Error when trying to align on OTUs

Technical Support
1

I recently made a script to obtain Otu-table and phylogenetic tree, however in the sequence alignment step #4, it happens that a lot of time passes and it doesn't leave this part, however the CPU and RAM usage is close to 95%. I don't know if this part of the script is wrong or if something is missing. Follow the entire script, if anyone can help me, or is it normal to take a long time to align OTUs???

My computer's specs are: Ryzen 7 (16 threads) and 16 GB RAM with 16 GB swap.

#!/bin/bash

echo -e "\033[0;32mSTARTING SCRIPT...\033[0m"

# Verificar se o número correto de parâmetros foi passado
if [ "$#" -ne 3 ]; then
  echo -e "\033[0;31mUso: $0 <path_manifest.csv> <classifier.qza> <output_directory>\033[0m"
  exit 1
fi

# Atribuir argumentos a variáveis
MANIFEST_PATH="$1"
CLASSIFIER_PATH="$2"
OUTPUT_DIR="$3"
DEMUX_OUTPUT="${OUTPUT_DIR}/demux.qza"

# Criar o diretório de saída se ele não existir
echo -e "\033[0;35mCreating output directory...\033[0m"
mkdir -p "$OUTPUT_DIR"
echo -e "\033[0;32mOutput directory successfully created.\033[0m"

# 1. Importar dados
echo -e "\033[0;35mImporting data.\033[0m"
qiime tools import \
  --type 'SampleData[SequencesWithQuality]' \
  --input-path "$MANIFEST_PATH" \
  --output-path "$DEMUX_OUTPUT" \
  --input-format SingleEndFastqManifestPhred33V2
echo -e "\033[0;32mImported successfully.\033[0m"

# 2. Visualizar dados importados
echo -e "\033[0;35mCreating visualization of imported data.\033[0m"
qiime demux summarize \
  --i-data "$DEMUX_OUTPUT" \
  --o-visualization "${OUTPUT_DIR}/demux-summary.qzv"
echo -e "\033[0;32mFinished, see ${OUTPUT_DIR}.\033[0m"

# 3. Desduplicar sequências
echo -e "\033[0;35mDereplicating sequences.\033[0m"
qiime vsearch dereplicate-sequences \
  --i-sequences "$DEMUX_OUTPUT" \
  --o-dereplicated-table "${OUTPUT_DIR}/table.qza" \
  --o-dereplicated-sequences "${OUTPUT_DIR}/rep-seqs.qza"
echo -e "\033[0;32mDereplication successfully.\033[0m"

# 4. Agrupar sequências em OTUs (97% de similaridade)
echo -e "\033[0;35mAligning OTUs.\033[0m"
qiime vsearch cluster-features-de-novo \
  --i-table "${OUTPUT_DIR}/table.qza" \
  --i-sequences "${OUTPUT_DIR}/rep-seqs.qza" \
  --p-perc-identity 0.97 \
  --o-clustered-table "${OUTPUT_DIR}/otu-table.qza" \
  --o-clustered-sequences "${OUTPUT_DIR}/otu-rep-seqs.qza"
echo -e "\033[0;32mAligning successfully.\033[0m"

# 5. Visualizar OTUs
echo -e "\033[0;35mGenerating OTU visualization files.\033[0m"
qiime feature-table summarize \
  --i-table "${OUTPUT_DIR}/otu-table.qza" \
  --o-visualization "${OUTPUT_DIR}/otu-table.qzv"

qiime feature-table tabulate-seqs \
  --i-data "${OUTPUT_DIR}/otu-rep-seqs.qza" \
  --o-visualization "${OUTPUT_DIR}/otu-rep-seqs.qzv"
echo -e "\033[0;32mFinished, see ${OUTPUT_DIR}.\033[0m"

# 6. Atribuir taxonomia para OTUs
echo -e "\033[0;35mSorting...\033[0m"
qiime feature-classifier classify-sklearn \
  --i-classifier "$CLASSIFIER_PATH" \
  --i-reads "${OUTPUT_DIR}/otu-rep-seqs.qza" \
  --o-classification "${OUTPUT_DIR}/taxonomy.qza"
echo -e "\033[0;32mTaxonomic assignment completed successfully, see ${OUTPUT_DIR}.\033[0m"

# 7. Visualizar resultados da taxonomia para OTUs
echo -e "\033[0;35mGenerating OTU visualization files.\033[0m"
qiime metadata tabulate \
  --m-input-file "${OUTPUT_DIR}/taxonomy.qza" \
  --o-visualization "${OUTPUT_DIR}/taxonomy.qzv"
echo -e "\033[0;32mFinished, see ${OUTPUT_DIR}."

# 8. Gerar árvore filogenética a partir de OTUs
echo -e "\033[0;35mGenerating phylogenetic tree.\033[0m"
qiime alignment mafft \
  --i-sequences "${OUTPUT_DIR}/otu-rep-seqs.qza" \
  --o-alignment "${OUTPUT_DIR}/aligned-otu-rep-seqs.qza"
echo -e "\033[0;32m1/4\033[0m"

qiime alignment mask \
  --i-alignment "${OUTPUT_DIR}/aligned-otu-rep-seqs.qza" \
  --o-masked-alignment "${OUTPUT_DIR}/masked-aligned-otu-rep-seqs.qza"
echo -e "\033[0;32m2/4\033[0m"

qiime phylogeny fasttree \
  --i-alignment "${OUTPUT_DIR}/masked-aligned-otu-rep-seqs.qza" \
  --o-tree "${OUTPUT_DIR}/unrooted-tree.qza"
echo -e "\033[0;32m3/4\033[0m"

qiime phylogeny midpoint-root \
  --i-tree "${OUTPUT_DIR}/unrooted-tree.qza" \
  --o-rooted-tree "${OUTPUT_DIR}/rooted-tree.qza"
echo -e "\033[0;32m4/4 Árvore filogenética gerada.\033[0m"

# 9. Exportar a árvore filogenética no formato .nwk
echo -e "\033[0;35mExporting the phylogenetic tree to .nwk format.\033[0m"
qiime tools export \
  --input-path "${OUTPUT_DIR}/rooted-tree.qza" \
  --output-path "${OUTPUT_DIR}/tree-output.nwk"
echo -e "\033[0;32mExport completed to ${OUTPUT_DIR}/tree-output.\033[0m"

# Fim do script
echo -e "\033[0;32mScript finished. Check all generated files in: ${OUTPUT_DIR}.\033[0m"
2

Hello @sanduixxxe,

If your CPU and RAM usage are both that high, then it is very likely that this step is just taxing your computer. How long have you let it run for? You may be better off attempting to run this command on a cluster if you have access to one.