16S amplicon profiling from extraction control-Batch 1
Microbial profiling using 16S-based amplicon sequencing was performed at LGC (Germany). PCRs were commenced running a pre-amplification for 20 cycles using GM3 and 1061R. One L of this PCR was transferred to the second 20 L PCR, which was done using the standard primers and was run for another 20 cycles. Subsequent PCRs included 5 ng of DNA extract, 15 pmol of each forward primer 341F 5-NNNNNNNNNNTCCTACGGGNGGCWGCAG and reverse primer 785R 5- NNNNNNNNNNTGACTACHVGGGTATCTAAKCC in 20 L volume of 1 x MyTaq buffer containing 1.5 Units MyTaq DNA polymerase (BioLine) and 2 L of BioStabII PCR Enhancer (Sigma). This provided coverage of the V3-V4 region for microbiome analysis. PCRs were carried out for 30 cycles using the following parameters: 2 min 96C pre-denaturation; 96C for 15 s, 50C for 30 s, 70C for 90 s. DNA concentration of amplicons of interest was determined by Gel electrophoresis. About 20 ng amplicon DNA of each sample was pooled for up to 48 samples carrying different barcodes. The amplicon pools were purified with one volume AMPure XP beads (Agencourt) to remove primer dimer and other small miss-priming products, followed by an additional purification on MinElute columns (Qiagen). Purified amplicon pools (100 ng) were used to construct Illumina libraries using the Ovation Rapid DR Multiplex System 1-96 (NuGEN). Illumina libraries were pooled, size-selected, and sequenced on an Illumina MiSeq platform using V3 Chemistry (Illumina).