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I am running a 48 h sequencing protocol on a FLO-MIN107, on MinION, with DNA library-prepped with SQK-RAD004. MinKNOW is installed on my Mac and controlling the MinION. I have found the following histogram in MinKNOW. What do the numbers mean? In particular, how many reads have been produced? I understood that the events are consecutive electrical signals and are then transformed into reads? Do I have 4029 reads? The run has started 24 hours ago. 4029 reads surely is really low, isn't it? What does 4,120,722 mean?

enter image description here

The Platform QC of the flow cell was as follows:

  • group 1: 480
  • group 2: 374
  • group 3: 201
  • group 4: 59
  • total = 1114
Biomagician
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1 Answers1

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That's indeed the number of reads and that's quite low. How was your pore occupancy (number of pores sequencing) and your flow cell QC (number of pores good enough for sequencing)? How was your library concentration?

Events is an approximation of nucleotides: it's a guess based on the current signal. As far as I know if you multiply this by 1.8 you get approximately your throughput in nucleotides.

The 4,120,722 is the number of events in the currently selected (mouse hovering?) bin of the histogram.

Wouter De Coster
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  • I added my platform QC in my question. Regarding the library concentration, I did not do one. I only used 400 ng input material based on Qubit Broad Range measurement. In the SQK-RAD004, there is no library concentration measurement. Should I measure the concentration of my library before loading it into the MinION? – Biomagician Feb 23 '18 at 13:54
  • Oh I've only done ligation preps. The FC looks quite good. How many pores were actually sequencing? – Wouter De Coster Feb 23 '18 at 16:05
  • How do I know how many pores were actually sequencing? Right now (28h after starting), there are only 2 single pores but I remember having hundreds at the beginning. – Biomagician Feb 23 '18 at 16:53
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    The "Physical Layout" tells you how many pores are sequencing, you need to look at the number in "Strand". The "pore occupancy" is the ratio between Strand / (Strand + single pore), meaning the total number of "good" pores which you are actually using. This is a parameter which tells you someting about how good your library is.

    See for example this screenshot: https://pbs.twimg.com/media/DU32ZAaXkAEojB9.jpg:large Showing 445 pores actually sequencing out of 445+39 = 92% pores occupied.

     – Wouter De Coster Feb 24 '18 at 17:34
  • very useful to know! So the single pores are the pores available and the strand pores are the single pores actually sequencing? My run is finished now and I don't remember how many 'strand' pores I had but I have the feeling that there were not many... Do you know if these parameters are stored somewhere after the run? – Biomagician Feb 24 '18 at 19:00
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    That's right, but no, I don't think those are stored. – Wouter De Coster Feb 24 '18 at 20:47