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7.2 years ago
Vasu
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770
Hello Everyone,
The Data I'm using is Count based. I used DESeq2 to find differential expressed genes between two Groups, Cancer and Normal. I want to identify Long non coding RNA's from these. Can anyone please tell me how can I go forward in this analysis?
Thank you
Check this link, A: Any One please provide protocol for Analysing long noncoding RNA illumina NGS da
Thank you. But the Mapping is already done with ref genome with total RNA's already. Now I have DE genes and their annotation too. So, If I have lncRNA annotation now I can filter from that right? Do you think this is fine?
If you are just interested in lncRNAs, why don't you process the data using lncRNA annotation from the beginning ?
Yes, I can do it from the beginning but I would like to know whether doing the other way is right or not? Could you please tell me about that. Thank you
If you would like to just highlight DE lncRNAs from your analysis, better to perform complete analysis with only lncRNAs.
If you want to highlight both lncRNAs and protein coding RNAs from your analysis, the way you mentioned is acceptable. But keep in mind since lncRNAs expressed in low levels and while performing normalization along with protein coding (PC) genes, the normalization will have major bias towards highly expressed PCs.
Yes, Thank you very much. I gt the point and dis makes sense.
@EagleEye I have another doubt. In your previous comment If I want to do in the other way like before normalization if I remove the pcRNAs and take only lncRNAs then do the normalization, Will dis be right way?
And Could you please tell me where I can get the annotation only for lncRNAs? Thank you
I would prefer using Gencode lncRNAs as reference for quantification and then differential expression analysis for those lncRNAs.
Which annotation you used, Ensembl or Gencode. Here are the different annotation resources (Gencode contains list of lncRNAs as GTF file).
A: How to download ncRNA
Ok. So from the Count matrix with genes and Samples I remove the pcRNA genes based on lncRNA annotation(Gencode or Ensembl), I have only lncRNAs for the normalization and DE analysis steps. Am I right?
If you take lncRNA annotation from Gencode, you will not find any protein coding RNAs.
Example: http://www.gencodegenes.org/releases/24.html
you can see "Long non-coding RNA gene annotation" from the above link. Use that annotation to create your gene matrix using some quantification tools like HtSeq or featureCounts.
Hello, could you please tell me which of these annotation files "GRCh37 or GRCh38" has the advantage for identifying lincRNA's? and also why?
I would prefer to use GRCh38 annotation since it is new and updated for genome HG38.
Hi EagleEye, although it's too late, I've got some RNA-seq count as CPM (count per million) for doing long-non coding RNA analysis. I separated this type of RNAs (lncRNA). However, as the CPM is a kind of normalized count, I'm not sure how to continue the analysis. Could you please let me know if I can use the CPM count of lncRNAs and edgeR package to find differentials? or I need the raw count for starting the analysis, my mean is if doing normalization step on the raw count of lncRNA has advantages over using CPM of lncRNA? If yes, please kindly let me know how to convert CPM to the raw count?
Many thanks