I am very new to programming in general, and I'm trying my best to teach myself R for analyzing RNA-seq data we have.
I am using this guide and have gotten to the step where I need to align to mapped reads. However, when I use the GTF file to align my reads, I don't get any alignments.
I suspect that I used the wrong file for the genome alignment, the wrong GTF, or both.
Can someone please confirm or deny?
My RNA seq data is from mouse samples. I used this link to download Mus_musculus.GRCm39.104.gtf.gz, and the reference genome I used is the cDNA sequence from here.
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cdeantoneo31 •
confirmed :)
in a nutshell: there are two approaches you can use: either using genome or transcriptome. When you go for the genome approach you need to get the genome fasta file (& index it for your mapper) and a gff/gtf file that corresponds to that genome. Then align your read to the genome and use the gff file to link those mappings to the features in your gff file. Alternatively you can for the transcriptome route and then you only need the fasta file of the transcripts (cDNA). Align your reads to the transcripts and afterwards use samtools or such to count how many reads align to each transcript no fancy counting thing needed and thus no gff file needed either.
In case you want to do what your described you can only get that to work if you have a gff file that indicates the start and stop on each cDNA.
Yes, If you use genome based method ,you can use subjunc to align fastq to your Genome and use featurecount to quantify read counts, at this time ,you may need genome sequence and GTF file.
if you use transcriptome based method ,only one thing you need to do is using salmon to quantify read counts, input file are fastq file, transcriptome sequence and GTF file.
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