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Old 01-29-2016, 06:39 PM   #1
user 31888
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Default crude DNA extract suitable for sequencing?

I would like to check the integrity of crude DNA and RNA samples extracted from tissues in order to know if it is worth keep going with library prep and sequencing (MiSeq or HiSeq).

(1) Is running the crude DNA extracts on BioAnalyzer informative to know if the samples are sequence-able?
(I know for RNA I could use the RIN values)
(2) If yes, what particular criteria should be taken into consideration (presence of sharp bands/smear in the gel; broad/sharp peaks on the electropherogram)?
(3) Any other possible approach than Bioanalyzer?

Thanks !
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Old 01-30-2016, 12:34 PM   #2
luc
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What do you consider crude extracts? Would you run additional cleanups before library preps?
Almost any DNA sample will be suitable - RNA samples however not.
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Old 01-30-2016, 02:52 PM   #3
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By "crude extracts" I mean the DNA and RNA material I obtain after cell lysis, separation and purification using a commercial kit (QIAGEN for instance).

The tissues the DNA and RNA come from can be very old and sometimes are FFPE.
DNA and RNA can be degraded, that's why I would like to know what Bioanalyzer profiles to expect from degraded and sequencing-suitable DNA?
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Old 01-31-2016, 12:44 PM   #4
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The ideal profile for your samples depends almost entirely on what you're trying to sequence and to what end. For instance, with whole genome shotgun sequencing you don't need to worry too much about DNA degradation since you'll likely be fragmenting your sample even further prior to library prep. For FFPE samples there are several options (e.g. NEB's Repair Mix) to help to get your DNA ready for library prep. If you have access to a TapeStation you can use the Genomic DNA ScreenTape assay, which will give you a "DNA Integrity Number" similar to the RIN score, although of course these are somewhat arbitrary assignments and should be considered relative to similar samples (i.e. tissue type, species of origin, etc. etc.). Of course, there are applications that require relatively high-integrity DNA, such as synthetic long read preps.
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Old 01-31-2016, 08:19 PM   #5
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Thanks Luc and adam.geber for your replies !

So your point is that everything is potentially sequenceable if I plan to incorporate a fragmentation step in the library prep, assuming that the concentration of material is sufficient for library prep?

In this case, only checking the concentration of the fragments with a size ≥X bp (if I plan to do a 2 x X bp sequencing) would be enough to decide if sequencing the samples or not? It does not seem very precise to me, no?

Note: I plan to do WES (2x75bp or 2x100bp) with these human samples
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Old 02-01-2016, 04:27 PM   #6
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If you want an easy way to check FFPE samples for NGS suitablity and don't have access to a TapeStation, this kit from QIAGEN is a good option:GeneRead DNA Quantimize (https://www.qiagen.com/us/products/n...antimize-kits/)

Provides you insight into the amount of amplifiable DNA (which is important for FFPE samples), and will let you know if you should continue with library prep.
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Old 02-01-2016, 06:36 PM   #7
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Hi User,

I would suggest to just run a bioanalyzer or even an agarose gel and post the traces or a gel image here - this would be better than all the hypothetical scenarios.
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Old 02-17-2016, 01:20 AM   #8
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For DNA I would start with a regular agarose gel, and Nanodrop, to get an estimate of the integrity, purity and amount. For more accurate estimation of concentration use a fluorometric method (Qubit, Picogreen...).

Last edited by JBKri; 02-17-2016 at 01:24 AM.
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