Bioanalyzer and a pippen prep necessary?

[adler89]

Hi guys,
we are currently thinking about purchasing the Ion PGM and but Ion torrent recommend to use an Bioanalyzer and a pippen prep for library preparation. But the stuff costs an arm and a leg, do you really need this stuff or are there cheap alternatives?

Thank you in advance
Chris

[jonathanjacobs]

It actually an either/or question - not both.

The idea behind using the BioAnalyzer is that you can assess your library's quality far far better than using a gel. When we first started running the PGM, using the BioAnalyzer was critical and we saved ourselves from wasting some runs by checking the samples beforehand. However, quite honestly, now that we have run the PGM a few dozen times it rarely is the case that our library preps fail to pass QC.

So I guess, IMHO, it's not required - but a really good idea to have one-especially at the start. Plus, a BioAnalyzer can. E used for all kinds of other really useful assays in the lab, so it's not something that should be thought of as solely for the PGM.

[SarahNGS]

Actually pippen prep is for what I call "preparative scale" analysis. this machine and others like it (LabChip XT for example) are designed to recover the sample. It is the automatic equivalent of running your whole sample on a gel, cutting out the bands, and recovering the DNA from the gel slice. As such it is not an absolute requirement. It really depends on how many samples you will be doing a day or week and if sample prep becomes a bottleneck.
The Bioanalyzer and its cousin LabChip GX are analytical scale tools to measure DNA size distribution. you could also run a gel for this but a gel is not as sensitive or informative. the size cutoffs for PGM are pretty strict and you need to be sure your preps are going to give you good results. I'd say it is a must-have.

[pmiguel]

I agree with SarahNGS, if you have the means, get the BioAnalyzer. If you don't, then you can, in principle, get by without one. Just go into "blind zen archer" mode until after an amplification. Then you can run an agarose or acrylamide gel and see whether you hit your target or not.

The Pippinprep is cool but, in practice, we really don't use it much. You can tinker around with double-sided AmPure to do most of what the Pippinprep does.

--
Phillip

[JamesH]

Has anyone tried this:

http://www.shimadzu.com/an/lifescien...e/multina.html

instead of the bioanalyzer or labchip?

Runs are cheaper but does anyone know if it's any good?

[Sandokhan]

We also did not buy them, since we are already without arms or legs to sell. We are just starting and have managed to get results without them, besides, reagents for them will cost you probably an eye.

[thomasblomquist]

We're using a bioanalyzer for analysis, and the invitrogen e-gel system for library fractionation (about $1 per sample) with very good success.

[HMorrison]

Originally Posted by pmiguel

I agree with SarahNGS, if you have the means, get the BioAnalyzer. If you don't, then you can, in principle, get by without one. Just go into "blind zen archer" mode until after an amplification. Then you can run an agarose or acrylamide gel and see whether you hit your target or not.

The Pippinprep is cool but, in practice, we really don't use it much. You can tinker around with double-sided AmPure to do most of what the Pippinprep does.

--
Phillip

Phillip, can you give an example of a double-sided Ampure protocol? Thanks,
Hilary

[snetmcom]

Originally Posted by HMorrison

Phillip, can you give an example of a double-sided Ampure protocol? Thanks,
Hilary

quite a few threads on this exist if you search this forum.

[HMorrison]

Originally Posted by snetmcom

quite a few threads on this exist if you search this forum.

I just did. Not finding a protocol, although I did find a posting where selection was for 220 (170-220). I am looking for the equivalent of a gel cut. Never mind. I will return to the search when I have more time to guess key words.

[ECO]

Originally Posted by HMorrison

Phillip, can you give an example of a double-sided Ampure protocol? Thanks,
Hilary

You will almost certainly have to test this in your own hands and optimize the sizes for your results, but one example would be to first use a very low ratio of AMPure :: Sample, for example 0.5x-0.6x, which should bind everything ~300 and larger to the beads. Then the supernatant is saved, and additional AMPure is added to the supernatant to increase the AMPure::Sample ratio to something that will bind fragments larger than ~150, like 1.3x.

