Which sequencing parameters are required for SGSeq analysis of splice events?
4
0
Entering edit mode
@andreabast-18099
Last seen 3.0 years ago

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

rna-seq alternative splicing SGSeq • 615 views
ADD COMMENT
0
Entering edit mode
@leonard-goldstein-6845
Last seen 12 months ago
United States

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 COMMENT
0
Entering edit mode
@andreabast-18099
Last seen 3.0 years ago

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 COMMENT
0
Entering edit mode
@leonard-goldstein-6845
Last seen 12 months ago
United States

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 COMMENT
0
Entering edit mode
@andreabast-18099
Last seen 3.0 years ago

Hi Leonard,

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

Best,

Andrea

ADD COMMENT
0
Entering edit mode

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 REPLY
0
Entering edit mode

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 REPLY

Login before adding your answer.

Traffic: 262 users visited in the last hour
Help About
FAQ
Access RSS
API
Stats

Use of this site constitutes acceptance of our User Agreement and Privacy Policy.

Powered by the version 2.3.6