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This recipe compares alignment before and after QC using two different tools, bwa and bowtie2

0 results • updated 2.5 years ago by Bioinformatics Admin • needs authorization

This recipe compares alignment before and after QC using two different tools, bwa and bowtie2

Requirements the bioinfo environment and:

pip bio install --upgrade

Download the recipe and run it at command line:

 bash Compare_alignment_rates.sh CP050134 SRR653447

The first parameter is the SRR accession number the second number is the GenBank id for the reference genome.

Interesting cases

 # Ebola virus, bwa better by default
  bash Compare_alignment_rates.sh AF086833  SRR1972739

 # Zika virus Bowtie works better by default
 bash Compare_alignment_rates.sh KY075939 SRR5122835

 # Tobacco etch virus genome
 bash Compare_alignment_rates.sh NC_001555 SRR3466599

 # The alignment is worse after trimming! Whoops!
 bash Compare_alignment_rates.sh VWSY01000001  SRR16249304

If you want to know more about each sample run:

bio search AF086833  SRR1972739

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# Short read aligner - SRA accession, reference GenBank accession
#
# Usage example:
#
#     bash align.sh CP050134 SRR653447
#

# Stop on error
set -uex

# Reference genome accession number.
ACC=$1

# SRA accession number.
SRR=$2

# Reference FASTA.
REF=ref/$ACC.fa

# Create the directory.
mkdir -p ref reads

# Get the reference accession.
bio fetch $ACC -format fasta > $REF

# Get FASTQ data from your query.
fastq-dump -X 10000 --split-files --outdir reads $SRR 1>> log.txt 

# Assign variable names to your FASTQ data.
R1=reads/${SRR}_1.fastq
R2=reads/${SRR}_2.fastq

# The trimmed reads.
TP1=reads/${SRR}_trimmed_1.fastq
TU1=reads/${SRR}_unpaired_1.fastq

TP2=reads/${SRR}_trimmed_2.fastq
TU2=reads/${SRR}_unpaired_2.fastq

# Index the two genomes
bwa index $REF > log.txt 2> log.txt
bowtie2-build $REF $REF > log.txt 2>>log.txt

# Align before doing any quality control on your FASTQ data.
bwa mem $REF $R1 $R2 > bwa.sam 2>> log.txt
bowtie2 -x $REF -1 $R1 -2 $R2 > bowtie.sam 2>> log.txt

# Define your adaptors.
echo ">illumina" > adapter.fa
echo "AGATCGGAAGAG" >> adapter.fa

# Trim off adaptors and low quality reads.
trimmomatic PE ${R1} ${R2} $TP1 $TU1 $TP2 $TU2 ILLUMINACLIP:adapter.fa:2:30:5 SLIDINGWINDOW:4:30 >> log.txt 2>> log.txt

# Align after doing any quality control on your FASTQ data.
bwa mem $REF $TP1 $TP2 > bwa_qc.sam 2>> log.txt
bowtie2 -x $REF -1 $TP1 -2 $TP2 > bowtie_qc.sam 2>> log.txt

# Display certain lines from flagstat output
ls -1 *.sam | parallel -n 1 '(echo {} && samtools flagstat {} | grep mapped)'

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