augustus error

[olechnwin]

Hi,
I installed Quast 5.0.2 using pip install quast.
When I tried to run BUSCO, I got this error in augustus.log:

"/gpfs0/home/lab/opt/quast5_gm_env/lib/python3.6/site-packages/quast_libs/augustus3.2.3/bin/augustus: /lib64/libc.so.6: version 'GLIBC_2.14' not found (required by /gpfs0/home/lab/opt/quast5_gm_env/lib/python3.6/site-packages/quast_libs/augustus3.2.3/bin/augustus)"

How do I fix this error?
Thanks!

[olechnwin]

some additional information. if I load glibc 2.14 before running Quast, the basic statistics processor doesn't even run.

[alexeigurevich]

Hi, @olechnwin !

Sorry for the late reply. Could you please elaborate on

if I load glibc 2.14 before running Quast, the basic statistics processor doesn't even run.

Basic statistics processor is a Python module and it should not be affected by glibc. Could you copy/attach the log in this case?

As for the primary topic. BUSCO depends on Augustus software and normally we download Augustus binaries if a user asks to run BUSCO. It seems that Augustus was compiled using newer version of glibc than your system glibc. One possible solution is to manually recompile required binaries using your system default libs. To do so, please execute:
cd /gpfs0/home/lab/opt/quast5_gm_env/lib/python3.6/site-packages/quast_libs/augustus3.2.3/src/ make --always-make etraining augustus
Note: we only need two binaries (etraining and augustus), so there is no need to recompile everything.

After that, try to simply rerun Quast with the same arguments as before.

Let me know if you have any problems or my suggestion is not working!

[Leo-alves]

Hey there,
I've got two situations that I am not sure what are. Could you guys provide me with some help?
I run quast...

quast.py -o out_quast_com_CIRCOS.v3 -r ref.fa.gz -g gene:../../ref.gff.gz --threads 15 -l BAC001 --circos --glimmer --rna-finding --conserved-genes-finding --contig-thresholds 0,500,1000,10000,25000,50000,100000,250000,500000 --plots-format png --no-sv contigs.fasta
...and everything was OK, except for Busco which is not working. However, I didn't get an error message. The quast log printed it

2019-03-26 15:33:33
Running BUSCO...
Logs and results will be saved under /mnt/Volume01/bioinfo/analises/montagens_genomas/Bsubtilis/spades/out_10-12-18/out_quast_com_CIRCOS.v3/busco_stats...
Done.

I only have two files within /busco_stats/ which are:
run_BAC001.log short_summary_BAC001.txt
run_BAC001.log reports errors:

more run_BAC001.log
[hmmsearch]     Error: Failed to open sequence file /mnt/Volume01/bioinfo/analises/montagens_genomas/Bsubtilis/spades/out_10-12-18/out_quast_com_CIRC
OS.v3/busco_stats/run_BAC001/augustus_output/extracted_proteins/POG091H0041.faa.1 for reading
...
[hmmsearch]     74 of 82 task(s) completed at 03/26/2019 15:33:51
[hmmsearch]     82 of 82 task(s) completed at 03/26/2019 15:33:51
Results:
C:0.0%[S:0.0%,D:0.0%],F:0.0%,M:100.0%,n:148
0 Complete BUSCOs (C)
0 Complete and single-copy BUSCOs (S)
0 Complete and duplicated BUSCOs (D)
0 Fragmented BUSCOs (F)
148 Missing BUSCOs (M)
148 Total BUSCO groups searched
BUSCO did not find any match. Do not forget to check the file /mnt/Volume01/bioinfo/analises/montagens_genomas/Bsubtilis/spades/out_10-12-18/out_quas
t_com_CIRCOS.v3/busco_stats/run_BAC001/augustus_output/augustus.log to exclude a problem regarding Augustus
[bash]  rm: cannot remove ‘/mnt/Volume01/bioinfo/analises/montagens_genomas/Bsubtilis/spades/out_10-12-18/out_quast_com_CIRCOS.v3/busco_stats/tmp/tem
p_BAC001_3142052648’: No such file or directory
BUSCO analysis done with WARNING(s). Total running time: 18.3304510117 seconds
Results written in /mnt/Volume01/bioinfo/analises/montagens_genomas/Bsubtilis/spades/out_10-12-18/out_quast_com_CIRCOS.v3/busco_stats/run_BAC001/

See that the error report points to a folder busco_stats/run_BAC001/ which it's not here because it didn't run. Well, I've found this thread and executed the comand as you suggested.

cd /home/.../quast-5.0.2/quast_libs/augustus3.2.3/src/ 
make --always-make etraining augustus 

Rerun quast but the error continues.
However, is it possible that BUSCO is also doing something with the fly genome as we can see the option "-sp fly" in the command? And that's why it is not working? I couldn't reproduce the command as the files seems to not be there anymore.

