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qqwang-berkeley / JUM

A tool for annotation-free differential analysis of tissue-specific pre-mRNA alternative splicing patterns
MIT License
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Tons of stderr output; problem with preparing_intron_retention_count_table_short_intron.pl #23

Closed acesnik closed 4 years ago

acesnik commented 5 years ago

Hi there! I'm excited to use your tool and am testing it out.

With some test files, I'm getting about 4,000,000 lines of stderr output like this:

Use of uninitialized value $readlength in numeric lt (<) at /home/JUM_2.0.2/profile_overhang_from_STAR_output_step_1.pl line 39, <IN1> line 1.
Use of uninitialized value $readlength in subtraction (-) at /home/JUM_2.0.2/profile_overhang_from_STAR_output_step_1.pl line 43, <IN1> line 1.
Use of uninitialized value $readlength in numeric lt (<) at /home/JUM_2.0.2/profile_overhang_from_STAR_output_step_1.pl line 45, <IN1> line 1.
Use of uninitialized value $readlength in subtraction (-) at /home/JUM_2.0.2/profile_overhang_from_STAR_output_step_1.pl line 49, <IN1> line 1.
Use of uninitialized value $readlength in numeric lt (<) at /home/JUM_2.0.2/profile_overhang_from_STAR_output_step_1.pl line 39, <IN1> line 2.
Use of uninitialized value $readlength in subtraction (-) at /home/JUM_2.0.2/profile_overhang_from_STAR_output_step_1.pl line 43, <IN1> line 2.
Use of uninitialized value $readlength in numeric lt (<) at /home/JUM_2.0.2/profile_overhang_from_STAR_output_step_1.pl line 45, <IN1> line 2.
Use of uninitialized value $readlength in subtraction (-) at /home/JUM_2.0.2/profile_overhang_from_STAR_output_step_1.pl line 49, <IN1> line 2.
Use of uninitialized value $readlength in numeric lt (<) at /home/JUM_2.0.2/profile_overhang_from_STAR_output_step_1.pl line 39, <IN1> line 3.
Use of uninitialized value $readlength in subtraction (-) at /home/JUM_2.0.2/profile_overhang_from_STAR_output_step_1.pl line 43, <IN1> line 3.
Use of uninitialized value $readlength in numeric lt (<) at /home/JUM_2.0.2/profile_overhang_from_STAR_output_step_1.pl line 45, <IN1> line 3.
Use of uninitialized value $readlength in subtraction (-) at /home/JUM_2.0.2/profile_overhang_from_STAR_output_step_1.pl line 49, <IN1> line 3.

and this:

Use of uninitialized value $threshold in numeric ge (>=) at /home/JUM_2.0.2/preparing_intron_retention_count_table_long_intron.pl line 73.
Use of uninitialized value $threshold in numeric ge (>=) at /home/JUM_2.0.2/preparing_intron_retention_count_table_long_intron.pl line 73.
Use of uninitialized value $threshold in numeric ge (>=) at /home/JUM_2.0.2/preparing_intron_retention_count_table_long_intron.pl line 73.
Use of uninitialized value $threshold in numeric ge (>=) at /home/JUM_2.0.2/preparing_intron_retention_count_table_long_intron.pl line 73.
Use of uninitialized value $threshold in numeric ge (>=) at /home/JUM_2.0.2/preparing_intron_retention_count_table_long_intron.pl line 73.
Use of uninitialized value $threshold in numeric ge (>=) at /home/JUM_2.0.2/preparing_intron_retention_count_table_long_intron.pl line 73.
Use of uninitialized value $threshold in numeric ge (>=) at

and then it fails with:

can't open input1 file: No such file or directory at /home/JUM_2.0.2/preparing_intron_retention_count_table_short_intron.pl line 18.

Any help would be appreciated. Thanks!

acesnik commented 5 years ago

In terms of setup, I did two-pass alignment with STAR, sorted the BAM files, converted them to SAM files to have those around, and changed the names of the files to follow your naming conventions.

qqwang-berkeley commented 5 years ago

Hi Anthony,

Could you provide me the commands you used, and also, the results of running "ls -l -t" from the directory where you ran JUM?

Thanks,

Qingqing

On Tue, Aug 13, 2019 at 1:12 PM Anthony notifications@github.com wrote:

Hi there! I'm excited to use your tool and am testing it out.

