Question

Why does Rsubread featureCounts with Ensemble GRCm38.96.GTF picks ensembl_exon_id, instead of gene_id

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4.9 years ago

akh22 ▴ 110

I run the following :

featureCounts(data.bam, isPairedEnd = T, isGTFAnnotationFile=T,annot.ext="Mus_musculus.GRCm38.96.gtf", GTF.attrType = "gene_id")

Even if I specify a GTF Type attribute to "gene_id", GeneID in the featureCounts output shows "ensembl_exon_id", instead of "ensembl_gene_id". Is there any way to tell featureCounts to use "ensembl_gene_id" ?

Thanks in advance.

RNA-Seq R assembly gene • 3.5k views

ADD COMMENT • link 4.9 years ago by akh22 ▴ 110

score 2 · Answer 1 · 2019-06-09

I sort of figured this out. I had run the same script in.another of my Macmini running Mojave and exactly the same versions of Rstudio and R (bioconductor packages) and worked as expected. I noticed Rstudio was acting wacky so I deleted and reinstalled Rstudio and after that, the featureCount script punched out gene_id rather than exon_ID as mentioned previously.

Anyway, thanks for all the help that pointed me to the right direction.

h.mon · Answer 2 · 2019-06-07

1

Entering edit mode

4.9 years ago

AB ▴ 360

Probably because your gtf file doesnt have a "gene_id". Try

featureCounts(data.bam, isPairedEnd = T, isGTFAnnotationFile=T,annot.ext="Mus_musculus.GRCm38.96.gtf", GTF.attrType = "ensembl_gene_id")

ADD COMMENT • link 4.9 years ago by AB ▴ 360

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Ensembl gtf annotations do include an "gene_id" attribute. So, unless akh22 edited the Ensembl gtf annotation somehow, or downloaded from another source, the issue must be elsewhere.

zcat Mus_musculus.GRCm38.96.gtf.gz | head

#!genome-build GRCm38.p6
#!genome-version GRCm38
#!genome-date 2012-01
#!genome-build-accession NCBI:GCA_000001635.8
#!genebuild-last-updated 2018-11
1 havana  gene    3073253 3074322 .   +   .   gene_id "ENSMUSG00000102693"; gene_version "1"; gene_name "4933401J01Rik"; gene_source "havana"; gene_biotype "TEC";
1 havana  transcript  3073253 3074322 .   +   .   gene_id "ENSMUSG00000102693"; gene_version "1"; transcript_id "ENSMUST00000193812"; transcript_version "1"; gene_name "4933401J01Rik"; gene_source "havana"; gene_biotype "TEC"; transcript_name "4933401J01Rik-201"; transcript_source "havana"; transcript_biotype "TEC"; tag "basic"; transcript_support_level "NA";
1 havana  exon    3073253 3074322 .   +   .   gene_id "ENSMUSG00000102693"; gene_version "1"; transcript_id "ENSMUST00000193812"; transcript_version "1"; exon_number "1"; gene_name "4933401J01Rik"; gene_source "havana"; gene_biotype "TEC"; transcript_name "4933401J01Rik-201"; transcript_source "havana"; transcript_biotype "TEC"; exon_id "ENSMUSE00001343744"; exon_version "1"; tag "basic"; transcript_support_level "NA";
1 ensembl gene    3102016 3102125 .   +   .   gene_id "ENSMUSG00000064842"; gene_version "1"; gene_name "Gm26206"; gene_source "ensembl"; gene_biotype "snRNA";
1 ensembl transcript  3102016 3102125 .   +   .   gene_id "ENSMUSG00000064842"; gene_version "1"; transcript_id "ENSMUST00000082908"; transcript_version "1"; gene_name "Gm26206"; gene_source "ensembl"; gene_biotype "snRNA"; transcript_name "Gm26206-201"; transcript_source "ensembl"; transcript_biotype "snRNA"; tag "basic"; transcript_support_level "NA";

ADD REPLY • link 4.9 years ago by h.mon 35k

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No I did not edit GTF at all. I just down loaded directly from Ensembl FTP.

