Nextera V2 sequences

[aleferna]

Hi does anybody have the sequences for the new Nextera V2 library, apparently they changed the sequences a bit from the old one? I ask their support but they sent me a very cryptic "letter to customer" pdf with a bunch of sequences for TruSeq and Nextera but I can't tell which is which.

[aleferna]

Just got off the phone from Illumina support, unlike V1, Nextera V2 does not provide the sequences of its primers/adapters. Extremely annoying, has anybody done Sanger Sequencing of these kits?

[james hadfield]

This is annoying as anyone that really wants the seqeunces will just get them off a seqeuncer. Illumina were very happy to release sequences over the last few years, although a while back they tried hard to keep the Y-shaped structure more secret. Hopefully someone higher up will see they are only creating bad feeling and release them.

[aleferna]

The worst part is that they send you a generic document "letter to customer" where they just through all the TruSeq sequences at you, and I wasted 3 days trying to figure out. I thought: well they know what they are doing so probably the seqs are there and I just can't figure out how the adapter fits into the transposon. I had to call tech support, and the guy there was really nice, he said: sorry this is a corporate issue I just cant give you the sequences because Illumina has chosen not to release them. So at least I know that I'm not crazy, the adapters they sent me won't fit the transposon. Now we have to sue the rep that sold us the machine because in the tender it says that we have to get all the sequences of adapters and primers required for the process. But its just so annoying, what are they hoping to achieve?
You know we wanted to buy a IonTorrent initially, but the tender they gave us was twice as much as the MiSeq (since we don't have a Covaris Sonicator) and the IonProton was coming out. So it was impossible to choose a soon to be outdated machine for twice as much as a proving working available system with Nextera which we really really like. Please somebody come up with a decent runner up to MiSeq so that Illumina doesn't rule us with this iron fist! NanoPore? Ion? Solid?

[aleferna]

my apologies for the rant... I needed to vent..

[MrGuy]

Covaris sonicator? What's that have to do with Ion Torrent? Library prep really has nothing to do with the sequencer aside from getting the adapters on so you can sequence it. How you fragment your template has nothing to do with it.

[aleferna]

Well, you have to fragment you sample to sequence it, we don't have a sonicator hence if we had a IonTorrent it would be useless?? Illumina provides a alternative way through Nextera, I guess if you already have a sonicator this is not an issue, but if you start from 0 like us then the sonicator you have to choose between MiSeq/Nextera or Ion/Covaris solution. A sequencer is useless unless you can prepare your sample no? Hey I'm no fan of Illumina, not today specially, but if you are NOT a sequencing facility, they still have the best and cheapest option by far. As I said I wanted to buy an Ion, I didn't buy it because it was more expensive than a MiSeq (due to the ridiculously server they sell with it) and because they didn't provide a complete solution, we had to get a sonicator that was as expensive as the sequencer... If somebody from Ion is listening, embed a stupid little computer in the sequencer (ala miseq) and give me a cd with the software to process the data or make it in the cloud, the server is killing your solution, its bulky, it takes too much space and time to manage and configure, and most of all figure out how to sell a solution that competes with Nextera.

[Vivek Todur]

hi....Can any one tell me, how exactly the phred quality score is assigned while sequencing....

[MrGuy]

You do know that Ion already has an enzyme solution:

http://www.invitrogen.com/1/1/16328-ion-xpress-plus-fragment-library-kit.html

I still don't understand why a covaris is required in your eyes...? There are so many other ways to fragment a library without buying the rolls royce of sonicators. Who gave you that idea? Besides, you could probably swap the oligos out of the nextera kit to use it for ion. Nextera was used for flx before illumina bought them a while back.

[aleferna]

Well, maybe you're right and this has changed, when I looked it up last time the only thing I could find was somebody saying that the kit was not working. Still is quite a new untested kit... I haven't heard too much about it, does it work? Any data on its performance against Nextera?

[rnaeye]

Well, you have to fragment you sample to sequence it, we don't have a sonicator hence if we had a IonTorrent it would be useless?? ...

Here is a cheap enzymatic solution to your problem:

http://www.neb.com/nebecomm/products/productM0348.asp

[pmiguel]

Just got off the phone from Illumina support, unlike V1, Nextera V2 does not provide the sequences of its primers/adapters. Extremely annoying, has anybody done Sanger Sequencing of these kits?

I think this may have just changed. (Today?) The new letter (down-loadable from illumina.com if you are registered for icom/MyIllumina, appears to be much more detailed (and diagrammatic) than earlier letters.

The date on the letter I downloaded from the site, is May 24th -- so it may just have appeared today?

--
Phillip

[ECO]

Here's the link directly to it, since their site makes it annoyingly over-complicated to find...https://icom.illumina.com/Download/S...tUCIhbHYc3NkUw

[steinmann]

Thanks a lot for the link! Quite a few people must have been waiting for this.

[aleferna]

yeap although according to support those are NOT the sequencing primers, those are the old ones, the new ones are still "secret". Why do they send this generic ill conceived document with all the sequences that I already had? Why not just Nextera - sequences, MiSeq - sequences, TruSeq ????? must they make everything so complicated?

We have to order some adapters for these sequences for a custom protocol and we've been fighting for this for a month and with all this versions I'm not sure which one to order.