16S metagenomics

16S analysis flowchart

Sequence variation in the 16S ribosomal RNA (rRNA) gene is widely used to characterize taxonomic diversity present in microbial communities. This gene is one of the most conserved among many organisms but contains nine hypervariable regions (V1-V9) that can provide species (or genus) -specific amplificates useful for bacterial identification.

For this reason, genes that encode the rRNA (rDNA) have been used extensively to determine taxonomy and to estimate rates of species divergence among bacteria. More in particular, primers binding to conserved regions are used to amplify with PCR and further sequence the hypervariable regions. However, in an heterogeneous sample, low frequency species can go undetected, unless an high amount of amplificates is sequenced.

Genechron Laboratory now offers the possibility to couple this extraordinary powerful tool with the sequencing depth offered by the Ion Torrent PGM machine. The use of V3-specific primers coupled with a robust bioinformatic pipeline ensures taxonomic assignation of the sequences that enables the reconstruction of the diversity present in the samples provided.

We offer:

  • quantification of DNA with Qubit 2.0
  • amplification of bacterial genomic DNA with V3-specific primers (if requested)
  • Ion Torrent sequencing
  • collaboration between the customer and our product specialist for the choice of the most effective sequencing strategy
  • optional bioinformatics:
    • Reads trimming and removal of low QV sequences
    • Detection and removal of chimeric sequences formed during amplification
    • Taxonomic assignation, including OTU prediction, rarefaction curves, estimation of species richness (ACE) and Shannon & Simpson diversity index

We request:

  • minimum of 200 ng of input DNA as starting material (minimal concentration 20 ng/uL), according to the analysis requested
  • quality check of DNA to be submitted for integrity and purity (Nanodrop or similar; agarose gel photo and/or Agilent BioAnalyzer electropherogram). Optionally, quality checks can be conducted by Genechron

Data is supplied as raw sequencing data (FASTQ) and/or as a table containing the taxonomic assignation derived.

Delivery time is dependent on when we receive the samples, run type and number of PGM chips, please inquire.

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