Following multiplex amplification, a second round of 12 parallel singleplex PCR reactions using primers for each individual target at a final concentration of 500 nM were performed to ensure robust amplification of product for primers with lower efficiency in multiplex. High fidelity Q5 Hot Start Polymerase and other PCR reagents were used as described above.
Singleplex reactions were amplified in a 7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA) for 15 cycles using the following conditions: 95 °C/2 min (Q5 polymerase activation); 15 cycles of 94 °C/10 s (denaturation), 65 °C/20 s, (annealing), and 72 °C/30 s (extension); a final extension 72 °C/2 min extension was performed to ensure complete extension of all products. Each singleplex PCR product was checked for quality and quantity with an Agilent 2100 Bioanalyzer using DNA Chips with DNA 1000 Kit reagents according to manufacturer protocol (Agilent Technologies, Deutschland GmbH, Waldbronn, Germany). Sample-specific singleplex reactions then were (a) mixed in equimolar amounts to ensure an equal balance of target reads among sequencing read counts and (b) column-purified using QIAquick PCR Purification Kit (Qiagen, Hilden, Germany) according to manufacturer protocol.
Singleplex reactions were amplified in a 7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA) for 15 cycles using the following conditions: 95 °C/2 min (Q5 polymerase activation); 15 cycles of 94 °C/10 s (denaturation), 65 °C/20 s, (annealing), and 72 °C/30 s (extension); a final extension 72 °C/2 min extension was performed to ensure complete extension of all products. Each singleplex PCR product was checked for quality and quantity with an Agilent 2100 Bioanalyzer using DNA Chips with DNA 1000 Kit reagents according to manufacturer protocol (Agilent Technologies, Deutschland GmbH, Waldbronn, Germany). Sample-specific singleplex reactions then were (a) mixed in equimolar amounts to ensure an equal balance of target reads among sequencing read counts and (b) column-purified using QIAquick PCR Purification Kit (Qiagen, Hilden, Germany) according to manufacturer protocol.