Agilent d1000 screentape assay

DNA was isolated from F1 and F2 faecal samples using the QIAamp DNA stool mini kit (Qiagen, UK). Microbiome sequencing was conducted on the V3-V4 region of the 16 S rRNA gene following the Illumina 16 S Metagenomic Sequencing Library Preparation protocol. 16 S rRNA amplicons were generated using forward 5’ (TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG) and Reverse 5’ GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC) primers, flanked by Illumina adapter-overhang sequences. Illumina dual index barcodes (Illumina XT Index Kit v2, Set A: FC-131-2001) were attached to each amplicon. AMpure XP beads (Beckman; A63882) were used for all PCR clean-up steps. Libraries were quantified using the Qubit Fluorometer and the Qubit dsDNA HS Kit (Thermo-Fisher Scientific). Library fragment-length distributions were analysed using the Agilent TapeStation 4200 and the Agilent D1000 ScreenTape Assay (Agilent; 5067–5582 and 5067–5583). Libraries were then pooled in equimolar amounts and the library pool was size-selected using the Blue Pippin (Sage Science) and a 1.5% Pippin Gel Cassette (Sage Science; BDF2010). Sequencing was performed on an Illumina MiSeq using a MiSeq Reagent Kit v3 (600 cycle) (Illumina; MS-102-3003) to generate 300 bp paired-end reads. Raw reads were processed by Qiime2 pipeline and trimmed. Greengenes version 13.8 was used in classification^{66 (link)}.

The PCR was composed of 12.5 μL of 2× KAPA HiFi HotStart ReadyMix (Kapa Biosystems), 200 nM 515F and 926R primers (Table 2), and 12.5 ng of template DNA in a 25 μL reaction volume. The PCR was performed with the following cycle conditions: 3 min at 95°C, followed by 25 cycles of 30 s at 95°C, 30 s at 55°C then 30 s at 72°C; and 5 min at 72°C. The final cycle ended with infinite hold at 4°C. Each reaction was generated in triplicates, then pooled and purified with SPRISelect magnetic beads (Beckman Coulter) with double size selection ratio of 0.85 to 0.56, according to the manufacturer’s instructions. The quality of the amplicons was validated with the Agilent 4200 TapeStation System, using the AgilentD1000 ScreenTape Assay (Agilent Technologies, Inc). Sequencing was performed with MiSeq Illumina 2 × 300 bp chemistry, and the sequence reads were analyzed through the DADA2 version 1.10.1 pipeline (67 (link), 68 (link)), performed in R version 3.5.3 (69 ) using Mac OS version 10.14.4.