Tophat-fusion error

[bounlu]

Hi,

In the mapping with tophat-fusion step, I get the below error:

$ clear_quant -1 206_1.fq.gz -2 206_2.fq.gz -g /library/hg19.fa -i /library/hg19.fa -j /library/hg19.fa -G /library/hg19.genes.gtf -o 206 -p 40
###Parameters:
Namespace(bowtie1='/library/hg19.fa', genome='/library/hg19.fa', gtf='/library/hg19.genes.gtf', hisat='/library/hg19.fa', m1='206_1.fq.gz', m2='206_2.fq.gz', output='206', thread='40')
###Parameters

###Start hisat2 mapping
# start to get sp sites for hisat mapping
# start to align to genome by hisat
# get mapped and unmapped reads
# sort bam file
# index bam file
###End hisat2 mapping

###Start tophat-fusion mapping
Traceback (most recent call last):
  File "/software/anaconda2/bin/clear_quant", line 11, in <module>
    load_entry_point('CLEAR==1.0.0', 'console_scripts', 'clear_quant')()
  File "build/bdist.linux-x86_64/egg/src/run.py", line 256, in main
  File "build/bdist.linux-x86_64/egg/src/run.py", line 159, in fusion_align
  File "/software/anaconda2/lib/python2.7/subprocess.py", line 223, in check_output
    raise CalledProcessError(retcode, cmd, output=output)
subprocess.CalledProcessError: Command '['tophat2', '-o', '206/fusion', '-p', '40', '--fusion-search', '--keep-fasta-order', '--bowtie1', '--no-coverage-search', '/library/hg19.fa', '206/hisat/unmapped.fq']' returned non-zero exit status 1

I am not sure what the error says and how to fix it. Can you help? @xingma

[zxclovezby]

Hi， I encountered the same error. I checked the log and found the following error. I have tried to replace different versions of tophat2, but it still hasn't been solved

###Start tophat-fusion mapping
Traceback (most recent call last):
  File "/usr/local/bin/clear_quant", line 11, in <module>
    load_entry_point('CLEAR==1.0.0', 'console_scripts', 'clear_quant')()
  File "build/bdist.linux-x86_64/egg/src/run.py", line 256, in main
  File "build/bdist.linux-x86_64/egg/src/run.py", line 159, in fusion_align
  File "/usr/lib/python2.7/subprocess.py", line 223, in check_output
    raise CalledProcessError(retcode, cmd, output=output)
subprocess.CalledProcessError: Command '['tophat2', '-o', '/data//fusion', '-p', '5', '--fusion-search', '--keep-fasta-order', '--bowtie1', '--no-coverage-search', '/data/databases/genome/bowtie1_index/ucsc.hg19', '/data/hisat/unmapped.fq']' returned non-zero exit status 1

[2020-12-16 14:04:17] Beginning TopHat run (v2.1.1)
-----------------------------------------------
[2020-12-16 14:04:17] Checking for Bowtie
                  Bowtie version:        1.2.2.0
[2020-12-16 14:04:17] Checking for Bowtie index files (genome)..
[2020-12-16 14:04:17] Checking for reference FASTA file
        Warning: Could not find FASTA file /data/databases/genome/bowtie1_index/ucsc.hg19.fa
[2020-12-16 14:04:17] Reconstituting reference FASTA file from Bowtie index
  Executing: /usr/bin/bowtie-inspect /data/databases/genome/bowtie1_index/ucsc.hg19 > /data/fusion/tmp/ucsc.hg19.fa
[2020-12-16 14:05:54] Generating SAM header for /data/databases/genome/bowtie1_index/ucsc.hg19
[2020-12-16 14:05:55] Preparing reads
         left reads: min. length=50, max. length=63, 95441 kept reads (17 discarded)
[2020-12-16 14:05:56] Mapping left_kept_reads to genome ucsc.hg19 with Bowtie
[sam_read1] missing header? Abort!
        [FAILED]
Error running bowtie:
Error while flushing and closing output

[zxclovezby]

The problem has been solved and can be run according to this version of the software

tophat2：tophat-2.0.12.Linux_x86_64---------------------http://ccb.jhu.edu/software/tophat/downloads/
samtools：0.1.18.0-------------------------
bowtie：1.0.0.0--------------------------https://sourceforge.net/projects/bowtie-bio/files/bowtie/1.0.0/bowtie-1.0.0-linux-x86_64.zip/download

[sharmi85]

Hello. I am trying to use CLEAR for my data set and running the following command: clear_quant -1 /userdata/sharmishtha/Hela/trimmedFastqFiles/trim_HeLa-AMT-1_R1.fastq.gz -2 /userdata/sharmishtha/Hela/trimmedFastqFiles/trim_HeLa-AMT-1_R2.fastq.gz -g /userdata/sharmishtha/ref_and_anno/hg38/hg38.fa -i /userdata/sharmishtha/IndexFiles/hg38/hisat2index/hg38_hisat2_index -j /userdata/sharmishtha/IndexFiles/hg38/bowtie1_index/bowtie1_index -G /userdata/sharmishtha/IndexFiles/hg38/hg38_kg.gtf -o HelaAMT1_output_dir

