iTag Microbiome Analysis

iTag Microbiome 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. 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.

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

We use the high-fidelity Phusion polymerase for amplification of marker genes. If extractions contain some carry-over inhibition or a high concentration of DNA, typically we test 1:1 and 1:10 dilutions and run the PCR products on gels for verification. PCR is done with dual-barcoded primers (Kozich et al. 2014) targeting either the 16S V4 (Bacteria), 18S V4 (Eukarya) or ITS2 (Fungi) regions. Our barcoding strategy enables multiplexing up to 384 samples per run. PCR products are verified visually by running a representative subset of samples on a gel. Samples with failed PCRs (or spurious bands) are re-amplified by optimizing PCR conditions. The PCR reactions are cleaned-up and normalized using the high-throughput SequalPrep 96-well Plate Kit. Samples are then pooled to make one library that is quantified accurately with the KAPA qPCR Library Quant kit.

Sequencing

Samples are loaded onto plates and run through the Illumina MiSeq using the corresponding primer sets for either 16S V4 (Bacteria), 18S V4 (Eukarya) or ITS2 (Fungi) regions. Samples are multiplexed using four 96-well plates, sequencing up to 384 samples per run.