mNGS of Mycobacterium shigaense
"Metagenomic shotgun sequencing of human skin sample: DNA libraries were prepared using automated nucleic acid extraction, enzymatic fragmentation, end repair, terminal adenylation, and adaptor ligation. Libraries were quantified by real-time PCR (KAPA) and pooled. Sequencing was performed on Illumina NextSeq with single-end 50bp reads (approximately 20 million reads per library). Human sequences were filtered out (GRCh38.p13), and remaining reads were aligned to a reference database (NCBI nt, GenBank, and in-house curated genomic database) to identify microbial species and read counts. Quality control included library concentration >50 pM, Q20 >85%, Q30 >80%, and negative control analysis (culture medium with 10 Jurkat cells/mL). Microbial reads were reported if RPM (sample)/RPM (negative control) 5."