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rrwick / Deepbinner

a signal-level demultiplexer for Oxford Nanopore reads
GNU General Public License v3.0
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DeepBinner on multi-fast5 followed by albacore on moved single fast5 fails. #26

Open jnesme opened 5 years ago

jnesme commented 5 years ago

Dear Ryan,

I was wondering if the issue I'm having was already reported. I'm using deepbinner realtime locally to demultiplex Rapid Barcoding run. I've updated deepbinner to be able to handle multi-fast5.

I then upload the whole folder with scp -r to my analysis server where I want to run Albacore using a for loop on each barcode folder. I would have then concatenated the obtained fastq where both software agrees. It was working fine until recent MinKNOW update and the multi-fast5 but now I'm having this error in Albacore where it seems it can't read the single read fast5 files produced by deepbinner/multi_to_single_fast5 output. Here's what I'm getting from Albacore pipeline.log:

2019-03-05 10:43:06,922 Submitting file "1e2ddcba-c011-4dca-99f3-9cddb76df821.fast5".
2019-03-05 10:43:06,943 Could not extract read data from file "1e2ddcba-c011-4dca-99f3-9cddb76df821.fast5".
2019-03-05 10:43:06,943 Submitting file "1e2e153f-fe84-476a-9e92-7797640ee07e.fast5".
2019-03-05 10:43:06,951 Could not extract read data from file "1e2e153f-fe84-476a-9e92-7797640ee07e.fast5".
2019-03-05 10:43:06,951 Submitting file "1e2f4690-6517-440d-b6d1-9c704a9b1900.fast5".
2019-03-05 10:43:06,963 Could not extract read data from file "1e2f4690-6517-440d-b6d1-9c704a9b1900.fast5".
2019-03-05 10:43:06,963 Submitting file "1e2f5b54-08f9-4771-9e1b-445917c8d3f6.fast5".
2019-03-05 10:43:06,983 Could not extract read data from file "1e2f5b54-08f9-4771-9e1b-445917c8d3f6.fast5".

Any idea of what might cause this issue or should I forget about the double demultiplex solution ? I was going for the double demultiplex because we did notice quite a lot of barcode crosstalk and sequencing only novel DNA templates. I guess I could just use Albacore 2.3.4 on multi-fast5 and forget the deepbinner signal level demultiplexing, although I liked the idea of being extremely stringent in the selection of reads incorporating the assembly - with Unicylcer of cours :) ...

Best regards and many thanks for those precious tools offered to the community.

jnesme commented 5 years ago

ok sorry for posting this irrelevant issue to DeepBinner, the problem lies with using deprecated Albacore.... Works like a charm with Guppy on deepbinner sorted reads. It just lack then the two layers of barcode demultiplexing.