A Novel Method for Complete Environmental Microbial Profiling

Targeted amplicon sequencing enables comparison of microbial communities across different time points, locations, or treatments. We present a novel approach that leverages NGS with microfluidics PCR allowing simultaneous amplification of bacterial and archaeal 16S, eukaryotic 18S, fungal ITS and functional-gene specific targets. The procedure results in barcoded, multi-amplicon libraries that are ready for sequencing on a MiSeq or HiSeq.

Selected primer pairs from rRNA and other targets plus sample DNAs are loaded on a Fluidigm Access Array Integrated Fluidic Circuit. Samples and primers are distributed through microfluidic channels in 2,304 parallel amplification chambers. A second primer set, included with each sample, adds tails to the PCR products with barcodes and Illumina-specific sequences. Following amplification, harvested products are quantified, run on a Fragment Analyzer to confirm sizes, pooled and gel-size selected. Up to 1,536 individually-barcoded samples, amplified with up to 48 different primer sets, can be combined into one pool for sequencing.

The pool is quantitated by qPCR, and sequenced on a MiSeq or HiSeq with paired-reads 250nt based on the number of sample/primer combinations to target at least 20k sequencing reads per unique sample plus primer set.

A custom pipeline is implemented to sort data by primer set and barcode. Final data is ready to be analyzed in QIIME, Mothur, IM-tornado, or other software packages.

This novel microbial profiling system is a fast, cost-effective, multi-targeted metagenomic approach for whole microbial community analysis. Surveying the entire microbial spectrum allows a more complete understanding of the complex nature of these communities.