Download bwa:

curl -L > bwa-0.7.15.tar.bz2

Unpack and build it:

tar xjvf bwa-0.7.15.tar.bz2
cd bwa-0.7.15

Install it:

sudo cp bwa /usr/local/bin

Downloading data

Now, go to a new directory and grab the data:

mkdir /mnt/mapping
cd /mnt/mapping

curl -O
curl -O

We will also need the assembly; rather than rebuilding it, you can download a copy that we saved for you:

curl -O
gunzip subset_assembly.fa

Next, you’ll need to index the assembly:

bwa index subset_assembly.fa

Splitting the reads

The reads are in paired-end/interleaved format, so you’ll need to split them -:

for i in *.pe.fq.gz
   gunzip -c $i | head -800000 | -1 $i.1 -2 $i.2 -

This will take the interleaved reads and produce .1 and .2 files from them.

Mapping the reads

Map the left reads:

for i in *.1
   bwa aln subset_assembly.fa $i > $(echo $i | cut -d. -f1)_1.sai

Map the right reads:

for i in *.2
   bwa aln subset_assembly.fa $i > $(echo $i | cut -d. -f1)_2.sai

Combine the paired ends with bwa sampe:

bwa sampe subset_assembly.fa SRR1976948_1.sai SRR1976948_2.sai SRR1976948.*.1 SRR1976948.*.2 > SRR1976948.sam
bwa sampe subset_assembly.fa SRR1977249_1.sai SRR1977249_2.sai SRR1977249.*.1 SRR1977249.*.2 > SRR1977249.sam

Converting to BAM to visualize

First, index the assembly for samtools:

samtools faidx subset_assembly.fa

Then, convert both SAM files to BAM files:

for i in *.sam
   samtools import subset_assembly.fa $i $i.bam
   samtools sort $i.bam $i.bam.sorted
   samtools index $i.bam.sorted.bam

Visualizing the read mapping

Find a contig name to visualize:

grep -v ^@ SRR1976948.sam | \
    cut -f 3 | sort | uniq -c | sort -n

Pick one e.g. k99_13588.

Now execute:

samtools tview SRR1976948.sam.bam.sorted.bam subset_assembly.fa -p k99_13588:400

(use arrow keys to scroll, ‘q’ to quit)

Look at it in both mappings:

samtools tview SRR1977249.sam.bam.sorted.bam subset_assembly.fa -p k99_13588:400

Why is the mapping so good??

Note: no strain variation :).

Grab some untrimmed data:

curl -O
curl -O

Now align this untrimmed data:

gunzip -c SRR1976948_1.fastq.gz | head -800000 > SRR1976948.1
gunzip -c SRR1976948_2.fastq.gz | head -800000 > SRR1976948.2

bwa aln subset_assembly.fa SRR1976948.1 > SRR1976948_1.untrimmed.sai
bwa aln subset_assembly.fa SRR1976948.2 > SRR1976948_2.untrimmed.sai

bwa sampe subset_assembly.fa SRR1976948_1.untrimmed.sai SRR1976948_2.untrimmed.sai SRR1976948.1 SRR1976948.2 > SRR1976948.untrimmed.sam

samtools import subset_assembly.fa $i $i.bam
samtools sort $i.bam $i.bam.sorted
samtools index $i.bam.sorted.bam

And now look:

samtools tview SRR1976948.untrimmed.sam.bam.sorted.bam subset_assembly.fa -p k99_13588:500

You can also use ‘Tablet’ to view the downloaded BAM file - see the Tablet paper.

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