How to to lncRNA analysis from WTS data ?

Question

[Sowmya Pulapet]

Hi,

I have WTS data with me and I have to do mRNA analysis, lncRNA analysis etc. using the data. Until now what I have done is map the pre-processed data to reference genome using Hisat2 and do quantification using htseq by setting -t gene. The counts file obtained was used to do mRNAseq DEG analysis.

For lncRNAseq, I will do transcript assembly using Stringtie and then merge the assembly files. After that filter the transcripts using the following criteria:

1. Select transcripts which are longer than 200 bp and have more than 2 exons
2. Compare with known mRNAs according to the class code
3. CPC2 tool to calculate coding potential and filter based on that

Then use the remaining transcripts to do differential expression analysis using cuffdiff.

I have certain doubts regarding this:

1. Is the method to to mRNA seq analysis from WTS data appropriate ?
2. Whenever I have used this method to filter lncRNAs I have got only very few and hardly one or two differentially expressed lncRNAs
3. Could you suggest me some useful tutorials or reference articles for lncRNA seq analysis ?

I believe I am missing something in my pipeline at the filteration part. Hope someone will help me to figure this out.

Thank you.