Mapping with BWA

Objectives

In this lab, you will explore a popular fast mapper called BWA. Simulated RNA-seq data will be provided to you; the data contains 75 bp paired-end reads that have been generated in silico to replicate real gene count data from Drosophila. The data simulates two biological groups with three biological replicates per group (6 samples total).  The objectives of this lab is mainly to:

Introduction

BWA (the Burrows-Wheeler Aligner) is a fast short read aligner. It's the successor to another aligner you might have used or heard of called MAQ (Mapping and Assembly with Quality). As the name suggests, it uses the burrows-wheeler transform to perform alignment in a time and memory efficient manner.

BWA Variants

BWA has three different algorithms:

• For reads upto 100 bp long:
  • BWA-backtrack : BWA aln/samse/sampe  
    
    
• For reads upto 1 Mbp long:
  • BWA-SW
  • BWA-MEM : Newer! Typically faster and more accurate.

Get your data

Six raw data files have been provided for all our further RNA-seq analysis:

• c1_r1, c1_r2, c1_r3 from the first biological condition
• c2_r1, c2_r2, and c2_r3 from the second biological condition

cds
cd my_rnaseq_course
cp -r /corral-repl/utexas/BioITeam/rnaseq_course_2015/bwa_exercise . &
cd bwa_exercise

Run BWA

Load the module:

module load bwa/0.7.7 

There are multiple versions of BWA on TACC, so you might want to check which one you have loaded for when you write up your awesome publication that was made possible by your analysis of next-gen sequencing data.

module spider bwa
module list
bwa

You can see the different commands available under the bwa package from the command line help:

bwa

Part 1. Create a index of your reference

NO NEED TO RUN THIS NOW- YOUR INDEX HAS ALREADY BEEN BUILT!

bwa index -a bwtsw reference/genome.fa

Part 2a. Align the samples to reference using bwa aln/samse/sampe

We will be using this set of commands (with options that you should try to figure out) in this order, on each sample:

    
    bwa aln
    bwa samse or sampe

Let's submit the bwa aln job

 *.sai file is a file containing "alignment seeds" in a file format specific to BWA.  We still need to extend these seed matches into alignments of entire reads, choose the best matches, and convert the output to SAM format. Do we use sampe or samse?

Lets submit the bwa sampe job, but have it be on hold till previous job is finished.

Part 2b. Align the samples to reference using bwa mem

nano commands.mem
 
bwa mem reference/genome.fa data/GSM794483_C1_R1_1.fq data/GSM794483_C1_R1_2.fq > C1_R1.mem.sam
bwa mem reference/genome.fa data/GSM794484_C1_R2_1.fq data/GSM794484_C1_R2_2.fq > C1_R2.mem.sam
bwa mem reference/genome.fa data/GSM794485_C1_R3_1.fq data/GSM794485_C1_R3_2.fq > C1_R3.mem.sam
bwa mem reference/genome.fa data/GSM794486_C2_R1_1.fq data/GSM794486_C2_R1_2.fq > C2_R1.mem.sam
bwa mem reference/genome.fa data/GSM794487_C2_R2_1.fq data/GSM794487_C2_R2_2.fq > C2_R2.mem.sam
bwa mem reference/genome.fa data/GSM794488_C2_R3_1.fq data/GSM794488_C2_R3_2.fq > C2_R3.mem.sam