Question

HTSeq-count ambiguous results - all transcripts vs. single transcript

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6.1 years ago

oriolebaltimore ▴ 190

My aim is to count reads aligned to introns of human BRCA1. I am using HTSeq-count. I made a GTF file by obtaining bed file of intronic regions (Hg38) from UCSC table browser. I converted BED file to GTF based on Ensembl data format.

Bed file:

17  43045802    43047642    NM_007294.3_intron_0_0_chr17_43045803_r 0   -
17  43047703    43049120    NM_007294.3_intron_1_0_chr17_43047704_r 0   -
17  43049194    43051062    NM_007294.3_intron_2_0_chr17_43049195_r 0   -
17  43051117    43057051    NM_007294.3_intron_3_0_chr17_43051118_r 0   -
17  43057135    43063332    NM_007294.3_intron_4_0_chr17_43057136_r 0   -
17  43063373    43063873    NM_007294.3_intron_5_0_chr17_43063374_r 0   -
17  43063951    43067607    NM_007294.3_intron_6_0_chr17_43063952_r 0   -
17  43067695    43070927    NM_007294.3_intron_7_0_chr17_43067696_r 0   -
17  43071238    43074330    NM_007294.3_intron_8_0_chr17_43071239_r 0   -

GTF file:

17  Refseq  intron  43045802    43047642    .   -   .   transcript_id "NM_007294"; intron_number "NM_007294_intron_0";
17  Refseq  intron  43047703    43049120    .   -   .   transcript_id "NM_007294"; intron_number "NM_007294_intron_1";
17  Refseq  intron  43049194    43051062    .   -   .   transcript_id "NM_007294"; intron_number "NM_007294_intron_2";
17  Refseq  intron  43051117    43057051    .   -   .   transcript_id "NM_007294"; intron_number "NM_007294_intron_3";
17  Refseq  intron  43057135    43063332    .   -   .   transcript_id "NM_007294"; intron_number "NM_007294_intron_4";
17  Refseq  intron  43063373    43063873    .   -   .   transcript_id "NM_007294"; intron_number "NM_007294_intron_5";
17  Refseq  intron  43063951    43067607    .   -   .   transcript_id "NM_007294"; intron_number "NM_007294_intron_6";
17  Refseq  intron  43067695    43070927    .   -   .   transcript_id "NM_007294"; intron_number "NM_007294_intron_7";
17  Refseq  intron  43071238    43074330    .   -   .   transcript_id "NM_007294"; intron_number "NM_007294_intron_8";

There are multiple transcripts for this gene. If I count reads aligned to introns of NM_007294, I get the result.

NM_007294_intron_0  28
NM_007294_intron_1  21
NM_007294_intron_2  31
NM_007294_intron_3  22
NM_007294_intron_4  30
NM_007294_intron_5  10
NM_007294_intron_6  96
NM_007294_intron_7  73
NM_007294_intron_8  42

Now when I run HTSeq on same bam file giving GTF file for all BRCA transcript, I cannot get the same result:

NM_007294_intron_0  0
NM_007294_intron_1  0
NM_007294_intron_10 0
NM_007294_intron_11 0
NM_007294_intron_12 0
NM_007294_intron_13 0
NM_007294_intron_14 0
NM_007294_intron_15 0
NM_007294_intron_16 0
NM_007294_intron_17 0
NM_007294_intron_18 0
NM_007294_intron_19 0
NM_007294_intron_2  0
NM_007294_intron_20 0
NM_007294_intron_21 0
NM_007294_intron_3  0
NM_007294_intron_4  0
NM_007294_intron_5  0
NM_007294_intron_6  0
NM_007294_intron_7  0
NM_007294_intron_8  0
NM_007294_intron_9  0
NM_007297_intron_0  0
NM_007297_intron_1  0
NM_007297_intron_10 0
NM_007297_intron_11 0
NM_007297_intron_12 0
NM_007297_intron_13 0
NM_007297_intron_14 0
NM_007297_intron_15 0
NM_007297_intron_16 0
NM_007297_intron_17 0
NM_007297_intron_18 0
NM_007297_intron_19 0
NM_007297_intron_2  0
NM_007297_intron_20 0
NM_007297_intron_3  0
NM_007297_intron_4  0
NM_007297_intron_5  0
NM_007297_intron_6  0
NM_007297_intron_7  0
NM_007297_intron_8  0
NM_007297_intron_9  0
NM_007298_intron_0  0
NM_007298_intron_1  0
NM_007298_intron_10 0
NM_007298_intron_11 0
NM_007298_intron_12 0
NM_007298_intron_13 0
NM_007298_intron_14 0
NM_007298_intron_15 0
NM_007298_intron_16 0
NM_007298_intron_17 0
NM_007298_intron_18 0
NM_007298_intron_19 0
NM_007298_intron_2  0
NM_007298_intron_20 0
NM_007298_intron_3  0
NM_007298_intron_4  0
NM_007298_intron_5  0
NM_007298_intron_6  0
NM_007298_intron_7  0
NM_007298_intron_8  0
NM_007298_intron_9  0
NM_007299_intron_0  0
NM_007299_intron_1  0
NM_007299_intron_10 0
NM_007299_intron_11 0
NM_007299_intron_12 0
NM_007299_intron_13 0
NM_007299_intron_14 0
NM_007299_intron_15 0
NM_007299_intron_16 0
NM_007299_intron_17 0
NM_007299_intron_18 0
NM_007299_intron_19 0
NM_007299_intron_2  0
NM_007299_intron_20 0
NM_007299_intron_3  0
NM_007299_intron_4  0
NM_007299_intron_5  0
NM_007299_intron_6  0
NM_007299_intron_7  0
NM_007299_intron_8  0
NM_007299_intron_9  0
NM_007300_intron_0  0
NM_007300_intron_1  0
NM_007300_intron_10 0
NM_007300_intron_11 0
NM_007300_intron_12 0
NM_007300_intron_13 0
NM_007300_intron_14 0
NM_007300_intron_15 0
NM_007300_intron_16 0
NM_007300_intron_17 0
NM_007300_intron_18 0
NM_007300_intron_19 0
NM_007300_intron_2  0
NM_007300_intron_20 0
NM_007300_intron_21 0
NM_007300_intron_22 0
NM_007300_intron_3  0
NM_007300_intron_4  0
NM_007300_intron_5  0
NM_007300_intron_6  0
NM_007300_intron_7  0
NM_007300_intron_8  0

