contigs contain 'NNNNNN' ??

[ye00ye]

Hello! I'm using OPERA-MS to assembly NGS and nanopore reads.

the key parameters were set as ''--short-read1 {R1} --short-read2 {R2} --long-read {TGS} --out-dir {out_dir} --no-polishing --no-ref-clustering --no-gap-filling''

the result was named as contigs.fasta, but many contigs contain 'N', so I want to know what really the result is, contigs or scaffolds ?

Thanks for your help

I found same problem was asked in #75, but I still don't know what the answer is

[jsgounot]

Hello ye00ye,

As you can see in the figure I shared in the other thread, OPERA-MS uses OPERA-LG to scaffolds contigs. So the contigs.fa file is indeed composed of scaffolds. Sorry for the misunderstanding, we kept the name to avoid compatibility issues. Because you used option no-gap-filling, you will have N parts generated from the scaffolding.

Edit, further quote is actually wrong. There are no bugs in OPERA-MS right now regarding this option.

Now the more puzzling part is that your issue made me find another bug in OPERA-MS where --no-gap-filling does not have the expected behavior. Meaning that in your run, you probably did run the gap-filling part, which should have fill the Ns gap, at least most of them.

Therefore, would you be able to share in this thread:

1. The stdout output of your OPERA-MS command line with the timeline.
2. What is the number of % of N in your final assembly?

Thank you very much!

[jsgounot]

Hello ye00ye,

I updated my previous answer, the option works as intended and there is actually no bug here. In your case, you used no-gap-filling, OPERA-MS therefore did not cover the gap generated with OPERA-LG scaffolding. If you do not use this option, you should not have N in your final file.

Hope it helps.
JS

[ye00ye]

thanks for your answer, that is what I want, i will have a try. | | ye--ye | | ***@***.*** | ---- Replied Message ---- | From | GOUNOT ***@***.***> | | Date | 7/4/2023 15:37 | | To | ***@***.***> | | Cc | ***@***.***> , ***@***.***> | | Subject | Re: [CSB5/OPERA-MS] contigs contain 'NNNNNN' ?? (Issue #81) | Hello ye00ye, I updated my previous answer, the option works as intended and there is actually no bug here. In your case, you used no-gap-filling, OPERA-MS therefore did not cover the gap generated with OPERA-LG scaffolding. If you do not use this option, you should not have N in your final file. Hope it helps. JS — Reply to this email directly, view it on GitHub, or unsubscribe. You are receiving this because you authored the thread.Message ID: ***@***.***>

[ye00ye]

