hoelzer-lab / ribap

A comprehensive bacterial core gene-set annotation pipeline based on Roary and pairwise ILPs
GNU General Public License v3.0
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RIBAP:filter_alignment failed #54

Closed bharat1912 closed 1 month ago

bharat1912 commented 10 months ago

Hi Martin, The following error occurred at the alignment step though the test data ran without failure. I also ran a small 8 strain dataset and that also ran without any errors. However, this 28 strain dataset which includes the 8 strains that I had run previously, failed at completion.

Any assistance provided is appreciated.


$nextflow run hoelzer-lab/ribap -r 1.0.0 --fasta '*.fasta' --cores 12 --max_cores 12 -profile local,docker --tree --bootstrap 1000 --outdir ribap-results -w ribap-work N E X T F L O W ~ version 23.04.1 Launching https://github.com/hoelzer-lab/ribap [confident_bhabha] DSL2 - revision: f5118310a3 [1.0.0]

Profile: local,docker

Current User: bharat Nextflow-version: 23.04.1 Starting time: 15-04-2023 06:51 UTC Workdir location (intermediate files): /media/bharat/volume1/Bacillaceaea_ref_July_2023/renamed_files/RIBAP_2/ribap-work Output dir name: results

INFORMATION: ILPs and their intermediate results are deleted to save disk space (use --keepILPs to keep them). executor > local (319) [ee/54328f] process > RIBAP:rename (14) [100%] 28 of 28 ✔ [08/3812ec] process > RIBAP:prokka (28) [100%] 28 of 28 ✔ [f6/30fad1] process > RIBAP:strain_ids [100%] 1 of 1 ✔ [5c/9676ca] process > RIBAP:roary (1) [100%] 5 of 5 ✔ [0b/13cbf5] process > RIBAP:mmseqs2 [100%] 1 of 1 ✔ [7b/14b0ad] process > RIBAP:mmseqs2tsv [100%] 1 of 1 ✔ [0e/8f72fb] process > RIBAP:ilp_refinement (1) [100%] 9 of 9 ✔ [39/937d29] process > RIBAP:combine_roary_ilp (1) [100%] 1 of 1 ✔ [63/64b14f] process > RIBAP:prepare_msa (1) [100%] 1 of 1 ✔ [ca/a9b0a3] process > RIBAP:mafft (87) [100%] 99 of 99 ✔ [db/6ace5b] process > RIBAP:fasttree (98) [ 92%] 92 of 99 [31/f1c012] process > RIBAP:nw_display (43) [ 40%] 37 of 92 [- ] process > RIBAP:generate_html - [63/3e7d48] process > RIBAP:generate_upsetr_input... [100%] 1 of 1 ✔ [ce/c7ea5a] process > RIBAP:upsetr (1) [100%] 1 of 1 ✔ [ee/db3760] process > RIBAP:filter_alignment [ 0%] 0 of 1 [- ] process > RIBAP:nexus_core - [- ] process > RIBAP:iqtree - ERROR ~ Error executing process > 'RIBAP:filter_alignment'

Caused by: Process RIBAP:filter_alignment terminated with an error exit status (1)

Command executed:

