16S rRNA amplicon-seq analysis¶

Preprocessing¶

Rename sequence IDs¶

Sequence ID in FASTQ files were renamed by USEARCH.

usearch \
   -fastx_relabel ${indir}/${file}.fastq \
   -prefix ${prefix}. \
   -fastqout ${outdir}/${prefix}_raw.fastq

Trim primer sequences¶

Primer sequence were trimmed by using TagCleaner.

# Trim primer sequences
forward=AGAGTTTGATCMTGGCTCAG
reverse=ACTCCTACGGGAGGCAGCA
${tagcleaner} \
   -fastq ${indir}/${file}.fastq \
   -out ${outdir}/${prefix}_trimmed \
   -out_format 3 \ # Return fastq file
   -nomatch 3 \ # Exclude reads not matching the tag at either end
   -tag5 ${forward} -tag3 ${reverse} \
   -minlen 200

Quality filtering & Truncation¶

All sequences were truncated into 270 bp as required by UPARSE.

# Check length distribution
${usearch} \
   -fastq_eestats2 ${outdir}/${prefix}_trimmed.fastq \
   -output ${outdir}/${prefix}.eestats \
   -length_cutoffs 200,300,10 \
   -fastq_qmax 47

# Quality filter & sequence truncation
${usearch} \
   -fastq_filter ${outdir}/${prefix}_trimmed.fastq \
   -fastq_maxee_rate 0.01 \
   -fastaout ${outdir}/${prefix}.fasta \
   -fastq_maxns 0 \
   -relabel ${prefix}. \
   -fastq_trunclen 270 \
   -fastq_qmax 47

OTU clustering¶

UPARSE¶

OTU clustering using UPARSE algorithm

# Merge all FASTA files
cat ${indir}/*fasta > filtered.fa

# Dereplication
usearch \
   -fastx_uniques filtered.fa \
   -sizeout \
   -relabel Uniq \
   -fastaout uniques.fa

# OTU clustering
usearch \
   -cluster_otus uniques.fa \
   -otus otus.fa \
   -relabel OTU

# Remapping
usearch \
   -otutab raw.fq \
   -otus otus.fa \
   -otutabout otutab.txt \
   -mapout map.txt

# Create OTU table
usearch \
   -otutab filtered.fa \
   -otus otus.fa \
   -otutabout otutab.qced_read_only.txt \
   -mapout map.qced_read_only.txt

Taxonomic classification¶

Assign taxonomy to the OTU with RDP classifier & USEARCH global alignment against SILVA database

# RDP classifier
classifier=/home/toru/bin/rdp_classifier_2.10.1/dist/classifier.jar
java -Xmx1g -jar \
   ${classifier} classify \
   -c 0.8 \
   -o otus.RDPclassifier.txt \
   otus.fa

Phylogenetic tree construction¶

Add outgroup sequence¶

Added archeal 16S sequence as outgroup to determine root of the phylogenetic tree

outgroup=/home/toru/Database/archaea_16S_outgroup.fasta
cat $outgroup otus.fa > otus.outgroup.fa

Multiple alignment¶

mafft otus.outgroup.fa > otus.outgroup.mafft.fa

Tree construction¶

FastTree -gtr -nt otus.outgroup.mafft.fa > otus.outgroup.mafft.FastTree.tre