Hello JS, I run the commend as follows without --no-gap-filling: nohup perl OPERA-MS.pl \ --contig-file bc2020_ngs.fasta --long-read bc2020_nanopore_plus.fasta \ --out-dir bc2020_nanopore_ngs --no-polishing --no-ref-clustering --num-processors 10 > bc2020_nanopore_ngs.log & it is disrupted, and the log file says: Error in during gap_filling. Please see /project/huangqiang/NTGS/opera-ms/bc2020_nanopore_ngs//intermediate_files/opera_long_read/GAPFILLING/gap_filling.err for details. and then the gap_filling.err says: /project/RD/bin/OPERA-MS//tools_opera_ms//perl /project/RD/bin/OPERA-MS//OPERA-LG/bin//extract_read_sequence.pl --edge-file /project/huangqiang/NTGS/opera-ms/bc2020_nanopore_ngs//intermediate_files/read_mapping/edge_read_info.dat --contig-file /project/huangqiang/NTGS/opera-ms/bc2020_nanopore_ngs//intermediate_files/user_assembly/contigs.fasta --opera-lr-dir /project/huangqiang/NTGS/opera-ms/bc2020_nanopore_ngs//intermediate_files/opera_long_read/GAPFILLING/../ --read-file /project/huangqiang/NTGS/opera-ms/bc2020_nanopore_plus.fasta --output-directory /project/huangqiang/NTGS/opera-ms/bc2020_nanopore_ngs//intermediate_files/opera_long_read/GAPFILLING 2> /project/huangqiang/NTGS/opera-ms/bc2020_nanopore_ngs//intermediate_files/opera_long_read/GAPFILLING/extract_read.err cat /project/huangqiang/NTGS/opera-ms/bc2020_nanopore_ngs//intermediate_files/opera_long_read/GAPFILLING/consensus_cmd.sh | xargs -L 1 -P 10 -I COMMAND sh -c "COMMAND" 2> /project/huangqiang/NTGS/opera-ms/bc2020_nanopore_ngs//intermediate_files/opera_long_read/GAPFILLING/consensus_cmd.sh.log mkdir /project/huangqiang/NTGS/opera-ms/bc2020_nanopore_ngs//intermediate_files/opera_long_read/GAPFILLING/TILLING rm /project/huangqiang/NTGS/opera-ms/bc2020_nanopore_ngs//intermediate_files/opera_long_read/GAPFILLING/TILLING/consensus.fa; for f in /project/huangqiang/NTGS/opera-ms/bc2020_nanopore_ngs//intermediate_files/opera_long_read/GAPFILLING/*opera_scaffold_*/racon.fa; do sed 's/Consensus_//' $f >> /project/huangqiang/NTGS/opera-ms/bc2020_nanopore_ngs//intermediate_files/opera_long_read/GAPFILLING/TILLING/consensus.fa; done rm: cannot remove ‘/project/huangqiang/NTGS/opera-ms/bc2020_nanopore_ngs//intermediate_files/opera_long_read/GAPFILLING/TILLING/consensus.fa’: No such file or directory sed: can't read /project/huangqiang/NTGS/opera-ms/bc2020_nanopore_ngs//intermediate_files/opera_long_read/GAPFILLING/*opera_scaffold_*/racon.fa: No such file or directory /project/RD/bin/OPERA-MS//tools_opera_ms//perl /project/RD/bin/OPERA-MS//OPERA-LG/bin//run_mummer_large_ref.pl /project/huangqiang/NTGS/opera-ms/bc2020_nanopore_ngs//intermediate_files/opera_long_read/GAPFILLING/TILLING/consensus.fa /project/huangqiang/NTGS/opera-ms/bc2020_nanopore_ngs//intermediate_files/opera_long_read/GAPFILLING/TILLING/REF /project/huangqiang/NTGS/opera-ms/bc2020_nanopore_ngs//intermediate_files/user_assembly/contigs.fasta /project/huangqiang/NTGS/opera-ms/bc2020_nanopore_ngs//intermediate_files/opera_long_read/GAPFILLING/TILLING/QUERY /project/huangqiang/NTGS/opera-ms/bc2020_nanopore_ngs//intermediate_files/opera_long_read/GAPFILLING /project/huangqiang/NTGS/opera-ms/bc2020_nanopore_ngs//intermediate_files/opera_long_read/GAPFILLING/TILLING/first_tilling.coords 10 /project/RD/bin/OPERA-MS//tools_opera_ms//MUMmer3.23/ > /project/huangqiang/NTGS/opera-ms/bc2020_nanopore_ngs//intermediate_files/opera_long_read/GAPFILLING/TILLING/tilling_1.out 2> /project/huangqiang/NTGS/opera-ms/bc2020_nanopore_ngs//intermediate_files/opera_long_read/GAPFILLING/TILLING/tilling_1.err Error during tilling generation. Please see /project/huangqiang/NTGS/opera-ms/bc2020_nanopore_ngs//intermediate_files/opera_long_read/GAPFILLING/TILLING/tilling_1.out and /project/huangqiang/NTGS/opera-ms/bc2020_nanopore_ngs//intermediate_files/opera_long_read/GAPFILLING/TILLING/tilling_1.err for details. | | ye--ye | | ***@***.*** | ---- Replied Message ---- | From | GOUNOT ***@***.***> | | Date | 7/4/2023 15:37 | | To | ***@***.***> | | Cc | ***@***.***> , ***@***.***> | | Subject | Re: [CSB5/OPERA-MS] contigs contain 'NNNNNN' ?? (Issue #81) | Hello ye00ye, I updated my previous answer, the option works as intended and there is actually no bug here. In your case, you used no-gap-filling, OPERA-MS therefore did not cover the gap generated with OPERA-LG scaffolding. If you do not use this option, you should not have N in your final file. Hope it helps. JS — Reply to this email directly, view it on GitHub, or unsubscribe. You are receiving this because you authored the thread.Message ID: ***@***.***>

[jsgounot]

Hi ye00ye,

Can you also share the content of the tilling_1.err file?

