Illuminas operating system

Kultamuna2 · 03-07-2018, 11:30 AM

Hi! Can anyone tell me, why Illumina's sequencing systems have two lasers (or in NovaSeq, four lasers)? I am a bit confused how the system works. Do those lasers change constantly wavelengths to identify all the four bases, or do they stay in a specific wavelength? Thanks already in advance!

kc3228 · 03-07-2018, 12:20 PM

The two lasers are red and green. Red is C, green is T. A is labeled with both red and green, so shows up in both. G is dark (unlabeled), so a lack of signal is interpreted as G.

GW_OK · 03-09-2018, 10:17 AM

The lasers excite at fixed wavelengths. The fluorophores emit at different respective wavelengths. The example given by kc3228 is for the dual color chemistries of the NextSeq and NovaSeq. The MiniSeq, Miseq, and Hiseq systems use a 4 color chemistry.

misterc · 03-09-2018, 10:54 AM

MiniSeq is essentially a single camera NextSeq, so 2-color SBS there (not 4).

GW_OK · 03-12-2018, 06:48 AM

Good to know.

I suppose the iSeq is single color, since it's got the CMOS in the flowcell.

GenoMax · 03-12-2018, 07:00 AM

iSeq is one-dye, two-chemistry and two-images per sequencing cycle. See page 3.

kmcarr · 03-12-2018, 07:13 AM

Here's another doc from Illumina which also describes the CMOS flow cell and compares the various detection chemistries (1, 2, 4 color) used by Illumina.

GenoMax · 03-12-2018, 07:20 AM

Nice find @kmcarr. Thanks for posting.

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03-07-2018, 11:30 AM	#1
Kultamuna2 Junior Member Location: Austria Join Date: Mar 2018 Posts: 5	Illuminas operating system Hi! Can anyone tell me, why Illumina's sequencing systems have two lasers (or in NovaSeq, four lasers)? I am a bit confused how the system works. Do those lasers change constantly wavelengths to identify all the four bases, or do they stay in a specific wavelength? Thanks already in advance!

03-07-2018, 12:20 PM	#2
kc3228 Junior Member Location: Missouri Join Date: May 2017 Posts: 5	The two lasers are red and green. Red is C, green is T. A is labeled with both red and green, so shows up in both. G is dark (unlabeled), so a lack of signal is interpreted as G.

03-09-2018, 10:17 AM	#3
GW_OK Senior Member Location: Oklahoma Join Date: Sep 2009 Posts: 411	The lasers excite at fixed wavelengths. The fluorophores emit at different respective wavelengths. The example given by kc3228 is for the dual color chemistries of the NextSeq and NovaSeq. The MiniSeq, Miseq, and Hiseq systems use a 4 color chemistry.

03-09-2018, 10:54 AM	#4
misterc Member Location: Livermore, CA Join Date: Jan 2016 Posts: 44	MiniSeq is essentially a single camera NextSeq, so 2-color SBS there (not 4).

03-12-2018, 06:48 AM	#5
GW_OK Senior Member Location: Oklahoma Join Date: Sep 2009 Posts: 411	Good to know. I suppose the iSeq is single color, since it's got the CMOS in the flowcell.

03-12-2018, 07:00 AM	#6
GenoMax Senior Member Location: East Coast USA Join Date: Feb 2008 Posts: 7,081	iSeq is one-dye, two-chemistry and two-images per sequencing cycle. See page 3.

03-12-2018, 07:13 AM	#7
kmcarr Senior Member Location: USA, Midwest Join Date: May 2008 Posts: 1,178	Here's another doc from Illumina which also describes the CMOS flow cell and compares the various detection chemistries (1, 2, 4 color) used by Illumina.

03-12-2018, 07:20 AM	#8
GenoMax Senior Member Location: East Coast USA Join Date: Feb 2008 Posts: 7,081	Nice find @kmcarr. Thanks for posting.