Question: Which sequencing parameters are required for SGSeq analysis of splice events?
0
gravatar for andrea.bast
12 months ago by
andrea.bast0 wrote:

We plan to do an RNA-Seq analysis of knockdown and control samples in order to analyse differences in alternative splicing patterns between the two groups. The analysis should be done using R package SGSeq. We would now like to know which parameters we have to choose for the RNA-Seq experiment that allow us to do the analysis with SGSeq lateron.
We know that we need strand-specific sequencing with long paired end reads (100bp-150bp). We are however unsure about the sequencing depth. Do you have suggestions for that?

Thank you for your help,
best regards,
Andrea

ADD COMMENTlink modified 12 months ago • written 12 months ago by andrea.bast0
Answer: Which sequencing parameters are required for SGSeq analysis of splice events?
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gravatar for Leonard Goldstein
12 months ago by
United States
Leonard Goldstein80 wrote:

Hi Andrea, 

Actually strand-specific sequencing is not a requirement (the package was written a few years ago when most available data sets were not strand-specific). Since SGSeq relies on split reads, longer reads are helpful. There is no requirement for a particular sequencing depth. More reads are better, especially when analyzing genes with low expression, but this has to be weighed against higher cost. Standard recommendations for transcriptome studies should be a good starting point. 

Best, 

Leonard

ADD COMMENTlink written 12 months ago by Leonard Goldstein80
Answer: Which sequencing parameters are required for SGSeq analysis of splice events?
0
gravatar for andrea.bast
12 months ago by
andrea.bast0 wrote:

Dear Leonhard,

thanks a lot for your reply. Good to know that we don't necessarily require strand-specific sequencing. Concerning the sequencing depth, do you think that 50M reads for each sample with 3 biological replicates per group would be ok?

Best,

Andrea 

ADD COMMENTlink written 12 months ago by andrea.bast0
Answer: Which sequencing parameters are required for SGSeq analysis of splice events?
0
gravatar for Leonard Goldstein
12 months ago by
United States
Leonard Goldstein80 wrote:

Hi Andrea, 

If you have the option to do strand-specific sequencing, this is still a good idea for transcriptome analysis (just not required for SGSeq). Read depth and replicates sound reasonable. 

Best, 

Leonard

ADD COMMENTlink written 12 months ago by Leonard Goldstein80
Answer: Which sequencing parameters are required for SGSeq analysis of splice events?
0
gravatar for andrea.bast
12 months ago by
andrea.bast0 wrote:

Hi Leonard,

ok, I think we will then perform the experiment like that. Thanks a lot for your help.

Best,

Andrea

ADD COMMENTlink written 12 months ago by andrea.bast0

Dear Leonhard,

one more question occured while discussing about the project. Is there a requirement for the length of the fragments generated in the library preparation? You also said that longer reads would be helpful. We would plan to do 2x150bp (40 mio reads per sample). Would you say that should work?

Best,

Andrea

ADD REPLYlink written 12 months ago by andrea.bast0

There is no specific requirement for fragment or read length. I would follow standard recommendations for library preparation. The sequencing specs and read length sound fine.

ADD REPLYlink written 12 months ago by Leonard Goldstein80
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