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OPERA-MS - Hybrid Metagenomic Assembler
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no contigs.fasta output, problem with Gap filling step #56

Closed DDavila10 closed 3 years ago

DDavila10 commented 3 years ago

Hi all,

I am currently using OPERA-MS. I run the test files and works perfectly but using my data I do not have a final contings.fasta, conting_info.txt or assembly.stats.

The log.err indicates a problem in Gap_filling step

/beegfs/group_dv/home/DDavila/OPERA-MS/opera_assembly_reference_metagenome/..//tools_opera_ms/perl /beegfs/group_dv/home/DDavila/OPERA-MS/opera_assembly_reference_metagenome/..//OPERA-LG/bin//gapfilling.pl /beegfs/group_dv/home/DDavila/OPERA-MS/opera_assembly_reference_metagenome/hybrid_reference_metagenome_by_opera_mouse0476//intermediate_files/opera_long_read/GAPFILLING /beegfs/group_dv/home/DDavila/OPERA-MS/opera_assembly_reference_metagenome/hybrid_reference_metagenome_by_opera_mouse0476//intermediate_files/megahit_assembly/final.contigs.fa /beegfs/group_dv/home/DDavila/mouse_metagenomes/adaptor_trimmed_mouse0476_metagenomics_nanopore.fa /beegfs/group_dv/home/DDavila/OPERA-MS/opera_assembly_reference_metagenome/hybrid_reference_metagenome_by_opera_mouse0476//intermediate_files/read_mapping 10 499 /beegfs/group_dv/home/DDavila/OPERA-MS/opera_assembly_reference_metagenome/..//OPERA-LG/bin/ /beegfs/group_dv/home/DDavila/OPERA-MS/opera_assembly_reference_metagenome/..//tools_opera_ms/ /beegfs/group_dv/home/DDavila/OPERA-MS/opera_assembly_reference_metagenome/..//tools_opera_ms/ /beegfs/group_dv/home/DDavila/OPERA-MS/opera_assembly_reference_metagenome/..//tools_opera_ms/ /beegfs/group_dv/home/DDavila/OPERA-MS/opera_assembly_reference_metagenome/..//tools_opera_ms//MUMmer3.23/ 2> /beegfs/group_dv/home/DDavila/OPERA-MS/opera_assembly_reference_metagenome/hybrid_reference_metagenome_by_opera_mouse0476//intermediate_files/opera_long_read/GAPFILLING/gap_filling.err

and the last step of OPERA-MS only mentioned

[Mon Apr 5 18:36:03 2021] Gap filling [8/8]

and then stops

Could you please help me with this problem? I would really appreciate it !

Best,

jsgounot commented 3 years ago

Hi Davila,

Thanks for using OPERA-MS ! Is /beegfs/group_dv/home/DDavila/OPERA-MS/opera_assembly_reference_metagenome/hybrid_reference_metagenome_by_opera_mouse0476//intermediate_files/opera_long_read/GAPFILLING/gap_filling.err empty ?

DDavila10 commented 3 years ago

Hi Jean-Sebastien,

Thank you so much for your prompt reply. gap_filling.err is not empty

/beegfs/group_dv/home/DDavila/OPERA-MS/opera_assembly_reference_metagenome/..//tools_opera_ms//perl /beegfs/group_dv/home/DDavila/OPERA-MS/opera_assembly_reference_metagenome/..//OPERA-LG/bin//extract_read_sequence.pl --edge-file /beegfs/group_dv/home/DDavila/OPERA-MS/opera_assembly_reference_metagenome/hybrid_reference_metagenome_by_opera_mouse0476//intermediate_files/read_mapping/edge_read_info.dat --contig-file /beegfs/group_dv/home/DDavila/OPERA-MS/opera_assembly_reference_metagenome/hybrid_reference_metagenome_by_opera_mouse0476//intermediate_files/megahit_assembly/final.contigs.fa --opera-lr-dir /beegfs/group_dv/home/DDavila/OPERA-MS/opera_assembly_reference_metagenome/hybrid_reference_metagenome_by_opera_mouse0476//intermediate_files/opera_long_read/GAPFILLING/../ --read-file /beegfs/group_dv/home/DDavila/mouse_metagenomes/adaptor_trimmed_mouse0476_metagenomics_nanopore.fa --output-directory /beegfs/group_dv/home/DDavila/OPERA-MS/opera_assembly_reference_metagenome/hybrid_reference_metagenome_by_opera_mouse0476//intermediate_files/opera_long_read/GAPFILLING 2> /beegfs/group_dv/home/DDavila/OPERA-MS/opera_assembly_reference_metagenome/hybrid_reference_metagenome_by_opera_mouse0476//intermediate_files/opera_long_read/GAPFILLING/extract_read.err

