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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!
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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. -
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.Comment
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MiniSeq is essentially a single camera NextSeq, so 2-color SBS there (not 4).Comment
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Good to know.
I suppose the iSeq is single color, since it's got the CMOS in the flowcell.Comment
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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.Comment
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Nice find @kmcarr. Thanks for posting.Comment
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Love this - good data definitely starts from good input, and poor input can only give relatively poor data. I particularly like the mention of Nanodrop/absorbance based methods for quantification. It's such a toss up if you'll get an accurate reading or what amounts to a randomly generated number, and a lot of library/sequencing related issues can be traced back to poor quant.Yesterday, 11:43 AM
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I’m not a sequencing expert. I’m a purification scientist who uses NGS to evaluate workflows my group develops. With this perspective, we think about the sample first and the NGS workflow second. The sequencer is an exceptionally honest reporter, but it can only report on what you give it, so whether you get clean, interpretable data from an NGS workflow is largely determined before you begin.
Here are nine questions we think about, in roughly the order they matter, before...
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