amplicon size for titanium?

[greigite]

Hi,
I have a sequencing project involving tiling of multiple amplicons across a genome. Each is sized approx 1-1.5 kb. Is it possible to sequence these amplicons directly using 454 titanium? I've been told that 1 kb is too small for fragmentation via nebulization, and too long for amplification in emPCR. Is my best bet to redesign the amplicons to ~500 bp and add the fusion primers to my target-specific primers? Or find restriction enzymes that will make my fragments smaller? Or is there a way to salvage the 1 kb amplicons directly? The region I'm targeting is highly variable so it's difficult to get perfectly spaced primer pairs.

[kmcarr]

The amplicon sequencing kit for Titanium is not released yet. It is supposed to be released this Fall. I don't have any concrete information so take what I say with a grain of salt. Assuming amplicon read lengths on Titanium will be similar to the shotgun reads (400-450nt) then I would think the maximum amplicon size you would want to generate is 700nt (including the template specific portions of the fusion primers). This way you could sequence each amplicon product using the II and III emulsion kits (sequencing from each end of the amplicon) with enough overlap to make certain you adequately cover the entire amplicon.

[greigite]

Thanks, kmcarr. I'm aware that there is no amplicon sequencing kit for Titanium yet, so I was planning on fragmenting the amplicons and using multiplex adapters. Do you know if nebulization works well on these smaller fragment sizes? I'm planning to try it myself, but am curious if anyone else has had success. Unfortunately we do not have a Covaris or Bioruptor.

[AKroxy]

Are the A and B adaptor sequences the same for titanium and FLX? If so, why would a specific titanium kit be needed?

[flxlex]

Latest we heard at the User Meeting in Lisbon is that the software seems to have problems handling amplicons sequenced using Titanium chemistry. We tried once and got suboptimal results. My advice is to wait for the official release.

BTW, A and B adapter sequences for Titanium are NOT the same as for FLX...

[454Sequencing]

Hi everyone,

Allow me to clear a few things up. 454 Life Sciences plans to release methods, reagents, and software for GS FLX Titanium series amplicon sequencing in the fall. Please stay tuned for a Technical Bulletin on the topic that will be available shortly. Included within will be amplicon design guidelines including the new fusion primer sequences, length considerations, and other related details.

Addressing flxlex's question: Rather than a problem with the existing software, it's simply that much as with Standard series a different analysis pipeline is required for Titanium series amplicon sequencing. Using the "normal" i.e. shotgun/paired end signal processing is not recommended for amplicons and may lead to poor results.

In the interim I would recommend following up with your local Roche representative if you have any questions.

Best regards,

Jason
Product Manager, 454 Life Sciences

[joa_ds]

hehe, we also tried amplicon sequencing on the Titanium. Poor results. Only 200k reads with poor quality. So should wait indeed for the titanium package.

[Hamid]

Hi Greigite,

You can indeed use the Covaris instrument to shear PCR products to smaller fragments. We have good experience with shearing PCR products down to 300-400bp for use library preparation. Feel free to contact me for the settings.

hamid

[kmcarr]

Originally Posted by flxlex

Latest we heard at the User Meeting in Lisbon is that the software seems to have problems handling amplicons sequenced using Titanium chemistry. We tried once and got suboptimal results. My advice is to wait for the official release.

BTW, A and B adapter sequences for Titanium are NOT the same as for FLX...

There's new information on this. Our FAS just sent us a Technical Bulletin which Roche/454 is distributing ahead of the (soon to be released) Titanium Amplicon kits. For amplicons the A and B adapters are different but only in the respect that they are extended at their 5' ends:

Titanium Fusion Primer-A: 5'-CGTATCGCCTCCCTCGCGCCATCAG-{MID}-{template-specific-seq}-3'
FLX Fusion Primer-A:            5'-GCCTCCCTCGCGCCATCAG-{MID}-{template-specific-seq}-3'

Titanium Fusion Primer-B: 5'-CTATGCGCCTTGCCAGCCCGCTCAG-{MID}-{template-specific-seq}-3'
FLX Fusion Primer-B:            5'-GCCTTGCCAGCCCGCTCAG-{MID}-{template-specific-seq}-3'

The Bulletin states that the new Amplicon kits and protocols will be backward compatible with amplicons created using the standard FLX adapters. However it strongly recommends that you redesign your amplicon primers as the older FLX ones, while they will work, perform sub-optimally producing fewer reads.

Recommended total amplicon size is 200-600bp (including the fusion primers). Products up to 800 bp are possible but you may encounter problems.

[jffkm]

I was wondering why there is a limitation to the size of the amplicons that can be sequenced. I know that larger (>600bp) amplicons will not get complete sequencing coverage from a one-sided read, but apart from that, I don't understand why there would be a physical reduction in the quality of the reads as emulsion PCR can be routinely done on amplicons exceeding 3kb (nature protocols, etc.). The extension times used in the emPCR procedure also provide more than sufficient rates for covering larger amplicons as well. Any input would be appreciated. thanks!

[sulfobus]

Is this kit for amplicon sequencing with titanium released yet?

[454Sequencing]

Dear sulfobus,

Yes, the emPCR kits that support GS FLX Titanium series sequencing of amplicons are available. You can find information on current products at our website, specifically at http://www.454.com/products-solutions/product-list.asp. In this case it is the Lib-A series of emPCR kits that you would want. Additional details are provided at http://www.454.com/products-solution...sequencing.asp and your local Roche representative will be able to supply further guidance.

If you are a current customer I would recommend the my454 customer portal (http://www.454.com/my454). Therein you will find documentation including current user manuals as well as other useful information.

Best regards,
Jason

Originally Posted by sulfobus

Is this kit for amplicon sequencing with titanium released yet?

[sudsf16]

All but one of our clients want unidirectional amplicon sequencing which we have been providing with the standard FLX chemistry. The new Titanium amplicon kits are bidirectional only. If you need unidirectional sequencing, Kit II or Kit III using FLX chemistry is what's available from Roche.

[kmcarr]

Originally Posted by sudsf16

All but one of our clients want unidirectional amplicon sequencing which we have been providing with the standard FLX chemistry. The new Titanium amplicon kits are bidirectional only. If you need unidirectional sequencing, Kit II or Kit III using FLX chemistry is what's available from Roche.

sudsf,

Jason (454Sequencing) addressed that issue in another thread. There will be enough of each reagent to do a unidirectional run.

Additional info:

O.K. I just went looking for Application Brief which Jason mentioned in that thread. It is posted on the 454 web site and is called APP001-2009_Lib-LunideirectionalAmplicons.pdf. Basically the strategy is to design your amplicon primers using the normal shotgun A & B adapter sequences and then do the emPCR with the Lib-L kit instead of the Lib-A. I believe that this method would not be backward compatible with amplicons originally designed for the standard FLX protocol. The Lib-A emPCR kit is backward compatible.

[laurahug]

Hi all,

I have the same question as greigite, two years later: has anything changed? Can I sequence a 1-1.5kb PCR amplicon with any of the currently available 454 technologies? I'm working with a gene family as well, and require the amplicon length to be that long to avoid hypervariable regions to which primer design is near-impossible.

I don't, however, need a full over-lapping sequence from the analysis, it is more like pyrotagging from an analysis standpoint.

So has emPCR overcome the ~800 bp limit? Do LV or MV kits make a difference in that respect?

Thanks for any information, I've looked everywhere.

Laura

[kmcarr]

Laura,

No nothing has changed. In fact Roche just release a Tech Note showing how poor longer amplicons work in the current system.