454 amplicon recurring problem - wide, large untrimmed distribution

[Indiana]

Hello,
We have done several amplicon sequencing projects lately that have yielded poor results. Link below is to a summary of thoses runs including Bioanalyzer traces, untrimmed and trimmed distributions for several of them (pword is "amplicon"):

https://slashtmp.iu.edu/files/download?FILE=jbford%2F73954FPONQF

Good runs were on:

April 22
June 7
July 31
Oct 19 (Region 2)

Then the weird distributions started (wide and too-high untrimmed, dismal trimmed):

Nov 3
Nov 7
Nov 18 (partial)
Nov 26 (partial)

We are upgraded to GSFLX-XL+, but are still using regular Titanium kits.

[pmiguel]

Hi Indiana,
When was your instrument upgraded to XL+?
Any issues with your shotgun runs?

What parameters are you using for signal processing? (You would probably have to get that info from your IT guys.)

--
Phillip

[pmiguel]

I am interested to know if there is some issue with your instrument running Titanium since you got the upgrade or, as I suspect, the runs are fine but the way your IT guys tuned your amplicon processing pipeline is not compatible with Titanium-run-on-a-FLX+ data.

--
Phillip

[LFMar]

Hi everybody, I just performed an amplicon run to sequence 16S RNA using fusion primers and the "one way read approach". The amplicons have ~700bp and the bioanalyzer profiles were perfect with no primer-dimer peak. Our machine was upgraded to GS FLX+ but we used XLR70 reagents. Guess what? I obtained a high % of short read/short primer, reads with low quality. The processing was made using amplicon and shotgun pipelines giving the same poor result. Amplicon sequencing using 454 shouldn't be allowed. I would appreciate any advise from you. Thanks in advance.

[RCJK]

LFMar, what emPCR conditions did you use?

[LFMar]

LFMar, what emPCR conditions did you use?

I used standard emPCR conditions

[RCJK]

Try using the emPCR conditions for long amplicons and see if that helps.

[pmiguel]

Hi everybody, I just performed an amplicon run to sequence 16S RNA using fusion primers and the "one way read approach". The amplicons have ~700bp and the bioanalyzer profiles were perfect with no primer-dimer peak.

ssDNA primer-dimers can anneal to the adapters of your 700 bp amplicons and effectively "hide" from size/molecular weight-based fractionation methods. I was hearing that people were getting better results on PCR products (which is what fusion amplicons are) when they were subjected to 2 cycles of AmPureXP clean-up.

AmPure is a size-based fractionation method, but perhaps by doing 2 cycles the primer-dimer strands exchange off the full length amplicons enough to get a good result.

Or, you could go old-school and run a denaturing acrylamide gel. That way all the strands run separately and the primer-dimers can't hide.

--
Phillip