NM_007300_intron_9  0

How is this possible. whe I give a gtf file with one transcript, I get counts, however when GTF file with all transcripts of BRCA1 the resulting counts are 0.

It cannot be chromosome mapping issue (chr17 vs. 17) or any other issue that I can think of.

Can anyone help understand this ambiguous behavior of HTSeq.

htseq-count -f bam -r name -s reverse -t intron -i intron_number myBAM.bam introns_NM_007294.gtf ---- WORKS.

htseq-count -f bam -r name -s reverse -t intron -i intron_number myBAM.bam introns_all_transcripts.gtf --- Doesn't WORK.

Thanks Adrian.

GTF file:

17      Refseq  intron  43045802        43047642        .       -       .       transcript_id "NM_007294"; intron_number "NM_007294_intron_0";
17      Refseq  intron  43047703        43049120        .       -       .       transcript_id "NM_007294"; intron_number "NM_007294_intron_1";
17      Refseq  intron  43049194        43051062        .       -       .       transcript_id "NM_007294"; intron_number "NM_007294_intron_2";
17      Refseq  intron  43051117        43057051        .       -       .       transcript_id "NM_007294"; intron_number "NM_007294_intron_3";
17      Refseq  intron  43057135        43063332        .       -       .       transcript_id "NM_007294"; intron_number "NM_007294_intron_4";
17      Refseq  intron  43063373        43063873        .       -       .       transcript_id "NM_007294"; intron_number "NM_007294_intron_5";
17      Refseq  intron  43063951        43067607        .       -       .       transcript_id "NM_007294"; intron_number "NM_007294_intron_6";
17      Refseq  intron  43067695        43070927        .       -       .       transcript_id "NM_007294"; intron_number "NM_007294_intron_7";
17      Refseq  intron  43071238        43074330        .       -       .       transcript_id "NM_007294"; intron_number "NM_007294_intron_8";
17      Refseq  intron  43074521        43076487        .       -       .       transcript_id "NM_007294"; intron_number "NM_007294_intron_9";
17      Refseq  intron  43076614        43082403        .       -       .       transcript_id "NM_007294"; intron_number "NM_007294_intron_10";
17      Refseq  intron  43082575        43090943        .       -       .       transcript_id "NM_007294"; intron_number "NM_007294_intron_11";
17      Refseq  intron  43091032        43091434        .       -       .       transcript_id "NM_007294"; intron_number "NM_007294_intron_12";
17      Refseq  intron  43094860        43095845        .       -       .       transcript_id "NM_007294"; intron_number "NM_007294_intron_13";
17      Refseq  intron  43095922        43097243        .       -       .       transcript_id "NM_007294"; intron_number "NM_007294_intron_14";
17      Refseq  intron  43097289        43099774        .       -       .       transcript_id "NM_007294"; intron_number "NM_007294_intron_15";
17      Refseq  intron  43099880        43104121        .       -       .       transcript_id "NM_007294"; intron_number "NM_007294_intron_16";
17      Refseq  intron  43104261        43104867        .       -       .       transcript_id "NM_007294"; intron_number "NM_007294_intron_17";
17      Refseq  intron  43104956        43106455        .       -       .       transcript_id "NM_007294"; intron_number "NM_007294_intron_18";
17      Refseq  intron  43106533        43115725        .       -       .       transcript_id "NM_007294"; intron_number "NM_007294_intron_19";
17      Refseq  intron  43115779        43124016        .       -       .       transcript_id "NM_007294"; intron_number "NM_007294_intron_20";
17      Refseq  intron  43124115        43125270        .       -       .       transcript_id "NM_007294"; intron_number "NM_007294_intron_21";
17      Refseq  intron  43045802        43047642        .       -       .       transcript_id "NM_007297"; intron_number "NM_007297_intron_0";
17      Refseq  intron  43047703        43049120        .       -       .       transcript_id "NM_007297"; intron_number "NM_007297_intron_1";
17      Refseq  intron  43049194        43051062        .       -       .       transcript_id "NM_007297"; intron_number "NM_007297_intron_2";
