Hello,
I am trying to run UROBORUS on reads simulated from human cdna of length 150bp.
I used this link to download UROBORUS:
wget https://github.com/WGLab/UROBORUS/archive/v2.0.0.tar.gz
I used TopHat2 and Bowtie2 to generate unmapped.bam and used samtools (Version: 0.1.19-44428cd) to generate unmapped.sam.
Then, I tried to run UROBORUS on unmapped.sam using command:
uro_path=/home/nagarjun/Downloads/UROBORUS-2.0.0/bin index_path=/data2/circrna/bow1index perl "$uro_path"/UROBORUS.pl -index "$index_path"/humbow1 -gtf "$index_path"/human.gtf -fasta "$index_path" unmapped.sam accepted_hits.bam
Then, I started to get series of errors; and the final circRNA list is empty.
[nagarjun@localhost tophat_out]$ perl "$uro_path"/UROBORUS.pl -index "$index_path"/humbow1 -gtf "$index_path"/human.gtf -fasta "$index_path" unmapped.sam accepted_hits.bam
[Mon Mar 18 14:26:05 2019] Preparing the reads..........finished!
[Mon Mar 18 14:26:08 2019] Now tophat..........
[2019-03-18 14:26:08] Beginning TopHat run (v2.1.1)
[2019-03-18 14:26:08] Checking for Bowtie
Bowtie version: 1.2.2.0
[2019-03-18 14:26:08] Checking for Bowtie index files (genome)..
[2019-03-18 14:26:08] Checking for reference FASTA file
[2019-03-18 14:26:08] Generating SAM header for /data2/circrna/bow1index/humbow1
[2019-03-18 14:26:09] Preparing reads
left reads: min. length=20, max. length=20, 489200 kept reads (56 discarded)
right reads: min. length=20, max. length=20, 489206 kept reads (50 discarded)
Warning: you have only one segment per read.
If the read length is greater than or equal to 45bp,
we strongly recommend that you decrease --segment-length to about half the read length because TopHat will work better with multiple segments
[2019-03-18 14:26:14] Mapping left_kept_reads to genome humbow1 with Bowtie
[FAILED]
Error running bowtie:
Error reading _plen[] array: 890800743, 4294967292
Command: /usr/bin/bowtie-align-s --wrapper basic-0 -v 2 -k 20 -m 20 -S -p 6 --sam-nohead --max /dev/null /data2/circrna/bow1index/humbow1 -
finished!
open: No such file or directory
[main_samview] fail to open "accepted_hits.bam" for reading.
[Mon Mar 18 14:26:14 2019] Filtering the mapped seeds...........finished!
[Mon Mar 18 14:26:16 2019] Locating the seeds in the genome...........finished!
[Mon Mar 18 14:26:16 2019] Locating the seed lab ...........finished!
[Mon Mar 18 14:26:16 2019] Extending the paired seeds...........[Mon Mar 18 14:26:16 2019]
finished!
[Mon Mar 18 14:26:16 2019] Extending the single seed...........[Mon Mar 18 14:26:16 2019]
finished!
[Mon Mar 18 14:26:16 2019] Now bowtie..........Warning: -M was specified w/o --best; automatically enabling --best
Error reading _plen[] array: 890800743, 4294967292
Command: /usr/bin/bowtie-align-s --wrapper basic-0 -p 6 -n 0 -M 20 -I 100 -X 100000 --ff -1 IMR_1.fastq -2 IMR_2.fastq -S --sam-nohead /data2/circrna/bow1index/humbow1
finished!
[Mon Mar 18 14:26:16 2019]
[Mon Mar 18 14:26:16 2019] Now filtering mapped IMR..........finished!
[Mon Mar 18 14:26:16 2019]
[Mon Mar 18 14:26:16 2019] Now sorting mapped IMR sam file..........Use of uninitialized value $line in split at /home/nagarjun/Downloads/UROBORUS-2.0.0/bin/UROBORUS.pl line 1561.
Use of uninitialized value $line in split at /home/nagarjun/Downloads/UROBORUS-2.0.0/bin/UROBORUS.pl line 1564.
