Mapping¶

Download bwa:

cd
curl -L https://sourceforge.net/projects/bio-bwa/files/bwa-0.7.15.tar.bz2/download > bwa-0.7.15.tar.bz2

Unpack and build it:

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

Install it:

sudo cp bwa /usr/local/bin

Downloading data¶

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

mkdir ~/mapping
cd ~/mapping

curl -O https://s3-us-west-1.amazonaws.com/dib-training.ucdavis.edu/metagenomics-scripps-2016-10-12/SRR1976948.abundtrim.subset.pe.fq.gz
curl -O https://s3-us-west-1.amazonaws.com/dib-training.ucdavis.edu/metagenomics-scripps-2016-10-12/SRR1977249.abundtrim.subset.pe.fq.gz

And extract the files:

for file in *fq.gz:
  do
  gunzip $file
done

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

curl -O https://s3-us-west-1.amazonaws.com/dib-training.ucdavis.edu/metagenomics-scripps-2016-10-12/subset_assembly.fa.gz
gunzip subset_assembly.fa

Mapping the reads¶

First, we will need to to index the megahit assembly:

bwa index subset_assembly.fa

to The reads are in paired-end/interleaved format, so you’ll need to add the -p flag to indicate to bwa that these are paired end data:

Map the reads:

for i in *fq
  do
  bwa mem -p subset_assembly.fa $i > ${i}.aln.sam
done

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

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 https://s3-us-west-1.amazonaws.com/dib-training.ucdavis.edu/metagenomics-scripps-2016-10-12/SRR1976948_1.fastq.gz
curl -O https://s3-us-west-1.amazonaws.com/dib-training.ucdavis.edu/metagenomics-scripps-2016-10-12/SRR1976948_2.fastq.gz

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

i=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.