Services

16S sequencing

Assay overview

We provide services to sequence various hypervariable regions (V1-V9) of 16S rRNA gene for bacterial taxonomy and phylogeny. Customer can select the hypervariable region/s based on their sample source and study aims.

A sketch of 16S rRNA gene and hypervariable regions:

 
 

For commonly studied regions such as: V3 & V4, we use Illumina Nextera recommended primer sets and protocol to amplify the targeted region/s and ligate illumina adapter & barcodes. We also offer in house developed protocol for full length 16S rRNA gene sequencing.

16S Amplicon PCR Forward Primer = 5'

TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG

16S Amplicon PCR Reverse Primer = 5'

GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC

Customer need to provide either original sample (to isolate DNA) or extracted genomic DNA representative of their sample. We recommend 100-500ng (20ng/ul conc.) high complexity DNA to generate high quality 16s sequencing libraries.  We use illumina recommended dual indexing to barcode sequencing libraries before pooling. QC pass libraries are precisely quantified using Q PCR before loading onto MiSeqDX illumina platform using 300 PE protocol.

 

Example of work flow: