title: 16S-seq skin lesion swab: cutaneous leishmaniasis
design: Swab specimens from lesions and contralateral skin were collected in sterile 0.15 M NaCl with 0.1% Tween 20 in PBS 1x and stored at -20oC. Thirty-two negative control swab samples were exposed to air in the same room where the patients were seen and are called environmental samples. These samples were collected throughout patient recruitment and inclusion in this study. Genomic DNA extraction from swabs, library preparation, and 16S rRNA amplicon sequencing from swab specimens were all performed at the CHOP High-throughput Sequencing and Analytical Core of the PennCHOP Microbiome Program. DNA was extracted using the QIACube HT robotic workstation for nucleic acid purification (Qiagen) together with the DNeasy 96 PowerSoil Pro QIAcube HT kit (Qiagen), per standard protocol. Amplification of the 16S rRNA gene V1V3 region was performed using primers FP 5-AGAGTTTGATCCTGGCTCAG-3 and RC 5-ATTACCGCGGCTGCTGG-3, followed by library preparation, quantification, and pooling as previously described (PMID: 34437510). Sequencing was performed on the Illumina MiSeq using 300 bp paired-end chemistry. Extraction blanks and DNA-free water were subjected to the same amplification and purification procedure to assess potential environmental contamination.