Hello,
I know that this topic has been massively covered in this forum, but I wasn't able to find the right answer for my questions.
Basically, I'm dealing with some bacterial RNA-seq data. I have two biological replicates and two conditions (so four libraries). I used bowtie for alignment of my reads and subsequently DESeq to calculate means/fold change/stats in order to compare my two conditions.
Now, I would like to calculate gene coverage per 1kb (relative) in order to compare absolute expression of every gene. I was thinking that if I take number of reads and I divide it by the gene length and then multiply by 1000 I will get some kind of "gene expression ratio per 1kb" which I could use to compare for all my genes. So, I could say that gene "A" is ten times more expressed then gene "B" just by comparing those ratios. Is this the right way of doing it, or perhaps I'm missing something?
Also, I sought to use BEDTools for this, but I don't know if I could insert multiple files (biological replicates) for my analysis. Anybody know how?
Finaly, anybody know how to extract gene length from a GFF or even GenBank file? BEDTools does it but I have duplicated values (for gene/CDS/exons).
Thanks in advance! I don't know what I would do without this forum...
TP
I know that this topic has been massively covered in this forum, but I wasn't able to find the right answer for my questions.
Basically, I'm dealing with some bacterial RNA-seq data. I have two biological replicates and two conditions (so four libraries). I used bowtie for alignment of my reads and subsequently DESeq to calculate means/fold change/stats in order to compare my two conditions.
Now, I would like to calculate gene coverage per 1kb (relative) in order to compare absolute expression of every gene. I was thinking that if I take number of reads and I divide it by the gene length and then multiply by 1000 I will get some kind of "gene expression ratio per 1kb" which I could use to compare for all my genes. So, I could say that gene "A" is ten times more expressed then gene "B" just by comparing those ratios. Is this the right way of doing it, or perhaps I'm missing something?
Also, I sought to use BEDTools for this, but I don't know if I could insert multiple files (biological replicates) for my analysis. Anybody know how?
Finaly, anybody know how to extract gene length from a GFF or even GenBank file? BEDTools does it but I have duplicated values (for gene/CDS/exons).
Thanks in advance! I don't know what I would do without this forum...
TP
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