Question: Low transrate assembly score with good bowtie2 mapping?
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gravatar for fgrazom
10 months ago by
fgrazom0
fgrazom0 wrote:

Hi Everyone!, this is my first question here, but i have been following you and getting tips from this community for a long time.

So, up to my question, i got data from 8 samples of a paired true-seq RNA-Seq experiment, i used sortmeRNA to delete rRNA, then used trimmomatic to clip adaptors, trim reads with a slidingwindow and removed all the reads below 50 nt. Then we used Trinity with default options (just as a first try), and got an assembled transcriptome with around 85k contigs, everything seem to be alright until i tried transrate to evaluate the assembly, it outputs an incredibly low score (0.04) and this seems to be caused because most of the reads are not aligning to the transcriptome, so i ran again trinity (i noticed i didn`t specify ss_libtype) specifying the ss_libtype and got another assembly that got a transrate score like the former.

I´ve been reading and looking for ways to improve the assembly, I was about to start all over again with the QC and then i tried running an alignment with bowtie2 to the first assembly using one of the samples and it turns out i got around 90% of the reads aligned to the assembly.

So my question here is, has anybody been in this type of situations?, and what would you do?, should i trust transrate or bowtie2?. i was thinking about trying to assemble with different kmer size and choose the best assembly, but now im stuck with this.

Edit: I'm using the first assembly, because i was sure it was strand specific but looking at the protocol used i noticed i was wrong, so i discarded the second assembly.

rna-seq alignment assembly • 446 views
ADD COMMENTlink modified 10 months ago • written 10 months ago by fgrazom0

until i tried transrate to evaluate the assembly, it outputs an incredibly low score (0.04) and this seems to be caused because most of the reads are not aligning to the transcriptome

And what is the mapping rate? Did you map the raw reads, or the trimmed, rRNA cleaned reads?

ADD REPLYlink written 10 months ago by h.mon25k

Hi!, the mapping rate is just 0.2, mapping the trimmed rRNA cleaned reads.

ADD REPLYlink written 10 months ago by fgrazom0

It is a known issue (Mapping of TransRate using SNAP, SNAP failing in v 1.0.3, SNAP failed with Transrate v 1.0.3), without an answer from the developers still unresolved.

You could try install SNAP manually, and check if it runs properly.

ADD REPLYlink modified 10 months ago • written 10 months ago by h.mon25k

i didn`t know that, i thought it was using salmon instead of SNAP.

I'll try installing SNAP and let you know the outcome.

Thank you very much!,

ADD REPLYlink written 10 months ago by fgrazom0
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