Thanks,
JS

[ye00ye]

sorry, the file tilling_1.err is not produced, and there is a file called nucmer.error, the contents is 20230706|181828| 2052| ERROR: mummer and/or mgaps returned non-zero | | ye--ye | | ***@***.*** | ---- Replied Message ---- | From | GOUNOT ***@***.***> | | Date | 7/5/2023 16:01 | | To | ***@***.***> | | Cc | ***@***.***> , ***@***.***> | | Subject | Re: [CSB5/OPERA-MS] contigs contain 'NNNNNN' ?? (Issue #81) | Hi ye00ye, Can you also share the content of the tilling_1.err file? Thanks, JS — Reply to this email directly, view it on GitHub, or unsubscribe. You are receiving this because you authored the thread.Message ID: ***@***.***>

[jsgounot]

You don't see a tilling_1.err file at this location: /project/huangqiang/NTGS/opera-ms/bc2020_nanopore_ngs//intermediate_files/opera_long_read/GAPFILLING/TILLING/tilling_1.err? That's weird. What about this file: /project/huangqiang/NTGS/opera-ms/bc2020_nanopore_ngs//intermediate_files/opera_long_read/GAPFILLING/TILLING/tilling_1.out?

Can you check you're able to run one of the Mummer executables? Like nucmer, should be here: /project/RD/bin/OPERA-MS//tools_opera_ms//MUMmer3.23/nucmer

If you did not find the file before, can you try to run the command line here: /project/RD/bin/OPERA-MS//tools_opera_ms//perl /project/RD/bin/OPERA-MS//OPERA-LG/bin//run_mummer_large_ref.pl /project/huangqiang/NTGS/opera-ms/bc2020_nanopore_ngs//intermediate_files/opera_long_read/GAPFILLING/TILLING/consensus.fa /project/huangqiang/NTGS/opera-ms/bc2020_nanopore_ngs//intermediate_files/opera_long_read/GAPFILLING/TILLING/REF /project/huangqiang/NTGS/opera-ms/bc2020_nanopore_ngs//intermediate_files/user_assembly/contigs.fasta /project/huangqiang/NTGS/opera-ms/bc2020_nanopore_ngs//intermediate_files/opera_long_read/GAPFILLING/TILLING/QUERY /project/huangqiang/NTGS/opera-ms/bc2020_nanopore_ngs//intermediate_files/opera_long_read/GAPFILLING /project/huangqiang/NTGS/opera-ms/bc2020_nanopore_ngs//intermediate_files/opera_long_read/GAPFILLING/TILLING/first_tilling.coords 10 /project/RD/bin/OPERA-MS//tools_opera_ms//MUMmer3.23/?

Thanks,
JS

[ye00ye]

