Illumina sample prep validation

[Scott Coutts]

Hi All,

Just curious how you're evaluating the success of your library prep
before cluster generation.

Thanks.

[Pauline F]

We typically use the Bioanalyzer for quality checks and the Qubit for quantification.  We have had discrepancies between expected cluster densities and sample concentration going into cluster generation using spectrophotometer quantification.

 

Pauline

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[Todd Slaby]

i've found that the bioanalyzer quantitation is close enough to the
qPCR validated libraries that i've generated to warrant just using the
bioanalyzer estimate for sample dilution. for these experiments my SE
cluster density on a GAIIx was 180,000-220,000 per lane when loading 8
pM of libraries. if your insert size is smaller than 200bp i would
recommend cloning and sequencing some fragments to make sure you don't
have self-ligated adapter concatemers in your library.

nanodrop quantitation and picogram quantitation both reproducibly
overestimated my samples' concentrations.

Todd

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Todd

[Scott Coutts]

Scott Coutts wrote:
> Hi All,
>
> Just curious how you're evaluating the success of your library prep
> before cluster generation.
>
> Thanks.
>
>

Does anyone measure the amount of DNA present before and/or after the
enrichment PCR (for those of you that still do an enrichment PCR)?

At what point do you decide that you're not going to sequence the
library (i.e. what constitutes a 'failed' library prep?)

Thanks.

[Val Vas]

I know that Illumina is coming up with a QC kit

[Kittler, Ellen Ph. D.]

We TOPO-TA clone an aliquot of the library, pick 20 colonies and do
standard sequencing to verify presence of adapters and attachment
sequences as well appropriate (ie expected) insert sequences. This has
intercepted some interesting goof ups too! One user built an entire
library of the size ladder (confused it with his sample band in the next
lane). Another one had several species represented in the insert
sequences ... from a lab that never changes the gel box buffer, they
just shake and run again (ugh)

We also check libraries on a BioAnalyzer with the DNA High Sensitivity
Chip, this has been much more reliable for quantification as well as
quality.

Ellie

Ellen....@umassmed.edu