PhyloFlash fails while creating database

[psampara]

Hello Phyloflashers! I have been trying to run PhyloFlash 3.4. However, the program phyloFlash_makedb.pl fails with error code 512. I installed PhyloFlash 3.4 by cloning the GitHub package and installed the rest of the dependencies. I am unable to install using Conda due to my cluster/server requirements. All dependencies have been installed and verified with perl phyloFlash.pl -check_env. Please let me know how to proceed with this issue, that would be a huge help!

I used the following command perl phyloFlash_makedb.pl --remote

After downloading SILVA and UniVec databases, I get the following error message:

[20:14:40] File ok [20:14:40] unpacking SILVA database [20:14:58] searching for LSU contamination in SSU RefNR [20:14:58] running subcommand: /scratch/pranavs/phyloFlash/barrnap-HGV/bin/barrnap_HGV --kingdom bac --threads 64 --evalue 1e-10 --gene lsu --reject 0.01 ./138.1/SILVA_SSU.fasta >tmp.barrnap_hits.bac.gff 2>tmp.barrnap_hits.bac.barrnap.out [20:14:59] FATAL: Tool execution failed!. Error was 'No such file or directory' and return code '512' Check log file tmp.barrnap_hits.bac.gff Check error log file tmp.barrnap_hits.bac.barrnap.out Aborting. [20:14:59] Saving log to file phyloFlash_log_on_error.

The full log is below

[20:14:58] This is barrnap_HGV 0.7 [20:14:58] Written by Torsten Seemann torsten.seemann@gmail.com [20:14:58] Obtained from https://github.com/Victorian-Bioinformatics-Consortium/barrnap [20:14:58] Detected operating system: linux [20:14:58] Using HMMER binary: /scratch/pranavs/phyloFlash/barrnap-HGV/bin/../binaries/linux/nhmmer [20:14:58] Will use 64 threads [20:14:58] Setting evalue cutoff to 1e-10 [20:14:58] Will tag genes < 0.8 of expected length. [20:14:58] Will reject genes < 0.01 of expected length. [20:14:58] Using database: /scratch/pranavs/phyloFlash/barrnap-HGV/bin/../db/lsu/bac.hmm [20:14:58] Scanning ./138.1/SILVA_SSU.fasta for lsu bac rRNA genes... please wait [20:14:58] Command: /scratch/pranavs/phyloFlash/barrnap-HGV/bin/../binaries/linux/nhmmer --cpu 64 -E 1e-10 --w_length 3878 -o /dev/null --tblout /dev/stdout \/scratch\/pranavs\/phyloFlash\/barrnap-HGV\/bin\/..\/db\/lsu\/bac.hmm .\/138.1\/SILVA_SSU.fasta sh: line 1: 17892 Aborted /scratch/pranavs/phyloFlash/barrnap-HGV/bin/../binaries/linux/nhmmer --cpu 64 -E 1e-10 --w_length 3878 -o /dev/null --tblout /dev/stdout \/scratch\/pranavs\/phyloFlash\/barrnap-HGV\/bin\/..\/db\/lsu\/bac.hmm .\/138.1\/SILVA_SSU.fasta 2>&1 [20:14:59] bad line in nhmmer output - Fatal exception (source file ../../easel/esl_sq.c, line 231):

[kbseah]

hello @psampara , sorry for the late response. Could you please try adding the option --CPUs 16? At the moment it is using all available processors, but I suspect that this results in too much memory being requested by nhmmer, hence the crash.

[acastills]

hi @kbseah , I'm running into the same issue and still got the same error using --CPUs 16 and 8. I also updated barrnap (via mamba), but no luck. Do you have other suggestions?

[kbseah]

hello @acastills could you please post the command you used, the log file phyloFlash_log_on_error, as well as the barrnap log file?

[acastills]

sure, the command was: phyloFlash_makedb.pl --silva_file arb_silva/SILVA_138.1_SSURef_NR99_12_06_20_opt.arb.gz --univec_file univec/UniVec --CPUs 16 phyloFlash_log_on_error is:

