Quant it picogreen dsdna detection kit

All DNA extraction and purification procedures were conducted in a white room where pressure, temperature, and humidity were controlled to minimize contamination. Individual discs were cut from the FTA cards using a sterile 5.0-mm single round hole punch, and total DNA was isolated using the QIAamp DNA Investigator Kit (Qiagen, Toronto, ON, Canada), according to the manufacturer’s protocol. DNA was quantified in duplicate using a Quant-iT PicoGreen dsDNA detection kit (Molecular Probes, Eugene OR, USA). Amplification of the V3–V4 region of the 16S ribosomal RNA (rRNA) gene and 16S gene amplicon sequencing for all DNA samples were performed at Centre d'Expertise et de Services, Génome Québec (Montréal, QC, Canada). Amplification used the universal primers 341F (5′-CCTACGGGNGGCWGCAG-3′) and 805R (5′-GACTACHVGGGTATCTAATCC-3′). Sequence libraries were prepared by Genome Quebec with the TruSeq DNA Library Prep Kit (Illumina, San Diego, CA, USA) and quantified using the Kapa Library Quantification Kit for Illumina platforms (Kapa Biosystems). Paired-end sequences were generated on a MiSeq platform PE300 (Illumina Corporation, San Diego, CA, USA) with the MiSeq Reagent Kit v3 using 600 cycles (Illumina, San Diego, CA, USA). Raw data files are publicly available on the NCBI Sequence Read Archive (PRJNA853332).

All DNA extraction and purification procedures were conducted in a clean room where pressure, temperature, and humidity were controlled to minimize contamination. Individual discs were cut from the FTA™ cards using a sterile 5.0 mm single round hole punch, and total DNA was isolated using the QIAamp DNA Investigator Kit (Qiagen, Toronto, ON, Canada) according to the manufacturer’s protocol. DNA was quantified in duplicate using a Quant-iT™ PicoGreen® dsDNA detection kit (Molecular Probes, Eugene OR, USA). Amplification of the V3–V4 region of the 16S rRNA gene and 16S rRNA gene amplicon sequencing for all DNA samples were performed at Centre d'Expertise et de Services Génome Québec (Montréal, QC, Canada) using the universal primers 341F (5′-CCTACGGGNGGCWGCAG-3′) and 805R (5′-GACTACHVGGGTATCTAATCC-3′). Sequence libraries were prepared by Genome Quebec with the TruSeq® DNA Library Prep Kit (Illumina, San Diego, CA, USA) and quantified using the KAPA Library Quantification Kit for Illumina platforms (Kapa Biosystems). Paired-end sequences were generated on a MiSeq platform PE300 (Illumina Corporation, San Diego, CA, USA) with the MiSeq Reagent Kit v3 600 cycles (Illumina, San Diego, CA, USA). Raw data files are publicly available in the NCBI Sequence Read Archive (PRJNA1015160).

Two rounds of PCR were performed to barcode the selected DNA libraries. In the first round, a forward primer and reverse primer were used to introduce a constant region that would serve as an annealing site in the second round of PCR. In the second round, the Nextera DNA CD index (Illumina) was used as the primers. The 5' end of the index was used for binding between the amplicon and the sequencing flow cell, the 3' end was used for annealing to the PCR product from the first round, and the middle 8 bases, i5 or i7, were used for barcoding different samples. DNA samples were quantified with the Quant-iT PicoGreen dsDNA detection kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. Sequencing was performed on an Illumina MiniSeq using a paired-end read kit following the manufacturer’s instructions.