With some test files, I'm getting about 4,000,000 lines of stderr output like this:

Use of uninitialized value $readlength in numeric lt (<) at /home/JUM_2.0.2/profile_overhang_from_STAR_output_step_1.pl line 39, line 1. Use of uninitialized value $readlength in subtraction (-) at /home/JUM_2.0.2/profile_overhang_from_STAR_output_step_1.pl line 43, line 1. Use of uninitialized value $readlength in numeric lt (<) at /home/JUM_2.0.2/profile_overhang_from_STAR_output_step_1.pl line 45, line 1. Use of uninitialized value $readlength in subtraction (-) at /home/JUM_2.0.2/profile_overhang_from_STAR_output_step_1.pl line 49, line 1. Use of uninitialized value $readlength in numeric lt (<) at /home/JUM_2.0.2/profile_overhang_from_STAR_output_step_1.pl line 39, line 2. Use of uninitialized value $readlength in subtraction (-) at /home/JUM_2.0.2/profile_overhang_from_STAR_output_step_1.pl line 43, line 2. Use of uninitialized value $readlength in numeric lt (<) at /home/JUM_2.0.2/profile_overhang_from_STAR_output_step_1.pl line 45, line 2. Use of uninitialized value $readlength in subtraction (-) at /home/JUM_2.0.2/profile_overhang_from_STAR_output_step_1.pl line 49, line 2. Use of uninitialized value $readlength in numeric lt (<) at /home/JUM_2.0.2/profile_overhang_from_STAR_output_step_1.pl line 39, line 3. Use of uninitialized value $readlength in subtraction (-) at /home/JUM_2.0.2/profile_overhang_from_STAR_output_step_1.pl line 43, line 3. Use of uninitialized value $readlength in numeric lt (<) at /home/JUM_2.0.2/profile_overhang_from_STAR_output_step_1.pl line 45, line 3. Use of uninitialized value $readlength in subtraction (-) at /home/JUM_2.0.2/profile_overhang_from_STAR_output_step_1.pl line 49, line 3.

and this:

Use of uninitialized value $threshold in numeric ge (>=) at /home/JUM_2.0.2/preparing_intron_retention_count_table_long_intron.pl line 73. Use of uninitialized value $threshold in numeric ge (>=) at /home/JUM_2.0.2/preparing_intron_retention_count_table_long_intron.pl line 73. Use of uninitialized value $threshold in numeric ge (>=) at /home/JUM_2.0.2/preparing_intron_retention_count_table_long_intron.pl line 73. Use of uninitialized value $threshold in numeric ge (>=) at /home/JUM_2.0.2/preparing_intron_retention_count_table_long_intron.pl line 73. Use of uninitialized value $threshold in numeric ge (>=) at /home/JUM_2.0.2/preparing_intron_retention_count_table_long_intron.pl line 73. Use of uninitialized value $threshold in numeric ge (>=) at /home/JUM_2.0.2/preparing_intron_retention_count_table_long_intron.pl line 73. Use of uninitialized value $threshold in numeric ge (>=) at

and then it fails with:

can't open input1 file: No such file or directory at /home/JUM_2.0.2/preparing_intron_retention_count_table_short_intron.pl line 18.

Any help would be appreciated. Thanks!

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acesnik commented 4 years ago

/home/tools/JUM_2.0.2/JUM_A.sh --Folder /home/tools/JUM_2.0.2 --JuncThreshold 5 --Condition1SampleName G1_1.,G1_2.,G1_3. --Condition1_fileNum_threshold 2 --Condition2SampleName S_1.,S_2.,S_3. --Condition2_fileNum_threshold 2 --Thread 2

I'm attaching the listed files here: lslt.txt

Thanks!

acesnik commented 4 years ago

I used samtools view -h /home/output/JUM_Analysis/G1_1.Aligned.out_sorted.bam > /home/output/JUM_Analysis/G1_1.Aligned.out.sam and such to get the SAM files for the tool. I output sorted BAMs by default from STAR, so I had to convert them.

qqwang-berkeley commented 4 years ago

From the command it looks like you did not specify a couple specific parameters:

Your original command /home/tools/JUM_2.0.2/JUM_A.sh --Folder /home/tools/JUM_2.0.2 --JuncThreshold 5 --Condition1SampleName G1_1.,G1_2.,G1_3. --Condition1_fileNum_threshold 2 --Condition2SampleName S_1.,S_2.,S_3. --Thread 2

should be: /home/tools/JUM_2.0.2/JUM_A.sh --Folder /home/tools/JUM_2.0.2 --JuncThreshold 5 --Condition1_fileNum_threshold X --Condition2_fileNum_threshold X --IRthreshold X --Readlength X --Thread 2 --Condition1SampleName G1_1.,G1_2.,G1_3. --Condition1_fileNum_threshold 2 --Condition2SampleName S_1.,S_2.,S_3.

X will a user chosen number.

On Wed, Aug 21, 2019 at 6:37 PM Anthony notifications@github.com wrote:

/home/tools/JUM_2.0.2/JUM_A.sh --Folder /home/tools/JUM_2.0.2 --JuncThreshold 5 --Condition1SampleName G1_1.,G1_2.,G1_3. --Condition1_fileNum_threshold 2 --Condition2SampleName S_1.,S_2.,S_3. --Thread 2

I'm attaching the listed files here: lslt.txt https://github.com/qqwang-berkeley/JUM/files/3527606/lslt.txt

Thanks!

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acesnik commented 4 years ago

Okay, that fixed this issue. I think the main thing that was missing from my command was IRthreshold.