inconsistent bedtools error

Question

[tnminh89]

Hi everyone,

I am trying to do bedtools coverage with my .bed and .bam files; however, only half of the .bed files worked, while the other half had this error:

Error: unable to open file or unable to determine types for file SRR14194209_minus_bottom_sorted_removed_opposite_gene_6kb.bed
- Please ensure that your file is TAB delimited (e.g., cat -t FILE).
- Also ensure that your file has integer chromosome coordinates in the expected columns (e.g., cols 2 and 3 for BED).

When I checked for tab delimited, cat -t FILE, here was the output example for all the files:

chr4^I119280127^I119286127^IID=FL_L1-1635^I.^I-^I119274114^I119280127^IL1.3_300bp5NoF^I6^I593^I6013^I0.0986197

Any insights of what might be happening? Thank you so much in advance!

[Pierre Lindenbaum]

what is the output of the following commands:

file SRR14194209_minus_bottom_sorted_removed_opposite_gene_6kb.bed

awk -F '\t' '(NF < 3 || $1=="" || int($2)<0 ||  int($3)<=0 || int($2)>int($3) )' SRR14194209_minus_bottom_sorted_removed_opposite_gene_6kb.bed | head

cut -f 1 SRR14194209_minus_bottom_sorted_removed_opposite_gene_6kb.bed | uniq | sort | uniq -c

[tnminh89]

Here is the output:

awk -F '\t' '(NF < 3 || $1=="" || int($2)<0 ||  int($3)<=0 || int($2)>int($3) )' SRR14194209_minus_bottom_sorted_removed_opposite_gene_6kb.bed | head

chrX    74040075        31097677        ID=FL_L1-5481   .       -       74033944        74040075        L1.3_300bp5NoF  457     6067    6131    0.9895613000000001
chr9    116515133       41061290        ID=FL_L1-3498   .       -       116509016       116515133       L1.3_300bp5NoF  108     5207    6117    0.8512343000000001
chr3    75489612        45755449        ID=FL_L1-1010   .       -       75483459        75489612        L1.3_300bp5NoF  91      4456    6153    0.7241996
chr2    40238627        28751640        ID=FL_L1-0457   .       -       40232601        40238627        L1.3_300bp5NoF  88      3982    6026    0.6608031999999999
chr1    49122342        26432282        ID=FL_L1-0045   .       -       49116226        49122342        L1.3_300bp5NoF  84      4525    6116    0.7398627
chr11   86830636        13921450        ID=FL_L1-3967   .       -       86824479        86830636        L1.3_300bp5NoF  77      3580    6157    0.5814520000000001
chr9    70212755        41061290        ID=FL_L1-3413   .       -       70206630        70212755        L1.3_300bp5NoF  55      3195    6125    0.5216327000000001
chr3    139571485       45755449        ID=FL_L1-1181   .       -       139565365       139571485       L1.3_300bp5NoF  54      3689    6120    0.6027778
chr19   37054500        1505022 ID=FL_L1-5098   .       -       37048722        37054500        L1.3_300bp5NoF  44      2851    5778    0.4934233
chr1    169257591       26432282        ID=FL_L1-0269   .       -       169251562       169257591       L1.3_300bp5NoF  33      2162    6029    0.3586001


cut -f 1 SRR14194209_minus_bottom_sorted_removed_opposite_gene_6kb.bed | uniq | sort | uniq -c
      5 chr1
      3 chr10
     11 chr11
      6 chr12
      2 chr13
      3 chr14
      3 chr15
      1 chr17
      3 chr19
     10 chr2
      1 chr21
      5 chr3
     10 chr4
      2 chr5
      4 chr7
      6 chr8
      4 chr9
      4 chrX

[Pierre Lindenbaum]

the output of the file command is missing;

[tnminh89]

Yes, so sorry, my bad. Here is the output:

SRR14194209_minus_bottom_sorted_removed_opposite_gene_6kb.bed: ASCII text

Answer 1

[Pierre Lindenbaum]

all your START positions are greater than the END positions. This is invalid for BED.

https://samtools.github.io/hts-specs/BEDv1.pdf

chromEnd: End position of the feature on the chromosome. chromEnd must be an integer greater than or equal to the value of chromStart ...

[tnminh89]

Ah I see, thank you so much!

[Pierre Lindenbaum]

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