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.
DNA Extraction & Quality Control
DNA is extracted from samples using the Mag-Bind® technology and hand-held 96 channel electronic pipette. The use of magnetics beads for DNA extraction is fundamental for automation and to specifically capture DNA while excluding organic inhibitors. In-house testing has shown that the DNA extraction kit used 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 pooled samples are 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.
Shotgun Sequencing
Paired reads are concatenated and then processed to remove adaptors, barcodes, and 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.
We provide sequencing on the Illumina MiSeq platform.
Custom reporting can be done after consultation with our bioinformaticians to understand your particular requirements.
