iTag amplicon sequencing is performed to determine the relative abundance of each taxa in a community, and to compare the taxonomic profiling between groups of interest. This level of analysis can help to address changes in the overall microbial profile over time, or between treatment groups. We can address taxonomic profiling for prokaryotic communities (16S V4, V1-V3 or V4_skin), eukaryotic communities (18S), fungal communities (ITS2), and archaeal communities (16S V4-V5).
Microbiome Insights now offers a unique 16S V4 protocol, V4_skin, optimized for skin samples. This pipeline provides:
- Up to 10 times lower error rate relative to the 16S V1-V3 region
- Better representation of p. acnes compared to common V4 primers
- Better detection of bacteria representative of the skin microbiome that are underdetected by sequencing the V1-V3 region
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 this kit to have a good balance of DNA yield and quality as demonstrated on a variety of environmental sample types.
DNA quantification and quality checks are done via Qubit.
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 prokaryotic (16S V4, V1-V3 or V4_skin), eukaryotic (18S), fungal (ITS2), or archaeal (16S V4-V5) 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.