16S & 18S rRNA Gene Sequencing
16S rRNA gene sequencing, or 16S amplicon sequencing, is performed to determine the relative abundance of taxa in a bacterial and archaeal 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.
The 18S protocol is designed to amplify eukaryotes broadly with a focus on microbial eukaryotic lineages. We have the following rRNA primers available:
16S V4 515F-806R
18S V9 1391F-1510R
Primers sequences (Forward, Read1, Index and reverse-barcoded) are described in earthmicrobiome.org as well as the reference protocols.
16S & 18S rRNA Gene Sequencing
16S rRNA gene sequencing, or 16S amplicon sequencing, is performed to determine the relative abundance of taxa in a bacterial and archaeal 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.
The 18S protocol is designed to amplify eukaryotes broadly with a focus on microbial eukaryotic lineages. We have the following rRNA primers available:
16S V4 515F-806R
18S V9 1391F-1510R
Primers sequences (Forward, Read1, Index and reverse-barcoded) are described in earthmicrobiome.org as well as the reference protocols.
DNA Extraction & Quality Control
Sample processing is the first and most crucial step in any metagenomics project. The DNA extracted should be representative of all cells present in the sample and sufficient amounts of high quality nucleic acids must be obtained for subsequent library preparation and sequencing.
DNA is extracted from samples using the Mag-Bind® technology and hand-held 96 channel electronic pipette. The use of magnetic beads for DNA extraction is fundamental for automation and to specifically capture DNA while excluding organic inhibitors. In-house testing has shown that the DNA extraction kit used has a good balance of DNA yield and quality, as demonstrated on a variety of environmental sample types.
DNA Quantification
DNA quantity and quality checks are done via Spectrophotometric and Fluorometric methods.
Library Preparation & Sequencing
Post-PCR samples are submitted to agarose gel electrophoresis and then purified using magnetics beads technology. Purified DNA are quantified and normalized for further pooling. Normalization and pooling are carried out using high-precision pipetting robot. The pool obtained are prepared and combined with PhiX control according to Illumina Library Preparation Guide for sequencing. The DNA sequencing is performed on the MiSeq Illumina Platform.
