16S Amplicon Sequencing Library Preparation

High throughput short read sequencing of 16S rRNA for the profiling of microbial communities is a cost-effective and efficient method that enables high precision taxonomic classification from phylum to species. The technique enables the study of metagenomes from microbial habitats ranging from soil and plants to the digestive pathways of humans and animals. The main targeted region in Metagenomic studies is the prokaryotic 16S ribosomal RNA (rRNA) gene which is approximately 1,500 bp in length and contains 9 variable (V1-V9) and conserved regions. Several library preparation protocols target single or multiple hyper-variable regions depending on the level of identification required and utilize sample barcode tags to allow massive multiplexed sequencing.

Sequences of the 16S rRNA gene have already been determined for a large number of microbes. These sequences are available in two main databases, namely Greengenes (1) and the Ribosomal Database Project (2). A common tool for analyzing metagenomics samples is Quantitative Insights into Microbial Ecology (QIIME) (3,4). Both the Roche 454 and Illumina MiSeq platform are suited for 16S rRNA amplicon sequencing. The MiSeq platform offers a faster turnaround time and greater number of reads, accommodating a larger number of samples and deeper scrutiny of metagenomic populations.

Genohub has a lot of experience helping design 16S sequencing projects. Contact us to take advantage of our complementary consultation service. You can also search for and identify providers who offer 16S rRNA and library prep services.

See Genohub's up-to-date list of available 16S Amplicon-Seq library prep services

16S Amplicon Sequencing Kits

Bioo Scientific NEXTflex 16S V4 Amplicon-Seq Kit

The NEXTflex 16S V4 kit is designed for sequencing of the 4th hypervariable domain of microbial 16S rRNA, a very specific phylogenetic marker. The kit utilizes long primers that not only flank the V4 domain but also contain Illumina’s flow cell sequencing read primer and flow cell binding sites. The required input of microbial DNA is 50 ng and the protocol can be completed in a single 2 hour long PCR step. Up to 288 barcode are available for sample indexing.

Protocol Overview:

  1. Bacterial DNA isolation
  2. Primer annealing and PCR amplification

Bioo Scientific NEXTflex 16S V1-V3 Amplicon-Seq Kit

The NEXTflex 16S V1-V3 kit is designed for sequencing a region between the first and fourth hypervariable domain of microbial 16S rRNA, a well established method for studying phylogeny and taxonomy in complex microbial environments. The 1st and 2nd PCR primers in the kit are designed to be fully compatible with Illumina sequencing chemistry, obviating the need for custom sequencing read primers. Users can start from as low as 1 ng of input DNA material and multiplex up to 384 samples in a single Illumina MiSeq lane.

Protocol Overview:

  1. Bacterial DNA isolation
  2. 1st PCR Primer annealing and amplification
  3. 2nd PCR Primer annealing and amplification

  • (1) Desantis TZ, Hugenholtz P, Larsen N, Rojas M, Brodie EL, et al. (2006) Greengenes, a chimera-checked 16S rRNA gene database and workbench compatible with ARB. Appl Environ Microbiol 72(7): 5069-5072.
  • (2) Cole JR, Wang Q, Cardenas E, Fish J, Chai B, et al. (2009) The Ribosomal Database Project : Improved alignments and new tools for rRNA analysis. Nucl Acids Res 37: D141-D145.
  • (3) Caporaso JG, Kuczynski J, Stombaugh J, Bittinger K, Bushman, FD, et al., (2010) QIIME allows analysis of high-throughput community sequencing data. Nat Methods 7(5) 335-336.
  • (4) http://qiime.org/tutorials/processing_illumina_data.html