Hello! I can't run featureCounts with gff3 file. Please, help!

My gff3 file structure is:

chr1    .   miRNA_primary_transcript    17369   17436   .   -   .   ID=MI0022705;Alias=MI0022705;Name=hsa-mir-6859-1
chr1    .   miRNA   17409   17431   .   -   .   ID=MIMAT0027618;Alias=MIMAT0027618;Name=hsa-miR-6859-5p;Derives_from=MI0022705
chr1    .   miRNA   17369   17391   .   -   .   ID=MIMAT0027619;Alias=MIMAT0027619;Name=hsa-miR-6859-3p;Derives_from=MI0022705

Command line:

./featureCounts -F -a ./../annotation/hsa_miR.gff3 -o ./../../../projects/vesicles/1_repeat_vesicles/counts_K.txt ./../../../projects/vesicles/1_repeat_vesicles/1_repeat_vesicles_NONCODE_K_miRNA.sam

Answer:

Warning: Unknown annotation format: -a. GTF format is used.

Please tell me what to do, I have already tried everything. Carefully studied the manual,

-F(isGTFAnnotationFile): Specify the format of the annotation file. Acceptable formats include ‘GTF’ and ‘SAF’ (see Section 6.2.2 for details). By default,C version of featureCounts program accepts a GTFformat annotation and R version accepts a SAF format annotation. In-built annotations in SAF format are provided.

tried to use cufflinks/gffread to change the file format, but nothing happened either.

./gffread ./../../tools/subread-1.6.4-Linux-x86_64/annotation/hsa_miR.gff3 -T -o hsa_miR.gtf

Please, help!

You did not use the -F option correctly - you need to provide a value to it. For your case, you can specify this option as -F 'GTF'. You actually do not need to specify this option at all as 'GTF' is the default value.

The 'GTF' value denotes both GTF format and compatible GFF formats such as GFF3 format used in your analysis. This is described in the screen description for this parameter (you can see this by just typing featureCounts at your prompt):

  -F <string>         Specify format of the provided annotation file. Acceptable
                      formats include 'GTF' (or compatible GFF format) and
                      'SAF'. 'GTF' by default.  For SAF format, please refer to
                      Users Guide.

Also the default values for attribute type and feature type parameters won't work for your annotation. You might use the following setting for these two parameters:

-t  'miRNA' -g 'ID'