# Show the system information ## Execute environment: LINUX cat /etc/lsb-release ### DISTRIB_ID=Ubuntu ### DISTRIB_RELEASE=20.04 ### DISTRIB_CODENAME=focal ### DISTRIB_DESCRIPTION="Ubuntu 20.04.4 LTS" ## Execute environment: QIIME2 qiime info ### System versions ### Python version: 3.8.12 ### QIIME 2 release: 2021.11 ### QIIME 2 version: 2021.11.0 ### q2cli version: 2021.11.0 ### Installed plugins ### alignment: 2021.11.0 ### composition: 2021.11.0 ### cutadapt: 2021.11.0 ### dada2: 2021.11.0 ### deblur: 2021.11.0 ### demux: 2021.11.0 ### diversity: 2021.11.0 ### diversity-lib: 2021.11.0 ### emperor: 2021.11.0 ### feature-classifier: 2021.11.0 ### feature-table: 2021.11.0 ### fragment-insertion: 2021.11.0 ### gneiss: 2021.11.0 ### longitudinal: 2021.11.0 ### metadata: 2021.11.0 ### phylogeny: 2021.11.0 ### picrust2: 2021.11 ### quality-control: 2021.11.0 ### quality-filter: 2021.11.0 ### rescript: 2021.11.0+3.g8aa880e ### sample-classifier: 2021.11.0 ### taxa: 2021.11.0 ### types: 2021.11.0 ### vsearch: 2021.11.0 ### Application config directory ### ${HOME}/anaconda3/envs/qiime2-2021.11/var/q2cli ### Getting help ### To get help with QIIME 2, visit https://qiime2.org #0 Preprocess # Define variables JOBS=$(($(grep cpu.cores /proc/cpuinfo | sort -u | sed 's/[^0-9]//g')/2)) THREADS=$(grep processor /proc/cpuinfo | wc -l) ## Input data directory **absolute path** INDIR="${HOME}/raw" ## Output directory name OUTDIR="${HOME}/$(date "+%y%m%d")_qiime2" ## Type of run TYPE='paired-end' ## Primer information FWD='CCTACGGGNBGCASCAG' REV='GACTACNVGGGTATCTAATCC' ## Reference database DB='silva-1381-SSU-Pro341F-Pro805R-classifier.qza' # Make output directories mkdir -p ${OUTDIR}/{manifest,qza,qzv,log} # Make "fastq manifest" file echo -e "sample-id\tforward-absolute-filepath\treverse-absolute-filepath" > ${OUTDIR}/manifest/manifest.tsv ls -1v ${INDIR} | perl -nle 'if(/_S[0-9]{1,3}_L001_R1/){print "$`"}' > ${OUTDIR}/manifest/sample-id.txt ls -1v ${INDIR}/*R1* > ${OUTDIR}/manifest/forward-absolute-filepath.txt ls -1v ${INDIR}/*R2* > ${OUTDIR}/manifest/reverse-absolute-filepath.txt paste -d "\t" \ ${OUTDIR}/manifest/sample-id.txt \ ${OUTDIR}/manifest/forward-absolute-filepath.txt \ ${OUTDIR}/manifest/reverse-absolute-filepath.txt \ >> ${OUTDIR}/manifest/manifest.tsv #1 Importing data qiime tools import \ --type 'SampleData[PairedEndSequencesWithQuality]' \ --input-path ${OUTDIR}/manifest/manifest.tsv \ --input-format PairedEndFastqManifestPhred33V2 \ --output-path ${OUTDIR}/input.qza # Quality check qiime demux summarize \ --i-data ${OUTDIR}/input.qza \ --o-visualization ${OUTDIR}/input.qzv qiime tools export \ --input-path ${OUTDIR}/input.qzv \ --output-path ${OUTDIR}/input-summary #2 Trimming primer and adapter sequences FWDRC=$(echo "${FWD}" | tr ACGTMRYKVHDBacgtmrykvhdb TGCAKYRMBDHVtgcakyrmbdhv | rev) REVRC=$(echo "${REV}" | tr ACGTMRYKVHDBacgtmrykvhdb TGCAKYRMBDHVtgcakyrmbdhv | rev) nohup qiime cutadapt trim-paired \ --i-demultiplexed-sequences ${OUTDIR}/input.qza \ --p-cores ${THREADS} \ --p-adapter-f ${REVRC} \ --p-adapter-r ${FWDRC} \ --p-front-f ${FWD} \ --p-front-r ${REV} \ --p-times 10 \ --p-match-read-wildcards \ --p-match-adapter-wildcards \ --p-minimum-length 100 \ --p-discard-untrimmed \ --o-trimmed-sequences ${OUTDIR}/sequences.qza \ --verbose \ &> ${OUTDIR}/cutadapt.log & wait # Quality check qiime demux summarize \ --i-data ${OUTDIR}/sequences.qza \ --o-visualization ${OUTDIR}/sequences.qzv qiime tools export \ --input-path ${OUTDIR}/sequences.qzv \ --output-path ${OUTDIR}/trimmed-summary #3 DADA2 ## Determine truncate parameters based on the quality score TRUNC1=280 TRUNC2=206 << COMMENT_OUT TRUNC: a truncated length after removing bases, which represent < threshold quality score (QS=20) at the first quartile, from 3' end with viewing quality-plot.html COMMENT_OUT ## Denoising, quality filtering, correcting reading errors, removing chimera, PhiX and singleton sequences, merging reads, and dereplicating nohup qiime dada2 denoise-paired \ --i-demultiplexed-seqs ${OUTDIR}/sequences.qza \ --p-n-threads 0 \ --p-trunc-len-f ${TRUNC1} \ --p-trunc-len-r ${TRUNC2} \ --p-max-ee-f 2 \ --p-max-ee-r 5 \ --o-table ${OUTDIR}/table.qza \ --o-representative-sequences ${OUTDIR}/rep-seqs.qza \ --o-denoising-stats ${OUTDIR}/denoising-stats.qza \ --verbose \ &> ${OUTDIR}/dada2.log & wait # Summarize a feature table qiime feature-table summarize \ --i-table ${OUTDIR}/table.qza \ --o-visualization ${OUTDIR}/table.qzv qiime tools export \ --input-path ${OUTDIR}/table.qzv \ --output-path ${OUTDIR}/table # Mapping feature IDs to representative sequences qiime feature-table tabulate-seqs \ --i-data ${OUTDIR}/rep-seqs.qza \ --o-visualization ${OUTDIR}/rep-seqs.qzv qiime tools export \ --input-path ${OUTDIR}/rep-seqs.qzv \ --output-path ${OUTDIR}/rep-seqs # Viewing representative sequences stats qiime metadata tabulate \ --m-input-file ${OUTDIR}/rep-seqs.qza \ --o-visualization ${OUTDIR}/rep-seqs-stats.qzv qiime tools export \ --input-path ${OUTDIR}/rep-seqs-stats.qzv \ --output-path ${OUTDIR}/rep-seqs-stats # Viewing denoizing stats qiime metadata tabulate \ --m-input-file ${OUTDIR}/denoising-stats.qza \ --o-visualization ${OUTDIR}/denoising-stats.qzv qiime tools export \ --input-path ${OUTDIR}/denoising-stats.qzv \ --output-path ${OUTDIR}/denoising-stats #4 Taxonomical assignment to ASV nohup qiime feature-classifier classify-sklearn \ --i-classifier ${DB} \ --i-reads ${OUTDIR}/rep-seqs.qza \ --p-n-jobs ${JOBS} \ --o-classification ${OUTDIR}/taxonomy.qza & wait qiime metadata tabulate \ --m-input-file ${OUTDIR}/taxonomy.qza \ --o-visualization ${OUTDIR}/taxonomy.qzv qiime tools export \ --input-path ${OUTDIR}/taxonomy.qzv \ --output-path ${OUTDIR}/taxonomy #5 Generating bar plot qiime taxa barplot \ --i-table ${OUTDIR}/table.qza \ --i-taxonomy ${OUTDIR}/taxonomy.qza \ --o-visualization ${OUTDIR}/taxa-bar-plots.qzv qiime tools export \ --input-path ${OUTDIR}/taxa-bar-plots.qzv \ --output-path ${OUTDIR}/taxa-bar-plots #6 Postprocess # Collect qza, qzv and log files mv ${OUTDIR}/*.qza ${OUTDIR}/qza mv ${OUTDIR}/*.qzv ${OUTDIR}/qzv mv ${OUTDIR}/*.log ${OUTDIR}/log exit 0