16s metagenomics library preparation protocol

The standard Illumina 16S metagenomics library preparation protocol was used (available at: https://support.illumina.com/content/dam/illumina-support/documents/documentation/chemistry_documentation/16s/16s-metagenomic-library-prep-guide-15044223-b.pdf). All amplification and sequencing steps were carried out at University Core DNA Services, Sequencing and Genetic Analysis Lab (University of Calgary, AB, Canada). The 16S Amplicon PCR forward primer (5’TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG) and reverse primer (5’GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC) were used to amplify the V3 and V4 regions of the 16S rRNA gene. Illumina sequencing adapters and dual-index barcodes were added to the amplicon target to allow for library pooling prior to sequencing. Briefly, 16S rRNA gene amplicons were generated using a KAPA HiFi HotStart ReadyMix Kit (Kapa Biosystems) with the following PCR conditions: a 3 min initial denaturation at 95 °C followed by 25 cycles of 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 30 s, with a final extension of 5 min at 72 °C. Amplicon were then purified with Agencourt AMPure XP beads (Beckman Coulter Inc., ON, Canada), and sequenced on an Illumina MiSeq system (Illumina Inc., Victoria, BC, Canada) using the 2 × 300 bp paired-end sequencing kit. Negative controls were included in triplicate during amplification and sequencing.

Total microbial community DNA was extracted from all sample types and analyzed by using the Quick-DNA Fecal/Soil Microbe miniprep (Zymo Research Corp, Irvine, CA, USA) following manufacturer's recommendations. Pure DNA was quantified using a Qubit 2.0 fluorometer. DNA samples at a 5 ng/μl concentration were used to prepare libraries using the Illumina 16S-metagenomics library preparation protocol. The V3–V4 hyper variable region of bacterial 16S rRNA gene was amplified by using the primers 341F (5′-CCTACGGGNGGCWGCAG-3′) and 805R (5′-GACTACHVGGGTATCTAATCC-3′) containing Illumina adaptors as illustrated in Klindworth et al. (19 (link)). The PCR amplicons were checked in agarose gels and purified using AMPure XP beads (Beckman Coulter Inc) as per manufacturer's recommendations. Purified amplicons were then indexed by using the Nextera UD index set (Illumina, San Diego, CA, USA). Libraries were quantified in triplicate using a Qubit 2.0 fluorometer with a dsDNA HS Assay kit (Invitrogen, Carlsbad, CA, USA), pooled at equal concentrations (4 nM) to generate equivalent number of raw reads, and diluted to a final concentration of 6 pM. Amplicon libraries were spiked with 5% PhiX control (Illumina, San Diego, CA, USA) according to manufacturer's recommendations. Samples were sequenced using a MiSeq Reagent kit v3 (600 cycle) on an Illumina MiSeq platform (Illumina, San Diego, CA, USA).