De novo assembly of RNA-Seq reads

De novo assembly of reads

First, we combine the raw reads from different conditions into a single FASTQ file (for each end) and use Trinity to generate a reference assembly.

* Assume the fq.gz files were already placed in the Trinity/RNASEQ_data folder

mkdir ~/LSLNGS2015/Trinity
mkdir ~/LSLNGS2015/Trinity/RNASEQ_data/

Execute

cd ~/LSLNGS2015/Trinity

zcat RNASEQ_data/*.left.fq.gz | gzip > RNASEQ_data/sp.left.fq.gz
zcat RNASEQ_data/*.right.fq.gz | gzip > RNASEQ_data/sp.right.fq.gz

bsub -q 16G -o ./trinity_reference.std -e ./trinity_reference.err -J Trinity \
"Trinity --seqType fq --SS_lib_type RF \
--left RNASEQ_data/sp.left.fq.gz --right RNASEQ_data/sp.right.fq.gz \
--CPU 10 --max_memory 16G --output trinity_reference >& trinity_reference.log"

You will get a message like Job <xxxxxx> is submitted to queue <16G>. to let you know your submission is successful.

You can use bjobs to check your job status.

JOBID   USER    STAT  QUEUE      FROM_HOST   EXEC_HOST   JOB_NAME   SUBMIT_TIME
993732  s00ycm0 RUN   16G        alps1       8*alps1-40  Trinity    Nov 18 22:38

Resource usage

ALPS Queue Name	CPU Time	Max Memory	Duration
16G	2097.24 sec.	2 GB	6 minutes 37 seconds

The assembled transcripts can be found at trinity_reference/Trinity.fasta.

Examine assembly stats

The TrinityStats.pl script will report the basic statistics about the assembly produced by Trinity. The numbers may vary slightly, as the assembly results are not deterministic.

Execute

Locate util/TrinityStats.pl in the trinityrnaseq-2.2.0 distribution, and run

PATH_TO_TRINITY/util/TrinityStats.pl trinity_reference/Trinity.fasta

Output

################################
## Counts of transcripts, etc.
################################
Total trinity 'genes':  377
Total trinity transcripts:      385
Percent GC: 38.65

########################################
Stats based on ALL transcript contigs:
########################################

        Contig N10: 3373
        Contig N20: 2605
        Contig N30: 2219
        Contig N40: 1936
        Contig N50: 1703

        Median contig length: 772
        Average contig: 1046.43
        Total assembled bases: 402875


#####################################################
## Stats based on ONLY LONGEST ISOFORM per 'GENE':
#####################################################

        Contig N10: 3373
        Contig N20: 2605
        Contig N30: 2219
        Contig N40: 1936
        Contig N50: 1682

        Median contig length: 765
        Average contig: 1038.27
        Total assembled bases: 391428