The Genomics Core provides 16S ribosomal RNA gene amplification and sequecing services. We can rpovide a variety of services including:
DNA extraction using Qiagen DNeasy PowerSoil HTP 96 Kit (former MoBio PowerSoil-htp 96-well Soil DNA Isolation Kit). Our laboratory is equipped with high-speed Qiagen TissueLyser that is compatible with deep well plates, two Caliper liquid handling workstation, and centrifuges that are able to spin 96-well plates.
16S ribosomal RNA gene can be amplified using V1-V3 regions primer set or V4 region using Earth Microbiome Project primer set.
We also offer a custom targeted amplicon sequencing protocol using two-step PCR approach. The first round of PCR involves adding a overhang sequence to the specific primers to amplify the selected marker and allow for barcoding during the second round of PCR. In the second round of PCR, Illumina Nextera barcode adaptors are added to each amplicon.
We also include a well defined community of 9 genomic DNAs, VCU mock community. This will allow us to quantify the amount of sequencing error and to detect run-to-run drift effects. PCR products are cleaned using the Agencourt AmpPure magnetic bead kit and quantified using Quant-iT PicoGreen dsDNA Assay Kit. Cleaned PCR products will then be pooled in equamolar quantities and sequenced on the Illumina MiSeq instrument.
16S ribosomal RNA gene amplicon is tipically sequenced on the Illumina MiSeq instrument. This instrument has the quickest run per cycle and the longest read length. The 600 cycles kit provides the ideal recipe for long amplicons (i.e. V1-V3 region and "longer" V4 region). The MiSeq instrument will generate ~24 million reads in both forward and reverse directions.
The Genomics Core offers comprehensive data analysis including RDP and Qiime platforms. The analysis includes filtering raw fastq files for primer and adapter dimer sequences, removing chimeric sequences, clustering sequences into OTUs using the OTU Qiime method. OTU annotation is provided using SILVA or greengenes databases.