empty overlap set!

[Adelaam]

Hello,

I am trying Racon for first time. I am getting this error when I try to polish my data. I have tried with the files provided for the test and I get same error.

[racon::Polisher::initialize] loaded target sequences [racon::Polisher::initialize] loaded sequences [racon::Polisher::initialize] error: empty overlap set!

Thank you very much.

[rvaser]

Hi, can you paste the command you are using?

Best regards, Robert

[Adelaam]

'/home/markwilks/racon/build/bin/racon' -t 4 '/home/markwilks/Desktop/M.abscessus/barcode02/barcode02new/barcode02silter2kbq7.fastq.gz' '/home/markwilks/Desktop/M.abscessus/barcode02/barcode02new/barcode02newminimap/barcode02newoverlap.paf.gz' '/home/markwilks/Desktop/M.abscessus/barcode02/barcode02new/barcode02newminimap/barcode02newminimap.contigs.fa' [racon::Polisher::initialize] loaded target sequences [racon::Polisher::initialize] loaded sequences [racon::Polisher::initialize] error: empty overlap set! I called the second overlap but is the one I got from minimap2 (reads-assembly). BW,

[rvaser]

Could you please copy the minimap2 command you used to get the .paf file?

[Adelaam]

'/home/markwilks/minimap2-2.13_x64-linux/minimap2' -t 4 -map-ont '/home/markwilks/Desktop/M.abscessus/barcode02/barcode02new/barcode02newminimap/barcode02newminimap.contigs.fa' '/home/markwilks/Desktop/M.abscessus/barcode02/barcode02new/barcode02silter2kbq7.fastq.gz' | gzip -1 > '/home/markwilks/Desktop/M.abscessus/barcode02/barcode02new/barcode02newminimap/barcode02newoverlap.paf.gz'

[Adelaam]

Yeah, I did a mistake with the output got from minimpa2.

Thank you!

[apoosakkannu]

Hi, I am encountering similar problem, empty overlap set. I am very new to the metagenomics as well as racon. My commands are following,

minimap2 -x map-ont 98ZLc_assembly_NP.fasta 98ZLc_nanopore_filt.fastq > 98ZLc_assembly_minimap_NP.paf

racon 98ZLc_nanopore_filt.fastq 98ZLc_assembly_minimap_NP.paf 98ZLc_nanopore_filt_ava_assembly.fasta > \ 98ZLc_nanopore_filt_racon.fasta

could you please help me to resolve this problem?

Thanks.

[rvaser]

Hi, it appears that you misplaced the reference file, i.e. in the minimap2 command you are mapping reads to 98ZLc_assembly_NP.fasta and in the racon command you want to polish 98ZLc_nanopore_filt_ava_assembly.fasta. Those two files should match.

Best regards, Robert

[apoosakkannu]

Thanks for your kind reply. It has worked. Now i am getting slightly different following error,

minimap2 -x sr 98ZLc_nanopore_assembly_racon_metka2.fasta 98ZLc_combineraw_IL.fastq > 98ZLc_mini_IL.paf

racon 98ZLc_nanopore_raw.fastq 98ZLc_mini_IL.paf 98ZLc_nanopore_assembly_racon_metka2.fasta > 98ZLc_nanopore_assembly_racon_metka2_raconIL.fa [racon::Polisher::initialize] loaded target sequences 1.170 s [racon::Polisher::initialize] loaded sequences 152.444 s [racon::Polisher::initialize] loaded overlaps 116.237 s [racon::Overlap::find_breaking_points] error: overlap is not transmuted!

Could you please give some insight on this error and any possibility to overcome.

[rvaser]

It appears again that you misplaced the files :D You are mapping 98ZLc_combineraw_IL.fastq to your assembly with minimap2 and in racon you are using different reads 98ZLc_nanopore_raw.fastq. These two files should also match :)

[apoosakkannu]

my bad. Thanks. But again some error so, anbu@bubulin:~/polyplax/downstreamanalysiswithvaclavassembly$ racon -t 19 98ZLc_combineraw_IL.fastq 98ZLc_mini_IL.paf 98ZLc_nanopore_assembly_racon_metka2.fasta > 98ZLc_nanopore_assembly_racon_metka2_raconIL.fa [racon::Polisher::initialize] loaded target sequences 1.175 s [racon::Polisher::initialize] loaded sequences 200.535 s [racon::Overlap::transmute] error: unequal lengths in sequence and overlap file for sequence A00419:60:H7KWLDRXX:2:2101:3522:1016!

[rvaser]

Was the Illumina file 98ZLc_combineraw_IL.fastq created by joining 2 paired end files?

[apoosakkannu]

yes, it is !

[rvaser]

The error is probably that reads from a pair have equal names up to the first white space. You can add 1 to the first read and 2 to the second read name. Here is a script I wrote a while back: https://github.com/isovic/racon/issues/68#issuecomment-386223150. Usage is here: https://github.com/isovic/racon/issues/68#issuecomment-453412183.

[apoosakkannu]

thanks. so i made script.py file and saved it my working directory and ran the script. got the following error, anbu@bubulin:~/polyplax/downstreamanalysiswithvaclavassembly$ python script.py 98ZLc_combineraw_IL.fastq Traceback (most recent call last): File "script.py", line 46, in if (valid): NameError: name 'valid' is not defined

am i doing something wrong?

[rvaser]

Hmmm that is weird, I tried with both python2 and python3 and it works.

[apoosakkannu]

i have fastq file, is it ok

[rvaser]

Yeah, It should be a fastq file. Did you by any chance mess up the indentation in the script?

[apoosakkannu]

i just copied it. ii have not changed anything.

[apoosakkannu]

if you have script.py file, could you please attach it. I will try with it.

[rvaser]

There you go.

rename.zip

[apoosakkannu]

Thanks. It is running now. i guess it will run for a while?

[rvaser]

If you have a big file it might take a while :)

[apoosakkannu]

it is 22.1 gb, i guess big enough.

[rvaser]

Indeed it is. I hope you run the command with piping to a new file!

[apoosakkannu]

no i did not do, i am new to scripting. could you give me how to do. it is running on the screen itself.

[rvaser]

python rename.py 98ZLc_combineraw_IL.fastq > 98ZLc_renamed_IL.fastq

[apoosakkannu]

thanks.

[apoosakkannu]

do i need to do the minimap with the renamed file before polishing with racon?

[rvaser]

Unfortunately yes.

[apoosakkannu]

great, thanks.