Microbial Diversity Analysis of NHCL Samples

10 NHCLs and 3 normal samples were tested for NGS analysis. DNA extracted from NHCL and normal samples was used as a template. The 16S rRNA amplicon libraries (V4 region) were prepared according to the Illumina 16S Metagenomic Sequencing Library Preparation protocol with the primers 515F (5′-TCGTCCGCCAGCGTCAGATGTGTATAAGAGACAG-GTGYCAGCMGCCGCGGTAA-3′) and 806R (5′-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGGACTACNVGGGTWTCTAAT-3′). The libraries were sequenced on the Illumina MiSeq platform to generate paired end (301 bp × 2) sequence reads. Phylogenetic analysis was performed using the Quantitative Insights into Microbial Ecology 2 (QIIME 2) platform version 2019.7 [29 (link)] Raw sequence data were demultiplexed denoised with DADA2 [30 (link)] using the parameters p-trim-left-f 10, p-trim-left-r 10, p-trunc-len-f 200, and p-trunc-len-r 150. Amplicon sequence variants (ASVs) were assigned taxonomy using the feature classifier classify-sklearn and the SILVA 132 99% 515F/806R reference sequences [31 (link)].