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Stringtie de novo VS reference guided (-G)

Hi Community,

I am currently preforming RNA-seq analysis of human dataset and my aim is to find novel transcripts and isoforms. I have aligned the sequences to the reference genome using Hisat2 and assembled the transcripts using Stringtie in both reference guided and de novo methods. When I looked at the number of assembled transcripts I see reference guided mode has assembled twice number of transcripts than de novo method:

stringtie denovo: exon 400375; transcript 59007

stringtie reference-guided (-G): exon 708407; transcript 121080

My question is: Is it normal to see this difference and if so could you please, help me in understanding the reason or refer to any article.

Thanks in advance.

reference-guided denovo stringtie rna-seq
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updated by Istvan Albert Istvan Albert • written by vasanthmandla • vasanthmandla

1 answer

The Stringtie manual states:

Although StringTie is primarily a genome-guided approach, it can borrow algorithmic techniques from de novo genome assembly to help with transcript assembly.

https://ccb.jhu.edu/software/stringtie/index.shtml?t=manual

The above implies that it will perform more effectively in guided mode, which is what you also observe. That being said what the difference should be will depend on a whole slew of properties of both your sequence data and the reference annotations.

I would recommend to investigate and compare the regions where one approach produces more transcripts than the other.

  • What kind of commonalities can you observe.
  • What kinds of tradeoffs can you observe
  • Which assembly appears to be more trustworthy
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written by Istvan Albert Istvan Albert

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