Anti-enriched Troubleshooting

[Anthony.287]

Hi all,
There is a note in the emPCR manuals that says you can collect the anti-enriched beads for troubleshooting. Does anyone have any recommendations for trouble shooting these beads? The only thing we've thought of is maybe qPCR, but is there a better way?
Thank you!!
Anthony

[Nicole 454 Sequencing]

Hi Anthony,

There are a few simple experiments these beads can be used for. Assuming you have little to no enrichment, troubleshooting the anti-enriched beads may narrow down where things went wrong:

-inefficient Melt step
-heat block not on during Enrichment Primer Annealing
-didn't add Enrichment Primer/ used wrong primer
-use of wrong buffer during Enrichment Primer Annealing
-wrong buffer during Enrichment Bead incubation
-too low copy per bead or thermocycling didn't work properly
-poor library construction

Repeating the enrichment process with these anti-enriched beads could recover beads with amplified target that were lost the first time through. If troubleshooting efforts don’t yield enriched beads, it’s likely the issue is upstream of Breaking and Enrichment.

Nicole
Technical Support Scientist
454 Life Sciences, A Roche Company

[MissDNA]

But to repeat the process with the anti-enriched beads, one would be wasting a kit in hopes to perhaps recover enriched beads. Wouldn´t be better start over again?
Also, if you do recover beads on the 2nd try, the answer about what went wrong would not be simple since there is a lot of variables to look for as you mentioned.

[pmiguel]

You can count your anti-enriched beads. Make sure you did not lose beads somewhere post-breaking.

We never do this, though.

--
Phillip

[MissDNA]

We don´t even save them...

[Anthony.287]

Yeah, I usually don't keep them, put recently a particular library was giving me trouble so I decided to save the anti-enriched beads just in case. If there isn't any practical way to troubleshoot with them, then I'll probably just toss them in the future. Unless it's critical to get that particular library enriched right then; then I might try to repeat the enrichment.
Thanks everyone!

[Nicole 454 Sequencing]

Hi Anthony,

Another method you might consider is saving the supernatant from the first Melt step. You could perform qPCR or run on the Bioanalyzer to see what if anything is being amplified during emPCR.

Nicole

Technical Support Scientist
454 Life Sciences, A Roche Company

[jasmineja]

HI all, We have recently experienced a similar problem we are running 454junior with amplicons (rapid lib) or genomic DNA (using LIB L) and after the emPCR reaction we get very low bead count , almost none . As part of troubleshooting we tried a new NaOH solution, the annealing temp is monitored , do you have any idea what could go wrong?

[WPAFB]

Hello. I am working with a GS Junior. As along with many other folks on this board, my group is barely getting a bead count after the emPCR. The pellet is usually barely visible and we rarely move on with the sequencing step. When we do decide to sequence, it never works. So I guess I have two questions.

1- How much DNA should be added to the capture beads before the thermocycling of the emPCR? We have troubleshot many things, and we think this is where we are making our biggest mistakes. Such as this scenario: If I have a 300 bp fragment of DNA, how many picograms of it should I add to my capture beads at the beginning of emPCR?

2- I have read on some of these threads that some people might have an excel spreadsheet to help calculate the question I asked above. Does anyone have anything like that they'd be willing to share? If so, please let me know and I will give you my private email address.

Thanks for any help you can give!!

[radekchr]

1- How much DNA should be added to the capture beads before the thermocycling of the emPCR? We have troubleshot many things, and we think this is where we are making our biggest mistakes. Such as this scenario: If I have a 300 bp fragment of DNA, how many picograms of it should I add to my capture beads at the beginning of emPCR?

2- I have read on some of these threads that some people might have an excel spreadsheet to help calculate the question I asked above. Does anyone have anything like that they'd be willing to share? If so, please let me know and I will give you my private email address.

Thanks for any help you can give!!

Hi WPAFB,

In our lab we calculate the # of DNA molecules per ul using the following equation:

{[conc. (in g/ul)] / (650 * size)} * 6.022E23

where the concentration is given in grams per microliter and the size is the average bp length of our library.

From there it's sort of a guessing game the first couple times for how much DNA to use in the emPCR, but once we get a good ratio of beads for a given amplicon our results are pretty consistent. I tend to start with going for a ratio of 1 molecule per bead, and usually get enrichment between 5-15% with that.
Use the formula in the emPCR protocol to figure out what concentration and volume of DNA you need to get to your desired molecules per bead. I always like to keep my input volume at 10 ul and have created an excel sheet to make those calculations very simple. If you give me your private email address I can send you a copy.

Hope this helps!

[ParkerKB]

Hi,

We are also having similar problems with our library preps for a GS Junior. We have proceeded to the enrichment step and had no enriched beads. Our roche technical rep suggested a PCR to amp the library BEFORE emPCR that targets the adapters to check for adapter ligation and also the size distribution of the library. We have repeatedly had some libraries that don't amplify (and thus have/would fail during emPCR), suggesting that the adapter ligation has failed. In contrast, libraries that amp always have successful emPCR. So I would think the problem lies primarily with adapter ligation and/or AMPure clean up afterwards, leading to poor input into emPCR, rather than a problem or mistake with enrichment. Has anyone had similar results or have suggestions on how to troubleshoot/increase the consistency of library prep?

[pmiguel]

Hi WPAFB,

In our lab we calculate the # of DNA molecules per ul using the following equation:

{[conc. (in g/ul)] / (650 * size)} * 6.022E23

where the concentration is given in grams per microliter and the size is the average bp length of our library.

If you are not near your computer or a calculator there are some rules of thumb to estimating the number of molecules in a solution from a known mass of DNA. (Also good for a "sanity check" of the result you get from your calculator or spreadsheet.) Notice that the MW of an "average" base pair used above "650" and the 6.02 of Avagadros number almost cancel to 1/100 (well, 1/93, but close enough) out so that if your DNA is 1000 bp, then

1 ug ~= 1 trillion (1E12) molecules
1 ng ~= 1 billion (1E9) molecules
1 pg ~= 1 million (1E6) molecules

Then you can just adjust for single stranded molecule (double) or shorter (more molecules) or longer (fewer molecules) as needed.

Also, recently, I like to think of Avogadro's number as 0.6E24 instead of 6.022E23 because then I can just use E24 (10^24) as an estimate and keep in the back of my head that it is really only 60% of that. This isn't really used for 454 protocols. But then it is handy to think that if I have 1 ul of 2 nM solution then I have E24-E6(ul)-E9(nM) or 2E9 molecules in that ul. (Get it? 24-6-9=9) 2 billion. or 2x602.2 million if I need additional accuracy.

You do want to memorize the prefixes vs the exponents:

m -3
u -6
n -9
p -12

and, for fun, if you like:

f -15
a -18

because then you know that your 1 aM solution has just under 1 molecule per ul. (.602) Not useful, but perhaps entertaining?

--
Phillip