Details
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Type:
Task
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Status: Closed (View Workflow)
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Priority:
Major
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Resolution: Done
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Affects Version/s: None
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Fix Version/s: None
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Labels:None
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Story Points:0.25
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Epic Link:
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Sprint:Fall 5 2021 Oct 11 - Oct 22
Description
Some confusions have emerged regarding the strandedness of RNA-Seq data sets from tomato. Investigate tooling that can detect this in a dataset.
Attachments
Activity
The software is installed on the system. To run it:
module load rseqc
It dumps you into a virtual environment for the software.
Retrieved SRA-stored data from a previous experiment and converted from sra to fastq using fastq-dump. However, the defline did not include anything about the read number. Needed to use a different option.
Trying fasterq-dump, with options:
-S|--split-files write reads into different files
-p|--progress show progress (not possible if stdout used)
-P|--print-read-nr include read-number in defline
Command line:
fasterq-dump -S -P -p SRR11284{116..139}
Because I need internet connection for this to work, ran it on the head node, in a tmux session. Started 14:52 2021-10-24.
Ref:
- Command-line number expansion syntax from "Bash tricks" by Julia Evans https://www.reddit.com/r/linux/comments/cn5ea3/bash_tricks_by_julia_evans/
Examples of running infer_experiment.py, with output:
Unstranded protocol:
$ infer_experiment.py -i VF36_C_R1.bam -r S_lycopersicum_Sep_2019.bed Reading reference gene model S_lycopersicum_Sep_2019.bed ... Done Loading SAM/BAM file ... Total 200000 usable reads were sampled This is PairEnd Data Fraction of reads failed to determine: 0.0087 Fraction of reads explained by "1++,1--,2+-,2-+": 0.4945 Fraction of reads explained by "1+-,1-+,2++,2--": 0.4969
Intepretation: About half of the time, read 1 has the same sequence as its gene's plus strand. The other half of the time, it matches the gene's minus strand.
Stranded synthesis protocol:
$ infer_experiment.py -i A_C_3S.bam -r O_sativa_japonica_Oct_2011.bed [W::hts_idx_load3] The index file is older than the data file: A_C_3S.bam.bai Reading reference gene model O_sativa_japonica_Oct_2011.bed ... Done Loading SAM/BAM file ... Total 200000 usable reads were sampled This is SingleEnd Data Fraction of reads failed to determine: 0.0201 Fraction of reads explained by "++,--": 0.0095 Fraction of reads explained by "+-,-+": 0.9704
Interpretation: Almost every sequenced read matches the antisense strand of a gene, never the sense strand of a gene.
Older BAM files (from SRP252265), aligned with hisat2 and run using the wrong strandedness parameter:
infer_experiment.py -i N3-S.bam -r S_lycopersicum_Sep_2019.bed [W::hts_idx_load3] The index file is older than the data file: N3-S.bam.bai Reading reference gene model S_lycopersicum_Sep_2019.bed ... Done Loading SAM/BAM file ... Total 200000 usable reads were sampled This is PairEnd Data Fraction of reads failed to determine: 0.0013 Fraction of reads explained by "1++,1--,2+-,2-+": 0.5170 Fraction of reads explained by "1+-,1-+,2++,2--": 0.4817
Options and arguments of "infer_experiment.py":
$ infer_experiment.py -h Usage: infer_experiment.py [options] Options: --version show program's version number and exit -h, --help show this help message and exit -i INPUT_FILE, --input-file=INPUT_FILE Input alignment file in SAM or BAM format -r REFGENE_BED, --refgene=REFGENE_BED Reference gene model in bed fomat. -s SAMPLE_SIZE, --sample-size=SAMPLE_SIZE Number of reads sampled from SAM/BAM file. default=200000 -q MAP_QUAL, --mapq=MAP_QUAL Minimum mapping quality (phred scaled) for an alignment to be considered as "uniquely mapped". default=30
RSeqQC is a suite of python programs for analyzing RNA-Seq data. One of these programs is "infer_experiment.py", which can detect whether a stranded library synthesis protocol was used.
To install and then use RSeqQC, use pip3 to install virtualenv for your user. Then start the virtual environment and install RSeqQC and its dependencies, using pip3.
pip3 install virtualenv --user mkdir ~/envs cd envs virtualenv rseqc source ~/envs/rseqc/bin/activate pip3 install RSeQC
Moving to done.