Building the transcriptome

The purpose of many RNAseq studies is to compare the gene expression patterns across different treatments. In order to do this, as I mentioned in the previous post, all the reads from each treatment (from the one plant) need to be combined first.

Trinity assembly software incorporates other tools as well as assembly, for example Trimmomatic can be run within the trinity pipeline. To do the trimming and assembly you would run a script like this (note no spaces or line breaks):

Trinity –seqType fq –max_memory 24G –CPU 7 –left AP0_R1.fastq,AP1_R1.fastq,AP2_R1.fastq –right AP0_R2.fastq,AP1_R2.fastq,AP2_R2.fastq–trimmomatic –output AP_trinity

This will build all the reads into a complete transcriptome for the plant labelled AP from RNA expressed pre-inoculation, at 24 hours and at 48 hours.

Depending on the success of the sequencing, ie. the number and length of raw reads, the assembly process will take around 1 to 3 days of wall-time. Below is the pbs script to run for trimmed reads.

#PBS -P (project name)
#PBS -N AP_assembly
#PBS -l nodes=1:ppn=24
#PBS -l walltime=100:00:00
#PBS -l pmem=24gb
#PBS -e ./AP_trinity/AP_assembly.txt
#PBS -M email@sydney.edu.au
#PBS -m abe

# Load modules
module load bowtie
module load java
module load trinity/2.1.1

# Working directory
cd /project/full path name to your working directory (where all the input files are)

# Run trinity
Trinity –seqType fq –max_memory 24G –CPU 24 –left AP0_R1.trimpaired.fastq,AP1_R 1.trimpaired.fastq,AP2_R1.trimpaired.fastq –right AP0_R2.trimpaired.fastq,AP1_R2.trimpaired.fastq,AP2_R2.trimpaired.fastq –output AP_trinity

NB: As I said previously, I am learning on the process as I go. Actually writing this bog is making me find the mistakes I have made along the way. Just found an error in the way I ran the above trinity script. It appears that the paired and unpaired trimmed reads are combined with the trinity assembly. I have only used my paired reads. At this point I am way down the pipeline so this is a big blow! I have decided to continue with my current pipeline as well as re-run the assemblies with both the paired and un-paired reads and then compare the outcomes.

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