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2.0 years ago
Roy.anupama11
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0
Hello, I have to search for DEGs from four samples of crop. I am following reference based mapping of reads to genome using HISAT2. I have completed till the generation of merged .gtf files for the samples using STRINGTIE. Since I am new to RNA seq analysis, I am not able to go forward and do counting and eventually find the DEGs. I hope you will help me.
Thank you
Anupama
Unless you are looking for new transcripts there is actually no need to use stringtie at all. It is overly complicated and whatever "novel" transcripts it calls need validation. I would quantify my BAM files against the reference GTF with something like featureCounts and then feed the generated count matrix into differential analysis frameworks such as DESeq2.
Instead of featureCounts and HTseq, what else can be used?
You can go back to the fastq files and use selective aligners such as salmon that do the quamtification. Why?
Salmon is being used for transcriptome mapped reads, as far as I know.
The quantification itself is transcriptome, that is correct, but you can easily aggregate the per-transcript abundance estimates into gene level counts with https://bioconductor.org/packages/devel/bioc/vignettes/tximport/inst/doc/tximport.html#Salmon
That is a standard pipeline that is widely used.
Okay let me proceed with Salmon. Thank you.