Question: Alternative splicing analysis of targeted RNASeq data
gravatar for ddzhangzz
2.9 years ago by
United States
ddzhangzz90 wrote:

My client has run targeted RNA sequencing for a list of genes from different chromosomes in human and wanted to perform alternative splicing analysis. I have never been working on these type of targeted data analysis and am wondering whether there is standard ways for this analysis. What programs I can use to do this analysis? Could somebody can give me some possible description of the procedure?

rna-seq • 1.2k views
ADD COMMENTlink modified 2.9 years ago by EagleEye6.5k • written 2.9 years ago by ddzhangzz90
gravatar for EagleEye
2.9 years ago by
EagleEye6.5k wrote:

Check out previous posts

A: workflow/tool for alternative splicing

Or if your are newbie try this workflow,

Are those total RNA (Transcriptome) sequencing samples? If so,



1) FastQC (Check quality of sequencing).

2) Trimmomatic or Cutadapt (if necessary, uncleaned reads for adapters)

3) STAR or HISAT2 (I would personally recommend STAR)

4) SAMstat (Check quality of alignment)

5) RSEM or Cufflinks (Check mapping quality, MAPQ using SAMstat from alignment before starting quantification)

6) Differential expression analysis: DESeq2 or edgeR or Ballgown (I personally not tried ballgown)

7) GeneSCF v1.1 (command-line)/Enrichr (Web-based) [Gene ontology or pathway analysis for differentially expressed isoforms related genes, this step is only applicable for protein coding genes]. GeneSCF is more reliable than other tools because it is real-time based and also check other advantages of using GeneSCF.

ADD COMMENTlink modified 2.9 years ago • written 2.9 years ago by EagleEye6.5k

Given that OP is talking about targeted RNA-seq data (which I interpret as including a probe-based capture step in library prep) I expect that assumptions made in common differential expression analysis tools are violated, e.g. DESeq2 and edgeR expecting most genes to be not differentially expressed for dispersion estimation.

ADD REPLYlink written 2.9 years ago by WouterDeCoster42k

If this is RNA Capture-seq, the 6th step is not necessary as mentioned by WouterDeCoster. Also check this article.

ADD REPLYlink modified 2.9 years ago • written 2.9 years ago by Santhilal Subhash (EagleEye)0

Thanks to @EagleEye. but what I am interested is how to do it for targeted RNASeq data in AS analysis (for selected genes).

ADD REPLYlink written 2.9 years ago by ddzhangzz90
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