raise BWAMethException("first run bwameth.py index %s" % fa)

[sengoku93]

After running this command:
/nfs/sw/bwameth/bwameth-0.2.0/bin/bwameth.py --reference methylation_cfDNA/resources/bwameth/hg19.fa trunc_test/trunc_test.R1.fastq trunc_test/trunc_test.R2.fastq --threads 4 > trunc_test_aligned.bam

And trying multiple versions of python:
python/2.7.10 python/2.7.14 python/2.7.8-museq python/3.6.1 python/3.6.7
python/2.7.11 python/2.7.3 python/3.4.5 python/3.6.2 python/3.7.1
python/2.7.11-shared python/2.7.5 python/3.5.1 python/3.6.4 python/ucs4
python/2.7.12 python/2.7.8 python/3.5.2 python/3.6.4.0

I am still getting this error:

Traceback (most recent call last):
File "/nfs/sw/bwameth/bwameth-0.2.0/bin/bwameth.py", line 4, in
import('pkg_resources').run_script('bwameth==0.2.0', 'bwameth.py')
File "/nfs/sw/bwameth/bwameth-0.2.0/lib/python2.7/site-packages/pkg_resources/init.py", line 735, in run_script
self.require(requires)[0].run_script(script_name, ns)
File "/nfs/sw/bwameth/bwameth-0.2.0/lib/python2.7/site-packages/pkg_resources/init.py", line 1652, in run_script
exec(code, namespace, namespace)
File "/nfs/sw/bwameth/bwameth-0.2.0/lib/python2.7/site-packages/bwameth-0.2.0-py2.7.egg/EGG-INFO/scripts/bwameth.py", line 450, in
main(sys.argv[1:])
File "/nfs/sw/bwameth/bwameth-0.2.0/lib/python2.7/site-packages/bwameth-0.2.0-py2.7.egg/EGG-INFO/scripts/bwameth.py", line 447, in main
set_as_failed=args.set_as_failed)
File "/nfs/sw/bwameth/bwameth-0.2.0/lib/python2.7/site-packages/bwameth-0.2.0-py2.7.egg/EGG-INFO/scripts/bwameth.py", line 260, in bwa_mem
raise BWAMethException("first run bwameth.py index %s" % fa)
main.BWAMethException: first run bwameth.py index
methylation_cfDNA/resources/bwameth/hg19.fa

Any suggestions?

[sengoku93]

Seems like reference isn't the issue and generated successfully:

[bwt_gen] Finished constructing BWT in 1308 iterations.
[bwa_index] 4514.01 seconds elapse.
[bwa_index] Update BWT... 23.31 sec
[bwa_index] Pack forward-only FASTA... 22.44 sec
[bwa_index] Construct SA from BWT and Occ...

[brentp]

I would say to make sure that methylation_cfDNA/resources/bwameth/hg19.fa (a relative path) actually exists and that all the .c2t and index files exist in the directory.

[sengoku93]

I provide full path in actual command, just did not want to post publicly. For hg19.fa, do you have an estimate of how big the .c2t files should be?

3.0G Aug 9 15:43 resources/bwameth/hg19.fa
5.9G Aug 14 10:13 resources/bwameth/hg19.fa.bwameth.c2t
5.8G Aug 15 12:43 resources/bwameth/hg19.fa.bwameth.c2t.bwt
1.5G Aug 15 12:44 resources/bwameth/hg19.fa.bwameth.c2t.pac
14K Aug 15 12:44 resources/bwameth/hg19.fa.bwameth.c2t.ann
13K Aug 15 12:44 resources/bwameth/hg19.fa.bwameth.c2t.amb
2.9G Aug 15 13:18 resources/bwameth/hg19.fa.bwameth.c2t.sa

[brentp]

that looks ok. my guess it that there's something odd with how you're specifying the path. maybe the part of the path you have elided has spaces or is incorrect somehow? or maybe it's NFS and not available on your compute node or something like this.

[sengoku93]

What does NSF mean?

[sengoku93]

NFS*

[brentp]

networked file system

[rmoran7]

This is probably resolved. However, I came across this issue and the problem was the timestamp of the .fa was newer than the c2t files. There was no changes in to the ref in my case but moving files after indexing caused the times to change. The code specifically has design for this for good reason, but a better error message would be nice.

[MagdalenaWinklhofer]

Error: "BWAMethException: first run bwameth.py index ... OR bwameth.py index-mem2 ... OR make sure the modification time on the generated c2t files is newer than on the .fa file"

Hi, I encountered the same problem but could not solve it. I am working on an HPC and indexed my genome in a separate slurm script (only done once) and struggle now with the alignment. I made sure that the .fa file is older than the new index files (.c2t) and that they are all stored in the same directory. The indexing worked fine, but I could not get the alignment to work.

