I am performing small rna analysis and using srna-workbench for data trimming (Adaptor trimming and length filtering). After length trimming (16-40 bases) i am observing the distict reads are maximum of 32 bp whereas for small rna peak is observed at 18-22bp. What could be the reason?
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bioinfo_ga •
2 answers
Depending on the protocol used (which you did not mention so it is hard to tell) you could have degradation products remaining from turn-over of endogenous mRNA.
However, a smallRNA peak at 18-22bp makes sense... at least in animals as this would correspond to high levels of miRNA and/or siRNA. Generally smallRNA have the following ballpark size ranges (you should investigate what is expected for your organism of interest):
- 21-22 nt siRNA
- 21-23 nt miRNA
- 24-31 nt piRNA
The smaller sizes could be the result of over-trimming, the mRNA byproducts as first mentioned, and/or minor degradation of your small reads. If the distribution of your read lengths (i.e. molecule lengths) matches what has been previously observed in the literature I wouldn't worry too much.
Here is an example for Drosophila from this paper:
Hi, as well as plotting the length, you can also check the biotype (ie species of RNA) of overlapping genes
I've written a Python library called PyReference that includes a command to do this:
# Install
python3 -m pip install pyreference
# Process GTF into a JSON file
pyreference_gff_to_json.py --gtf gencode.hg38.gtf
# Create a setup file (see website for details)
# Run against your BAM file
pyreference_biotype.py ${BAM_FILE}
This generates a graph:
As well as a CSV of read counts per biotype for each length
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Hi ,
How do your performed your trimming step with list of adaptor sequence ?
What tool do you used ?
Are you sure you can t have 32 bp RNA possible ? I was thinking in Piwi RNA for exemple .
Best
I have used small rna workbench tool and used inbuilt adapter (LMN_3). I went through published papers of which almost all have reported the same trend (18-22bp). I checked for piwi RNA also but it is <0.02% in my data.
ok may be just check with fastq if you have a correct nuclear acid repartition in your trimmed reads.
You would not expect very much piRNA unless you are deriving your sequencing samples from the core reproductive organs.*
*Assuming you aren't researching plants
Hello bioinfo_ga,
What's your experience with srna-workbench? I am trying to analyze my plant srna seq data with it. But I don't know how to install it in my windows system :(. Could you share your experience with me? Appreciate sincerely.
Wei Xu