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I run the MinION MinKNOW without the live base-calling option. We know there is Metrichor and Albacore to perform base-calling after this process. However, I have not done any base-calling yet.

My question is: Is it possible to use directly the fast5 files with poretools to extract a fastq file without doing any previous base-calling process? I tried it and I get an empty fastq file.

My reads directory only contains the fast5 folder and I run:

poretools fastq fast5/ > output.fastq

Any ideas why do I get an empty file? What is the difference between base-called fast5 and non-base-called fast5 files?

Now I am trying to do base-calling with Albacore to see if I get a fastq file.

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  • $\begingroup$ You’ve edited the title - I should add that poretools can work with non base-called FAST5 files with some of its options, but not the fastq option. Some of the metadata options will function. $\endgroup$ – Scot Dec 5 '17 at 4:54
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No, poretools does not do basecalling. The poretools fastq command can be used to extract the FASTQ information from the basecalled FAST5 file (via MinKNOW live-basecalling or albacore). Alternatively, both of these basecallers can export a FASTQ file directly if desired.

The difference between a basecalled FAST5 and a nonbasecalled FAST5 is the presence of the FASTQ within the file. This can be directly viewed using an HDF5 viewer, like h5dump from the commandline, or HDFView from the GUI.

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Albacore can be used to create a fastq, directly from your fast5 files. As such there is no more reason to use poretools, expect perhaps for getting some metadata. With regards to metadata I would like to add that the sequencing_summary.txt generated by albacore also contains a lot of metadata.

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