Whole Genome Sequencing with Single Base Extension (Ilumina Ifinium)

July 10, 2020 1:47 AM

I'm trying to understand how this exactly works but I don't get it. According to this figure every SNP gets one bead in the array, the DNA hybridizes with it and you get a red signal if it's AA, yellow AB and green if it's BB. But how do they assign the correct Nucleotide? If the two oligos on the bead end in G and T, you can distinguish CC, AA, AC. But if there is no signal for that bead, it can be either G ,T or GT? What am I missing, I have a feeling it's quite obvious... Paper is here.
posted by SweetLiesOfBokonon to Science & Nature (4 answers total)

This paper describes genotyping , not sequencing. Genotyping is an array whereby millions of probes homologous to known SNPs are hybridized to a fragmented DNA sample and thus map out the genome based on the library of SNPs defined in the array. Here is a brief explanation of how genotyping and sequencing are different.
posted by waving at 4:01 AM on July 10, 2020

Best answer: This paper describes a proof of concept experiment that's not entirely complete. You're correct in that it can't discriminate all possible SNP classes in a single assay with the color scheme they used. Basically, they tested it on a known set of SNPs that could be discriminated with only two labeled nucleotide probes.

From the paper:

Using a two-color SBE format, four out of six SNP classes can be genotyped in any given reaction. With judicious assignment of the two colors to the labeled bases (that is, A and T, red; C and G, green), over 83.5% of possible SNP assays in database SNP can be genotyped in a single reaction per array (Supplementary Table 1 online). The remaining 16.5% inaccessible SNPs, (A/T, and C/G), can be scored with SBE biochemistry by designing ASPE probes to these SNPs, and ratioing the probe pairs in the appropriate color channel (dependent on 3' base adjacent to SNP)

So two probe colors can cover the majority of known SNPs (A/G, A/C, T/G, T/C), and the less common SNPs (A/T and C/G) can be covered in a second reaction with different dye combinations.

That seems to be what Illumina was doing fairly recently: technical note.
posted by fountainofdoubt at 6:06 AM on July 10, 2020 [1 favorite]

Best answer: This is an interesting look back at Illumina technology before "next gen" sequencing hit the scene. 2006 is a bit before my area of expertise, but I deal with contemporary Illumina seq today so I took a quick look back at how the llumina Infinitum microarrays worked to see if I could answer your question. (I have not read the paper, just looked at the figure you linked.) Each spot/bead has many copies of an oligo that will hybridise to a known sequence for a human SNP, as you describe, and then the chemistry happening is to extend one nucleotide over using a polymerase mix with fluorescent nucleotides, to read out the next base where there's frequently a difference in human populations. Looking at this figure, I think what you're missing is that while there are only two colors of fluorescence being used and measured, if you label ALL the nucleotides with those two colors, you can introduce them one at a time (or two colors at a time?) to read out the next base for each spot/SNP. Here I'm just speculating based on how current Illumina tech works. But if you add a mix of green G and red T and take a picture, then wash the array to clear those nucleotides out, then add green C and red A and take another picture, then analyze both images, you'd be able to distinguish between all the bases. In contrast, current Illumina sequencing chemistry labels A, C and T all different colors, with G as dark, mixes them all in at the same time and takes one picture to read out whatever the next base in a chain of nucleotides, then washes and repeats to read out the subsequent base, repeat for 150 nucleotides. But here you only need to read out the terminal base, so I'd guess putting the nucleotides in sequentially and taking multiple pictures would work. Hopefully someone else can come along & confirm or correct me.
posted by deludingmyself at 6:16 AM on July 10, 2020

Oh, and I see fountainofdoubt read the paper and the answer is just that they're not distinguishing all possible SNPs, ha!
posted by deludingmyself at 6:20 AM on July 10, 2020