Gut Microbiome Perturbations in Pediatric Patients with Hidradenitis Suppurativa

We collected stool samples from 8 patients with Hidradenitis Suppurativa (HS) and 8 healthy volunteers (control), which included 4 pediatric HS patients and 5 pediatric controls. All subjects provided written and verbal informed consent through the Boston University Aram V. Chobanian & Edward Avedisian School of Medicine institutional review board-approved protocol (H-38273). Exclusion criteria included gastrointestinal illness, use of systemic antibiotics or biologic therapies in the past 4 weeks, and history of IBD. Study subjects and controls were matched for age, gender, and ethnicity. Fecal samples were collected, sequenced, and analyzed as previously reported (Kam et al., 2021; DOI: 10.1016/j.jid.2020.04.017), with further details below. Sample collection Fecal samples from the central portion of the specimen were collected using the OMNIgene GUT kits to minimize contamination by adjacent cutaneous microbiota. Patients performed sample collection at home and stored samples at 4C until they were ready to transport the samples to the investigator. Upon receival, the investigators immediately stored stool samples at -80C. When all samples were received, stool samples were sent to MR DNA to microbiota sequencing. Sequencing The 16S rRNA gene V4 variable region PCR primers 515/806 were used in a 30-35 cyclePCR using the HotStarTaq Plus Master Mix Kit (Qiagen, USA) under the following conditions: 95C for 5 minutes, followed by 30-35 cycles of 95C for 30 seconds, 53C for 40 seconds and 72C for 1 minute, after which a final elongation step at 72C for 10 minutes was performed. After amplification, PCR products are checked in 2% agarose gel to determine the success of amplification and the relative intensity of bands. Samples are multiplexed using unique dual indices and are pooled together (e.g., 100 samples) in equal proportions based on their molecular weight and DNA concentrations. Pooled samples are purified using calibrated Ampure XP beads. Then the pooled and purified PCR product is used to prepare an Illumina DNA library. Sequencing was performed at MR DNA (www.mrdnalab.com, Shallowater, TX, USA) on a MiSeq following the manufacturer guidelines. Sequence data were processed using MR DNA analysis pipeline (MR DNA, Shallowater, TX, USA). In summary, sequences are joined, sequences less than 150bp removed, and sequences with ambiguous base calls removed. Sequences were quality filtered using a maximum expected error threshold of 1.0 and dereplicated. The dereplicated or unique sequences are denoised; unique sequences identified with sequencing and/or PCR point errors and removed, followed by chimera removal, thereby providing a denoised sequence or zOTU. Final zOTUs were taxonomically classified using BLASTn against a curated database derived from NCBI (www.ncbi.nlm.nih.gov). Data processing The Q25 sequence data derived from the sequencing process was processed using the MR DNA ribosomal and functional gene analysis pipeline (www.mrdnalab.com , MR DNA, Shallowater, TX). Sequences are depleted of primers, short sequences less than 150bp are removed, and sequences with ambiguous base calls removed. Sequences are quality filtered using a maximum expected error threshold of 1.0 and dereplicated. The dereplicated or unique sequences are denoised; unique sequences identified with sequencing or PCR point errors are removed, followed by chimera removal, thereby providing a denoised sequence or zOTU. Final zOTUs were taxonomically classified using BLASTn against a curated database derived from NCBI (www.ncbi.nlm.nih.gov) and compiled into each taxonomic level into both counts and percentage files. Counts files contain the actual number of sequences while the percent files contain the relative (proportion) percentage of sequences within each sample that map to the designated taxonomic classification.