Although Roche/454 hasn't released Titanium amplicon sequencing yet, are there any 454 users here who have developed protocols/workarounds to sequence amplicons with Titanium chemistry?
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robhall -- You can sequence PCR products (amplicons) with the Titanium General Library method but employing their Low Molecular Weight (LMW) DNA variation. Basically you take your PCR product (70 - 800 bp), skip the fragmentation and size selection steps, polish the ends and then proceed with the standard library prep as normal. (There may be some minor variations or notes for LMW DNA so read the manual carefully before starting.) With this procedure vs. the standard amplicon procedure (using fusion primers) your PCR product is randomly oriented relative to the A and B adapters so you will get sequence reads from both ends of your PCR product.
para_seq -- The GS FLX Amplicon sequencing allows you to select either the A or B adapter to sequence from. There are two different emulsion PCR kits available which alternately orient the amplicon for sequencing; they are called emPCR Kit II (Primer A) and emPCR Kit III (Primer B). (emPCR Kit I is for shotgun libraries).
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thanks kmcarr--we can give that a try. We are planning to multiplex and hoped to sequence from both directions at once, but I believe Roche/454 does have a tech bulletin for designing multiplex adapters with Titanium. We just wouldn't be able to control how equally we are sequencing in each orientation per amplicon.
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