How can get very low counts in RNA-seq ?

Question

[sabaghianamir70]

Hi every one. i did rna seq analysis. in PER3 gene and PER4 pseudogene i didnt get any counts, but in IGV i saw some small peaks which means they have some counts, even very low, but still they have !!. i want to know is there any way i can get any counts from that ? were my options in my aligner wrong or what else ?

i used blood samples. and used with trimmomatic, STAR, featureCounts

[Pierre Lindenbaum]

i want to know is there any way i can get any counts from that ?

may be there is a low MAPQ for those reads . Use samtools view in.bam "chr1:234-567" to explore the reads in the region of the gene.

[ATpoint]

Duplicate of What is the best tool for estimate the counts of genes with very low expression rate ?

[sabaghianamir70]

i know these two are similar. but im make my point more clear this time. thank you anyway

[ATpoint]

Did you try salmon? I think it is still the best option to make use of problematic reads such as multimappers.

[sabaghianamir70]

since i working in galaxy plat form . working with galaxy is a little confusing and different from other tools i used. because it using data without needing to aligner. what the exact steps of Salmon ? i mean after trimming. we use salmon and featureCounts? i dont know what workflow should i use

[swbarnes2]

Why don't you just try it first? It's right there on Galaxy. Try it and then you can come back with specific questions.

Answer 1

[swbarnes2]

Your read counter might be refusing to count reads that do not align uniquely. Pierre's command will let you count reads in the region, even if your read counter won't assign them to a gene.