standard gff3 file format is incompatible with count_reads.R

[mpgriesh]

Hi, I would like to use BactSeq with de novo assemblies rather than those available from NCBI. I have a .gff3 file from Bakta annotation and I would prefer to use that as the ref_ann file.

I have tried converting it to .bed, .gff, and even converted it to .gtf. Regardless, I get the following error thrown by count_reads.R. I have adjusted the GTF.featureType option to be 'CDS' rather than 'gene'.

ERROR: no features were loaded in format GTF. The annotation format can be specified by the 'isGTFAnnotationFile' option, and the required feature type can be specified by the 'GTF.featureType' option.

[adamd3]

Hi,
Could you share your GFF file (or just a few lines from it)? there are often subtle differences between sources that could cause the GFF parsing to fail.

note the example in the test_data directory here:
https://raw.githubusercontent.com/adamd3/BactSeq/refs/heads/main/test_data/Mabs.gff3

[mohitsharma-123]

Hi im using gff3 file from bakta and another time gff file from prokka but got error please help me

i also tried to convert gff3 file from bakta to gtf file using GFFRead and then tried gtf file but still getting error

(gffread) mohitsharma@deep:~/5sb_myxobacteria_completed/genome_processing$ nextflow run BactSeq --data_dir /data/mohitsharma/transcriptomics/test/ --sample_file /home/mohitsharma/5sb_myxobacteria_completed/bactseq/sample_test.tsv --ref_genome /home/mohitsharma/5sb_myxobacteria_completed/genome_processing/input_genomes/5SB.fna --ref_ann /home/mohitsharma/5sb_myxobacteria_completed/genome_processing/5SB.gtf -profile singularity --cont_tabl /home/mohitsharma/5sb_myxobacteria_completed/bactseq/contrasts_test.tsv --outdir test_BactSeq_output --paired

N E X T F L O W ~ version 24.10.3

Launching https://github.com/adamd3/BactSeq [nostalgic_leavitt] DSL2 - revision: 699649e [main]

executor > local (7)
[a6/49eb98] process > MAKE_META_FILE (sample_test.tsv) [100%] 1 of 1 ✔
[fc/689084] process > TRIMGALORE (5SB_30) [100%] 2 of 2 ✔
[a2/33200a] process > MAKE_BWA_INDEX (5SB.fna) [100%] 1 of 1 ✔
[ff/030be5] process > BWA_ALIGN (5SB_0) [100%] 2 of 2 ✔
[cd/d598f6] process > COUNT_READS (5SB.gtf) [ 0%] 0 of 1
[- ] process > NORMALISE_COUNTS -
[- ] process > PCA_SAMPLES -
[- ] process > DIFF_EXPRESSION -
ERROR ~ Error executing process > 'COUNT_READS (5SB.gtf)'

Caused by:
Process COUNT_READS (5SB.gtf) terminated with an error exit status (1)

Command executed:

count_reads.R -p TRUE -s reverse -m sample_metadata.tsv -g 5SB.gtf -t 12

Command exit status:
1

Command output:

      ==========     _____ _    _ ____  _____  ______          _____  
      =====         / ____| |  | |  _ \|  __ \|  ____|   /\   |  __ \ 
        =====      | (___ | |  | | |_) | |__) | |__     /  \  | |  | |
          ====      \___ \| |  | |  _ <|  _  /|  __|   / /\ \ | |  | |
            ====    ____) | |__| | |_) | | \ \| |____ / ____ \| |__| |
      ==========   |_____/ \____/|____/|_|  \_\______/_/    \_\_____/
     Rsubread 2.20.0

//========================== featureCounts setting ===========================\
|| ||
|| Input files : 2 BAM files ||
|| ||
|| 5SB_0.bam ||
|| 5SB_30.bam ||
|| ||
|| Paired-end : yes ||
|| Count read pairs : yes ||
|| Annotation : 5SB.gtf (GTF) ||
|| Dir for temp files : . ||
|| Threads : 12 ||
|| Level : meta-feature level ||
|| Multimapping reads : counted (fractional) ||
|| Multi-overlapping reads : not counted ||
|| Min overlapping bases : 1 ||
|| ||
\============================================================================//

//================================= Running ==================================\
|| ||
|| Load annotation file 5SB.gtf ... ||
ERROR: no features were loaded in format GTF. The annotation format can be specified by the 'isGTFAnnotationFile' option, and the required feature type can be specified by the 'GTF.featureType' option.

No counts were generated.

