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

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

Run BWA

Load the module:

module load bwa

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

bwa index -a bwtsw reference/genome.fa

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

You will need to run this set of commands (with options that you should try to figure out) in this order, on each sample:

bwa index
bwa aln
bwa samse or sampe

What's going on at each step?

Since this will take a while to run, you can look at already generated results at: /corral-repl/utexas/BioITeam/rnaseq_course/bwa_exercise/results

 What is a *.sai file? It's a file containing "alignment seeds" in a file format specific to BWA. Many programs produce this kind of "intermediate" file in their own format and then at the end have tools for converting things to a "community" format shared by many downstream programs.

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?

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

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