A specific gene is not shown up in assembled transcriptome

[lincw]

I had assembled a non-model system plant's RNA-seq reads by Trinity packages (without reference). After annotated the transcriptome by blast against model plant arabidopsis, I fount there was a specific gene (A) missed in the assembled transcriptome. Previous, other colleagues in the lab had checked the expression of A gene, and they did get some results.

In order to know why I couldn't get the A gene in the assembled transcriptome, I extracted the raw read sequences from FastQ file and blat against the published A-homologue sequence. There were only two reads could match with the homologue sequence, and there was a huge gap between them.

Have you ever meet such situation, and how to explain it? Does it mean the quality of that RNA-seq is not good enough? But I can get some other key genes, and their expression patterns were matched other RT-PCR results.

Many thanks,
CW

[Wallysb01]

Is this RT-qPCR on the same RNA that was sequenced?

What was the ct-value for the gene?

How many reads did get for this sample?

There is going to be a pretty big difference between detectable by RT-qPCR and assemble-able from RNA-seq. Personally, I’ve had a hard time finding genes I couldn’t pick up by RT-qPCR at ct of 35+, so if that ct is high, I’d be inclined to think it hardly expressed and isn’t likely to assemble.

[lincw]

Thank you for your response.

The RNA used in RT-PCR and sequencing was not the same batch.

We used RT-PCR to check the expression pattern of those genes, not qPCR. The PCR cycles and the agarose exposure time under UV light box of this gene were higher and longer than others.

I think this may be the reason why I can't get the gene in RNA-seq assembled result.