Command error:

      ==========     _____ _    _ ____  _____  ______          _____  
      =====         / ____| |  | |  _ \|  __ \|  ____|   /\   |  __ \ 
        =====      | (___ | |  | | |_) | |__) | |__     /  \  | |  | |
          ====      \___ \| |  | |  _ <|  _  /|  __|   / /\ \ | |  | |
            ====    ____) | |__| | |_) | | \ \| |____ / ____ \| |__| |
      ==========   |_____/ \____/|____/|_|  \_\______/_/    \_\_____/
     Rsubread 2.20.0

//========================== featureCounts setting ===========================\
executor > local (7)
[a6/49eb98] process > MAKE_META_FILE (sample_test.tsv) [100%] 1 of 1 ✔
[fc/689084] process > TRIMGALORE (5SB_30) [100%] 2 of 2 ✔
[a2/33200a] process > MAKE_BWA_INDEX (5SB.fna) [100%] 1 of 1 ✔
[ff/030be5] process > BWA_ALIGN (5SB_0) [100%] 2 of 2 ✔
[cd/d598f6] process > COUNT_READS (5SB.gtf) [100%] 1 of 1, failed: 1 ✘
[- ] process > NORMALISE_COUNTS -
[- ] process > PCA_SAMPLES -
[- ] process > DIFF_EXPRESSION -
ERROR ~ Error executing process > 'COUNT_READS (5SB.gtf)'

Caused by:
Process COUNT_READS (5SB.gtf) terminated with an error exit status (1)

Command executed:

count_reads.R -p TRUE -s reverse -m sample_metadata.tsv -g 5SB.gtf -t 12

Command exit status:
1

Command output:

      ==========     _____ _    _ ____  _____  ______          _____  
      =====         / ____| |  | |  _ \|  __ \|  ____|   /\   |  __ \ 
        =====      | (___ | |  | | |_) | |__) | |__     /  \  | |  | |
          ====      \___ \| |  | |  _ <|  _  /|  __|   / /\ \ | |  | |
            ====    ____) | |__| | |_) | | \ \| |____ / ____ \| |__| |
      ==========   |_____/ \____/|____/|_|  \_\______/_/    \_\_____/
     Rsubread 2.20.0

//========================== featureCounts setting ===========================\
|| ||
|| Input files : 2 BAM files ||
|| ||
|| 5SB_0.bam ||
|| 5SB_30.bam ||
|| ||
|| Paired-end : yes ||
|| Count read pairs : yes ||
|| Annotation : 5SB.gtf (GTF) ||
|| Dir for temp files : . ||
|| Threads : 12 ||
|| Level : meta-feature level ||
|| Multimapping reads : counted (fractional) ||
|| Multi-overlapping reads : not counted ||
|| Min overlapping bases : 1 ||
|| ||
\============================================================================//

//================================= Running ==================================\
|| ||
|| Load annotation file 5SB.gtf ... ||
ERROR: no features were loaded in format GTF. The annotation format can be specified by the 'isGTFAnnotationFile' option, and the required feature type can be specified by the 'GTF.featureType' option.

No counts were generated.

Command error:

      ==========     _____ _    _ ____  _____  ______          _____  
      =====         / ____| |  | |  _ \|  __ \|  ____|   /\   |  __ \ 
        =====      | (___ | |  | | |_) | |__) | |__     /  \  | |  | |
          ====      \___ \| |  | |  _ <|  _  /|  __|   / /\ \ | |  | |
            ====    ____) | |__| | |_) | | \ \| |____ / ____ \| |__| |
      ==========   |_____/ \____/|____/|_|  \_\______/_/    \_\_____/
     Rsubread 2.20.0

//========================== featureCounts setting ===========================\
|| ||
|| Input files : 2 BAM files ||
|| ||
|| 5SB_0.bam ||
|| 5SB_30.bam ||
|| ||
|| Paired-end : yes ||
|| Count read pairs : yes ||
|| Annotation : 5SB.gtf (GTF) ||
|| Dir for temp files : . ||
|| Threads : 12 ||
|| Level : meta-feature level ||
|| Multimapping reads : counted (fractional) ||
|| Multi-overlapping reads : not counted ||
|| Min overlapping bases : 1 ||
|| ||
\============================================================================//

//================================= Running ==================================\
|| ||
|| Load annotation file 5SB.gtf ... ||
ERROR: no features were loaded in format GTF. The annotation format can be specified by the 'isGTFAnnotationFile' option, and the required feature type can be specified by the 'GTF.featureType' option.

No counts were generated.
Execution halted

Work dir:
/home/mohitsharma/5sb_myxobacteria_completed/genome_processing/work/cd/d598f66fe78efbc60a1e283269999e

Container:
/home/mohitsharma/5sb_myxobacteria_completed/genome_processing/./adamd3-bactseq-latest.img

Tip: when you have fixed the problem you can continue the execution adding the option -resume to the run command line

-- Check '.nextflow.log' file for details