barcoded pyrosequencing uising titanium kit

[biohumin]

Hi :
we are going to evalute the bacteical diversity in the sea using 16S rDNA bacterial tag-encoded amplicon pyrosequencing. I found the GS20 and FLX kit have the same 454 primer A & primer B sequences which use in emPCR. I am wondering if the titanium kit has the same primeers' sequences and could use in barcoded sequencing, because it has longer read length and more reads.

[454Sequencing]

Hi biohumin,

You will find some useful information in the following post:

http://seqanswers.com/forums/showpost.php?p=8662&postcount=9

-as well as in my earlier post in the same thread.

If you've not yet designed fusion primers your best bet is to use the Titanium Series primer design. The associated emPCR kits will shortly be released to enable sequencing using Titanium Series XLR70 kits. Importantly, the Titanium Series amplicon data analysis pipeline will be made available at the same time.

If you have any further questions please don't hesitate to contact your local Roche representative.

Best regards,
Jason

Technical Product Manager
454 Life Sciences, A Roche Company

[SeqMonster]

Hi,

We are planning to sequence a series of 16s using titanium chemistry and I was reading through the latest released titanium amplicon emPCR manual. Can anyone clarify whether the emulsion cup A and cup B are the same (I assumed not since it says "Do not mix DNA capture beads A and B") and do they come as a pair when you ordered it? Why are there 2 different emulsion cup now? Since I only have the MID tag on my fusion primer A, would it still be sequence from both emulsion set-up? And I guess I will use the amplification Primer A for emPCR?

Thanks and it will be great if someone could help.

SeqMonster

[454Sequencing]

Hi SeqMonster,

The Emulsion Oil cups as provided in the kit are identical. However, the emPCR Reagent Kit (Lib-A) contains reagents for both A- and B- side amplicon sequencing. In other words, there are separate aliquots of A and B DNA Capture Beads, Enrichment Primer, Amplification Primer, and Sequencing Primer. Supplying all of these materials in a single kit enables bidirectional amplicon sequencing. You can certainly choose to sequence from one side only if that meets your particular experimental needs.

That said, stay tuned as we'll be releasing some documentation shortly that will outline some additional experimental design options for amplicon sequencing.

Best regards,
Jason

Hi,

We are planning to sequence a series of 16s using titanium chemistry and I was reading through the latest released titanium amplicon emPCR manual. Can anyone clarify whether the emulsion cup A and cup B are the same (I assumed not since it says "Do not mix DNA capture beads A and B") and do they come as a pair when you ordered it? Why are there 2 different emulsion cup now? Since I only have the MID tag on my fusion primer A, would it still be sequence from both emulsion set-up? And I guess I will use the amplification Primer A for emPCR?

Thanks and it will be great if someone could help.

SeqMonster

[kmcarr]

The Emulsion Oil cups as provided in the kit are identical. However, the emPCR Reagent Kit (Lib-A) contains reagents for both A- and B- side amplicon sequencing. In other words, there are separate aliquots of A and B DNA Capture Beads, Enrichment Primer, Amplification Primer, and Sequencing Primer. Supplying all of these materials in a single kit enables bidirectional amplicon sequencing. You can certainly choose to sequence from one side only if that meets your particular experimental needs.

Jason,

Does that mean that the Lib-A kit has enough of each reagent to prepare beads sufficient for a full plate of sequencing from just the A or B end, or would we have to burn through two kits of we wanted to do two large regions of just the A end?

[454Sequencing]

Hi kmcarr,

Yes, it is possible to do a full run of only A- or B- amplicon sequencing with a single Lib-A emPCR kit. However, the minimum emPCR enrichment required for each gasket format must be taken into consideration to ensure you will have sufficient beads to load at the maximum recommended density.

That said, one of the (shortly) forthcoming documents that I mentioned in my earlier post will be an Application Brief that describes the use of an additional approach for this very situation. I think that anyone who primarily/exclusively sequences amplicons from one adaptor only will be interested in this Brief.

Best regards,
Jason

Jason,

Does that mean that the Lib-A kit has enough of each reagent to prepare beads sufficient for a full plate of sequencing from just the A or B end, or would we have to burn through two kits of we wanted to do two large regions of just the A end?

[SeqMonster]

Hi Jason,

Thanks for the reply. Do you know when is the bulletin that you mentioned will be releasing?

/SeqMonster

[jffkm]

Amplicon sequencing titanium

I'd also be interested in obtaining the official Roche documentation on amplicon sequencing for titanium chemistry. I know users have been experimenting with this and have gotten successful runs. Also, the application bulletin describing the slightly modified tags for titanium fusion primers hints that everything is ready to go. Please let us know where we can find the docs. Thanks

[454Sequencing]

Hi jffkm,

If you are a customer you can obtain these documents directly from the my454 customer portal (http://www.454.com/my454). All of the user manuals as well as other documentation may be found there. The kits to support amplicon sequencing using GS FLX Titanium series chemistry are in fact available.

Best regards,
Jason

I'd also be interested in obtaining the official Roche documentation on amplicon sequencing for titanium chemistry. I know users have been experimenting with this and have gotten successful runs. Also, the application bulletin describing the slightly modified tags for titanium fusion primers hints that everything is ready to go. Please let us know where we can find the docs. Thanks