fast5 file is not open.

[bradabramson]

I'm getting this error (see below). I used ont_fast5_api to demultiplex the multi-fast5s and then ran poreplex on those fast5s. Any input would be helpful. Thank you.

2019-01-31 14:21:01,777 Command line: /usr/local/projdata/8520/projects/PANGENOME/localenv/anaconda/envs/ONTIll/bin/poreplex -i ./demultiplex_fast5/ -o ./demultiplex_fast5/Poreplex --trim-adapter --barcoding --basecall --parallel 40 --live 2019-01-31 14:21:01,777 == Analysis settings ====================================== 2019-01-31 14:21:01,777 * Input: ./demultiplex_fast5/ (live, 60 sec delay) 2019-01-31 14:21:01,777 * Output: ./demultiplex_fast5/Poreplex 2019-01-31 14:21:01,777 * Processes: 40 2019-01-31 14:21:01,777 * Presets: rna-r941.cfg 2019-01-31 14:21:01,777 * Basecall on-the-fly: Yes (albacore 2.3.3) 2019-01-31 14:21:01,777 * Trim 3' adapter: Yes 2019-01-31 14:21:01,777 * Filter concatenated read: No 2019-01-31 14:21:01,778 * Separate by barcode: Yes 2019-01-31 14:21:01,778 * Real-time alignment: No 2019-01-31 14:21:01,778 * FASTQ in output: Yes 2019-01-31 14:21:01,778 * FAST5 in output: No 2019-01-31 14:21:01,778 * Basecall table in output: No 2019-01-31 14:21:01,778 =========================================================== 2019-01-31 14:21:01,778 2019-01-31 14:24:21,105 [signal_analyzer.py:96] (37/cfe32c71-be0b-448f-bf2e-aa5099d51cfe.fast5) Unhandled exception OSError: Fast5 file is not open: ./demultiplex_fast5/37/cfe32c71-be0b-448f-bf2e-aa5099d51cfe.fast5 Traceback (most recent call last): File "/usr/local/projdata/8520/projects/PANGENOME/localenv/anaconda/envs/ONTIll/lib/python3.6/site-packages/poreplex/signal_analyzer.py", line 96, in process npread = prepare_loading(f5file) File "/usr/local/projdata/8520/projects/PANGENOME/localenv/anaconda/envs/ONTIll/lib/python3.6/site-packages/poreplex/signal_loader.py", line 79, in prepare_loading npread = NanoporeRead(filename, self.fast5prefix) File "/usr/local/projdata/8520/projects/PANGENOME/localenv/anaconda/envs/ONTIll/lib/python3.6/site-packages/poreplex/signal_loader.py", line 124, in init self.load() File "/usr/local/projdata/8520/projects/PANGENOME/localenv/anaconda/envs/ONTIll/lib/python3.6/site-packages/poreplex/signal_loader.py", line 205, in load self.channel_info = fast5.get_channel_info() File "/usr/local/projdata/8520/projects/PANGENOME/localenv/anaconda/envs/ONTIll/lib/python3.6/site-packages/ont_fast5_api/fast5_file.py", line 126, in get_channel_info self.assert_open() File "/usr/local/projdata/8520/projects/PANGENOME/localenv/anaconda/envs/ONTIll/lib/python3.6/site-packages/ont_fast5_api/fast5_file.py", line 57, in assert_open raise IOError("Fast5 file is not open: {}".format(self.filename)) OSError: Fast5 file is not open: ./demultiplex_fast5/37/cfe32c71-be0b-448f-bf2e-aa5099d51cfe.fast5

[hyeshik]

Hi @bradabramson, Thank you very much for the reporting. I think this error could be fixed in the newest release. Could you check if the problem persists in the latest build?

[bradabramson]

Hello, thank you for the quick reply. I only just now tried it and I received this error from the python 3.6 version on the multi-fast5 and also the single-fast5 after I used “multi_to_single_fast5”.

########################################################################## (LE=ONTIll)[babramso@bigmem-1:/usr/local/scratch/SYNBIO/babramson/RNA]$ poreplex -i /usr/local/scratch/SYNBIO/babramson/RNA/ -o /usr/local/scratch/SYNBIO/babramson/poreplex_V272/ --trim-adapter --barcoding --filter-chimera --basecall --parallel 40

Poreplex version 0.4 by Hyeshik Chang [email protected]

• Cuts nanopore direct RNA sequencing data into bite-size pieces for RNA Biology

Output directory /usr/local/scratch/SYNBIO/babramson/poreplex_V272/ is not empty. Clear it? (y/N) y

== Analysis settings ======================================

• Input: /usr/local/scratch/SYNBIO/babramson/RNA/
• Output: /usr/local/scratch/SYNBIO/babramson/poreplex_V272/
• Processes: 40
• Presets: rna-r941.cfg
• Basecall on-the-fly: Yes (albacore 2.3.3)
• Trim 3' adapter: Yes
• Filter concatenated read: Yes
• Separate by barcode: Yes
• Real-time alignment: No
• FASTQ in output: Yes
• FAST5 in output: No
• Basecall table in output: No ===========================================================

==> Processing FAST5 files ERROR: [signal_analyzer.py:48] Unhandled exception FileNotFoundError: File b'/usr/local/projdata/8520/projects/PANGENOME/localenv/anaconda/envs/ONTIll/lib/python3.6/site-packages/poreplex/kmer_models/r9.4_180mv_70bps_5mer_RNA/template_median69pA.model' does not existTask exception was never retrieved future: <Task finished coro=<ProcessingSession.run_process_batch() done, defined at /usr/local/projdata/8520/projects/PANGENOME/localenv/anaconda/envs/ONTIll/lib/python3.6/site-packages/poreplex/pipeline.py:183> exception=CancelledError()> concurrent.futures._base.CancelledError

From: Hyeshik Chang [email protected] Sent: Wednesday, February 13, 2019 12:58 AM To: hyeshik/poreplex [email protected] Cc: Abramson, Bradley [email protected]; Mention [email protected] Subject: Re: [hyeshik/poreplex] fast5 file is not open. (#14)

Hi @bradabramsonhttps://github.com/bradabramson, Thank you very much for the reporting. I think this error could be fixed in the newest releasehttps://github.com/hyeshik/poreplex/releases/tag/v0.4. Could you check if the problem persists in the latest build?

— You are receiving this because you were mentioned. Reply to this email directly, view it on GitHubhttps://github.com/hyeshik/poreplex/issues/14#issuecomment-463114352, or mute the threadhttps://github.com/notifications/unsubscribe-auth/AsNc0hpZ4r3KxTbLHJJY_tcMQ3u_IVlDks5vM9OhgaJpZM4adSxy.

[hyeshik]

Hi @bradabramson, There were some missing files from the binary package for v0.4. I just release a new minor release v0.4.1 which should fix the problem. Sorry for the inconvenience.

[bradabramson]

the kmer folder is not being installed when using pip repo (PYPI) but the git repo works.