Summary: Efficient use of NGS sequencing is highly dependent upon accurate measurement of both the quality and the quantity of DNA in the library. Library quality is assessed on a Bioanalyser or similar instrument; library DNA quantity is best measured by qPCR. qPCR is the only quantification method which measures just those DNA library fragments which have both (P5 and P7) adapter sequences ligated on either end, which will subsequently form clusters on a flowcell for sequencing. Other methods are either not specific for DNA (spectrophotometry at 260 nM, e.g Nanodrop™), or measure all DNA molecules and not just those containing P5 and P7 adapters (fluorimetry with a dsDNA specific dye, e.g. Qubit®, and electrophoretic methods e.g. Bioanalyser). qPCR quantification of NGS DNA libraries has a much wider dynamic range than other methods (typically a 7 log range), and is much more sensitive and so requires much less of the library sample.
Accurate quantification of amplifiable library DNA loaded into a flow cell is one of the most critical steps in the NGS workflow. Loading insufficient adapter-ligated DNA will result in low cluster density and inefficient use of the flow cell, while too much adapter-ligated DNA may lead to densely-packed clusters which are too difficult to call.
NGS library quantification standard
Each vial contains 1.8 fmoles of a pure, dried DNA standard suitable for use in quantifying Illumina Next Generation Sequencing (NGS) libraries. When reconstituted in 90 uL of nuclease-free water, the concentration of the standard is 20 pM.
Standard for Illumina NGS library quantification
90 uL of 20 pM standard for Illumina NGS library quantification
● Sufficient for up to 15 standard curves
● Quantify up to 315 libraries per pack
● Use with any qPCR mix and qPCR machine
Product code I_NGS_001
Price: £150 – free delivery within the UK
To order, please email your requirements to finance@qstandard.co.uk
Real-time qPCR quantification using the qStandard NGS library standard is:
Accurate – provides a reliable, high purity, double-stranded DNA for qPCR measurement of only the adapter-ligated DNA in the library which is amplifiable on the flow cell. DNA fragments containing a single ligated adapter or no adapters will not be measured. Adapter-dimers, if present, have a different melting peak from the NGS standard product and can be identified. The qPCR efficiency is ~98%.
Sensitive – uses only a very small amount of the DNA library as a typical library can be diluted 10,000 to 100,000-fold prior to quantification because the qStandard assay is linear and sensitive to 0.0002pM.
Rapid, high throughput – 20 libraries can be measured in one run on a single 96-well plate (assuming duplicates of NTC/standards and 2 dilutions of libraries). Library concentration data are generated directly in the qPCR assay, requiring a simple multiplication to correct for library dilution and account for ratio of average library size/standard size.
Convenient – contains a single dried DNA standard, which is easy to reconstitute fully, simple to dilute to provide up to 6 dilutions (0.0002pM – 20pM) to generate a linear standard curve (R2>0.995) covering a broad library concentration range.
Economical – contains only the qStandard NGS DNA standard. The standard provided will allow 10 standard curves (20 μL reactions) providing quantification of 200 libraries on 10 plates (assuming duplicate reactions for 2 dilutions of each library). We don’t want to inflate your assay costs by selling you a small aliquot of qPCR enzyme SYBR Green master mix and P5/P7 compatible qPCR primers. You may already have these, or could buy them cheaply in bulk if necessary. So, we don’t provide the qPCR primers but we do provide their sequences with the standard so that you can order them cheaply at whatever scale suits your needs from any reputable supplier of DNA oligos.