17      Refseq  intron  43051117        43057051        .       -       .       transcript_id "NM_007297"; intron_number "NM_007297_intron_3";
17      Refseq  intron  43057135        43063332        .       -       .       transcript_id "NM_007297"; intron_number "NM_007297_intron_4";
17      Refseq  intron  43063373        43063873        .       -       .       transcript_id "NM_007297"; intron_number "NM_007297_intron_5";
17      Refseq  intron  43063951        43067607        .       -       .       transcript_id "NM_007297"; intron_number "NM_007297_intron_6";
17      Refseq  intron  43067695        43070927        .       -       .       transcript_id "NM_007297"; intron_number "NM_007297_intron_7";
17      Refseq  intron  43071238        43074330        .       -       .       transcript_id "NM_007297"; intron_number "NM_007297_intron_8";
17      Refseq  intron  43074521        43076487        .       -       .       transcript_id "NM_007297"; intron_number "NM_007297_intron_9";
17      Refseq  intron  43076614        43082403        .       -       .       transcript_id "NM_007297"; intron_number "NM_007297_intron_10";
17      Refseq  intron  43082575        43090943        .       -       .       transcript_id "NM_007297"; intron_number "NM_007297_intron_11";
17      Refseq  intron  43091032        43091434        .       -       .       transcript_id "NM_007297"; intron_number "NM_007297_intron_12";
17      Refseq  intron  43094860        43095845        .       -       .       transcript_id "NM_007297"; intron_number "NM_007297_intron_13";
17      Refseq  intron  43095922        43097243        .       -       .       transcript_id "NM_007297"; intron_number "NM_007297_intron_14";
17      Refseq  intron  43097289        43099774        .       -       .       transcript_id "NM_007297"; intron_number "NM_007297_intron_15";
17      Refseq  intron  43099880        43104121        .       -       .       transcript_id "NM_007297"; intron_number "NM_007297_intron_16";
17      Refseq  intron  43104261        43104867        .       -       .       transcript_id "NM_007297"; intron_number "NM_007297_intron_17";
17      Refseq  intron  43104956        43106455        .       -       .       transcript_id "NM_007297"; intron_number "NM_007297_intron_18";
17      Refseq  intron  43106533        43124016        .       -       .       transcript_id "NM_007297"; intron_number "NM_007297_intron_19";
17      Refseq  intron  43124115        43125276        .       -       .       transcript_id "NM_007297"; intron_number "NM_007297_intron_20";
17      Refseq  intron  43045802        43047642        .       -       .       transcript_id "NM_007298"; intron_number "NM_007298_intron_0";
17      Refseq  intron  43047703        43049120        .       -       .       transcript_id "NM_007298"; intron_number "NM_007298_intron_1";
17      Refseq  intron  43049194        43051062        .       -       .       transcript_id "NM_007298"; intron_number "NM_007298_intron_2";
17      Refseq  intron  43051117        43057051        .       -       .       transcript_id "NM_007298"; intron_number "NM_007298_intron_3";
17      Refseq  intron  43057135        43063332        .       -       .       transcript_id "NM_007298"; intron_number "NM_007298_intron_4";
17      Refseq  intron  43063373        43063873        .       -       .       transcript_id "NM_007298"; intron_number "NM_007298_intron_5";
17      Refseq  intron  43063951        43067607        .       -       .       transcript_id "NM_007298"; intron_number "NM_007298_intron_6";

RNA-Seq HTSeq gene • 1.5k views

ADD COMMENT • link 6.1 years ago by oriolebaltimore ▴ 190

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Thanks genomax for very clean formatting my question. I could not figure out how to do it.Thanks Adrian

ADD REPLY • link 6.1 years ago by oriolebaltimore ▴ 190

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Counting programs do not count multimapping reads by default. Have you looked into that as a reason for getting 0 counts?

For future reference: You can use the formatting bar (especially the code option) by highlighting text you want to format and then clicking the button in red-box below.