CAP1_CKO_PostTAM_Deteriorated_Lung_16s_Rep2
DNA extraction and PCR amplification:Total microbial genomic DNA was extracted from lung samples using the FastPure Stool DNA Isolation Kit. DNA quality and concentration were assessed via agarose gel electrophoresis and a NanoDrop ND-2000 spectrophotometer, and stored at -80 . The hypervariable V3-V4 region of the 16S rRNA gene was amplified using primers 338F and 806R with a T100 Thermal Cycler. The PCR mixture contained Fast Pfu buffer, dNTPs, primers, polymerase, template DNA, and ddH2O. Amplification included initial denaturation, 27 cycles of denaturing, annealing, and extension, followed by purification of the products from agarose gel and quantification.Illumina sequencing and subsequent processing and analysis:Purified amplicons were pooled and paired-end sequenced on an Illumina NextSeq 2000 PE300 platform. After demultiplexing, sequences were quality filtered using fastp and merged with FLASH. High-quality sequences were denoised using the DADA2 plugin in the Qiime2 pipeline, resulting in amplicon sequence variants (ASVs). To mitigate sequencing depth effects on diversity measures, samples were rarefied to 20,000 sequences, achieving an average Goods coverage of 97.90%. Taxonomic assignment of ASVs was conducted using the Naive Bayes classifier in Qiime2 with the SILVA 16S rRNA database.
SUB14768327
70 16s