There are three safe stopping points in the protocol for storing the library:

1. After the barcoding reaction,
2. After the library pooling and purification,
3. And after the adapter attachment.

In case of points 1 and 2, the recommended storage conditions are 4°C for up to 1 month and for long-term storage, we recommend storage temperatures less than -80°C.

Although libraries should be immediately sequenced after the attachment of the adapter (point 3), they can also be stored at 4°C for up to 1 week if needed.,

After adding sequencing buffer and loading beads to the library, there’s no option for storing the library. Such library must be immediately loaded to the flow cell.

If something happens which prevents to start the sequencing and the library has been already loaded to the flow cell it is safe to store the flow cell in the fridge (4-8°C) for max 24 hours.

Fast5 is a format of raw data coming out from the sequencer. Fast5 file is like a bucket collecting reads from the sequencer. Its capacity is defined by the user in MinKNOW during the run setup. Basecaller translates these reads into fastq files whose capacity is defined again by the user during the run setup.
Example 1: Fast5 = 4000 reads, Fastq = 4000 reads. In this example, 1 fast5 will generate 1 fastq file. So if you collect 20 fast5 files per barcode, it will generate 20 fastq files.
Example 2: Fast5 = 4000 reads, Fastq = 500k reads. In this example, multiple fastq5 files will generate one fastq file. So if you collect 125 fast5 files per barcode, it will generate 1 fastq file.
Example 3: Fast5 4000 reads, Fastq = 2000 reads. In this example, 1 fast5 will generate 2 fastq files. So if you collect 20 fast5 files per barcode, it will generate 40 fastq files.

As long as your flow cell has >800 pores at the beginning of sequencing, the translocation speed, Q score and temperature are within the limits, the run will generate enough data for successful genotyping. The minimum reads required per sample is 50000-60000.