Amplicon sequencing, or rRNA gene sequencing, is performed to determine the relative abundance of taxa in a bacterial community, and to compare between groups of interest. This level of analysis can help to address changes in the overall microbial profile over time, or between treatment groups. Depending on the sample type, we have a variety of amplicons we can address:
- Prokaryotic communities (16S V4, V1-V3)
- Eukaryotic communities (18S)
- Fungal communities (ITS2)
- Archaeal communities (16S V4-V5)
We suggest speaking to a Microbiome Insights representative to better understand the differences in sequencing methods and which one may be best suited for your study.
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.
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), 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.