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Old 06-21-2012, 03:26 AM   #1
Nu2NGS
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Default eGel, Qubit, Bioanalyzer: when and why?

Hello all,

Happy Thursday. I am clearly new at NGS given this line of questioning. Thank you for you responses and guidance in advance.

We are about to purchase a PGM and Life tells me we would (at a minimum) need an eGel system, Qubit, and Bioanalyzer. When in the entire process (extraction (FFPE) to sequencing) would I need to use these and why? Would I need to use them more than once in the process at various stages? Do I need more than one set?

Thank you and have a great one
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Old 06-21-2012, 03:55 AM   #2
TonyBrooks
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Quote:
Originally Posted by Nu2NGS View Post
Hello all,

Happy Thursday. I am clearly new at NGS given this line of questioning. Thank you for you responses and guidance in advance.

We are about to purchase a PGM and Life tells me we would (at a minimum) need an eGel system, Qubit, and Bioanalyzer. When in the entire process (extraction (FFPE) to sequencing) would I need to use these and why? Would I need to use them more than once in the process at various stages? Do I need more than one set?

Thank you and have a great one
You'll need the following bits of kit at the following stages
1) Genomic DNA quantification - Qubit.
The Nanodrop is not accurate enough. You can replace the Qubit here with another method of fluorescence measurement (PicoGreen on a fluorometer)
2) Library size selection - eGel.
You need to select your library at some point. eGel is the easiest way, but you can probably replace with a standard agarose gel selection
3) Library QC - Bioanalyser
This is probably the most valuable kit here. You can QC on a gel if your yields are high enough, but it won't be as sensitive as a Bioanalyser. There are alternative capillary gel eletrophoresis machines you could used instead Shimadzu, Caliper).
4) emPCR enrichment check - Qubit
This stage is optional. We don't do it as it would require a QubitV2 and we only have a QubitV1.
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Old 06-21-2012, 05:41 AM   #3
pmiguel
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Originally Posted by TonyBrooks View Post
3) Library QC - Bioanalyser
This is probably the most valuable kit here. You can QC on a gel if your yields are high enough, but it won't be as sensitive as a Bioanalyser. There are alternative capillary gel eletrophoresis machines you could used instead Shimadzu, Caliper).
The most valuable, but also the most expensive. If you are strapped for cash, you might want to assess your current agarose gel electrophoresis/gel documentation system. You can replace, in a pinch, many of the items on the list by obtaining a "dark reader" illumination source (UV transilluminators damage DNA and RNA) and deploy sensitive dyes like "SYBR gold" for staining.

Also the quality of your "photographic" set up is an issue. Getting a set up that is both inexpensive and sensitive is possible, but may require time and skills you don't have.

Ultimately, you will probably not be able to approach the pg sensitivity of Agilent pico RNA and High Sensitive DNA chips. But you pay quite a bit for these and the kits go bad fairly quickly -- often in less than 1 year. So if you are not going to run more than 20 chips of a given type per year, this an issue and will entail additional expense.

Just to be fair, as an Illumina core, we would not even think of trying to operate with a Bioanalyzer. But there you are feeding a > $500,000 sequencer, so the ~$20,000 outlay for a Bioanalyzer is unquestionably worth it. But just to feed a <$100,000 instrument? It could be considered an extravagance. If you, or others in your group/department have additional uses for it, definitely get one. If not -- it is a judgement call.

--
Phillip
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Old 06-21-2012, 05:52 AM   #4
Nu2NGS
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Phillip and Tony,

Thank you for the rapid responses. Very helpful.
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Old 06-21-2012, 05:42 PM   #5
geneart
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I am a ametuer NGS person , prepping my samples using the truseq kit. I have very low yields of product ,according to agilent check. My peaks are good for some at 150bp and I do have the shoulder peaks anywhere from then on to 300bp region.My concern is when I ran agilent I got the data which provides the Molarity of the samples in pmols/L. I was required to run the samples on qubit I have samples that range from <.5ng/ml to 17.9ng/ml. Ideally,I am required to have a conc at 10nM for GA. But I heard that the concentrations at the end in each cell will be in pmols. Is this true???? That does not make sense to me at all ! Can anyone please explain whats going on in here and how much do we need to have to load on a GA to get good number of reads,please? Much appreciated !!!

Last edited by geneart; 06-21-2012 at 06:28 PM. Reason: need to add more
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Old 06-21-2012, 06:31 PM   #6
geneart
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Also I need to note that all my samples are from exosomal extractions, so I am not really surprised that there is not a huge amount of starting material comparable to a whole cell extract of RNA. Exosomal RNA extracted was ligated and RT/PCRd using the TRUSeq protocol. Having said this , would I still need that 10nM to load on GA????What is a minimum amount that I get down to and still get decent reads? Thanks again
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Old 06-22-2012, 01:54 AM   #7
TonyBrooks
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The recommended protocol from Illumina is to store your library in a final concentration of 10nM. Low concentration libraries are less stable (especially after multiple freeze thaws).
When running the GAIIx, you dilute your sample down to 2nM (10µL) and denature for 5 minutes with an equal volume of 0.1N NaOH to get a 20µL of a 1nM solution. This is then diluted again with Hyb Buffer HT1 (which also buffers out the NaOH) to a working concentration, usually around 12pM (12µL of denatured library plus 988µL HT1).
The problem with low concentration libraries is that the ratio of denatured sample to HT1 can become too high. If the final concentration of NaOH after dilution in HT1 is >2.5mM, it isn't buffered out properly and this leads to low cluster numbers - the NaOH interferes with the hybridisation to template to grafted oligos on the flowcell.
There are protocols mentioned on this site that are not officially Illumina supported bu.t allow you to denature at a much lower concentration. The trick is to add an equal volume and molarity of HCl to the sample after the 5 minute NaOH incubation before adding HT1. This neutralises the NaOH.

For example, consider a 200pM library that we want to hybridise to a GA flowcell.
Take 10µL of library and add 6µL of water
Add 2µL 2N NaOH, mix well and incubate for 5 minutes to denature.
Add 2µL 2N HCL and mix well. This should leave you with 20µL at 100pM with neutral(ish) pH.
Add 88µL HT1 to 12µL denatured sample. This then gives you a 12pM library in HT1 buffer which can be hybridised to a flowcell.

We've used the prototol a few times and it seems to be fine.
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