I am using StringTie v3.0.0 to discover novel transcripts from short-read RNA-seq data. After quantification, I noticed that some transcripts/exons have a coverage (cov) of 0, even though reads are aligned to these regions according to samtools view. Pipeline Used:

1-Transcript assembly per sample:

`stringtie -p 8 -c 2 -j 2 "$BAM_FILE" -G $GTF_FILE -o "$OUTPUT_GTF"`

2-Merging assembled transcripts:

`stringtie --merge $INPUT_DIR/*reconstructed.gtf -o $OUT_DIR/stringtie_merged_without_ref_output.gtf`

3-Quantification of transcripts:

`stringtie -p 12 -e -B $BAM_FILE -G $GTF_merged -o $OUTPUT_GTF/*_quantified.gtf`

Issue Observed:

For example, the transcript MSTRG.7.2 has zero coverage in t_data.ctab and _quantified.gtf despite read alignments. Results from grep in the .gtf file:

grep MSTRG.7.2 X2-WT_quantified.gtf

1   StringTie   transcript  4561613 4567577 .   -   .   gene_id "MSTRG.7"; transcript_id "MSTRG.7.2"; cov "0.0"; FPKM "0.000000"; TPM "0.000000";
1   StringTie   exon    4561613 4562891 .   -   .   gene_id "MSTRG.7"; transcript_id "MSTRG.7.2"; exon_number "1"; cov "0.0";
1   StringTie   exon    4563995 4564086 .   -   .   gene_id "MSTRG.7"; transcript_id "MSTRG.7.2"; exon_number "2"; cov "0.0";
1   StringTie   exon    4565359 4566165 .   -   .   gene_id "MSTRG.7"; transcript_id "MSTRG.7.2"; exon_number "3"; cov "0.0";
1   StringTie   exon    4566514 4567577 .   -   .   gene_id "MSTRG.7"; transcript_id "MSTRG.7.2"; exon_number "4"; cov "0.0";

Check in t_data.ctab:

grep MSTRG.7.2 t_data.ctab

t_id    chr strand  start   end t_name  num_exons   length  gene_id gene_name   cov FPKM
9   1   -   4561613 4567577 MSTRG.7.2   4   3242    MSTRG.7 .   0.000000    0.000000

Check in e_data.ctab (exon coverage):

    e_id    chr strand  start   end rcount  ucount  mrcount cov cov_sd  mcov    mcov_sd
    46  1   -   4561613 4562891 56  56  56.00   2.6654  1.8036  2.6654  1.8036
    43  1   -   4563995 4564086 1   1   1.00    2.8043  1.5965  2.8043  1.5965
    44  1   -   4565359 4566165 18  18  18.00   1.4597  1.3443  1.4597  1.3443
    45  1   -   4566514 4567577 6   6   6.00    0.3327  0.5931  0.3327  0.5931

Read counts verification with samtools:

samtools view -c X2-WT_S21Aligned.sortedByCoord.out.bam 1:4561613-4562891
58
samtools view -c X2-WT_S21Aligned.sortedByCoord.out.bam 1:4563995-4564086
6
samtools view -c X2-WT_S21Aligned.sortedByCoord.out.bam 1:4565359-4566165
21
samtools view -c X2-WT_S21Aligned.sortedByCoord.out.bam 1:4566514-4567577
6

We can see that reads are aligned to these exons, but cov remains 0 in t_data.ctab and _quantified.gtf. Questions: Why does StringTie assign a coverage of 0 even though reads are present in samtools view?