for file in group1070_mafft.aln group1071_mafft.aln group1072_mafft.aln group1073_mafft.aln group1074.1_mafft.aln group1074_mafft.aln group1075.1_mafft.aln group1075.2_mafft.aln group1075.3_mafft.aln group1075.4_mafft.aln ........................................................................................................................ group2666_mafft.aln group2667.1_mafft.aln group2667_mafft.aln group2668_mafft.aln group266_mafft.aln group267.1_mafft.aln group2671_mafft.aln group2673_mafft.aln group2675_mafft.aln group2676.1_mafft.aln group2676.2_mafft.aln group2676.3_mafft.aln group2676.4_mafft.aln group2676.5_mafft.aln group267_mafft.aln group2905_mafft.aln group2908_mafft.aln group2909_mafft.aln group2910_mafft.aln group2911_mafft.aln group2912_mafft.aln group2913_mafft.aln group2914_mafft.aln group2915_mafft.aln group2919_mafft.aln group2920_mafft.aln group2922_mafft.aln group2923_mafft.aln group2924_mafft.aln group2926.1_mafft.aln group2926_mafft.aln group2928_mafft.aln group292_mafft.aln group293.1_mafft.aln group2933_mafft.aln group2934_mafft.aln group2935_mafft.aln group2936_mafft.aln group2937_mafft.aln group2938_mafft.aln group293_mafft.aln group2940_mafft.aln group2942_mafft.aln group2943_mafft.aln group2944_mafft.aln group2948_mafft.aln group2949_mafft.aln group294_mafft.aln group295.1_mafft.aln group295.2_mafft.aln group295.3_mafft.aln group2951_mafft.aln group2953_mafft.aln group2954_mafft.aln group2955_mafft.aln group2956_mafft.aln group2957_mafft.aln group295_mafft.aln group2962_mafft.aln group2963_mafft.aln group2966_mafft.aln group2967_mafft.aln group2969_mafft.aln group296_mafft.aln group2971_mafft.aln group2166_mafft.aln group2167_mafft.aln group2168.1_mafft.aln group2168_mafft.aln group2169_mafft.aln group2171.1_mafft.aln group2171_mafft.aln group2172_mafft.aln group2173_mafft.aln group2174_mafft.aln group2175.1_mafft.aln group2175.2_mafft.aln group2175_mafft.aln group2176_mafft.aln group2177.1_mafft.aln group2177_mafft.aln 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group2382.6_mafft.aln group2382_mafft.aln group2383_mafft.aln group2384_mafft.aln group2385.1_mafft.aln group2385_mafft.aln group2386.1_mafft.aln group2386.2_mafft.aln group2386.3_mafft.aln group2386.4_mafft.aln group2386.5_mafft.aln group2386.6_mafft.aln group2386_mafft.aln group2387_mafft.aln group2388_mafft.aln group2389_mafft.aln group2390_mafft.aln group2391.1_mafft.aln group2391.2_mafft.aln group2391.3_mafft.aln group2391_mafft.aln group2392_mafft.aln group2393_mafft.aln group2394_mafft.aln group2395_mafft.aln group2396_mafft.aln group2397_mafft.aln group2398_mafft.aln group2399_mafft.aln group239_mafft.aln group240.1_mafft.aln group240.2_mafft.aln group240.3_mafft.aln group240.4_mafft.aln group240_mafft.aln group24_mafft.aln group2328.2_mafft.aln group2328_mafft.aln group2329_mafft.aln group2330_mafft.aln group2331_mafft.aln group2332_mafft.aln group2333_mafft.aln group2334.1_mafft.aln group2334_mafft.aln group2335_mafft.aln group2337_mafft.aln group2338_mafft.aln 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group3732_mafft.aln group3734_mafft.aln group3737_mafft.aln group373_mafft.aln group3745_mafft.aln group3754_mafft.aln group3755_mafft.aln group375_mafft.aln group376.10_mafft.aln group376.11_mafft.aln group376.12_mafft.aln group376.1_mafft.aln group376.2_mafft.aln group376.3_mafft.aln group376.4_mafft.aln group376.5_mafft.aln group376.6_mafft.aln group376.7_mafft.aln group376.8_mafft.aln group376.9_mafft.aln group3762_mafft.aln group3763_mafft.aln group3764_mafft.aln group3765_mafft.aln group3766_mafft.aln group3767_mafft.aln group3768_mafft.aln group3769_mafft.aln group376_mafft.aln group3770_mafft.aln group3774_mafft.aln group3775_mafft.aln group3777_mafft.aln group377_mafft.aln group3784_mafft.aln group3789_mafft.aln group378_mafft.aln group3795_mafft.aln group379_mafft.aln group243.22_mafft.aln group243.23_mafft.aln group243.3_mafft.aln group243.4_mafft.aln group243.5_mafft.aln group243.6_mafft.aln group243.7_mafft.aln group243.8_mafft.aln group243.9_mafft.aln group2430_mafft.aln 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group525.17_mafft.aln group525.18_mafft.aln group525.19_mafft.aln group525.1_mafft.aln group525.20_mafft.aln group525.21_mafft.aln group525.22_mafft.aln group525.23_mafft.aln group525.24_mafft.aln ................................................... group657.78_mafft.aln group657.79_mafft.aln group657.7_mafft.aln group657.80_mafft.aln group657.81_mafft.aln group657.82_mafft.aln group657.83_mafft.aln group657.84_mafft.aln group657.85_mafft.aln group657.86_mafft.aln group657.87_mafft.aln group657.8_mafft.aln; do NUM=$(grep -c '>' $file) STRAINS=$(cat strain_ids.txt | wc -l) if [ $NUM -eq $STRAINS ]; then cp ${file} "$(basename ${file} .aln)"core.aln sed -r -i '/>/ s/[^_]+$//' "$(basename ${file} .aln)"_core.aln sed -r -i '/^[^>]/ s/-/X/g' "$(basename ${file} .aln)"_core.aln fi done

