Ciriquant not configuring hisat2 indexed files
Entering edit mode
6 weeks ago
Atul K. • 0

Why I am getting this error when I have my hisat2 indexed genome is in the desired folder and th path to the folder is correct.

While running the ciriquant i am getting this error i.e.

CIRIquant --config /home/akk/circ_analysis/new_analysis/config.yaml -1 /home/akk/circ_analysis/new_analysis/SRR_1.fastq -2 /home/akk/circ_analysis/new_analysis/SRR_2.fastq
Traceback (most recent call last):
  File "/home/akk/software_library/CIRIquant_env/bin/CIRIquant", line 8, in <module>
  File "/home/akk/software_library/CIRIquant_env/lib/python2.7/site-packages/CIRIquant/", line 97, in main
    config = check_config(check_file(args.config_file))
  File "/home/akk/software_library/CIRIquant_env/lib/python2.7/site-packages/CIRIquant/", line 115, in check_config
    raise ConfigError('Could not find hisat2 index with suffix: *.[1-8].ht2 or *.[1-8].ht2l, please check your configuration')
CIRIquant.utils.ConfigError: Could not find hisat2 index with suffix: *.[1-8].ht2 or *.[1-8].ht2l, please check your configuration

the config file i am using is as follows :

# configuration for CIRIquant

# Input files
read1: /home/akk/circ_analysis/new_analysis/SRR_1.fastq
read2: /home/akk/circ_analysis/new_analysis/SRR_2.fastq

# Output directory
output_dir: ./

# Prefix for output files
output_prefix: prefix_for_output_files

# Paths to tools
  bwa: /home/akk/software_library/CIRIquant_env/bin/bwa
  hisat2: /home/akk/software_library/CIRIquant_env/bin/hisat2
  stringtie: /home/akk/software_library/CIRIquant_env/bin/stringtie
  samtools: /home/akk/software_library/CIRIquant_env/bin/samtools

# Paths to reference files
  fasta: /home/akk/hg38_genome/hg38.fa
  gtf: /home/akk/hg38_genome/hg38.refGene.gtf
  bwa_index: /home/akk/hg38_genome/hg38/hg38
  hisat_index: /home/akk/hg38_genome/hg38hisat

# Other parameters
threads: 4
anchor: 5
library_type: 0

And the contents of the folder hg38hisat are as follows:

akk@kashyap:~/hg38_genome/hg38hisat$ ls -lash
total 4.4G
4.0K drwxrwxr-x 2 akk akk 4.0K Apr 10 16:42 .
4.0K drwxrwxr-x 6 akk akk 4.0K Apr 10 15:05 ..
974M -rw-rw-r-- 1 akk akk 974M Apr 10 16:42 hg38.1.ht2
728M -rw-rw-r-- 1 akk akk 728M Apr 10 16:42 hg38.2.ht2
 16K -rw-rw-r-- 1 akk akk  15K Apr 10 15:41 hg38.3.ht2
728M -rw-rw-r-- 1 akk akk 728M Apr 10 15:41 hg38.4.ht2
1.3G -rw-rw-r-- 1 akk akk 1.3G Apr 10 16:49 hg38.5.ht2
741M -rw-rw-r-- 1 akk akk 741M Apr 10 16:49 hg38.6.ht2
4.0K -rw-rw-r-- 1 akk akk   12 Apr 10 15:41 hg38.7.ht2
4.0K -rw-rw-r-- 1 akk akk    8 Apr 10 15:41 hg38.8.ht2

It is pretty clear to me that the path to hisat2 indexed genome is correct but i do not know why it is not able to find it. Please help, i am missing something very basic ??

ciriquant • 788 views
Entering edit mode
6 weeks ago

Try replacing this line:

hisat_index: /home/akk/hg38_genome/hg38hisat


hisat_index: /home/akk/hg38_genome/hg38hisat/hg38
Entering edit mode

it worked. Thank you. Now my terminal is crashing, seems 16gb of RAM and 4 cores isn't enough. Can you suggest something?

