Illumina output questions

SDPA_Pet · 08-05-2019, 06:10 PM

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

https://www.illumina.com/systems/seq...elect-language

It says the running time is 4–55 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?

Thanks,
Ben

Meyana · 08-05-2019, 06:50 PM

The time of run will depend on the read length.

Check this link for run times for different read lengths:
https://www.illumina.com/systems/seq...fications.html

microgirl123 · 08-06-2019, 09:21 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, 10:03 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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08-05-2019, 06:10 PM	#1
SDPA_Pet Senior Member Location: US Join Date: Apr 2013 Posts: 222	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 https://www.illumina.com/systems/seq...elect-language It says the running time is 4–55 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? Thanks, Ben

08-05-2019, 06:50 PM	#2
Meyana Member Location: Japan Join Date: Sep 2017 Posts: 40	The time of run will depend on the read length. Check this link for run times for different read lengths: https://www.illumina.com/systems/seq...fications.html

08-06-2019, 09:21 AM	#3
microgirl123 Senior Member Location: New England Join Date: Jun 2012 Posts: 200	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.

08-06-2019, 10:03 AM	#4
GenoMax Senior Member Location: East Coast USA Join Date: Feb 2008 Posts: 7,080	@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.