Tombo resquiggle error

[q1134269149]

Hi, After basecalling with guppy, I using tombo to resquiggle the fast5 files of nanopore direct RNA-seq data. However, I checked the log and got the result as follows:

Final unsuccessful reads summary (100.0% reads unsuccessfully processed; 1512917 total reads): 89.2% (1349733 reads) : Unexpected error 10.8% ( 163147 reads) : Alignment not produced 0.0% ( 37 reads) : Reference mapping contains non-canonical bases (transcriptome reference cannot contain U bases)

I next checked the "unexpected_tombo_errors.5558.err" file, and found the same error for all single fast5 file as follows:

**_BaseCalled_template:::/mouse/qinhang_backup/guppy324/Calu_2_Infected/workspace/366/58719684-9a20-46b3-bf1a-9d95fd6185b1.fast5 ::: Traceback (most recent call last): File "/home/qinhang1/anaconda3/envs/tombo/lib/python3.7/site-packages/tombo/resquiggle.py", line 1570, in _resquiggle_worker map_res = adjust_map_res(map_res) File "/home/qinhang1/anaconda3/envs/tombo/lib/python3.7/site-packages/tombo/resquiggle.py", line 1523, in adjust_map_res map_res.raw_signal, DEFAULT_STALL_PARAMS)) File "/home/qinhang1/anaconda3/envs/tombo/lib/python3.7/site-packages/tombo/tombo_stats.py", line 313, in identify_stalls stall_metric[:] = np.NAN ValueError: cannot convert float NaN to integer

BaseCalled_template:::/mouse/qinhang_backup/guppy324/Calu_2_Infected/workspace/366/67226511-134b-4016-847f-5a2a2326ff11.fast5 ::: Traceback (most recent call last): File "/home/qinhang1/anaconda3/envs/tombo/lib/python3.7/site-packages/tombo/resquiggle.py", line 1570, in resquiggle_worker map_res = adjust_map_res(map_res) File "/home/qinhang1/anaconda3/envs/tombo/lib/python3.7/site-packages/tombo/resquiggle.py", line 1523, in adjust_map_res map_res.raw_signal, DEFAULT_STALL_PARAMS)) File "/home/qinhang1/anaconda3/envs/tombo/lib/python3.7/site-packages/tombo/tombo_stats.py", line 313, in identify_stalls stall_metric[:] = np.NAN ValueError: cannot convert float NaN to integer**

What's the problem, please? Thanks! hqin

[q1134269149]

This the log file. unexpected_tombo_errors.5558.err.txt

[JBerthelier]

Hi sorry to come to the conversation, I have the same issue by trying to use your DENA tool. Best,

[diegochavesm]

So after:

1. multi_to_single (Multi fast5 to single fast5)

i got the same behavior, most of my reads are ending up in the "Unexpected Error"

From the re-squiggling output 55.7% ( 3399 reads) : Unexpected error 44.4% ( 2708 reads) : Alignment not produced

all thought almost 3k reads were sorted in "Unexpected error" in the *.err 4 files aren't all of them, per file 50 reads (200 ca).

fast5 structure looks like:

/ Group /Analyses Group /Analyses/Basecall_1D_000 Group /Analyses/Basecall_1D_000/BaseCalled_template Group /Analyses/Basecall_1D_000/BaseCalled_template/Fastq Dataset {SCALAR} /Analyses/Basecall_1D_000/BaseCalled_template/Move Dataset {4697} /Analyses/Basecall_1D_000/BaseCalled_template/Trace Dataset {4697, 8} /Analyses/Basecall_1D_000/Summary Group /Analyses/Basecall_1D_000/Summary/basecall_1d_template Group /Analyses/RawGenomeCorrected_000 Group /Analyses/Segmentation_000 Group /Analyses/Segmentation_000/Summary Group /Analyses/Segmentation_000/Summary/segmentation Group /Raw Group /Raw/Reads Group /Raw/Reads/Read_1684 Group

any idea, what could be?

[q1134269149]

Hi sorry to come to the conversation, I have the same issue by trying to use your DENA tool. Best,

I created an environment of conda, and solved this problem by changing the version of python from 3.8 to 3.6, and the version of numpy from 1.23 to 1.19.

Best, hqin

[hengjwj]

Thanks hqin! It worked for me once I downgraded to numpy<1.20:

numpy 1.19.5 py37h3e96413_3 conda-forge python 3.7.12 hf930737_100_cpython conda-forge