16s DNA-Seq of stool: healthy adult male 23 years
DNA samples were submitted for 16S rRNA gene sequencing at the Integrated Microbiome Resource (IMR) at Dalhousie University (Halifax, Canada). Variable regions V3-V4 of the bacterial 16S rRNA gene were amplified from all purified DNA samples using a set of primers 341F: 5- CCTACGGGNGGCWGCAG -3 and 805R: 5- GACTACHVGGGTATCTAATCC -3 [13] and sequenced on an Illumina MiSeq using paired-end 300 bp sequencing [14, 15]. The samples were multiplexed at equal volumes with custom 16S fusion primers. The fusion primers contained Illumina Nextera adapters and barcodes for dual-indexing at both the forward and reverse paired ends of the fragments. Amplifications were performed using two different dilutions (undiluted and 1:10). 25L reactions contained: 5L of 5xHF PCR Buffer, 0.5L dNTPs (40mM), 5L forward and 5L reverse primer (1M), 0.25L Phusion polymerase (2 U L1; Thermo Scientific), 2L template and 7.25L PCR-grade water. Cycling conditions were: 98C (30s), followed by 30 cycles of 98C (10s), 55C (30s) and 72C (30s). Final extension was performed for 4.5min at 72C. The PCR product quality was verified using the E-gel 96-well high-throughput system (InvitrogenTM, Thermo Fisher Scientific Inc.). Amplicons were cleaned up and normalized simultaneously via the high-throughput Invitrogen SequalPrep 96-well plate kit (InvitrogenTM, Thermo Fisher Scientific Inc.). The samples and negative controls were pooled into one library. The library was quantified with Qubit (InvitrogenTM, Thermo Fisher Scientific Inc.) and loaded into the Illumina MiSeq platform as a 20 pM final denatured library according to the manufacturers protocol using 2300bp Pair-End v3 chemistry suitable for overlap and stitching together of paired amplicon reads into one full-length read of higher quality.