end reads.
The number of samples multiplexed depends on the sample type (i.e. samples that contain more host DNA or complex communities requiring more sequencing depth need consideration). For fecal samples, we recommend at least 1 Gigabase per sample to achieve shallow sequencing, which provides basic taxonomic functional profiling. For applications that require genome reconstruction, more sequencing is required.
