featureCount with custom GTF file

[haritz_irizar]

Hi all:

My name is Haritz Irizar and I am a postdoctoral fellow at the Icahn School of Medicine at Mount Sinai.

Working with RNAseq data, our group routinely uses featurecounts to obtain both gene-level and exon-level count matrices from .bam files.

In our last analysis, and with the objective to obtain the read counts not only for the genes of the host but also for the bacteria of the microbiome from the RNAseq data obtained from mouse colon biopsies, we have built a “superGenome” reference composed of around 1500 microbial genomes and created a custom GTF annotations file for that superGenome.

After some problems, we have managed to do the alignment with STAR using both the mouse genome and the superGenome, but when we have tried to use featurecounts on the resulting .bam files, we get an error concerning the GTF file.

To build the count matrix, we have concatenated the mouse annotation GTF and the superGenome annotation GTF files and fed the concatenated GTF to featurecounts and this is the error we get for several lines of the concatenated GTF file that correspond to the microbial annotations:

Warning: the feature on the 1570797-th line has zero coordinate or zero lengths


Warning: the feature on the 1570798-th line has zero coordinate or zero lengths

…

Any idea of what could be wrong?

Could please any of you have a look at the error logs of the featurecounts run (attached in pdf format) and at the extract of the GTF annotation file for the microbial genomes (in .xml format)?

Thanks a lot,

Haritz

[GenoMax]

Can you post the output of

$ sed -n '1570797,1570798p' your_gtf_file

so we can see that is in those two lines that generates an error?

[haritz_irizar]

Hi GenoMax:

These are 2 of the lines (lines 1570797 and 1570798) of the concatenated GTF file that give an error:

SuperGenome custom exon 2422121283 2423917566 . + . gene_id "Leuconostoc"; transcript_id "Leuconostoc_trans_784"; exon_id "Leuconostoc citreum KM20, complete genome";
SuperGenome custom exon 2423927567 2425821065 . + . gene_id "Leuconostoc"; transcript_id "Leuconostoc_trans_785"; exon_id "Leuconostoc gelidum JB7, complete genome";

Thanks for your help,

Haritz