Can not generate .utr.bed file using identifyDistal3UTR and predictAPA

[Yao-Chung Chen]

Hello Dr. Ye,

I have installed apatrap to analyze 3'utr in human caner cell line (control vs. treatment), and I run identifyDistal3UTR and predictAPA by writing a bash script and run it using tmux.
However, I cannot generated utr.bed file from using identifyDistal3UTR. Therefore, after using predictAPA, it generates an empty file. Can you help me or give me some ideas? Thank you.

Yao-Chung

Treatment bedgraph

(base) urameshi@avis:~/projects/3utr/HFN53$ head -n 5 HFN53_treatment.bedgraph
chr4    165341916       165342067       42
chr4    165341760       165341911       42
chr20   51597689        51597821        42
chr20   51597689        51597821        42
chr11   68438501        68438652        42

Control bedgraph

(base) urameshi@avis:~/projects/3utr/HFN53$ head -n 5 HFN53_control.bedgraph
chr20   44506836        44506987        42
chr11   66810610        66810761        42
chr11   66810596        66810747        42
chr3    10126460        10126600        42
chr3    10126460        10126600        42

annotation bed file

(base) urameshi@avis:~/projects/3utr/HFN53$ head -n 5 ~/annotation/hg38_ucsc.bed
chr1    11873   12227   NR_046018.2     0.000000        +       hg38_ncbiRefSeq exon    .       gene_id "NR_046018.2"; transcript_id "NR_046018.2";
chr1    12612   12721   NR_046018.2     0.000000        +       hg38_ncbiRefSeq exon    .       gene_id "NR_046018.2"; transcript_id "NR_046018.2";
chr1    13220   14409   NR_046018.2     0.000000        +       hg38_ncbiRefSeq exon    .       gene_id "NR_046018.2"; transcript_id "NR_046018.2";
chr1    14361   14829   NR_024540.1     0.000000        -       hg38_ncbiRefSeq exon    .       gene_id "NR_024540.1"; transcript_id "NR_024540.1";
chr1    14969   15038   NR_024540.1     0.000000        -       hg38_ncbiRefSeq exon    .       gene_id "NR_024540.1"; transcript_id "NR_024540.1";

My bash script

(base) urameshi@avis:~/projects/3utr/HFN53/apatrap_result$ cat apatrap.sh
#!/usr/bin/bash


cd ~/projects/3utr/HFN53/

# refine annotated 3utrs and identify novel 3utr using identifyDistal3UTR
identifyDistal3UTR -i HFN53_control.bedgraph \
HFN53_treatment.bedgraph \
-m ~/annotation/hg38_ucsc.bed \
-o ~/projects/3utr/HFN53/apatrap_result/HFN53.utr.bed

# infer all potential APA sites and estimate their correspoinding usage using predictAPA
predictAPA -i HFN53_control.bedgraph \
HFN53_treatment.bedgraph \
-g 2 -n 1 1 \
-u ~/annotation/hg38_ucsc.bed \
-o ~/projects/3utr/HFN53/apatrap_result/HFN53.APA.txt

[Congting Ye]

Hi Yao-Chung,

You used an incorrect annotation bed file in identifyDistal3UTR, you could find a correct annotation bed file from this link http://genome.ucsc.edu/cgi-bin/hgTables?hgsid=787774005_ABQYAdCOlXgJmK8wIcJqn5Hwc2ad&clade=mammal&org=Human&db=hg38&hgta_group=genes&hgta_track=refSeqComposite&hgta_table=refGene&hgta_regionType=genome&position=chr1%3A11%2C102%2C837-11%2C267%2C747&hgta_outputType=bed&hgta_outFileName=test.txt

Congting Ye

[Yao-Chung Chen]

Hello Dr. Ye,

Thank you for your suggestion. I download the bed file from UCSC table browser which you provided. However, as I re-run the idenfigyDistal3UTR, I recieve this message in my terminal:

UTR identification [|     ] 1% doneModification of non-creatable array value attempted, subscript -18459924 at script/identifyDistal3UTR.pl line 424.

The first four rows of the hg38 annotation bed file I download

chr1    201283451       201332993       NM_000299       0       +       201283702       201328836       0       15      453,104,395,145,208,178,63,115,156,177,154,187,85,107,2920,      0,10490,29714,33101,34120,35166,36364,36815,38526,39561,40976,41489,42302,45310,46622,
chr1    67092165        67134970        NM_001276351    0       -       67093004        67127240        0       8       1439,187,70,113,158,92,86,41,   0,3069,4086,23186,33586,35000,38976,42764,
chr1    67092165        67134970        NM_001276352    0       -       67093579        67127240        0       9       1439,70,145,68,113,158,92,86,41,        0,4086,11072,19411,23186,33586,35000,38976,42764,
chr1    201283505       201332989       NM_001005337    0       +       201283702       201328836       0       14      399,104,395,145,208,178,115,156,177,154,187,85,107,2916,0,10436,29660,33047,34066,35112,36761,38472,39507,40922,41435,42248,45256,46568,

I am not sure what is wrong. Can you briefly describe the content of annotation bed file which is needed for using APAtrap?
I also like to ask where can I find the script/identifyDistal3UTR.pl file which mention in the message.

Thank you for your help.

Yao-Chung

[Congting Ye]

Hi Yao-Chung,

I have run the APAtrap with the annotation bed file downloaded from the above link and the test data (https://sourceforge.net/projects/apatrap/files/Test_Data.zip/download), and find out it could run successfully, could you check if there is anything wrong with your bedgraph file?

The source code of APAtrap could be find at this website (https://sourceforge.net/projects/apatrap/files/Source%20Codes/).

Congting Ye

[Thomas Bradley]

Hello,

Just to add to this discussion, these type of errors tend to occur when your bedgraph file is not properly sorted.

For example:

chr1 100 200 5
chr1 90 180 6

Will generate this type of error because chr1 is not correctly sorted.

EDIT:

Looking at your bedgraph file - I can see this is not correctly sorted:

chr4 165341916 165342067 42
chr4 165341760 165341911 42

For the same sequence feature , the first row end position can not be greater than the second row start position - as this would imply contradictory information for a set of bases in the interval between those two values

The correct entry for this should be:

chr4 165341760 165342067 42

Many thanks,
Thomas