Shotgun Metagenome Analysis

Shotgun Metagenome Analysis

Sample Collection

For information on sample collection, storage and transport please see our sample handling guidelines in the support section. 

DNA extraction and quality control

DNA is extracted from samples using the MO BIO PowerSoil DNA Kit optimized for the KingFisher robot. This kit uses magnetic beads to specifically capture DNA while excluding organic inhibitors. In-house testing has shown this kit to have a good balance of DNA yield and quality as demonstrated on a variety of environmental sample types.

The MO BIO Powersoil DNA protocol is followed, with the addition of a bead beating step to facilitate lysis of particularly robust microorganisms. DNA quantification and quality checks are done via Qubit.

Library preparation and sequencing

Whole DNA is prepared for sequencing using the Illumina Nextera XT DNA library prep kit. Samples are barcoded and mixed together for sequencing (up to 96 samples). 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).

Sequencing

Paired reads are concatenated and then processed to remove adaptors, barcodes, low-quality reads as well as contaminant sequences (typically host-DNA). Sequences are then compared to phylogenetic and functional databases to obtain taxonomic and functional profiles.