but tilling_1.err is empty

Could you please help me to solve this problem?

Thanks a lot in advance

Best,

DDavila10 commented 3 years ago

extract_read.err contains:

Starting the sequence extraction rm -rf /beegfs/group_dv/home/DDavila/OPERA-MS/opera_assembly_reference_metagenome/hybrid_reference_metagenome_by_opera_mouse0476//intermediate_files/opera_long_read/GAPFILLING/LOG mkdir -p /beegfs/group_dv/home/DDavila/OPERA-MS/opera_assembly_reference_metagenome/hybrid_reference_metagenome_by_opera_mouse0476//intermediate_files/opera_long_read/GAPFILLING/LOG Number of edges selected for gapfilling 119896 /beegfs/group_dv/home/DDavila/OPERA-MS/opera_assembly_reference_metagenome/hybrid_reference_metagenome_by_opera_mouse0476//intermediate_files/opera_long_read/GAPFILLING/contig_extention.log *** Extract gap sequences from read file /beegfs/group_dv/home/DDavila/mouse_metagenomes/adaptor_trimmed_mouse0476_metagenomics_nanopore.fa

Is it helpful for you?

Best,

jsgounot commented 3 years ago

Did you mean extract_read.err instead of tilling_1.err ? If not, is it also empty ?

Regards,

jsgounot commented 3 years ago

That's unusual, in this context your file /beegfs/group_dv/home/DDavila/mouse_metagenomes/adaptor_trimmed_mouse0476_metagenomics_nanopore.fa is supposed to contained reads. Is this the case ? Can you share the command line you used to run OPERA-MS ?

DDavila10 commented 3 years ago

Thanks for the prompt answer! sorry for the misunderstanding.

extract_read.err is not empty tilling_1.err is empty.

/beegfs/group_dv/home/DDavila/mouse_metagenomes/adaptor_trimmed_mouse0476_metagenomics_nanopore.fa contains all the nanopore sequences.

This is the script that I used for running OPERA-MS

!/bin/bash

SBATCH --cpus-per-task=18

SBATCH --mem=15gb

SBATCH --time=5-24

SBATCH -p hooli

SBATCH -o mouse1.kneaddata.opera.assembly.out

singularity exec /beegfs/common/singularity/bioinformatics_software.v3.0.7.sif /bin/bash << SHI

!/bin/bash

source ~/.bashrc

module load perl

perl ../OPERA-MS.pl \ --short-read1 all_paired1_kneaddata_mouse0476.fastq \ --short-read2 all_paired1_kneaddata_mouse0476.fastq \ --long-read ~/mouse_metagenomes/adaptor_trimmed_mouse0476_metagenomics_nanopore.fa \ --no-ref-clustering \ --out-dir hybrid_reference_metagenome_by_opera_mouse0476 2> log.err

In our server, we use images and Singularity. I used the same script for the test_files and works perfectly.