The steps untill tophat fusion worked, but got an error after Tophat fusion:

Start circRNA annotation

Error: exonFrames field is being added, but I found a gene (ENST00000602051.5) with CDS but no valid frames. This can happen if program is invoked with -genePredExt but no valid frames are given in the file. If the 8th field of GFF/GTF file is always a placeholder, then don't use -genePredExt. Traceback (most recent call last): File "/userdata/sharmishtha/tools/anaconda3/envs/myenv/bin/clear_quant", line 11, in load_entry_point('CLEAR==1.0.0', 'console_scripts', 'clear_quant')() File "build/bdist.linux-x86_64/egg/src/run.py", line 262, in main File "build/bdist.linux-x86_64/egg/src/run.py", line 173, in circ_annot File "/userdata/sharmishtha/tools/anaconda3/envs/myenv/lib/python2.7/subprocess.py", line 223, in check_output raise CalledProcessError(retcode, cmd, output=output) subprocess.CalledProcessError: Command '['gtfToGenePred', '-genePredExt', '/userdata/sharmishtha/IndexFiles/hg38/hg38_kg.gtf', 'HelaAMT1_output_dir/circ/genePred.tmp']' returned non-zero exit status 255

I used te Circ explorer2 command to get the gtf file: cut -f2-11 hg38_ref.txt|genePredToGtf file stdin hg38_ref.gtf

So I dont know whats going on. Why is the gtf file is giving the error. kindly help

[zxclovezby]

Maybe you can try another GTF file，I've met the same thing.

[sharmi85]

i tried the reference gtf didn't work. When you had the same problem what did you do?

On Mon, Dec 28, 2020 at 12:03 PM xczhang notifications@github.com wrote:

Maybe you can try another GTF file，I've met the same thing.

— You are receiving this because you commented. Reply to this email directly, view it on GitHub https://github.com/YangLab/CLEAR/issues/10#issuecomment-751597813, or unsubscribe https://github.com/notifications/unsubscribe-auth/ASJAFP43JATIKQBCHAZC3ZLSXARCTANCNFSM4NTMW5WA .

-- Regards

Sharmishtha Shyamal, PhD Research Associate RNA Biology Lab Institute of Life Science-DBT Bhubaneshwar, Odisha India

[lbwfff]

The problem has been solved and can be run according to this version of the software

tophat2：tophat-2.0.12.Linux_x86_64---------------------http://ccb.jhu.edu/software/tophat/downloads/
samtools：0.1.18.0-------------------------
bowtie：1.0.0.0--------------------------https://sourceforge.net/projects/bowtie-bio/files/bowtie/1.0.0/bowtie-1.0.0-linux-x86_64.zip/download

Hi, zxclovezby I got the same error as you subprocess.CalledProcessError: Command '['tophat2', '-o', '206/fusion', '-p', '40', '--fusion-search', '--keep-fasta-order', '--bowtie1', '--no-coverage-search', '/library/hg19.fa', '206/hisat/unmapped.fq']' returned non-zero exit status 1 I changed tophat to 2.0.12 but got the same error, do I need to change the version of samtools and bowtie?

[z-bluesky]

Traceback (most recent call last): File "/public/home/z/miniconda3/envs/CIRCexplorer3/bin/clear_quant", line 11, in load_entry_point('CLEAR==1.0.1', 'console_scripts', 'clear_quant')() File "/public/home/z/miniconda3/envs/CIRCexplorer3/lib/python2.7/site-packages/CLEAR-1.0.1-py2.7.egg/src/run.py", line 263, in main args.bowtie1, fusion_dir, args.thread) File "/public/home/z/miniconda3/envs/CIRCexplorer3/lib/python2.7/site-packages/CLEAR-1.0.1-py2.7.egg/src/run.py", line 160, in fusion_align stderr=subprocess.STDOUT) File "/public/home/z/miniconda3/envs/CIRCexplorer3/lib/python2.7/subprocess.py", line 223, in check_output raise CalledProcessError(retcode, cmd, output=output) subprocess.CalledProcessError: Command '['tophat2', '-o', 'output_dir/fusion', '-p', '5', '--fusion-search', '--keep-fasta-order', '--bowtie1', '--no-coverage-search', '../gene_annotation-reference_genome_sequence_file/bowtie1_index', 'output_dir/hisat/unmapped.fq']' returned non-zero exit status 1

Although I have installed tophat 2.0.12, samtools -0.1.18, and bowtie-1.0.0.0, I also encounter the same problem