Use of uninitialized value $out_line in print at /home/nagarjun/Downloads/UROBORUS-2.0.0/bin/UROBORUS.pl line 1580.
finished!
[Mon Mar 18 14:26:16 2019] Now locating mapped IMR..........finished!
[Mon Mar 18 14:26:16 2019]
[Mon Mar 18 14:26:16 2019] Now mating the reads...........finished!
[Mon Mar 18 14:26:16 2019]
[Mon Mar 18 14:26:16 2019] Now filtering the accepted reads...........open: No such file or directory
[bam_sort_core] fail to open file accepted_hits.sorted.bam
open: No such file or directory
[main_samview] fail to open "accepted_hits.sorted.bam" for reading.
Use of uninitialized value $1 in print at /home/nagarjun/Downloads/UROBORUS-2.0.0/bin/UROBORUS.pl line 1778.
Use of uninitialized value $1 in print at /home/nagarjun/Downloads/UROBORUS-2.0.0/bin/UROBORUS.pl line 1778.
Use of uninitialized value $1 in print at /home/nagarjun/Downloads/UROBORUS-2.0.0/bin/UROBORUS.pl line 1778.
Use of uninitialized value $1 in print at /home/nagarjun/Downloads/UROBORUS-2.0.0/bin/UROBORUS.pl line 1778.
Use of uninitialized value $1 in print at /home/nagarjun/Downloads/UROBORUS-2.0.0/bin/UROBORUS.pl line 1778.
Use of uninitialized value $1 in print at /home/nagarjun/Downloads/UROBORUS-2.0.0/bin/UROBORUS.pl line 1778.
Use of uninitialized value $1 in print at /home/nagarjun/Downloads/UROBORUS-2.0.0/bin/UROBORUS.pl line 1778.
Use of uninitialized value $1 in print at /home/nagarjun/Downloads/UROBORUS-2.0.0/bin/UROBORUS.pl line 1778.
Use of uninitialized value $1 in print at /home/nagarjun/Downloads/UROBORUS-2.0.0/bin/UROBORUS.pl line 1778.
[Mon Mar 18 14:27:12 2019]
[Mon Mar 18 14:27:12 2019] Merging the results...........finished!
[Mon Mar 18 14:27:12 2019] Making the statistics...........finished!
[Mon Mar 18 14:27:12 2019] Output the circular RNA list!
[Mon Mar 18 14:27:12 2019] Completed successfully! The time elapsed: about 0.01 hours.
-rw-rw-r-- 1 nagarjun nagarjun 188 Mar 16 15:04 prep_reads.info
-rw-rw-r-- 1 nagarjun nagarjun 563 Mar 16 23:30 align_summary.txt
-rw-rw-r-- 1 nagarjun nagarjun 421592 Mar 16 23:30 insertions.bed
-rw-rw-r-- 1 nagarjun nagarjun 874653 Mar 16 23:30 deletions.bed
-rw-rw-r-- 1 nagarjun nagarjun 26194280 Mar 16 23:30 junctions.bed
-rw-rw-r-- 1 nagarjun nagarjun 7520512819 Mar 17 00:34 accepted_hits.bam
drwxrwxr-x 2 nagarjun nagarjun 4096 Mar 17 00:34 logs
-rw-rw-r-- 1 nagarjun nagarjun 49666683 Mar 17 00:35 unmapped.bam
-rw-rw-r-- 1 nagarjun nagarjun 167132556 Mar 18 11:26 unmapped.sam
drwxrwxr-x 4 nagarjun nagarjun 97 Mar 18 14:26 seed_mapped_out
-rw-rw-r-- 1 nagarjun nagarjun 0 Mar 18 14:26 accepted_hits.sorted.sam
-rw-rw-r-- 1 nagarjun nagarjun 0 Mar 18 14:26 PI_list.txt
drwxrwxr-x 3 nagarjun nagarjun 29 Mar 18 14:26 tmp_3_18_14_26
-rw-rw-r-- 1 nagarjun nagarjun 0 Mar 18 14:27 circRNA_list.txt`
Please help.
I also want to mention that initially it was giving error of not finding genome index file with humbow1..ebwt
I checked and found that the indexed files have extension .ebwtl
I renamed the files to *.ebwt and the error was gone. IS this the right approach?
