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SDPA_Pet 08-05-2019 05:10 PM

Illumina output questions
Hello, I am gonna do some Illumina 2X300bp amplicon sequencing. I suppose the platform (MiSeq) will run 600 cycles and time fixed. I just recently notice on the official website

It says the running time is 455 hours? It seems to me that the running time range is quite large? What's the difference if you run 4 hours or 55 hours. some questions:

1>Let's both 4 hours and 55 hours will give same length of reads.(In my case is 300bp). However, 55 hours will generate more reads (total number) than 4 hours.

2> Or 4 hours will give you shorter reads (e.g., 100bp) and lower number of reads.

3>Would 4 hours and 55hours running time used same protocols and same amount of regents?

4>Do you know how long normally a sequencing center will run? I would like them to run longer? Would be cost more? How should I tell them? Just simply say run as long as you could?


Meyana 08-05-2019 05:50 PM

The time of run will depend on the read length.

Check this link for run times for different read lengths:

microgirl123 08-06-2019 08:21 AM

The number of reads (molecules sequenced, also called clusters) is determined by the concentration of library added. There is an optimal number the sequencing center will be aiming for depending on the type of library you have.

The length of the reads is determined by the kit you decide to use. If you have an insert between your Illumina adapters of 600 bp, you could use a 600 cycle kit and read 300 bp in each direction. You could not use a 600 cycle kit with an insert size of 50 bp, since you would be reading far past the end of your insert into the Illumina adapter and flow cell.

The amount of time a MiSeq run takes is dependent on the length of the reads. Library hydridization and cluster formation takes a couple of hours. After that each read (cycle) takes around 5 minutes. So a 4 hour run is likely just 2x25 bp. A 55 hour run would be the full 300x300 bp run. The cost of the run is dependent on how many cycles you want performed - 2x25 bp kits contain less reagents and are cheaper than 2x300 bp kits.

GenoMax 08-06-2019 09:03 AM

@Ben: MiSeq nano flowcells produce a small amount of data and can run astonishingly quickly. That 4 h figure is for one of these FC with a short 50 cycle run. These FC only produce about 1-1.5M reads and they are mainly good for QC of libraries for larger NovaSeq runs or cases where one needs small amount of data/quick turn-around.

Don't worry about the run times you have no control over those. Select the length of sequencing you need and that should be it.

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