Qubit high sensitivity assay kit

DNA was extracted using the PowerSoil DNA Isolation Kit (MO-BIO), which has previously been found to be well-suited for fecal samples [35 (link)]. Negative controls were included from DNA extraction [36 (link)]. All plates included a mock community to serve as an internal control for calibration of the bioinformatical pipeline.
The 16S rDNA V3-V4 region was targeted using the primers S-D-Bact-0341-b-S-17 and S-D-Bact-0785-a-A-21 [37 (link)]. The following PCR program was used: 98 °C for 30 sec, 25x (98 °C for 10 s, 55 °C for 20 s, 72 °C for 20 s) and 72 °C for 5 min. Amplification was verified by running the products on an agarose gel. Indices were added in a subsequent PCR using the Nextera Index Kit V2 (Illumina), with the following PCR program: 98 °C for 30 sec, 8x (98 °C for 10 s, 55 °C for 20 s, 72 °C for 20 s) and 72 °C for 5 min. The attachment of barcodes was verified by running the products on an agarose gel.
Products from the nested PCR were pooled and the resulting library was cleaned with the Agencourt AMPure XP PCR purification kit (Beckman Coulter). The DNA concentration of pooled libraries was measured on a Qubit fluorometer using the Qubit High Sensitivity Assay Kit (Thermo Fisher Scientific). Sequencing was done on an Illumina MiSeq desktop sequencer using the MiSeq Reagent Kit V3 (Illumina) for 2x 300 bp paired-end sequencing.

DNA extraction was performed on 16 isolates from Patient 1 and 16 isolates from Patient 2 (Figure 1; Table 2), as previously described [9 (link)], with the addition of a bead beating step. The total DNA concentration was determined using the Qubit high-sensitivity assay kit (Thermofisher) and a sequencing library was prepared from 50 ng of DNA using the Nextera Library Preparation kit (Illumina). The post-PCR clean-up was carried out using Ampure XP beads (Beckman). The library size was validated using the Agilent 2200 TapeStation with the Agilent D1000 ScreenTape System, and 150 bp paired-end reads were sequenced on the Illumina NextSeq 550 system. The raw sequencing reads have been deposited on the European Nucleotide Archive (Study Accession PRJEB28875), as have 2 assemblies used as de novo references (see below).

Genomic DNA was isolated as described above. Following genomic DNA isolation, 1 μL of the isolated DNA (1–10 ng) was used as input for the first of two PCR reactions. Genomic loci were amplified in PCR1 using PhusionU polymerase (Thermo Fisher Scientific). PCR1 primers for genomic loci are listed in Table S2 under the HTS_fwd and HTS_rev columns. PCR1 was performed as follows: 95 °C for 3 min; 30–35 cycles of 95 °C for 15 s, 61 °C for 20 s, and 72 °C for 30s; 72°C for 1 min. PCR1 products were confirmed on a 1% agarose gel. 1 μL of PCR1 was used as an input for PCR2 to install Illumina barcodes. PCR2 was conducted for nine cycles of amplification using a Phusion HS II kit (Life Technologies). Following PCR2, samples were pooled and gel purified in a 1% agarose gel using a Qiaquick Gel Extraction Kit (Qiagen). Library concentration was quantified using the Qubit High-Sensitivity Assay Kit (Thermo Fisher Scientific). Samples were sequenced on an Illumina MiSeq instrument (paired-end read, read 1: 200–280 cycles, read 2: 0 cycles) using an Illumina MiSeq 300 v2 Kit (Illumina).