Best,

jsgounot commented 3 years ago

Could you also looked the content of this file ? /beegfs/group_dv/home/DDavila/OPERA-MS/opera_assembly_reference_metagenome/hybrid_reference_metagenome_by_opera_mouse0476//intermediate_files/opera_long_read/GAPFILLING/edge_err

thanks,

DDavila10 commented 3 years ago

yes, the content looks like

k99_1575792-vs-k99_92999 01c989c7-2f88-42f1-96f9-dfd2fdfe4c15 NA 2758 2659 99 OVERLAP k99_551112-vs-k99_587244 3068709b-66e0-4867-a8d8-dede2ccd07f7 NA 980 880 100 OVERLAP k99_512847-vs-k99_731839 6acedaa1-9025-4a0e-b73f-8476167c5337 0 1518 1985 467 GAP k99_1788660-vs-k99_185894 4a415099-186b-4737-9dc1-048e91878a0c NA 588 576 12 OVERLAP k99_1136312-vs-k99_1338282 546970f7-6a4e-4968-82af-0575606149e0 0 10945 11166 221 GAP k99_266455-vs-k99_680457 1a37ee91-3e6e-4273-9bfd-8c95b77ab60e 0 10659 12551 1892 GAP k99_1002566-vs-k99_2081854 0ac42566-d74e-423b-9621-6d78618bf8c6 NA 4931 4834 97 OVERLAP k99_1244537-vs-k99_669254 0ac42566-d74e-423b-9621-6d78618bf8c6 NA 3418 3329 89 OVERLAP k99_2081854-vs-k99_669254 0ac42566-d74e-423b-9621-6d78618bf8c6 NA 4356 4257 99 OVERLAP k99_2104342-vs-k99_836226 0ac42566-d74e-423b-9621-6d78618bf8c6 NA 646 544 102 OVERLAP k99_1244537-vs-k99_2104342 0ac42566-d74e-423b-9621-6d78618bf8c6 NA 2491 2390 101 OVERLAP k99_2101708-vs-k99_399170 d926bedb-70ba-430b-b027-2d2742df5494 NA 4841 4743 98 OVERLAP k99_1607702-vs-k99_956367 9f9b6b18-d96f-4a48-b914-dc75dd8ec8f9 NA 2465 2372 93 OVERLAP k99_1781482-vs-k99_2431936 15e106a1-da3f-4d9e-9f81-3d6477ce3b01 1 1418 2269 851 GAP k99_2431936-vs-k99_933394 15e106a1-da3f-4d9e-9f81-3d6477ce3b01 1 3176 10297 7121 GAP k99_1578627-vs-k99_1670263 0c9256b6-1998-4d56-ae33-10efbd033e7c NA 6834 6736 98 OVERLAP k99_1578627-vs-k99_5172 0c9256b6-1998-4d56-ae33-10efbd033e7c NA 10680 10584 96 OVERLAP k99_119743-vs-k99_2204764 09971716-1c0e-435e-95f3-c0ab1a25175c NA 1050 948 102 OVERLAP k99_1343127-vs-k99_786594 07aea4a4-00ea-421f-8fbf-2427e596218b NA 759 661 98 OVERLAP k99_1732075-vs-k99_906783 c9dc1130-b543-4132-bdb0-eb4e9d68c3aa 1 8065 8795 730 GAP k99_457650-vs-k99_734317 2552358d-b184-4a97-b98d-b213ee967981 NA 10244 10148 96 OVERLAP k99_404361-vs-k99_874010 2552358d-b184-4a97-b98d-b213ee967981 NA 5467 5368 99 OVERLAP k99_2019235-vs-k99_874010 2552358d-b184-4a97-b98d-b213ee967981 0 7031 7275 244 GAP k99_2019235-vs-k99_734317 2552358d-b184-4a97-b98d-b213ee967981 0 8940 8983 43 GAP k99_138485-vs-k99_748446 2552358d-b184-4a97-b98d-b213ee967981 0 15247 15912 665 GAP

Best,

jsgounot commented 3 years ago

Ok then it's really weird and I will be unable to debug this without your input files. Is it possible for you to mail them to me ? Sorry for inconvenience.

DDavila10 commented 3 years ago

thanks for the valuable help. I can share part of the input files. Where should I send it to?

jsgounot commented 3 years ago

jsgounot+github@gmail.com

DDavila10 commented 3 years ago

Hi Jean-Sebastien,

After checking, I had a problem with the nanopore.fa file, so I used the fastq version of the same reads and works!! Thanks a lot for the valuable help, support and prompt response!

Best!