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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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In amplicon sequencing using 454 GS FLX, does sequencing ONLY start from the end with Linker B? Thank you. -
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).Comment
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kmcarr--
Thanks a lot.Comment
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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.Comment
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please also have a look at this thread:
flxlexComment
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Hi kmcarr--Have you tried this? I'm curious if it has worked. My only worry is that the ligations won't work as well as they need to from PCR product ends, but I guess the end-polishing should take care of that?
-robhallComment
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robhall -- No we have not done any LMW preps. But as you say, the end polishing should work as well on PCR products as fragmented DNA.Comment
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Love this - good data definitely starts from good input, and poor input can only give relatively poor data. I particularly like the mention of Nanodrop/absorbance based methods for quantification. It's such a toss up if you'll get an accurate reading or what amounts to a randomly generated number, and a lot of library/sequencing related issues can be traced back to poor quant.07-01-2026, 11:43 AM
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I’m not a sequencing expert. I’m a purification scientist who uses NGS to evaluate workflows my group develops. With this perspective, we think about the sample first and the NGS workflow second. The sequencer is an exceptionally honest reporter, but it can only report on what you give it, so whether you get clean, interpretable data from an NGS workflow is largely determined before you begin.
Here are nine questions we think about, in roughly the order they matter, before...
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