Entering edit mode

I don't know for sure, but I think that decreasing the amount of threads (1 or 2 instead of 4) will decrease the memory requirements of the job so I suggest trying that.

Entering edit mode

Thank you. Decreased the threads to 2 (changed the threads: 2 in the config file; but it was somehow using 4). It did run for sometime but the following error came :

(/home/akk/software_library/CIRIquant_env) akk@kashyap:~/circ_analysis/new_analysis$ CIRIquant --config /home/akk/circ_analysis/new_analysis/config.yaml -1 /home/akk/circ_analysis/new_analysis/SRR14150856_1.fastq -2 /home/akk/circ_analysis/new_analysis/SRR14150856_2.fastq
[Fri 2024-04-19 16:40:07] [INFO ] Input reads: SRR14150856_1.fastq,SRR14150856_2.fastq
[Fri 2024-04-19 16:40:07] [INFO ] Library type: unstranded
[Fri 2024-04-19 16:40:07] [INFO ] Output directory: /home/akk/circ_analysis/new_analysis/SRR14150856, Output prefix: SRR14150856
[Fri 2024-04-19 16:40:07] [INFO ] Config: /home/akk/circ_analysis/new_analysis/config.yaml Loaded
[Fri 2024-04-19 16:40:07] [INFO ] 4 CPU cores availble, using 4
[Fri 2024-04-19 16:40:07] [INFO ] Align RNA-seq reads to reference genome ..
[Fri 2024-04-19 16:49:53] [INFO ] Estimate gene abundance ..
[Fri 2024-04-19 16:51:06] [INFO ] No circRNA information provided, run CIRI2 for junction site prediction ..
[Fri 2024-04-19 16:51:06] [INFO ] Running BWA-mem mapping candidate reads ..
[Fri 2024-04-19 17:23:23] [INFO ] Running CIRI2 for circRNA detection ..
[Sat 2024-04-20 06:28:11] [INFO ] Extract circular sequence
[Sat 2024-04-20 06:28:11] [100% ] [##################################################]
[Sat 2024-04-20 06:28:11] [INFO ] Building circular index ..
[Sat 2024-04-20 06:28:12] [INFO ] De novo alignment for circular RNAs ..
[Sat 2024-04-20 06:28:19] [INFO ] Detecting reads containing Back-splicing signals
Traceback (most recent call last):
  File "/home/akk/software_library/CIRIquant_env/bin/CIRIquant", line 8, in <module>
  File "/home/akk/software_library/CIRIquant_env/lib/python2.7/site-packages/CIRIquant/", line 198, in main
    args.no_fsj, args.bsj_read_file)
  File "/home/akk/software_library/CIRIquant_env/lib/python2.7/site-packages/CIRIquant/", line 658, in proc
    cand_bsj = proc_denovo_bam(denovo_bam, thread, circ_info, anchor, lib_type)
  File "/home/akk/software_library/CIRIquant_env/lib/python2.7/site-packages/CIRIquant/", line 306, in proc_denovo_bam
    sam = pysam.AlignmentFile(bam_file, 'rb')
  File "pysam/libcalignmentfile.pyx", line 736, in pysam.libcalignmentfile.AlignmentFile.__cinit__
  File "pysam/libcalignmentfile.pyx", line 985, in pysam.libcalignmentfile.AlignmentFile._open
ValueError: file has no sequences defined (mode='rb') - is it SAM/BAM format? Consider opening with check_sq=False
Entering edit mode

Looks like the bam file pysam is trying to read is not properly formated. I don't know if it is a bug from CIRI2 or something else. Perhaps you could try asking a separate question on the forum. See also similar errors: pysam alignment error and pysam error when reading .bam file ValueError: file has no sequences defined (mode='rb') - is it SAM/BAM format?

Entering edit mode

Thank you.


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