Hello, thank you for the great video. I am a non biologist trying to teach myself ngs analysis. I have 2 questions that might have obvious answers however I am not able to understand: 1) why to bother with "a base and color define the next base" when one can use 16 colors instead of 4 ? 2) what is the use of the 3 middle universal bases in the probes? i.e why not just ligate all di-bases at one cycle (instead of the offset 5 times...) and decode the colors ? thanks in advance!
hello Thanks for the excellent video. Could you please clarify something? The fragments of all unique DNA sequences are flanked by the P1 and P2 primers. Hence when they are binding to the beads, how is it ensured that each bead carries unique DNA sequences. These interactions are with the adaptor sequences right? Hence how is this possible since all DNA fragments carry same adaptor sequences?Thanks.
There are different adaptors for different fragments of DNA. The beads carrying DNA sequences(primers/ oligonucleotides/amplicons)are made by emulsion PCR and one bead is made in such a way that all the primers attached to it carry sequences complementary to any one type of adaptor, among many others. So, suppose there are 10 types of adaptors used to flank the fragmented DNA, there would be 10 different beads containing complementary sequence primers for those 10 adaptors respectively. Hope this helps.
Could you explain in more detail how exactly polony formation takes place? Does the adapter-bound DNA curves as in bridge amplification or is it somehow unbound after the 1st amplification round only to serve as primer in an adjacent adapter?
