Technical data

5 Post-Capture Sample Processing for Multiplexed Sequencing
Step 5. Optional: Quantify captured library pools by QPCR
72 SureSelect
XT2
Target Enrichment System for Illumina
Step 5. Optional: Quantify captured library pools by QPCR
For accurate determination of the DNA concentration in each captured
library pool, use the QPCR NGS Library Quantification Kit (for Illumina).
Refer to the protocol that is included with the QPCR NGS Library
Quantification Kit (p/n G4880A) for more details to do this step.
1 Prepare a standard curve using the quantification standard included in
the kit, according to the instructions provided in the user guide.
2 Dilute each captured library pool such that it falls within the range of
the standard curve.
Typically this corresponds to approximately a 1:1000 to 1:10,000
dilution of the captured DNA.
3 Prepare the QPCR master mix with Illumina adaptor-specific PCR
primers according to instructions provided in the kit.
4 Add an aliquot of the master mix to PCR tubes and add template.
5 On a QPCR system, such as the Mx3005p, run the thermal profile
outlined in the QPCR NGS Library Quantification kit user guide. Use
the SYBR Green instrument setting.
6 Use the standard curve to determine the concentration of each
unknown captured library pool, in nM.
The concentration will be used to accurately pool samples for
multiplexed sequencing.
In most cases, the cycle numbers in Table 28 will produce an adequate yield for sequencing
without introducing bias or non-specific products. If yield is too low or non-specific
products are observed, adjust the number of cycles accordingly with the remaining
captured DNA template.
NOTE