How to obtain unspliced tRNAs from FASTQ file

Question

[nkabo]

Dear all,

I have RNA-seq data coming from peripheral blood leukocytes. I have FASTQ file and I was asked to obtain unspliced tRNAs from that file. I am a beginner in RNA-Seq analysis and I do not know how I could extract unspliced tRNAs from FASTQ file. I tried to find a workflow or tool, but I could not detect any. I would like to ask:

• Is it possible to obtain unspliced tRNAs from a FASTQ file?
• If it is possible, could you suggest me a way/tool/workflow for such task?

Thanks in advance.

[lieven.sterck]

theoretically it might be possible (see swbarnes2 answer on how), but :

• RNAseq is usually processed RNA which means it is in most cases already spliced (unspliced reads are rather an exceptions/artefact)
• dependant on the type of RNA data you have, you should check how the RNA libraries were constructed as most often (default bulk rna-seq) is poly-A captured, meaning it is focussed on mRNA molecules and would thus omit most of the other RNA types

[nkabo]

Dear community,

@swbarnes2 suggested me to obtain sequences of unspliced tRNA genes and do the aligning afterwards. However, I could find regular tRNA sequences to do the alignment, but I could not see any groups of sequences, specifically belonging to unspliced tRNAs. A colleague also suggested to use "misc" part in NCBI, but I could not quite catch how it could be used in this case.

Could you propose a way to find sequences of unspliced tRNA genes?

Thanks in advance!

Answer 1

[swbarnes2]

Here's what I'd try:

1) Find sequences of unspliced tRNA genes, make a reference fasta

2) Align your fastqs to that reference.

[nkabo]

Thank you for your reply, I will try this approach. I think , after obtaining sequences for unspliced tRNA genes, I need to align the unprocessed version of RNAseq to these unspliced tRNA genes; since such small genes could be eliminated during the processing as lieven.sterck mentioned. I am looking for a source for unspliced tRNA genes, but I am not quite sure how I could detect unspliced tRNA genes and get their sequences. Could you suggest me a source for such aim? Thanks in advance.