Hi i meet a tombo resquiggle error：Reads do not to contain basecalls. Check --basecall-group option if basecalls are stored in non-standard location or use tombo annotate_raw_with_fastqs to add basecalls from FASTQ files to raw FAST5 files. which do you use version of tombo?

[gaoyubang]

tombo v1.5 you can use "tombo annotate_raw_with_fastqs" to add basecalls from FASTQ files to raw FAST5 files.

[LegendZDY]

Thank you for your advice! I checked the files，which contains basecalls. i also used Tombo v1.5 and Tombo does not support multi-read FAST5 format read data files. Tombo resquiggle was ok,when i used single-read FAST5.

[xj-Liang-Olga]

Hello, I would like to ask if the FASTQ file you mentioned was converted from downloading SRA file by NCBI. If not, how did you get the FASTQ file? Thanks

[gaoyubang]

I recommend you re basecalled it with guppy (version 3.6.1). In view of the big fast5 size, the uploaded fast5 file may not contained the newer basecalled information.

[xj-Liang-Olga]

I recommend you re basecalled it with guppy (version 3.6.1). In view of the big fast5 size, the uploaded fast5 file may not contained the newer basecalled information.

Thanks for your reply! I will have a try.

[xj-Liang-Olga]

Hello, I have run the 'tombo resquiggle' successfully, but there are 11.7% reads which unsuccessfully processed:

tombo resquiggle --overwrite --basecall-group Basecall_1D_000 nanopore_data/P.trichocarpa/0 refseq/ref_Ptrichocarp/Ptrichocarpa_533_v4.1.transcript.fa --processes 12 [14:46:01] Loading minimap2 reference. [14:46:03] Getting file list. [14:46:03] Loading default canonical RNA model. [14:46:03] Re-squiggling reads (raw signal to genomic sequence alignment). 100%|███████████████████████████████████████████████████████████████████████████████████████████████████████████████████████████████| 8000/8000 [01:31<00:00, 87.28it/s] [14:47:35] Final unsuccessful reads summary (11.7% reads unsuccessfully processed; 934 total reads): 8.1% ( 647 reads) : Alignment not produced
3.5% ( 283 reads) : Poor raw to expected signal matching (revert with tombo filter clear_filters) 0.1% ( 4 reads) : Read event to sequence alignment extends beyond bandwidth
[14:47:35] Saving Tombo reads index to file.

I would like to ask whether the unprocessed reads will affect the subsequent operation, and if so, how to solve it ? Looking forward to your reply!

[gaoyubang]

The unsuccessfully reads will not been analysised in the subsequent steps.

[xj-Liang-Olga]

The unsuccessfully reads will not been analysised in the subsequent steps.

OK, thanks for your answer!

[zjzace]

Hi,

I have run the guppy 5.07 with the fast5 out. Then, I split the fast5 file into single fast5 and run the tombo resquiggle and got this error. i tried to use tombo annotate_raw_with_fastqs, but it still didn't work.

Does anyone know how to solve it? Looking forward to your reply!

[gaoyubang]

What's wrong? You can post it。