How many cycles you can get out of the 36 cycle kit version 2?

[schönblick]

anyone ever tried more than 36 cycles?

[monad]

Not really, but based on volume left, you may extend 3 more cycles, 5 more would be a big risk as both cleavage mix and scan mix is limiting. v1 allows 46 cycle, but not available anymore.

[swbarnes2]

People in close contact with Illumina are trying out 75 cycles, and more. So longer reads are coming down the pike...

We got one run with good 48 cycles of data, with the older kits, but now, we get more like 40.

[monad]

People in close contact with Illumina are trying out 75 cycles, and more. So longer reads are coming down the pike...

We got one run with good 48 cycles of data, with the older kits, but now, we get more like 40.

I think original question is how many maximum cycle out of v2 seq kit.
You may set up with max cycle of your best guess, but if reagents ran out, it will pump air into the system.

Regarding super long read, we are routinely getting 2 x 88 and now moving onto 2 x 108 (35 +35+36)

[monad]

Official release will be 2 x 75.

[kmcarr]

Out of curiosity Monad are shortening any of the recipe times? By my reckoning 2X108 cycles would be ~12 days.

[monad]

no shortening of run time available yet, so it takes two days for every 36 cycles plus > half day for turnaround chemistry. Illumina is working on shorter recipe.

[suludana]

Index - 36+7

Hi,
the illumina protocol for multiplex recommends 36 + 7 cycles and to buy the sequencing kit for 36 cycles plus the sequencing kit for 18 cycles. What do you think about that? Do you think it is posible to do the read1 and the index read with an unique sequencing kit for 36 cycles? I'm talking about GAII.
Thanks

[graveley]

Hi,

my sales rep told me that version 3 of the sequencing kits are available for order starting Monday! Same pricing as v2 kits, but provide more accurate sequence so you can do 50-100 bp reads.

[james hadfield]

we get 44bp routinley from the 36 kit if something runs out then we trim the read to compensate.

[das]

Solexa longer reads

Hi:
With those longer reads - how is the quality of bases at the end of the reads.
With 50 bp reads we throw out bases 45-50. What is happening with 75 bp reads?

Thanks

[james hadfield]

We get 42bp from a 36 kit and run that routinely. It is just possible to get a multiplex index from a kit if you are doing a 36bp run but safer to top up with some leftover reagent from another kit (as long as it has not been sitting around obviously).
I still have not had any confirmation on the format of the next kits and was under the impression that Ilumina would just sell 18, 36 and 50bp kits. If you want longer then combine kits is what we were told. My preference woud be for 150bp in a bottle to put on the instrument and then just run samples until it runs out. However we do not have a gap between flowcells.
I had also heard about the shorter-cycle chemistry, but I did not think it was ready yet. There are two changes coming; one will be a high-fidelity polymerase to increase the quality of sequencing, the other is a faster sequencing enzyme which allows each cycle to decrease by 20-30%. Three flow cells a week are coming!
I am interested to hear what other people have to say.
James.

[BaCh]

Hi:
With those longer reads - how is the quality of bases at the end of the reads.
With 50 bp reads we throw out bases 45-50. What is happening with 75 bp reads?

On my 72 mers (well, 76mers with a 4 base tag), I regularly have these numbers:
- 0.5% of the reads are completely unusable
- 5.5% need a left trim
- 20% need a right trim

Which means that a bit less than 75% of all reads are error free on the whole length.

Trimming removes overall ~6.5% of all bases.

After trimming
- ~90% of all reads map without error to the reference
- ~7% map with one error
- ~1.2% map with two errors
- the remaining map with more errors (or not at all)

HTH
B.