DNA Analysis Methods Available for Water and Wastewater

16s

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 % relative abundance of each.

16s quantation

Bacteria & Archaea Analysis with Quantitation

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. qPCR quantitation measures the total quantity of 16S rRNA genes, which is extrapolated to each identified microbial group, measured in 16S rRNA gene copies per milliliter of sample. In short, we identify who’s there in the Bacteria and Archaea kingdoms, quantify the % relative abundance of each, and estimate how many of each are present, measured as 16S gene copies/mL of sample.

 

18s

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 % relative abundance of each.

18s quantitation

Eukarya Analysis with Quantitation

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. qPCR quantitation measures the total quantity of 18S rRNA genes, which is extrapolated to each identified microbial group, measured in 18S rRNA gene copies per milliliter of sample. In short, we identify who’s there in the Eukarya kingdom, quantify the % relative abundance of each, and estimate how many of each are present, measured as 18S gene copies/mL of sample.

16s 18s quantitation

Bacteria, Archaea & Eukarya Analysis with Quantitation

Our most robust analysis is used to map your system’s microbiome of bacteria, archaea, and eukarya. 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. qPCR quantitation measures the total quantity of 16S rRNA genes, which is extrapolated to each identified microbial group, measured in 16S rRNA gene copies per milliliter of sample. DNA amplicon sequencing of the 18S rRNA gene identifies 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. qPCR quantitation measures the total quantity of 18S rRNA genes, which is extrapolated to each identified microbial group, measured in 18S rRNA gene copies per milliliter of sample.

mcra

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.

DNA Analysis Methods, Scientific Summary

Quantitation

Real-time PCR (qPCR) is used to detect and quantify copies of the targeted gene per sample. The Bac2F/Bac2R and universal SSUF/SSUR primer-pairs are used to target prokaryotic (bacteria and archaea) and eukaryotic microorganisms (fungi, algae, amoeba, etc.) respectively. qPCR of prokaryotic organisms is completed using the Taqman Universal PCR Master Mix (Applied Biosystems) with 1 ul of template DNA as input onto the StepOne Plus Real-Time PCR System (Applied Biosystems). DNA from E. coli is used as a standard. qPCR of eukaryotic organisms is completed using the PowerUpTM SYBR® Green Master Mix (Applied Biosystems) with 1 ul of template DNA as input onto the StepOne Plus Real-Time PCR System (Applied Biosystems). DNA from Saccharomyces cerevisiae is used as a standard. The estimated quantity of gene copies per sample volume for each identified prokaryote group is estimated by multiplying the quantity of prokaryotes by the percentage relative abundance of each identified prokaryote group. The respective quantity of gene copies per sample volume for each identified eukaryote group is estimated by multiplying the quantity of eukaryotes by the percentage relative abundance of each identified eukaryote group.

dna sequencer

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).

Your Microbiome Analyst will provide guidance on the DNA analysis methods that will meet your needs and objectives.