Thank You in advance
Hello, I am trying to run UROBORUS on reads simulated from human cdna of length 150bp. I used this link to download UROBORUS: wget https://github.com/WGLab/UROBORUS/archive/v2.0.0.tar.gz I used TopHat2 and Bowtie2 to generate unmapped.bam and used samtools (Version: 0.1.19-44428cd) to generate unmapped.sam. Then, I tried to run UROBORUS on unmapped.sam using command:
uro_path=/home/nagarjun/Downloads/UROBORUS-2.0.0/bin index_path=/data2/circrna/bow1index perl "$uro_path"/UROBORUS.pl -index "$index_path"/humbow1 -gtf "$index_path"/human.gtf -fasta "$index_path" unmapped.sam accepted_hits.bamThen, I started to get series of errors; and the final circRNA list is empty.
[nagarjun@localhost tophat_out]$ perl "$uro_path"/UROBORUS.pl -index "$index_path"/humbow1 -gtf "$index_path"/human.gtf -fasta "$index_path" unmapped.sam accepted_hits.bam [Mon Mar 18 14:26:05 2019] Preparing the reads..........finished! [Mon Mar 18 14:26:08 2019] Now tophat.......... [2019-03-18 14:26:08] Beginning TopHat run (v2.1.1)
[2019-03-18 14:26:08] Checking for Bowtie Bowtie version: 1.2.2.0 [2019-03-18 14:26:08] Checking for Bowtie index files (genome).. [2019-03-18 14:26:08] Checking for reference FASTA file [2019-03-18 14:26:08] Generating SAM header for /data2/circrna/bow1index/humbow1 [2019-03-18 14:26:09] Preparing reads left reads: min. length=20, max. length=20, 489200 kept reads (56 discarded) right reads: min. length=20, max. length=20, 489206 kept reads (50 discarded) Warning: you have only one segment per read. If the read length is greater than or equal to 45bp, we strongly recommend that you decrease --segment-length to about half the read length because TopHat will work better with multiple segments [2019-03-18 14:26:14] Mapping left_kept_reads to genome humbow1 with Bowtie [FAILED] Error running bowtie: Error reading _plen[] array: 890800743, 4294967292 Command: /usr/bin/bowtie-align-s --wrapper basic-0 -v 2 -k 20 -m 20 -S -p 6 --sam-nohead --max /dev/null /data2/circrna/bow1index/humbow1 -
finished! open: No such file or directory [main_samview] fail to open "accepted_hits.bam" for reading. [Mon Mar 18 14:26:14 2019] Filtering the mapped seeds...........finished! [Mon Mar 18 14:26:16 2019] Locating the seeds in the genome...........finished! [Mon Mar 18 14:26:16 2019] Locating the seed lab ...........finished! [Mon Mar 18 14:26:16 2019] Extending the paired seeds...........[Mon Mar 18 14:26:16 2019] finished! [Mon Mar 18 14:26:16 2019] Extending the single seed...........[Mon Mar 18 14:26:16 2019] finished! [Mon Mar 18 14:26:16 2019] Now bowtie..........Warning: -M was specified w/o --best; automatically enabling --best Error reading _plen[] array: 890800743, 4294967292 Command: /usr/bin/bowtie-align-s --wrapper basic-0 -p 6 -n 0 -M 20 -I 100 -X 100000 --ff -1 IMR_1.fastq -2 IMR_2.fastq -S --sam-nohead /data2/circrna/bow1index/humbow1 finished! [Mon Mar 18 14:26:16 2019] [Mon Mar 18 14:26:16 2019] Now filtering mapped IMR..........finished! [Mon Mar 18 14:26:16 2019] [Mon Mar 18 14:26:16 2019] Now sorting mapped IMR sam file..........Use of uninitialized value $line in split at /home/nagarjun/Downloads/UROBORUS-2.0.0/bin/UROBORUS.pl