Aligning the reads to the transcriptome

Armed with the clustered fasta file and the trimmed raw reads you can now determine the read hits per transcript.

Alignment functions are supported within the Trinity platform, thereby incorporating the use of these third party tools, providing they are installed. Installed software required include; Bowtie (I used v1.1.2) and SAMtools (v1.2) to give outputs of bam files. Also required is Perl and of course Trinity – if running within the streamlined utilities.

#PBS -P project name
#PBS -N AP0_alignment
#PBS -l nodes=1:ppn=20
#PBS -l walltime=20:00:00
#PBS -l pmem=24gb
#PBS -e ./AP0_alignment.txt
#PBS -M email@sydney.edu.au
#PBS -m abe

# Load modules
module load perl
module load bowtie
module load java
module load samtools/1.2
module load trinity/2.1.1

# Working directory
cd /path to working directory

# Run bowtie script
/usr/local/trinity/2.1.1/util/bowtie_PE_separate_then_join.pl –seqType fq \
–left AP0_R1_pairedwithunpaired.trim.fq –right AP0_R2_pairedwithunpaired.trim.fq \
–target Syzygium.fasta –aligner bowtie \
— -p 4 –all –best –strata -m 300

From this will be output a folder called bowtie containing bam files and indexed bams. For the next step you will use the bam files from all samples and all times to determine the counts per transcript.I rename the bam files according the plant/time eg. AP0_coordSorted.bam and AP0_coordSorted.bam.bai

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