[17:42:55] Checking for required tools.
[17:42:55] Using grep found at "/bin/grep".
[17:42:55] Using bowtiebuild found at
       "/bioinf/home/acastill/mambaforge/envs/pf/bin/bowtie-build".
[17:42:55] Using bbmap found at
       "/bioinf/home/acastill/mambaforge/envs/pf/bin/bbmap.sh".
[17:42:55] Using barrnapHGV found at
       "/bioinf/home/acastill/mambaforge/envs/pf/lib/phyloFlash/barrnap-HGV/bin/barrnap_HGV".
[17:42:55] Using bbduk found at
       "/bioinf/home/acastill/mambaforge/envs/pf/bin/bbduk.sh".
[17:42:55] Using vsearch found at
       "/bioinf/home/acastill/mambaforge/envs/pf/bin/vsearch".
[17:42:55] Using bbmask found at
       "/bioinf/home/acastill/mambaforge/envs/pf/bin/bbmask.sh".
[17:42:55] All required tools found.
[17:42:55] using local copy of univec: univec/UniVec
[17:42:55] using local copy of Silva SSU RefNR:
       arb_silva/SILVA_138.1_SSURef_NR99_12_06_20_opt.arb.gz
[17:42:55] unpacking SILVA database
[17:42:55] File ./138.1//SILVA_SSU.fasta exists, not overwriting
[17:42:55] searching for LSU contamination in SSU RefNR
[17:42:55] running subcommand:
       /bioinf/home/acastill/mambaforge/envs/pf/lib/phyloFlash/barrnap-HGV/bin/barrnap_HGV
         --kingdom bac --threads 16 --evalue 1e-10 --gene lsu --reject
       0.01 ./138.1/SILVA_SSU.fasta  >tmp.barrnap_hits.bac.gff
       2>tmp.barrnap_hits.bac.barrnap.out
[17:42:56] FATAL: Tool execution failed!.
       Error was 'No such file or directory' and return code '512'
       Check log file tmp.barrnap_hits.bac.gff
       Check error log file tmp.barrnap_hits.bac.barrnap.out
       Aborting.
[17:42:56] Saving log to file phyloFlash_log_on_error

and tmp.barrnap_hits.bac.barrnap.out is:

[17:42:55] Checking for required tools.
[17:42:55] Using grep found at "/bin/grep".
[17:42:55] Using bowtiebuild found at
       "/bioinf/home/acastill/mambaforge/envs/pf/bin/bowtie-build".
[17:42:55] Using bbmap found at
       "/bioinf/home/acastill/mambaforge/envs/pf/bin/bbmap.sh".
[17:42:55] Using barrnapHGV found at
       "/bioinf/home/acastill/mambaforge/envs/pf/lib/phyloFlash/barrnap-HGV/bin/barrnap_HGV".
[17:42:55] Using bbduk found at
       "/bioinf/home/acastill/mambaforge/envs/pf/bin/bbduk.sh".
[17:42:55] Using vsearch found at
       "/bioinf/home/acastill/mambaforge/envs/pf/bin/vsearch".
[17:42:55] Using bbmask found at
       "/bioinf/home/acastill/mambaforge/envs/pf/bin/bbmask.sh".
[17:42:55] All required tools found.
[17:42:55] using local copy of univec: univec/UniVec
[17:42:55] using local copy of Silva SSU RefNR:
       arb_silva/SILVA_138.1_SSURef_NR99_12_06_20_opt.arb.gz
[17:42:55] unpacking SILVA database
[17:42:55] File ./138.1//SILVA_SSU.fasta exists, not overwriting
[17:42:55] searching for LSU contamination in SSU RefNR
[17:42:55] running subcommand:
       /bioinf/home/acastill/mambaforge/envs/pf/lib/phyloFlash/barrnap-HGV/bin/barrnap_HGV
         --kingdom bac --threads 16 --evalue 1e-10 --gene lsu --reject
       0.01 ./138.1/SILVA_SSU.fasta  >tmp.barrnap_hits.bac.gff
       2>tmp.barrnap_hits.bac.barrnap.out
[17:42:56] FATAL: Tool execution failed!.
       Error was 'No such file or directory' and return code '512'
       Check log file tmp.barrnap_hits.bac.gff
       Check error log file tmp.barrnap_hits.bac.barrnap.out
       Aborting.
[17:42:56] Saving log to file phyloFlash_log_on_error

[kbseah]

Thanks for supplying the log info. Looks like the SILVA input file was not a Fasta file. The correct file should be: https://www.arb-silva.de/fileadmin/silva_databases/current/Exports/SILVA_138.1_SSURef_NR99_tax_silva_trunc.fasta.gz

There are many similarly-named files on the SILVA site, so it's easy to get mixed up. We recommend using the --remote option to download automatically, but I know it's not always possible because of firewalls.

Do let me know if you still encounter problems.

[acastills]

Oops. It's working fine now, thank you!

[kbseah]

welcome!