$ more short_summary_BAC001.txt
# BUSCO version is: 3.0.2
# The lineage dataset is: bacteria_odb9 (Creation date: 2016-11-01, number of species: 3663, number of BUSCOs: 148)
# To reproduce this run: python <undefined_entry_point> -i /mnt/Volume01/bioinfo/analises/montagens_genomas/Bsubtilis/spades/out_10-12-18/out_quast_c
om_CIRCOS.v3/quast_corrected_input/BAC001.fasta -o BAC001 -l /usr/lib/python2.7/site-packages/quast-5.0.2-py2.7.egg/quast_libs/busco/bacteria/ -m gen
ome -c 15 -t /mnt/Volume01/bioinfo/analises/montagens_genomas/Bsubtilis/spades/out_10-12-18/out_quast_com_CIRCOS.v3/busco_stats/tmp/ **-sp fly** --august
us_parameters ''''
#
# Summarized benchmarking in BUSCO notation for file /mnt/Volume01/bioinfo/analises/montagens_genomas/Bsubtilis/spades/out_10-12-18/out_quast_com_CIR
COS.v3/quast_corrected_input/BAC001.fasta
# BUSCO was run in mode: genome

        C:0.0%[S:0.0%,D:0.0%],F:0.0%,M:100.0%,n:148

        0       Complete BUSCOs (C)
        0       Complete and single-copy BUSCOs (S)
        0       Complete and duplicated BUSCOs (D)
        0       Fragmented BUSCOs (F)
        148     Missing BUSCOs (M)
        148     Total BUSCO groups searched

I would be very gratefull if you could give any advice on that.
Leo

[alexeigurevich]

Hello, Leo!
Actually, BUSCO was most probably run on your assembly and the folder busco_stats/run_BAC001/ existed at the time of the run. However, Quast removes all intermediate folders when the analysis is finished, that is why you don't see it. Please run your Quast command with --debug option to keep all intermediate folders.
After that you would be able to check /mnt/Volume01/bioinfo/analises/montagens_genomas/Bsubtilis/spades/out_10-12-18/out_quas t_com_CIRCOS.v3/busco_stats/run_BAC001/augustus_output/augustus.log as the Busco log suggests. Most probably the issue is there. Note the message

[hmmsearch]     Error: Failed to open sequence file /mnt/Volume01/bioinfo/analises/montagens_genomas/Bsubtilis/spades/out_10-12-18/out_quast_com_CIRCOS.v3/busco_stats/run_BAC001/augustus_output/extracted_proteins/POG091H0041.faa.1 for reading

in the beginning of your log.

If you still is not be able to find the problem/solution, please post the Augustus log here and reopen the issue!

[liu-xingliang]

@alexeigurevich

I actually got same error message:

[hmmsearch]     Error: Failed to open sequence file /my_run/QUAST_BUSCO/quast_results/results_2019_09_25_15_40_13/busco_stats/run_dtgapfix/augustus_output/extracted_proteins/EOG09370UEC.faa.2 for reading
[hmmsearch]     Error: Failed to open sequence file /my_run/QUAST_BUSCO/quast_results/results_2019_09_25_15_40_13/busco_stats/run_dtgapfix/augustus_output/extracted_proteins/EOG09370XFG.faa.1 for reading
[hmmsearch]     Error: Failed to open sequence file /my_run/QUAST_BUSCO/quast_results/results_2019_09_25_15_40_13/busco_stats/run_dtgapfix/augustus_output/extracted_proteins/EOG09371BIR.faa.1 for reading
[hmmsearch]     Error: Failed to open sequence file /my_run/QUAST_BUSCO/quast_results/results_2019_09_25_15_40_13/busco_stats/run_dtgapfix/augustus_output/extracted_proteins/EOG093704HC.faa.1 for reading
[hmmsearch]     Error: Failed to open sequence file /my_run/QUAST_BUSCO/quast_results/results_2019_09_25_15_40_13/busco_stats/run_dtgapfix/augustus_output/extracted_proteins/EOG09370OLY.faa.1 for reading
[hmmsearch]     Error: Failed to open sequence file /my_run/QUAST_BUSCO/quast_results/results_2019_09_25_15_40_13/busco_stats/run_dtgapfix/augustus_output/extracted_proteins/EOG09370UID.faa.1 for reading
[hmmsearch]     Error: Failed to open sequence file /my_run/QUAST_BUSCO/quast_results/results_2019_09_25_15_40_13/busco_stats/run_dtgapfix/augustus_output/extracted_proteins/EOG093708TP.faa.1 for reading
[hmmsearch]     Error: Failed to open sequence file /my_run/QUAST_BUSCO/quast_results/results_2019_09_25_15_40_13/busco_stats/run_dtgapfix/augustus_output/extracted_proteins/EOG09371088.faa.2 for reading
[hmmsearch]     Error: Failed to open sequence file /my_run/QUAST_BUSCO/quast_results/results_2019_09_25_15_40_13/busco_stats/run_dtgapfix/augustus_output/extracted_proteins/EOG09370RJN.faa.1 for reading
[hmmsearch]     Error: Failed to open sequence file /my_run/QUAST_BUSCO/quast_results/results_2019_09_25_15_40_13/busco_stats/run_dtgapfix/augustus_output/extracted_proteins/EOG0937167R.faa.1 for reading
...

I was wondering I need to download those *.faa.1 files in advance or not, and where should I download them from.

Thank you!

UPDATE: the problem is actually because of augustus binary need higher version of glibc to run.

Outdated version: stdlibc++.so.6.19

Updated it to stdlibc++.so.6.26 by adding my anaconda lib folder to LD_LIBRARY_PATH:

export LD_LIBRARY_PATH=/my/anaconda3/lib:$LD_LIBRARY_PATH

The problem got solved.

[glichtenstein]

@liuxl18-hku Thanks!

in my case I added this line in my bash script, because I used miniconda

export LD_LIBRARY_PATH=~/miniconda3/lib/:$LD_LIBRARY_PATH