This is a test version of Biostars. For the public version, visit https://www.biostars.org.
gene length for calculating TPM values

I want to convert count to TPM similar to this post Calculating TPM Values . However my question is can we use the width column from the GTF file as gene length for TPM calculation?

 library("GenomicFeatures")
 gtf_txdb <- makeTxDbFromGFF("gencode.v33.annotation.gtf.gz")
 gene_list <- genes(gtf_txdb)
 gene_list <- as.data.frame(gene_list)
 gene_list[1:3, 1:4]

                   seqnames     start       end width
ENSG00000000003.15     chrX 100627108 100639991 12884
ENSG00000000005.6      chrX 100584936 100599885 14950
ENSG00000000419.12    chr20  50934867  50958555 23689
rna-seq sequencing tpm genome
• 11,608 views • link
written by DNA2RNA • DNA2RNA

3 answers

For future reference the answere is at Extract Total Non-Overlapping Exon Length Per Gene With Bioconductor

library(GenomicFeatures) 
txdb <- makeTranscriptDbFromGFF("yourFile.gtf",format="gtf")
exons.list.per.gene <- exonsBy(txdb,by="gene")
exonic.gene.sizes <- as.data.frame(sum(width(reduce(exons.list.per.gene))))
• 0 views • link
written by DNA2RNA • DNA2RNA

That's the sum of all the exons for that genes in your gtf. What if in your sample, all the reads are coming from a transcript which is half that length?

• 0 views • link
updated by Ram Ram • written by swbarnes2 swbarnes2

Attention to function name :

makeTxDbFromGFF

and not

makeTranscriptDbFromGFF

for version 1.40 and older of GenomicFeatures,at least.

• 0 views • link
updated by Ram Ram • written by mariakondili13 • mariakondili13

The width is calculated in IRanges as start-end, which gives you the whole length of the gene. I think TPM would depend on total CDS/exon length, not total gene length - intron lengths should not be accounted for. After all, transcripts don't have introns.

• 0 views • link
written by Ram Ram

Here the width column includes any existing introns, so you can't use the width from this particular table to calculate TPM. The gencode.v33.annotation.gtf likely has the intron ranges, which you can use for your calculation.

• 0 views • link
written by geneticatt • geneticatt

Log in to answer this question.

More posts like this