sorry, I found the tilling_1.err rm -r /project/huangqiang/NTGS/opera-ms2/bc2020_nanopore_ngs_reads_no_gap_filling//intermediate_files/opera_long_read/GAPFILLING/TILLING/QUERY /project/huangqiang/NTGS/opera-ms2/bc2020_nanopore_ngs_reads_no_gap_filling//intermediate_files/opera_long_read/GAPFILLING/TILLING/REF rm: cannot remove ‘/project/huangqiang/NTGS/opera-ms2/bc2020_nanopore_ngs_reads_no_gap_filling//intermediate_files/opera_long_read/GAPFILLING/TILLING/QUERY’: No such file or directory rm: cannot remove ‘/project/huangqiang/NTGS/opera-ms2/bc2020_nanopore_ngs_reads_no_gap_filling//intermediate_files/opera_long_read/GAPFILLING/TILLING/REF’: No such file or directory mkdir /project/huangqiang/NTGS/opera-ms2/bc2020_nanopore_ngs_reads_no_gap_filling//intermediate_files/opera_long_read/GAPFILLING/TILLING/REF *** split /project/huangqiang/NTGS/opera-ms2/bc2020_nanopore_ngs_reads_no_gap_filling//intermediate_files/opera_long_read/GAPFILLING/TILLING/consensus.fa Use of uninitialized value $genome in print at /project/RD/bin/OPERA-MS//OPERA-LG/bin//run_mummer_large_ref.pl line 152. mkdir /project/huangqiang/NTGS/opera-ms2/bc2020_nanopore_ngs_reads_no_gap_filling//intermediate_files/opera_long_read/GAPFILLING/TILLING/QUERY *** split /project/huangqiang/NTGS/opera-ms2/bc2020_nanopore_ngs_reads_no_gap_filling//intermediate_files/user_assembly/contigs.fasta *** run the mummer mapping rm -r /project/huangqiang/NTGS/opera-ms2/bc2020_nanopore_ngs_reads_no_gap_filling//intermediate_files/opera_long_read/GAPFILLING/mummer rm: cannot remove ‘/project/huangqiang/NTGS/opera-ms2/bc2020_nanopore_ngs_reads_no_gap_filling//intermediate_files/opera_long_read/GAPFILLING/mummer’: No such file or directory mkdir /project/huangqiang/NTGS/opera-ms2/bc2020_nanopore_ngs_reads_no_gap_filling//intermediate_files/opera_long_read/GAPFILLING/mummer cat /project/huangqiang/NTGS/opera-ms2/bc2020_nanopore_ngs_reads_no_gap_filling//intermediate_files/opera_long_read/GAPFILLING/mummer/cmd_delta.txt | xargs -I cmd --max-procs=10 bash -c cmd > /dev/null Error in during nucmer. | | ye--ye | | ***@***.*** | ---- Replied Message ---- | From | GOUNOT ***@***.***> | | Date | 7/7/2023 09:34 | | To | ***@***.***> | | Cc | ***@***.***> , ***@***.***> | | Subject | Re: [CSB5/OPERA-MS] contigs contain 'NNNNNN' ?? (Issue #81) | You don't see a tilling_1.err file at this location: /project/huangqiang/NTGS/opera-ms/bc2020_nanopore_ngs//intermediate_files/opera_long_read/GAPFILLING/TILLING/tilling_1.err? That's weird. What about this file: /project/huangqiang/NTGS/opera-ms/bc2020_nanopore_ngs//intermediate_files/opera_long_read/GAPFILLING/TILLING/tilling_1.out? Can you check you're able to run one of the Mummer executables? Like nucmer, should be here: /project/RD/bin/OPERA-MS//tools_opera_ms//MUMmer3.23/nucmer If you did not find the file before, can you try to run the command line here: /project/RD/bin/OPERA-MS//tools_opera_ms//perl /project/RD/bin/OPERA-MS//OPERA-LG/bin//run_mummer_large_ref.pl /project/huangqiang/NTGS/opera-ms/bc2020_nanopore_ngs//intermediate_files/opera_long_read/GAPFILLING/TILLING/consensus.fa /project/huangqiang/NTGS/opera-ms/bc2020_nanopore_ngs//intermediate_files/opera_long_read/GAPFILLING/TILLING/REF /project/huangqiang/NTGS/opera-ms/bc2020_nanopore_ngs//intermediate_files/user_assembly/contigs.fasta /project/huangqiang/NTGS/opera-ms/bc2020_nanopore_ngs//intermediate_files/opera_long_read/GAPFILLING/TILLING/QUERY /project/huangqiang/NTGS/opera-ms/bc2020_nanopore_ngs//intermediate_files/opera_long_read/GAPFILLING /project/huangqiang/NTGS/opera-ms/bc2020_nanopore_ngs//intermediate_files/opera_long_read/GAPFILLING/TILLING/first_tilling.coords 10 /project/RD/bin/OPERA-MS//tools_opera_ms//MUMmer3.23/? Thanks, JS — Reply to this email directly, view it on GitHub, or unsubscribe. You are receiving this because you authored the thread.Message ID: ***@***.***>

[jsgounot]

Hi,

this might be an installation issue (see other issues). You need to open the cmd_delta.txt file, try to run one command and see what happen.

Regards,
Jean-Sebastien