line 1561. Use of uninitialized value $line in split at /home/nagarjun/Downloads/UROBORUS-2.0.0/bin/UROBORUS.pl line 1564. Use of uninitialized value $out_line in print at /home/nagarjun/Downloads/UROBORUS-2.0.0/bin/UROBORUS.pl line 1580. finished! [Mon Mar 18 14:26:16 2019] Now locating mapped IMR..........finished! [Mon Mar 18 14:26:16 2019] [Mon Mar 18 14:26:16 2019] Now mating the reads...........finished! [Mon Mar 18 14:26:16 2019] [Mon Mar 18 14:26:16 2019] Now filtering the accepted reads...........open: No such file or directory [bam_sort_core] fail to open file accepted_hits.sorted.bam open: No such file or directory [main_samview] fail to open "accepted_hits.sorted.bam" for reading. Use of uninitialized value $1 in print at /home/nagarjun/Downloads/UROBORUS-2.0.0/bin/UROBORUS.pl line 1778. Use of uninitialized value $1 in print at /home/nagarjun/Downloads/UROBORUS-2.0.0/bin/UROBORUS.pl line 1778. Use of uninitialized value $1 in print at /home/nagarjun/Downloads/UROBORUS-2.0.0/bin/UROBORUS.pl line 1778. Use of uninitialized value $1 in print at /home/nagarjun/Downloads/UROBORUS-2.0.0/bin/UROBORUS.pl line 1778. Use of uninitialized value $1 in print at /home/nagarjun/Downloads/UROBORUS-2.0.0/bin/UROBORUS.pl line 1778. Use of uninitialized value $1 in print at /home/nagarjun/Downloads/UROBORUS-2.0.0/bin/UROBORUS.pl line 1778. Use of uninitialized value $1 in print at /home/nagarjun/Downloads/UROBORUS-2.0.0/bin/UROBORUS.pl line 1778. Use of uninitialized value $1 in print at /home/nagarjun/Downloads/UROBORUS-2.0.0/bin/UROBORUS.pl line 1778. Use of uninitialized value $1 in print at /home/nagarjun/Downloads/UROBORUS-2.0.0/bin/UROBORUS.pl line 1778. [Mon Mar 18 14:27:12 2019] [Mon Mar 18 14:27:12 2019] Merging the results...........finished! [Mon Mar 18 14:27:12 2019] Making the statistics...........finished! [Mon Mar 18 14:27:12 2019] Output the circular RNA list! [Mon Mar 18 14:27:12 2019] Completed successfully! The time elapsed: about 0.01 hours.
-rw-rw-r-- 1 nagarjun nagarjun 188 Mar 16 15:04 prep_reads.info -rw-rw-r-- 1 nagarjun nagarjun 563 Mar 16 23:30 align_summary.txt -rw-rw-r-- 1 nagarjun nagarjun 421592 Mar 16 23:30 insertions.bed -rw-rw-r-- 1 nagarjun nagarjun 874653 Mar 16 23:30 deletions.bed -rw-rw-r-- 1 nagarjun nagarjun 26194280 Mar 16 23:30 junctions.bed -rw-rw-r-- 1 nagarjun nagarjun 7520512819 Mar 17 00:34 accepted_hits.bam drwxrwxr-x 2 nagarjun nagarjun 4096 Mar 17 00:34 logs -rw-rw-r-- 1 nagarjun nagarjun 49666683 Mar 17 00:35 unmapped.bam -rw-rw-r-- 1 nagarjun nagarjun 167132556 Mar 18 11:26 unmapped.sam drwxrwxr-x 4 nagarjun nagarjun 97 Mar 18 14:26 seed_mapped_out -rw-rw-r-- 1 nagarjun nagarjun 0 Mar 18 14:26 accepted_hits.sorted.sam -rw-rw-r-- 1 nagarjun nagarjun 0 Mar 18 14:26 PI_list.txt drwxrwxr-x 3 nagarjun nagarjun 29 Mar 18 14:26 tmp_3_18_14_26 -rw-rw-r-- 1 nagarjun nagarjun 0 Mar 18 14:27 circRNA_list.txt`
Please help. I also want to mention that initially it was giving error of not finding genome index file with humbow1..ebwt I checked and found that the indexed files have extension .ebwtl I renamed the files to *.ebwt and the error was gone. IS this the right approach? Thank You in advance