for file in *_core.aln; do cd-hit -i $file -o "$file"_TMP -c 1.0 RECORDS=$(grep -c ">" "$file"TMP) if [ $RECORDS -ne 1 ]; then cp $file FINAL$file sed -r -i '/^[^>]/ s/X/-/g' FINAL_$file fi
done

Command exit status: 1

Command output:

Program: CD-HIT, V4.8.1 (+OpenMP), Apr 07 2021, 10:57:21 Command: cd-hit -i _core.aln -o _core.aln_TMP -c 1.0

Started: Thu Aug 17 06:33:33 2023

                          Output                              

Command error:

executor > local (319) [ee/54328f] process > RIBAP:rename (14) [100%] 28 of 28 ✔ [08/3812ec] process > RIBAP:prokka (28) [100%] 28 of 28 ✔ [f6/30fad1] process > RIBAP:strain_ids [100%] 1 of 1 ✔ [5c/9676ca] process > RIBAP:roary (1) [100%] 5 of 5 ✔ [0b/13cbf5] process > RIBAP:mmseqs2 [100%] 1 of 1 ✔ [7b/14b0ad] process > RIBAP:mmseqs2tsv [100%] 1 of 1 ✔ [0e/8f72fb] process > RIBAP:ilp_refinement (1) [100%] 9 of 9 ✔ [39/937d29] process > RIBAP:combine_roary_ilp (1) [100%] 1 of 1 ✔ [63/64b14f] process > RIBAP:prepare_msa (1) [100%] 1 of 1 ✔ [ca/a9b0a3] process > RIBAP:mafft (87) [100%] 99 of 99 ✔ [b2/870419] process > RIBAP:fasttree (99) [100%] 92 of 92 [31/f1c012] process > RIBAP:nw_display (43) [ 43%] 37 of 86 [- ] process > RIBAP:generate_html - [63/3e7d48] process > RIBAP:generate_upsetr_input... [100%] 1 of 1 ✔ [ce/c7ea5a] process > RIBAP:upsetr (1) [100%] 1 of 1 ✔ [ee/db3760] process > RIBAP:filter_alignment [100%] 1 of 1, failed: 1 ✘ [- ] process > RIBAP:nexus_core - [- ] process > RIBAP:iqtree - ERROR ~ Error executing process > 'RIBAP:filter_alignment'

Caused by: Process RIBAP:filter_alignment terminated with an error exit status (1)

Command executed:

