The origin of transcripts with putative frameshift in the de novo assembled transcriptome?

Question: The origin of transcripts with putative frameshift in the de novo assembled transcriptome?

5.5 years ago by

seta • 1.3k

Sweden

seta • 1.3k wrote:

Dear all,

I've performed a denovo transcriptome assembly of a non-model plants on trimmed and almost high quality illumina reads. I used Trapid tool to fast evaluation of assembled transcriptome, this tool reported high percentage of transcript with putative frameshift that is not desired. Could you please let me know why there is many transcript with putative frameshifte in the transcriptome based on Trapid prediction, and how I can solve this problem? Thanks for your points and tips.

rna-seq trapid frame-shift next-gen assembly • 1.5k views

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modified 5.4 years ago by Michael Dondrup ♦ 47k • written 5.5 years ago by seta • 1.3k

I suggest this might be an artifact, it is just difficult to exactly say why. One source might be the transcriptome assembly, which tool and parameters were used? Whatever you used, try a different assembly in addition. Another option is the predictions are bogus. Take some of those predicted frameshift containing transcripts and BlastX. Does the result show frameshifts as well: multiple HSPs in different frames for the best hits?

ADD REPLY • link modified 5.4 years ago • written 5.5 years ago by Michael Dondrup ♦ 47k

Thanks. I used CLC software for assembly of about 300 million PE reads. The high-quality assembly, in terms of N50 (1270bp), contig number and percentage of mapped back read (89%) was obtained in K-mer 64. However, Trapid tool detected many (about 60%) transcript with putative framshiftes. I'm really concern about this results. Could you please share me other tools to detect putative framshiftes?

ADD REPLY • link written 5.5 years ago by seta • 1.3k

I remember there was a similar post where we concluded that there was no evidence for a frame shift from blastx, correct? I think then we can regard this as resolved?

ADD REPLY • link written 5.4 years ago by Michael Dondrup ♦ 47k

Hi Michael, yeah and you kindly suggested me check the frame shift using blastx and

Retain all best hits or all hits with score > some threshold
Retain all hits with more than one HSP
Retain all hits where at least one HSP has different frame from the others
All queries that pass these filters are frame shift candidates

You was supposed to share a script to evaluate the above parameters from blastx, could you please put it here? Thanks in advance

ADD REPLY • link modified 5.4 years ago • written 5.4 years ago by seta • 1.3k

I'll attach the script to the other question: Tool to detecte transcript with putative framshifts in the de novo assembled transcriptome

ADD REPLY • link written 5.4 years ago by Michael Dondrup ♦ 47k

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