DNA Analysis Methods Available for Water and Wastewater

Bacteria & Archaea Analysis

Includes DNA amplicon sequencing of the 16S rRNA gene, identifying nearly all Bacteria and Archaea at the genus level or above and their relative percentage abundance. In short, we identify who’s there in the Bacteria and Archaea kingdoms and quantify the percentage relative abundance of each. Quantitation is available as an add on.

Eukarya Analysis

Includes DNA amplicon sequencing of the 18S rRNA gene, identifying nearly all Eukarya at the genus level or above and their relative percentage abundance. This includes the higher life forms in your system such as Protozoa, Metazoa, Fungi, Algae, and such. In short, we measure who’s there in the Eukaryotic kingdom and the percentage relative abundance of each. Quantitation is available as an add on.

Methanogen Analysis

Methanogens are the key microbes responsible for methane production in anaerobic digesters and other biogas systems. Our McrA gene DNA amplicon sequencing provides the most accurate DNA analysis technique for characterizing methanogen communities. It includes identification at the species level and measurement of percentage relative abundance of nearly all methanogens in your system. Quantitation is available as an add on.

DNA Analysis Methods, Scientific Summary

Quantitation

Sequencing

The 16S rRNA gene is PCR-amplified and sequenced to identify prokaryotic microorganisms (bacteria and archaea) and quantify the percent relative abundance of each identified organism. The 18S rRNA gene is PCR- amplified and sequenced to identify eukaryotic microorganisms (fungi, algae, amoeba, etc.) and quantify the percent relative abundance of each identified organism.

The 16s and 18s rRNA genes are PCR-amplified using the 515F/806R and euk1391F/eukBr primers, respectively. PCR is completed using the HotStarTaq Plus Master Mix Kit (Qiagen, USA) under the following conditions: 95°C for 5 minutes, followed by 30-35 cycles of 95°C for 30 seconds, 53°C for 40 seconds, and 72°C for 1 minute, after which a final elongation step at 72°C for 10 minutes is completed. The amplified DNA from each sample is loaded onto a 2% agarose gel to verify the presence/absence and determine the relative strength of DNA amplification. Samples are pooled together in equal proportions based on their molecular weight and DNA concentrations. Pooled samples are purified using calibrated SPRI beads, and then the pooled and purified DNA is used to prepare an Illumina DNA library.

Sequencing is performed on the Illumina NovaSeq sequencing platform following the manufacturer’s guidelines. Sequence data is processed using a proprietary analysis pipeline, where sequences <150bp are removed, and sequences with ambiguous base calls are removed. Sequences are filtered using a maximum expected error threshold of 1.0 and dereplicated. The dereplicated or unique sequences are denoised; unique sequences with sequencing and/or PCR point errors are identified and removed, followed by chimera removal; thereby providing a denoised sequence or zOTU. Final zOTUs are taxonomically classified using BLASTn against a curated database derived from NCBI (www.ncbi.nlm.nih.gov).