for file in group1070_mafft.aln group1071_mafft.aln group1072_mafft.aln group1073_mafft.aln group1074.1_mafft.aln group1074_mafft.aln group1075.1_mafft.aln group1075.2_mafft.aln group1075.3_mafft.aln group1075.4_mafft.aln group1075_mafft.aln group1076.1_mafft.aln group1076.2_mafft.aln group1076.3_mafft.aln group1076_mafft.aln group1077.1_mafft.aln group1077.2_mafft.aln group1077_mafft.aln group1078_mafft.aln group1079_mafft.aln group107_mafft.aln group108.1_mafft.aln group1080_mafft.aln group1082_mafft.aln group1084_mafft.aln group1085_mafft.aln group1086_mafft.aln group1087.1_mafft.aln group1087.2_mafft.aln group1087.3_mafft.aln group1087.4_mafft.aln group1087.5_mafft.aln group1087.6_mafft.aln group1087.7_mafft.aln group1087.8_mafft.aln group1087.9_mafft.aln group1087_mafft.aln group1088_mafft.aln group108_mafft.aln group1090_mafft.aln group1094.1_mafft.aln group1094.2_mafft.aln group1094_mafft.aln group1096_mafft.aln group1097.1_mafft.aln group1097_mafft.aln group1098_mafft.aln group1099_mafft.aln group109_mafft.aln group11_mafft.aln group1100_mafft.aln group1101_mafft.aln group1102.1_mafft.aln group1102.2_mafft.aln group1102_mafft.aln group1104_mafft.aln group1105_mafft.aln group1106_mafft.aln group1107_mafft.aln group1109_mafft.aln group110_mafft.aln group1110_mafft.aln group1112_mafft.aln group1113_mafft.aln group1114_mafft.aln group1116_mafft.aln group1117.1_mafft.aln group1117_mafft.aln group1119_mafft.aln group111_mafft.aln group112.1_mafft.aln group112.2_mafft.aln group112.3_mafft.aln group112.4_mafft.aln group112.5_mafft.aln group112.6_mafft.aln group112.7_mafft.aln group112.8_mafft.aln group112.9_mafft.aln group1122_mafft.aln group1123.1_mafft.aln group1123_mafft.aln group1124_mafft.aln group1127_mafft.aln group1128_mafft.aln group1129_mafft.aln group112_mafft.aln group1130_mafft.aln group1132.1_mafft.aln group1132.2_mafft.aln group1132.3_mafft.aln group1132.4_mafft.aln group1132.5_mafft.aln group1132.6_mafft.aln group1132.7_mafft.aln group1132_mafft.aln group1133_mafft.aln group1134_mafft.aln group1135.1_mafft.aln group113_mafft.aln group1135_mafft.aln group1136_mafft.aln group1137_mafft.aln group1138_mafft.aln group1139_mafft.aln group1140_mafft.aln group1141_mafft.aln group1142_mafft.aln group1143_mafft.aln group1144_mafft.aln group1148.1_mafft.aln group1148_mafft.aln group1150_mafft.aln group1151_mafft.aln group1153_mafft.aln group1155_mafft.aln group1156_mafft.aln group1157.1_mafft.aln group1157_mafft.aln group1158_mafft.aln group1159_mafft.aln group115_mafft.aln group1161.1_mafft.aln group1161_mafft.aln group1163_mafft.aln group1167_mafft.aln group1168_mafft.aln group1169_mafft.aln group117.1_mafft.aln group117.2_mafft.aln group117.3_mafft.aln group117.4_mafft.aln group117.5_mafft.aln group1170_mafft.aln group1171_mafft.aln group1172_mafft.aln group1173_mafft.aln group1175_mafft.aln group1176_mafft.aln group1177.1_mafft.aln group1177.2_mafft.aln group1177.3_mafft.aln group1177.4_mafft.aln group1177_mafft.aln group1178_mafft.aln group1179_mafft.aln group117_mafft.aln group118.1_mafft.aln group118.2_mafft.aln group118.3_mafft.aln group118.4_mafft.aln group1181_mafft.aln group1182_mafft.aln group1183_mafft.aln group1184_mafft.aln group1185_mafft.aln group1186.1_mafft.aln group1186_mafft.aln group1187_mafft.aln group1188_mafft.aln group1189_mafft.aln group118_mafft.aln group1190_mafft.aln group1191_mafft.aln group1192_mafft.aln group1193_mafft.aln ......................................................................................................... ............................................................................ mafft.aln group657.83_mafft.aln group657.84_mafft.aln group657.85_mafft.aln group657.86_mafft.aln group657.87_mafft.aln group657.8_mafft.aln; do NUM=$(grep -c '>' $file) STRAINS=$(cat strain_ids.txt | wc -l) if [ $NUM -eq $STRAINS ]; then cp ${file} "$(basename ${file} .aln)"core.aln sed -r -i '/>/ s/[^_]+$//' "$(basename ${file} .aln)"_core.aln sed -r -i '/^[^>]/ s/-/X/g' "$(basename ${file} .aln)"_core.aln fi done

