DNA Extraction and Quality Control
For our sequencing protocols, 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. 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.
Targeted Metabolite Analyses
We provide quantitative information for short chain fatty acids (SCFAs, bacterial products of fermentation with a role in gut health) and Calprotectin (intestinal inflammation biomarker). For both SCFA and Calprotecting analyses, samples are aliquoted and extracted independently of the above protocol.
Calprotectin is extracted from stool according to the BÜHLMANN fCAL ELISA Calprotectin assay, and measured fluorometrically in a microtiter.
Short chain fatty acids (SCFAs) are extracted from stool in an aqueous solution, analyzed in a gas chromatograph (GC). We report data for acetic, propionic, butyric, valeric, isovaleric, caproic, and heptanoic acids.
Samples are multiplexed on 96-well plates to generate amplicon libraries (16S V4, ITS2, and 18S V4). All samples are visualized using gel electrophoresis to ensure successful library preparation. The libraries are pooled following normalization using the ‘Applied Biosciences Sequal Prep Normalization kit’. The amplicon libraries are then quality checked through the Agilent Bioanalyzer, using a High Sensitivity DNA assay, and accurately quantified using quantitative PCR to ensure optimal cluster density during sequencing. Multiple amplicons (16S, ITS2, and 18S) can be sequenced simultaneously on a single MiSeq lane.
Samples are sequenced using the MiSeq V2 chemistry for all microbiome applications producing an average of 15 000 raw sequences per sample (250 bp x2). This chemistry is less error-prone than newer chemistries (Miseq V3) which produces longer reads (300 bp x2).
List of Services
Who is there?
The most common and economical approach for analysis of composition of microbial communities. Based on high-throughput sequencing of PCR amplicons for prokaryotic communities (16S V4 region), fungal communities (ITS2), and eukaryotic communities (18S). Click here for more info
How many are there?
Quantitation of total microbial density or specific microbial populations. Quantitative PCR targets total prokaryotes, bacteria, archaea, fungi as well as other taxonomic or functional groups by request. Click here for more info