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 is an unspliced mapper. 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
cd day_1_partB/bwa_exercise

Lets look at the data files and reference files

ls ../data
ls ../reference
 
#transcriptome
head ../reference/transcripts.fasta 
#see how many transcripts there are in the file
grep -c '^>' ../reference/transcripts.fasta
 
#genome
head ../reference/genome.fa
#see how many sequences there are in the file
grep -c '^>' ../reference/genome.fa
 
 
#annotation
head ../reference/genes.formatted.gtf
#see how many entries there are in this file
wc -l ../reference/genes.formatted.gtf

Load the module:Run BWA

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

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

All the files starting with the prefix transcripts.fasta are your BWA index files.

bwa index -a bwtsw reference/transcripts.fasta

Part 2. Align the samples to reference using bwa mem

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