This is a video on Illumina Sequencing that I really like:
https://www.youtube.com/watch?v=fCd6B5HRaZ8
However, I still have some technical doubts and I do appreciate your expert comments on any of these:
1) After preparing the sample DNA it is introduced on the lanes of the Illumina flow cell. How is it ensured that DNA will be uniformly distributed and not bind preferentially around to the point of injection?
2) My understanding is that the number of DNA bridge amplification cycles will affect the size of the clusters generated on the flow cell. How many cycles of bridge amplification are necessary? What impact does this have on the results?
3) With the introduction of different fluorescent tags on different nucleotides, can all nucleotides be present in the flow cell simultaneously? How can then be ensured that the polymerization of all the strands in a cluster occurs in a synchronized manner to measure a unique signal?
Thank you very much for your help!
1) This is based on the microfluidics and chip design which is designed and tested for the purpose. Furthermore, DNA is not solid, thus in aqueous phase the DNA / suspension would easily distributed in the flow cell.
2) Cluster size relates to the depth of sequencing, means increasing the probability of sequencing the less frequent amplicon in the sample.
3) The clusters are formed to make the signal strong enough to be detected, and the signal is for whole cluster, not for one sequence/amplicon in the cluster. Thus, it is already a synchronised signal. There might be some errors in individual amplicon, but the cluster signal does not.
The answer that Abhijeet provides is accurate, but let me add a point of clarification. To make more effective use of the flow cell surface space, Illumina created the patterned flow cell with distinct nanowells for cluster generation. Patterned flow cells are produced using semiconductor manufacturing technology, that ensures that DNA clusters only form within the nanowells, providing even, consistent spacing between adjacent clusters and allowing accurate resolution of clusters during imaging.
I have attached the link if you would like to read more on this subject: https://www.illumina.com/content/dam/illumina-marketing/documents/products/technotes/patterned-flow-cell-technology-technical-note-770-2015-010.pdf
Iryna Goraichuk Thank you for improving the discussion and refining the information.
Thank you Abhijeet and Iryna for your very valuable input. I still do not understand how the signal for the cluster is maintained in sync. I may have some misunderstanding about some point of the process:
Each DNA strand within the cluster will have its polymerase working on it and nucleotides will be incorporated (more or less) independently trough a stochastic process (there is no reversible capping of the growing DNA polymers, right?).
The signal from the last nucleotide incorporated will be what contributes (a very small contribution) to the cluster signal. Different strands may differ on what was the last nucleotide that they incorporated because, stochastically, some may get ahead or behind the others.
On average, the last nucleotide incorporated by most polymerases in the cluster may be the same, but this is an average rather than an exact synchronization. Is this the case? Does this effect impose a limit on the read length?
Thank you very much for your help!
Vincent Blay
? Each DNA strand within the cluster will have its polymerase working on it and nucleotides will be incorporated (more or less) independently trough a stochastic process (there is no reversible capping of the growing DNA polymers, right?). The signal from the last nucleotide incorporated will be what contributes (a very small contribution) to the cluster signal.
>> YES! and that is no problem as you see in the video (3.04 sec), different clusters are not synchronised and in principal can not be. But the average signal from an individual cluster is read as signal.
? Is this the case?
>> YES!
? Does this effect impose a limit on the read length?
>> YES! but not actually the read length only, but also the quality. The avaerage read length of different illumina platforms are different because the process and chemistry involved. Moreover, the reads always have a bad quality near the ends that is because the detector can not read the signal good enough, when the read grow over certain threshold in length.
This might be due to the chip designing/nanowell inconsistency or the detector. It seems more like a detector problem because the reverse reads not perform as good as forward reads. And there is no proper explanation to this problem.
Thank you all for your comments. Just to clarify the last point, Illumina does use a proprietary reversible terminator technology. After measuring every nucleotide addition, the fluorophore must be cleaved and the 3' end must be deblocked to add the next labelled nucleotide.
Do you know if the decapping and deblocking reagents are present all the time in the flow cell or there is a cyclical flow to introduce them and wash them off after every nucleotide addition?
I would like to ask: how does Illumina ensure unique fragment amplification in a cluster? There is no way to physically separate distinct DNA molecules. It is a completely random process. How then is PCR duplication (presence of the same DNA fragment in two different clonal clusters) kept to a minimum? I read a post by Eric Minikel (see link below) but am still not clear. Any clarification will be highly appreciated. Thanks!
https://www.cureffi.org/2012/12/11/how-pcr-duplicates-arise-in-next-generation-sequencing/
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