for file in *_core.aln; do cd-hit -i $file -o "$file"_TMP -c 1.0 RECORDS=$(grep -c ">" "$file"TMP) if [ $RECORDS -ne 1 ]; then cp $file FINAL$file sed -r -i '/^[^>]/ s/X/-/g' FINAL_$file fi
done

Command exit status: 1

Command output:

Program: CD-HIT, V4.8.1 (+OpenMP), Apr 07 2021, 10:57:21 Command: cd-hit -i _core.aln -o _core.aln_TMP -c 1.0

Started: Thu Aug 17 06:33:33 2023

                          Output                              

Command error:

Fatal Error: Failed to open the database file Program halted !!

================================================================ Program: CD-HIT, V4.8.1 (+OpenMP), Apr 07 2021, 10:57:21 Command: cd-hit -i _core.aln -o _core.aln_TMP -c 1.0

Started: Thu Aug 17 06:33:33 2023

                          Output                              

Work dir: /media/bharat/volume1/Bacillaceaea_ref_July_2023/renamed_files/RIBAP_2/ribap-work/ee/db37603abceec37bc9c0b422842ae2

Tip: view the complete command output by changing to the process work dir and entering the command cat .command.out

-- Check '.nextflow.log' file for details

hoelzer commented 10 months ago

hey @bharat1912 thanks for using RIBAP and the error report!

Hm, the error code 1 you get does us unfortunately not tell much. It might be that there is some problem with the CDHIT clustering command.

Can you check what is the content of this file:

cat /media/bharat/volume1/Bacillaceaea_ref_July_2023/renamed_files/RIBAP_2/ribap-work/ee/db37603abceec37bc9c0b422842ae2/.command.log

or

cat /media/bharat/volume1/Bacillaceaea_ref_July_2023/renamed_files/RIBAP_2/ribap-work/ee/db37603abceec37bc9c0b422842ae2/.command.err

(might be the same)

Besides, can you share the 28 genomes or is this confidential data? So I could try it and see what happens exactly. I could also sent you an upload link, if you can share them.

bharat1912 commented 10 months ago

Hi martin Thanks for your rapid response.

  1. I reran the same strains with revision 1.0.1 and got the same error.

  2. The last part of the log from .nextflow.log is: Command exit status: 1

Command output:

Program: CD-HIT, V4.8.1 (+OpenMP), Apr 07 2021, 10:57:21 Command: cd-hit -i _core.aln -o _core.aln_TMP -c 1.0

Started: Fri Aug 18 04:49:49 2023

                          Output                              

Command error:

Fatal Error: Failed to open the database file Program halted !!

================================================================ Program: CD-HIT, V4.8.1 (+OpenMP), Apr 07 2021, 10:57:21 Command: cd-hit -i _core.aln -o _core.aln_TMP -c 1.0

Started: Fri Aug 18 04:49:49 2023

                          Output                              

Work dir: /media/bharat/volume1/Bacillaceaea_ref_July_2023/renamed_files/RIBAP_2/ribap-work/c0/8c856210150aab580f47ae98c68302

Tip: when you have fixed the problem you can continue the execution adding the option -resume to the run command line Aug.-18 14:49:50.525 [Task monitor] DEBUG nextflow.Session - Session aborted -- Cause: Process RIBAP:filter_alignment terminated with an error exit status (1) Aug.-18 14:49:50.554 [Task monitor] DEBUG nextflow.Session - The following nodes are still active: [process] RIBAP:nw_display status=ACTIVE port 0: (queue) OPEN ; channel: nwk port 1: (cntrl) - ; channel: $

