Miseq sequencer

For the Vac cohort, HLA genotyping was performed with the HLA-Expert kit (DNA-Technology LLC) through the amplification of exons 2 and 3 of the HLA-A/B/C genes and exon 2 of the HLA-DRB1/3/4/5/DQB1/DPB1 genes. Prepared libraries were run on an Illumina MiSeq sequencer using a standard flow-cell with 2 × 250 paired-end sequencing. Reads were analyzed using HLA-Expert Software (DNA-Technology LLC) and the IPD-IMGT/HLA database 3.41.0 (10.1093/nar/gkz950).
For CP_trial, Vac_trial, HD-2021, and CP donors, HLA-genotyping was performed using the One Lambda ALLType kit (Thermo Fisher Scientific), which uses multiplex PCR to amplify full HLA-A/B/C gene sequences, and from exon 2 to the 3′ UTR of the HLA-DRB1/3/4/5/DQB1 genes. Prepared libraries were run on an Illumina MiSeq sequencer using a standard flow-cell with 2 × 150 paired-end sequencing. Reads were analyzed using One Lambda HLA TypeStream Visual Software (TSV), version 2.0.0.27232, and the IPD-IMGT/HLA database 3.39.0.0. Two donors (p1305, p1329) were HLA genotyped by Sanger sequencing for loci HLA-A, HLA-B, HLA-C, DRB1, and DQB1, using Protrans S4 and Protrans S3 reagents. The PCR products were prepared for sequencing with BigDye Terminator v1.1 (Thermo Fisher Scientific). Capillary electrophoresis was performed on a Genetic Analyzer Nanophore05.

Due to the advances in sequencing technology that became available over the study period, different protocols were used to obtain short-read sequences, as described before (17 (link), 27 (link), 29 (link)). In brief, early isolates were sequenced using 2 × 50 bp on an Illumina HiSeq 2000 sequencer (17 (link)) or using 2 × 300 bp on an Illumina MiSeq sequencer (29 (link)) or using 2 × 250 bp on a Illumina MiSeq sequencer (27 (link)). Table S2 provides a detailed overview of the sequencing protocols applied.

The sample of the library, with length 717 bp, was prepared for “shotgun” sequencing on a high-throughput Illumina^® MiSeq sequencer. For each sample, 3.75 ng of library DNA was prepared in a 50 μl reaction with 0.5 μl of Illumina^® Nextera and 25 ul TD buffer at 55°C for 5 minutes. This reaction was then purified using a 0.6x SPRI magnetic bead-based purification. The resulting material appears on a gel as a smear of DNA with varying length between ~100–717 bp. The prepared DNA was used as template in a 50 μl index PCR with NEBNext^® master mix and Illumina^® indexing primers; and re-purified using magnetic bead-based purification. In final preparation, each sample was diluted to 7 pM, before running on an Illumina^® MiSeq sequencer using reagents from an Illumina^® MiSeq Reagent Kit V2, to produce 150 base paired-end reads. The reference sequence of the library used for this study appears in the supporting information (S2b Fig).

Total DNA extraction was performed in strict accordance with the kit instructions of the FastDNA Spin Kit for Soil. DNA concentration and purity were measured using a NanoDrop 2000 ultra-micro spectrophotometer (Thermo Fisher Scientific), and DNA extraction quality was measured using 1% agarose gel electrophoresis. Polymerase chain reaction (PCR) was performed with 338F (5′-ACTCCTACGGGAGGCAGCAG-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′) primers for the V3-V4 hypervariable regions of the 16S ribosomal RNA (rRNA) gene, using an ABI GeneAmp 9700 PCR instrument (ABI, Foster City, CA, USA). The amplification procedures were 95 °C pre-denaturation for 3 min, 27 cycles (95 °C denaturation for 30 s, 55 °C annealing for 30 s, and 72 °C extension for 30 s), and 72 °C extension for 10 min. The amplification system was 20 μL, which included 4 μL FastPfu buffer (5×), 2.5 mM dNTPs, 0.8 μL primers (5 μM), 0.4 μL FastPfu polymerase, and 10 ng of DNA template. The PCR products were recovered using a 2% agarose gel, purified using the AxyPrep DNA Gel Extraction Kit, eluted with Tris-HCl, and detected by 2% agarose electrophoresis. Detection and quantification were performed using QuantiFluor-ST. Purified amplicons were combined in equimolar ratios and assessed for library quality. Paired-end sequencing (2 × 300 bp) was performed on the Illumina MiSeq sequencer (Illumina, San Diego, CA, USA).

Preparation of a cDNA library and Illumina MiSeq sequencing were carried out as described previously [30 (link), 33 (link)]. Briefly, a 200 bp fragment library ligated with bar-coded adapters was constructed for strain SKT-27 using an NEBNext Ultra RNA Library Prep Kit for Illumina v1.2 (New England Biolabs) according to the manufacturer’s instructions. Library purification was performed using Agencourt AMPure XP magnetic beads (Beckman Coulter). The quality of the purified cDNA library was assessed on an Agilent 2100 Bioanalyzer (Agilent Technologies). Nucleotide sequencing was performed on an Illumina MiSeq sequencer (Illumina) using a MiSeq Reagent Kit v2 (Illumina) to generate 151 paired-end reads. Data analysis was carried out using CLC Genomics Workbench v8.0.1 (CLC Bio). Contigs were assembled from the obtained sequence reads by de novo assembly. Using the assembled contigs as query sequences, the Basic Local Alignment Search Tool (BLAST) non-redundant nucleotide database was searched to obtain the full-length nucleotide sequence of each gene segment of strain SKT-27. The nucleotide sequences were translated into amino acid sequences using GENETYX v11 (GENETYX).

DNA extraction was carried out using the cultured cells protocol supplied with the DNeasy Blood and Tissue Kit (Qiagen, Germany) in a laminar flow hood to avert contamination. The concentration of extracted DNA was tested by the Nanodrop ND-1000 spectrophotometer (Thermo Electron Corporation, USA). Specific primer sets for V3-V4 regions were chosen to perform PCR amplification of 16S rDNA. To assess the contribution of extraneous DNA from reagents, extraction negative controls (no urine) and PCR negative controls (no template) were included as the blank controls. Qiaquick PCR purification kit (Qiagen, Valencia, CA) was selected for purifying the final PCR products from unincorporated nucleotides and primers. Purified samples were normalized to equal DNA concentration and sequenced by Illumina Miseq sequencer (Illumina, Inc., USA).