Open laudycherry opened 6 months ago
Hi @laudycherry
If columns 3 and 4 have similar counts, it likely means that the library is not stranded. Then, the only way to infer strand is by using spiced intron motifs. STAR should work fine with pre-trimmed reads, provided that the trimmer keeps the reads in the same order in Read1 and Read2.
Dear @alexdobin, I appreciate your answer, I have checked the library used, it is trueseq stranded, still I am getting similar counts in columns 3 and 4. Honestly I cannot understand why I am getting similar counts in both. what shall I do in this case? is it acceptable to run the data from column 2 in Deseq2 then? On the other hand I run cufflinks after running Star to determine the strands, unfortunately, Cufflinks does not provide the ensembl gene id, is there any tool that permits to convert cufflink ids to ensembl gene ids? Thank you in advance for your response,
Hi @laudycherry It is possible that the library is unstranded - a mistake made by the sequencing provider. It is acceptable to use unstranded counts in this case, but you will miss the genes that overlap on opposite strands.
Hello,
I am analyzing some RNA samples and I have noticed an issue with the quantification part using Star.
The data I have was generated by an external company using TruSeq3-PE-2 library, which as I know it is a stranded library kit. To do the alignment and quantification by STAR I have used the following script:
STAR --genomeDir ~/genome/STAR \ --runThreadN 8 \ --readFilesCommand zcat \ --readFilesIn /home/laudy/data/raw/Sample_1.fq.gz /home/laudy/data/raw/Sample_2.fq.gz \ --quantMode GeneCounts \ --sjdbGTFfile ~/genome/Mus_musculus.GRCm39.110.gtf \ --outFileNamePrefix /home/laudy/data/ReadCounts/S18_Musmusculus.GRCm39.110