The Lung Microbiota in Korean Patients with Non-Tuberculous Mycobacterial
Pulmonary Disease: Program commands and scripts to analyze 16S rRNA sequencing data
This page provides program commands and scripts that were used to analyze 16S rRNA sequencing data for the article "The Lung Microbiota in Korean Patients with Non-Tuberculous Mycobacterial Pulmonary Disease".
Merging paired reads using FLASH ver. 1.2.11
$ flash /path/to/paired/reads/forward/MATE_PAIRS_1.fastq /path/to/paired/reads/reverse/MATE_PAIRS_2.fastq -M 100 -d /path/to/merged/reads/ -o SAMPLE_ID 2>&1 | tee --append /path/to/merged/reads/flash_merged.log
Chimera detection and OTU clustering using CD-HIT ver. 4.5.4
$ cd-hit-otu-all-single.pl -i /path/to/merged/reads/SAMPLE_ID.extendedFrags.fastq -o /path/to/output/cdhit/ 2>&1 | tee --append cdhit_results.log
Sequence alignment using the QIIME PyNAST script
$ parallel_align_seqs_pynast.py -i /path/to/output/cdhit/OTU -t /path/to/template/file/template_fasta.fna -o /path/to/output/aligned/representatives/
Filtering highly variable regions from the aligned sequence using QIIME scripts
$ filter_alignment.py -i /path/to/output/aligned/representatives/aligned_cdhit_OTU.fasta -o /path/to/output/filtered/alignment/ -g 0.80 -e 0.10
Build phylogeny information for downstream analysis using QIIME scripts
$ make_phylogeny.py -i /path/to/output/filtered/alignment/variable_region_filtered.fasta -o /path/to/phylogeny/representative_tree.tre -l /path/to/phylogeny/representative_tree_log.txt
Taxonomy assignment for the sequence data file using QIIME scripts
$ assign_taxonomy.py -i /path/to/output/cdhit/OTU -r /path/to/SILVA/reference/sequences/otus.fasta -t /path/to/SILVA/reference/taxonomy/otu_taxonomy.txt -o /path/to/taxonomic/assigned/output
Building an OTU table using QIIME scripts
$ make_otu_table.py -i /path/to/cdhit/converted_OTU.nr2nd.clstr -o /path/to/output/OTU_table.biom -t /path/to/taxonomic/assigned/output/assignments.txt
Calculating alpha diversity using QIIME scripts
$ alpha_diversity.py -i /path/to/output/OTU_table.biom -o /path/to/alpha/diversity -m chao1,PD_whole_tree,observed_otus -t /path/to/phylogeny/representative_tree.tre
Calculating beta diversities with the prepared OTU table
$ beta_diversity.py -i /path/to/output/OTU_table.biom -o /path/to/beta/diversity -m unweighted_unifrac,weighted_unifrac -t /path/to/phylogeny/representative_tree.tre