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De novo assembly of RNA-Seq reads

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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/

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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.

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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.

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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.

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Execute

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

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

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Output

PreviousDe novo assembly using TrinityNextCompare de novo reconstructed transcripts to reference annotations

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