Cluster regeneration

[Andrew_Slatter]

I'm trying to figure out how the MiSeq clustering works with respect to sequencing. Firstly, after the initial cluster generation, both strands are present on the flow cell (i.e. both templates for sequencing read 1 and read 2). What i'm not sure about is how these clusters are processed as the various sequencing primers are added. Are half of the strands (i.e. the template for Read 2) cleaved from the flow cell before read 1 SBS starts ? - if so, how are the clusters converted to having read 2 template before read 2 SBS starts. Anyone know the details ?

[bbeitzel]

The read 1 primer will only anneal to one of the strands, and the read 2 (and index) primer will only anneal to the other strand.

[Andrew_Slatter]

So the clusters can be interrogated without stripping away either strand ?

[bbeitzel]

I'm not 100% sure, but I don't think the newly synthesized read 1 complementary strand is stripped away prior to starting the index and read 2 reads. If it was being stripped away, I would assume that you would have to add fresh NaOH to the MiSeq reagent cassette prior to loading. Then again, I guess you could denature by heating the flow cell...

[microgirl123]

Hi Andrew, have you looked at the MiSeq training videos on the Illumina website? They have a lot of information that might help.
I'm a bit rusty on how it all works, but the template for Read 1 and Read 2 is the same strand of DNA. After the first cluster generation, the first primer anneals to the end and SBS starts, heading downward into your cluster. When Read 1 is complete, the synthesized strands are stripped off and the next primer is hybridized. Read 2 is synthesized heading upward into your cluster.

[csquared]

If you want all of the details for the process, the Bentley et al Nature paper from 2008 is the best resource for exactly how all of the chemistry works for paired-end sequencing.

http://www.nature.com/nature/journal/v456/n7218/abs/nature07517.html

In short summary, data read 1 and the first index read are on one strand of DNA. At the conclusion of those reads, the DNA that was synthesized during the sequencing reaction is stripped off and clusters are regenerated (commonly called the turn). Data read 2 (the paired end read) is then performed on the newly synthesized strand.

There is a video here: http://www.illumina.com/technology/s...echnology.ilmn but it doesn't go into any details on cluster regeneration.

[kcchan]

I think the term "cluster regeneration" may be a bit misleading. There is no amplification step during the Read 2 turnaround. The complementary strand of every molecule in the flow cell is resynthesized. The original strand is then cleaved at the ends and denatured before read 2 sequencing.

[yaximik]

Simple testing of each well in MiSeq reagent cassette with pH paper indicates lack of NaOH. Most likely formamide at elevated temp is used for denaturation and stripping. Two anchored primers contain different cleavage points (dU and oxo-G) thus allowing two independent cleavage events for paired reads. Cleavage 1 removes one DNA strand, but still leaves the primer attached, which can be used to flip-over each DNA fragment in clusters for read 2. Cleavage 2 removes both the DNA strand and the primer, so flip-over is impossible.

[csquared]

I think the term "cluster regeneration" may be a bit misleading. There is no amplification step during the Read 2 turnaround. The complementary strand of every molecule in the flow cell is resynthesized. The original strand is then cleaved at the ends and denatured before read 2 sequencing.

"resynthesis" vs "regeneration" is a fine line. More importantly, there are 15 cycles alternating between formamide and amplification mix during the turn, very similar to exactly what happens during cluster generation. Everyone can draw their own conclusions as to if this constitutes "amplifcation" or not but it's certainly not a single cycle of amplification mix.

[Andrew_Slatter]

Thanks for all of your comments. The new training videos on the illumina website explain it very nicely. Dual index read strategy is particularly interesting.