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