Question: Paired end illumina RNAseq reads - running trinity with paired trimmed files and adding unpaired as single read?
0
gravatar for sdbaney
9 weeks ago by
sdbaney10
sdbaney10 wrote:

We have paired end Illumina HiSeq 4000 reads that we are working to remove adaptors and trim bases below our phred cutoff. Trimmomatic has 4 output files from our two input files. Two that are paired and then two that are unpaired. I was originally going to toss the unpaired trimmed files and just run trinity de novo assembly using the paired trimmed files but my co-advisor mentioned that we may want to use both. He is saying that trimmomatic when cutting the adapter sequences, may also cut the complementary sequence to that adaptor on the paired strand leading to removal of sequences that we don't want removed. I would assume that trimmomatic would only identify the actual adapter sequence within the designated forward or reverse read but now I am having trouble finding the answer to whether this is the case or not. He mentioned using both the paired and unpaired reads in the trinity de novo assemble, that we may be able to recover erroneously trimmed segments.

What are people's opinions/experiences on this matter?

rna-seq trinity • 320 views
ADD COMMENTlink modified 8 weeks ago • written 9 weeks ago by sdbaney10
0
gravatar for Jennifer Hillman Jackson
8 weeks ago by
United States
Jennifer Hillman Jackson25k wrote:

Hello,

For the Trimmomatic question, I haven't heard of that. It is very commonly used tool. You could test the trimming functionality out and how it changes your specific data by running the data through and comparing the results.

If your advisor wants to use a different trimming tool, Galaxy has several wrapped already, but really any tool could be wrapped. The public servers have some loaded and even more are in the ToolShed https://usegalaxy.org/toolshed for use in a local/docker/cloud Galaxy. https://galaxyproject.github.io/

If you want to include the unpaired reads, then place all of the forward reads in one dataset and all of the reverse reads in another. The tool Concatenate can be used if you decide to stick with a trimming tool that outputs the data based with a paired-or-not filter. Order the stack on the tool form so that the pairs are above the singles.

Also, you might try assembling with just pairs and then again with the singles included, and compare the results. The assembly comparison would be informative. Singles can create complex/spurious results. That doesn't mean it isn't useful data -- just be aware of what you have content-wise. This should all be discussed with your advisor if you are not clear on the final objective/goals.

Thanks, Jen, Galaxy team

ADD COMMENTlink written 8 weeks ago by Jennifer Hillman Jackson25k
0
gravatar for sdbaney
8 weeks ago by
sdbaney10
sdbaney10 wrote:

Thank you!! This gives me a lot of information. Thank you for being so helpful!

ADD COMMENTlink written 8 weeks ago by sdbaney10
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