Using featureCounts with SAF annotation file gives "Error: only GTF files contain additional attributes"
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atan • 0
@86ae790f
Last seen 30 minutes ago
United States

I can't figure out how to get featureCounts to generate a count matrix. I have a gff3 file that I've converted to a SAF. Regardless of whether I use the gff3 or the SAF (and whether the SAF is referenced as a .cvs, .tsv, or a data frame), I get an output of "Error: only GTF files contain additional attributes." I don't have a gtf file available for the version of the genome that I'm working with.

counts <- featureCounts(
  annot.ext = "~/Desktop/Spurp_reference_files/Spurp_genome/Sp5_genome_new_download/sp5_0_GCF.saf",
  isPairedEnd = TRUE,
  "C10St14_R-final.bam", "C10St18_R-final.bam", "C11F14_R-final.bam",
  "C11F18_R-final.bam", "C12F14_R2-final.bam", "C12F18_R-final.bam",
  "C13F14_R-final.bam", "C13F18_R-final.bam", "C8St14_R2-final.bam",
  "C8St18_R-final.bam", "C9St14_R2-final.bam", "C9St18_R-final.bam"
)

Output is:

ERROR: only GTF files contain additional attributes
No counts were generated.
Warning messages:
1: In paste("readSummary", ann, files_C, fout, .flatten.and.numeric(countReadPairs),  :
  NAs introduced by coercion
##followed by four more of the same warning

Head of my SAF looks like:

   GeneID            Chr    Start      End Strand
185/333 NW_022145545.1   759692   759767      +
185/333 NW_022145545.1   760175   761104      +
4e-bp NW_022145595.1 15804524 15804558      -
4e-bp NW_022145595.1 15805788 15805952      -
4e-bp NW_022145595.1 15808337 15808478      -
ABCB1 NW_022145604.1  6642470  6642583      +

I've also tried running featureCounts via command line but that gave an error that "no features loaded in format SAF" and terminated.

RNASeq Rsubread • 767 views
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SAF files must not have a header.

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I tried reading in the .csv version of the SAF with header = F and I'm still getting an error. I'm running:

counts <- featureCounts(
  annot.ext = inSaf,
  isPairedEnd = TRUE,
  isGTFAnnotationFile = F,
  "C10St14_R-final.bam", "C10St18_R-final.bam", "C11F14_R-final.bam",
  "C11F18_R-final.bam", "C12F14_R2-final.bam", "C12F18_R-final.bam",
  "C13F14_R-final.bam", "C13F18_R-final.bam", "C8St14_R2-final.bam",
  "C8St18_R-final.bam", "C9St14_R2-final.bam", "C9St18_R-final.bam"
)

and it's returning:

Error in .get.annotation.file(annot.inbuilt, annot.ext, isGTFAnnotationFile) : 
  One or more required columns are missing in the provided annotation data. Please refer to help page for annotation format.

Although, if I remove the "GeneID", etc. as column names, R puts in V1, etc. in place? That still gives the same error of the "required columns are missing."

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I was talking about the command line version.

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For some reason, that's still not working for me on command line.

I ended up figuring out another way around, though, by converting the gff3 to gtf and then double checking which GTF feature type needed to be listed in the options.

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Your bam file names should be stored in a vector and then provided to the files parameter.

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Thank you! I ended up running it with a gtf on command line, partially because I couldn't figure out how to list multiple files in R for input. I'll note this for future reference.

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