#!genome-build GRCm38.p6
#!genome-version GRCm38
#!genome-date 2012-01
#!genome-build-accession NCBI:GCA_000001635.8
#!genebuild-last-updated 2018-11
1   havana  gene    3073253 3074322 .   +   .   gene_id "ENSMUSG00000102693"; gene_version "1"; gene_name "4933401J01Rik"; gene_source "havana"; gene_biotype "TEC";
1   havana  transcript  3073253 3074322 .   +   .   gene_id "ENSMUSG00000102693"; gene_version "1"; transcript_id "ENSMUST00000193812"; transcript_version "1"; gene_name "4933401J01Rik"; gene_source "havana"; gene_biotype "TEC"; transcript_name "4933401J01Rik-201"; transcript_source "havana"; transcript_biotype "TEC"; tag "basic"; transcript_support_level "NA";
1   havana  exon    3073253 3074322 .   +   .   gene_id "ENSMUSG00000102693"; gene_version "1"; transcript_id "ENSMUST00000193812"; transcript_version "1"; exon_number "1"; gene_name "4933401J01Rik"; gene_source "havana"; gene_biotype "TEC"; transcript_name "4933401J01Rik-201"; transcript_source "havana"; transcript_biotype "TEC"; exon_id "ENSMUSE00001343744"; exon_version "1"; tag "basic"; transcript_support_level "NA";
1   ensembl gene    3102016 3102125 .   +   .   gene_id "ENSMUSG00000064842"; gene_version "1"; gene_name "Gm26206"; gene_source "ensembl"; gene_biotype "snRNA";
1   ensembl transcript  3102016 3102125 .   +   .   gene_id "ENSMUSG00000064842"; gene_version "1"; transcript_id "ENSMUST00000082908"; transcript_version "1"; gene_name "Gm26206"; gene_source "ensembl"; gene_biotype "snRNA"; transcript_name "Gm26206-201"; transcript_source "ensembl"; transcript_biotype "snRNA"; tag "basic"; transcript_support_level "NA";
1   ensembl exon    3102016 3102125 .   +   .   gene_id "ENSMUSG00000064842"; gene_version "1"; transcript_id "ENSMUST00000082908"; transcript_version "1"; exon_number "1"; gene_name "Gm26206"; gene_source "ensembl"; gene_biotype "snRNA"; transcript_name "Gm26206-201"; transcript_source "ensembl"; transcript_biotype "snRNA"; exon_id "ENSMUSE00000522066"; exon_version "1"; tag "basic"; transcript_support_level "NA";
1   ensembl_havana  gene    3205901 3671498 .   -   .   gene_id "ENSMUSG00000051951"; gene_version "5"; gene_name "Xkr4"; gene_source "ensembl_havana"; gene_biotype "protein_coding";

ADD REPLY • link updated 4.9 years ago by h.mon 35k • written 4.9 years ago by akh22 ▴ 110

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Can you show a snippet of the featureCounts output?

counts <- featureCounts(data.bam, isPairedEnd = T, isGTFAnnotationFile=T,annot.ext="Mus_musculus.GRCm38.96.gtf", GTF.attrType = "gene_id")
head( counts, n =10 )

ADD REPLY • link 4.9 years ago by h.mon 35k

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here we go;

$counts
                   Skin.25.IT.1.21.19.bam Skin.26.IT.1.21.19.bam Skin.27.IT.1.21.19.bam Skin.28.IT.1.21.19.bam Skin.29.IT.1.21.19.bam
ENSMUSE00001343744                      0                      0                      0                      0                      0
ENSMUSE00000522066                      0                      0                      0                      0                      0
ENSMUSE00000866652                      0                      1                      1                      0                      0
ENSMUSE00000858910                      0                      0                      0                      0                      0
ENSMUSE00000867897                      0                      0                      0                      0                      0
ENSMUSE00000863980                      0                      0                      0                      1                      0
ENSMUSE00000448840                      1                      0                      1                      1                      0
ENSMUSE00000449517                      1                      2                      2                      0                      1
ENSMUSE00000485541                      6                      4                      3                      1                      8
ENSMUSE00001339323                      1                      0                      0                      0                      0
ENSMUSE00001343189                      0                      0                      0                      1                      0
ENSMUSE00001343686                      0                      0                      0                      0                      0
ENSMUSE00001337180                      0                      0                      0                      0                      0
ENSMUSE00000869502                      0                      0                      0                      0                      0
ENSMUSE00000864479                      0                      0                      0                      0                      0
ENSMUSE00001345667                      0                      0                      0                      0                      0
ENSMUSE00001343235                      0                      0                      2                      0                      0
ENSMUSE00001343966                      0                      1                      1                      0                      0
ENSMUSE00001339227                     33                     24                     23                     15                     29

ADD REPLY • link updated 4.9 years ago by h.mon 35k • written 4.9 years ago by akh22 ▴ 110