"Error: no fast5 file found" When indexing

[SanityTree]

Hello,

I managed to run through the example provided so I know the code is working. But when I used a bit of sample data I generated a while back it gave me this error when I tried to index the data. Any suggestions to correct it?

I suspect maybe it is to do with the nature of my data? My Nanopore run was comparing a 3500 bp DNA with various Methyltransferases, which I barcoded and ran multiple samples together. I usually use Tombo to get resquiggles and examine the methylation sites, but wanted to try out Nanopolish with the same data to see what it would output.

[hasindu2008]

What's the directory structure and file name and extensions if your input files like?

[mzwaig]

Hi,

I'm having the same issue. My merged fastq and sequencing summary are in the directory I'm running the code in and the fast5s are in a subdirectory within it. I made sure I have rwx permissions on all files including the fast5s.

fast5_pass 2673_Tumor_PAG56305.fastq.index.fai 2673_Tumor_PAG56305.fastq 2673_Tumor_PAG56305.fastq.index.gzi 2673_Tumor_PAG56305.fastq.index Nanopolish_sequencing_summary_2673_Tumor_PAG56305.txt

I'm passing the sample name to nanopolish with variables. nanopolish index -d ${PROJDIR}/${Readset}/fast5_pass -s ${PROJDIR}/${Readset}/Nanopolish_sequencing_summary_${Readset}.txt ${PROJDIR}/${Readset}/${Readset}.fastq

Thanks, Melissa

[Violeta-de-Anca]

Hej, I am having a similar issue, when not inputing the summary file I also get the same error.

nanopolish index -v -d /home/viole/nanopore/fast5 mitochondria.normal.basecalled.fastq

Output from nanopolish:

[readdb] indexing /home/viole/nanopore/fast5 Error: no fast5 files found

When inputing the summary file from guppy it indexed, but only 32 reads out of 121162.

nanopolish index -v -s sequencing_summary_violeta_run.txt -d /home/viole/nanopore/fast5 mitochondria.normal.basecalled.fastq

Output from nanopolish:

[readdb] indexing /home/viole/nanopore/fast5 [readdb] num reads: 121162, num reads with path to fast5: 32

Any idea why this might be happening?

P.D: I wanted to add also that I have grep one read that was correctly indexed:

FAW45780_pass_7fb25cdd_59cf256b_22.fast5 b96cfb36-79da-4b6c-a1b1-4647d2cd5f27 59cf256be47a641beb65be04367cb11db5b86801 19 281 3 9021.770750 19.639750 3807 TRUE 9021.950750 15567 19.459750 6097 11.973799 2.802943 74.434624 11.493582 74.434624 11.493582

And another one that was not:

FAW45780_pass_7fb25cdd_59cf256b_31.fast5 0000921b-cf58-4262-a5b6-5f1d34e1f25f 59cf256be47a641beb65be04367cb11db5b86801 18 28 2 62319.420000 8.420000 1169 TRUE 62319.420000 6736 8.420000 1767 8.391960 2.410708 73.704872 12.588209 73.704872 12.588209

And I cannot see any difference between the both of them.

Thanks, Violeta de Anca

[SEVEN-XYCHEN]

I have also encountered this problem. Have you resolved it?

[hasindu2008]

Can you try the f5c index (compatible with nanopolish index) to see if it gives any warnings or errors? https://github.com/hasindu2008/f5c/

Suggest using a command like below (avoiding sequencing sequencing summary based option) f5c index -t8 --iop 8 a.fastq -d fast5dir

[SEVEN-XYCHEN]

Hi, @hasindu2008 Thank you! I have solved this problem! Best, Chen