[process] RIBAP:generate_html status=ACTIVE port 0: (queue) OPEN ; channel: - port 1: (value) bound ; channel: roary port 2: (value) bound ; channel: individual_annotations port 3: (value) OPEN ; channel: tree_svg port 4: (cntrl) - ; channel: $

Aug.-18 14:49:50.556 [main] DEBUG nextflow.Session - Session await > all processes finished Aug.-18 14:49:50.556 [main] DEBUG nextflow.Session - Session await > all barriers passed Aug.-18 14:49:50.565 [Task monitor] DEBUG n.processor.TaskPollingMonitor - Task completed > TaskHandler[id: 368; name: RIBAP:nw_display (38); status: COMPLETED; exit: 0; error: -; workDir: /media/bharat/volume1/Bacillaceaea_ref_July_2023/renamed_files/RIBAP_2/ribap-work/de/67762899c4ebb6114870b802f99d2e] Aug.-18 14:49:50.574 [main] WARN n.processor.TaskPollingMonitor - Killing running tasks (11) Aug.-18 14:49:50.574 [Task monitor] DEBUG n.processor.TaskPollingMonitor - Task completed > TaskHandler[id: 369; name: RIBAP:nw_display (39); status: COMPLETED; exit: 0; error: -; workDir: /media/bharat/volume1/Bacillaceaea_ref_July_2023/renamed_files/RIBAP_2/ribap-work/5e/fa22828f7e6c2f617dd654c1a8eb22] Aug.-18 14:49:50.628 [main] DEBUG nextflow.trace.WorkflowStatsObserver - Workflow completed > WorkflowStats[succeededCount=369; failedCount=1; ignoredCount=0; cachedCount=0; pendingCount=45; submittedCount=1; runningCount=-1; retriesCount=0; abortedCount=10; succeedDuration=8d 20h 30m 7s; failedDuration=43.7s; cachedDuration=0ms;loadCpus=-1; loadMemory=0; peakRunning=12; peakCpus=12; peakMemory=0; ] Aug.-18 14:49:50.787 [main] DEBUG nextflow.cache.CacheDB - Closing CacheDB done Aug.-18 14:49:50.803 [main] DEBUG nextflow.script.ScriptRunner - > Execution complete -- Goodbye

  1. You have suggested that It might be that there is some problem with the CDHIT clustering command. I had an issue with CD-HIT (ver 4.8.1), installed as a part of a different pipeline referenced here -- https://github.com/weizhongli/cdhit/issues/105. However, I am not sure why the test dataset does not fail at this point or a set of my 8 genomes but only fils with an extended data set as explained previously.

  2. No problems, I can upload the genome fasta files to your nominated site. Please let me know.

Thanks

Bharat

hoelzer commented 10 months ago

Hey Bharat, okay, please share the genome FASTAs with me and then I can have a deeper look.

You can use https://wetransfer.com/ which should be easy and fast. As my email adress you can set h*.m@gmail.com

thx!

hoelzer commented 10 months ago

Hey @bharat1912 I can reproduce your error.

The problem is that we do not find any core RIBAP group that has all of your 28 species in it.

Overall, we found 4956 RIBAP groups with varying numbers of species. In the filter_alignment step we look specifically for RIBAP groups with one gene from each input species: "core" RIBAP groups. But there is non for your 28 input genomes. However, there are 1756 RIBAP groups with 27/28 species.

From these core RIBAP groups, we remove all that have 100% identical sequences, and for the rest, we build multiple sequence alignments and the tree.

It seems we never had the case that for an input there was no core gene found in all input genomes. Because of that, cdhit is then started on an empty FASTA file and produces the error.

I am thinking what would be a good fix for that. We could, after looking for RIBAP groups with 28 genes, go down and look for groups with 27 genes if there is nothing found for all species. But then I am not sure how this will affect the MSA and tree calculations.

I think for now, you could run RIBAP without the --tree flag to prevent the error. But then you dont get a core gene tree.

We will keep you posted when we came up with a fix (that could be also to catch the error and tell the user that there is no core gene tree possible).

hoelzer commented 1 month ago

A solution is provided via https://github.com/hoelzer-lab/ribap/releases/tag/1.0.2