"bwameth.py --reference $REF some_R1.fastq.gz some_R2.fastq.gz > some.output.sam"

For the "--reference", I tried to specify the directory of the indexes (relative path), and I gave the program only two sequences (like in the example) to test it. The indexes are not in the same directory as the sequences. Could that be the issue?

My command looks like this:
"bwameth.py --reference .../directory_of_the_index/ .../file_R1.fq.gz .../file_R2.fq.gz -t 4 > test_aligned.bam"

Could you let me know what could be wrong or what else I could test to get the program running?

[brentp]

the argument to --reference should be the original .fasta. so e.g.

--reference hg38.fa

[ulazcanoCICbioGUNE]

Good morning,
I'm encountering the same error and none of the previous answers worked for me.

"File "/NewCluster_Software/conda_envs/bwameth/bin/bwameth.py", line 349, in bwa_mem
    raise BWAMethException("first run bwameth.py index %s OR bwameth.py index-mem2 %s OR make sure the modification time on the generated c2t files is newer than on the .fa file" % (fa, fa))"

I am also working on an HPC and indexed my genome in a separate slurm script. When trying to do the alignment with a .fastq file I get the error. I have alo checked that the reference genome file .fa.gz is older than the index files and are in the same directory.

Here is the command I'm trying to run:

"bwameth.py --reference /Genomes/Ref_genome/Homo_sapiens.GRCh38.dna.primary_assembly_110.fa.gz /FASTQ/SRR8261134.fastq > test_SRR8261134.bam"

Thanks in advanced for your help!

Uxue

[brentp]

@ulazcanoCICbioGUNE I think you'll need to decompress /Genomes/Ref_genome/Homo_sapiens.GRCh38.dna.primary_assembly_110.fa.gz and then re-run the indexing with bwameth.py index /Genomes/Ref_genome/Homo_sapiens.GRCh38.dna.primary_assembly_110.fa

[ulazcanoCICbioGUNE]

Hello, I have followed your recomendations but the error, even if it has changed a bit, its the same:

Traceback (most recent call last):
  File "/NewCluster_Software/conda_envs/bwameth/bin/bwameth.py", line 563, in <module>
    main(sys.argv[1:])
  File "/NewCluster_Software/conda_envs/bwameth/bin/bwameth.py", line 554, in main
    bwa_mem(args.reference, conv_fqs_cmd, ' '.join(map(str, pass_through_args)),
  File "/NewCluster_Software/conda_envs/bwameth/bin/bwameth.py", line 349, in bwa_mem
    raise BWAMethException("first run bwameth.py index %s OR bwameth.py index-mem2 %s OR make sure the modification time on the generated c2t files is newer than on the .fa file" % (fa, fa))
BWAMethException: first run bwameth.py index /Genomes_Rocky/Ref_genome/Homo_sapiens.GRCh38.dna.primary_assembly_110.fa OR bwameth.py index-mem2 /Genomes_Rocky/Ref_genome/Homo_sapiens.GRCh38.dna.primary_assembly_110.fa OR make sure the modification time on the generated c2t files is newer than on the .fa file

This is how I generated the Index:

[ulazcano@gn12 Ref_genome]$ bwameth.py index-mem2 Homo_sapiens.GRCh38.dna.primary_assembly_110.fa
converting c2t in Homo_sapiens.GRCh38.dna.primary_assembly_110.fa to Homo_sapiens.GRCh38.dna.primary_assembly_110.fa.bwameth.c2t
indexing with bwa-mem2: Homo_sapiens.GRCh38.dna.primary_assembly_110.fa.bwameth.c2t
Looking to launch executable "/NewCluster_Software/conda_envs/bwameth/bin/bwa-mem2.avx512bw", simd = .avx512bw
Launching executable "/NewCluster_Software/conda_envs/bwameth/bin/bwa-mem2.avx512bw"
[bwa_index] Pack FASTA... 28.07 sec
* Entering FMI_search
init ticks = 330480707462
ref seq len = 12399002872
binary seq ticks = 343408854256
build suffix-array ticks = 3739215915280
pos: 1549875360, ref_seq_len__: 1549875359
build fm-index ticks = 1126909408080
Total time taken: 2238.0656

Many many thanks for your rapid answere, is my first time working with BS-seq data and I really appreciate your help.

Uxue

[brentp]

you could try:

touch Homo_sapiens.GRCh38.dna.primary_assembly_110.fa.pac
touch Homo_sapiens.GRCh38.dna.primary_assembly_110.fa.amb

to make sure their mod times are newer than the .fa file.