In the second step you have your desired fragments on the beads, having effectively performed a gel free size cut. Not as clean as a gel, but infinitely more automatable.

[HMorrison]

Originally Posted by ECO

You will almost certainly have to test this in your own hands and optimize the sizes for your results, but one example would be to first use a very low ratio of AMPure :: Sample, for example 0.5x-0.6x, which should bind everything ~300 and larger to the beads. Then the supernatant is saved, and additional AMPure is added to the supernatant to increase the AMPure::Sample ratio to something that will bind fragments larger than ~150, like 1.3x.

In the second step you have your desired fragments on the beads, having effectively performed a gel free size cut. Not as clean as a gel, but infinitely more automatable.

Thanks so much, Eric. We'll experiment with MW ladder and try to add it to the repertoire. In fact, we'd like to eliminate very large fragments as well as the ones under 300. Standard Ampure clean up tends to overenrich the undesirable large products!

[pmiguel]

Hi Hilary,

Some detail in:

Rodrigue S, Materna AC, Timberlake SC, Blackburn MC, Malmstrom RR, et al. (2010) Unlocking Short Read Sequencing for Metagenomics. PLoS ONE 5(7): e11840. doi:10.1371/journal.pone.0011840

and

Lennon N, Lintner R, Anderson S, Alvarez P, Barry A, Brockman W et al. (2010). A scalable, fully automated process for construction of sequence-ready barcoded libraries for 454. Genome Biol 11: R15.
doi:10.1186/gb-2010-11-2-r15


But I guess ECO is right -- we just recently got around to trying it ourselves. At this juncture it looks pretty easy to get a not very tight size distribution.

Not sure what the effect of Ampure on bubble products is, though.

--
Phillip

[snetmcom]

Originally Posted by HMorrison

I just did. Not finding a protocol, although I did find a posting where selection was for 220 (170-220). I am looking for the equivalent of a gel cut. Never mind. I will return to the search when I have more time to guess key words.

Ampure will not be equivalent to a gel cut. It's quite broad for size selection, but it is easy to automate.

[aahardikar]

We use the bioanalyzer as well for all runs and AmPure for size selection. How do you get the e-gels to work?

[lisazgreen]

Attached is experiment I performed last year comparing E-Gel® SizeSelect™ to PippinPrep (SAGE Science) for the size selection step in Ion LC, prior to library amplification.

SizeSelect Run Conditions:

50 bp DNA Ladder Cat#10416-014

Run SizeSelect™ 2% (program 8)

Time for 185bp DNA target to Reach Reference= 15:04 min
Time for 185bp DNA target to Reach Collection Well= 18:00 min

[thomasblomquist]

Originally Posted by aahardikar

We use the bioanalyzer as well for all runs and AmPure for size selection. How do you get the e-gels to work?

You put the sample you want gel-purified in the starting well. Press the "Go" button. Then pipette out your band size of interest ~10-15 minutes later (depending on size required). Cost is $13 per gel. And you can run up to 8 library purifications at a time. About $1-2 per library.

I have been using the e-gel for about 4 months now, and have received very consistent library preps from them. Yes, the yield is low nM, but compared to what you need for Ion Torrent Library prep, this is many-fold magnitude more than what you will need for down stream applications. Highly recommend the e-gel for cost, time and reproducibility for NGS library prep.

[aahardikar]

Thanks for your reply....
Do believe it is as simple as you explain. Do you use the elution buffer for e-gels to elute the bands of interest? Any cross-contamination issues with loading?

[thomasblomquist]

Originally Posted by aahardikar

Thanks for your reply....
Do believe it is as simple as you explain. Do you use the elution buffer for e-gels to elute the bands of interest? Any cross-contamination issues with loading?

I haven't had any cross-contamination in my controls as of yet. RNA-ase free H2O is the elution buffer. Simple enough to swap out for some sort of tris edta buffer if you wish. It's a very simple system. Google: Invitrogen e-gel size select