Dynamag 96 side

Ten μl of 10 mg/ml streptavidin coated magnetic beads (Dynabeads M-270 streptavidin; Invitrogen) was used for each LR-PCR reaction. Beads were pooled in a 1.5 ml microcentrifuge tube and washed twice in 1 ml of 1X binding and washing buffer containing 5 mM Tris-HCL, pH7.5, 0.5 mM EDTA, and 1 M NaCl, with separation from the buffer accomplished with a DynaMag-2 (Invitrogen, Oslo, Norway). Following the washes, beads were re-suspended in 20 μl of 2X binding and washing buffer (10 mM Tris-HCL, pH7.5, 1 mM EDTA, 2 M NaCl) per reaction, and 20 μl of suspension added to each LR-PCR tube. Tubes were incubated at room temperature for 45 min, with gentle mixing by inversion every 5 min. Following binding, magnetic beads were washed two times with 100 μl of 1X binding and washing buffer, with mixing and separation accomplished using a DynaMag-96 Side (Invitrogen, Oslo, Norway). The samples were re-suspended in 100 μl of 10 mM Tris buffer, then 50 μl transferred to each of 2 real-time PCR tubes. Using the magnet, buffer was removed from the tubes, leaving the DNA bound beads as template for the second round real-time PCR.

In the final step, each library was diluted to 10 nM in Low TE, and then all libraries were pooled at 10 nM and diluted to 4 nM. Finally, the pooled libraries were denatured and diluted at 12 pM and loaded on a MiSeq Reagent Nano Kit v2 (300-cycles) cartridge with 12.5 pM PhiX (1.5%) sequencing control V3 (Illumina). Libraries were then sequenced on Illumina MiseqDX (Illumina) instrumentation in RUO mode. The whole process also required the use of a Veriti 96 thermal cycler (Applied Biosystem) for the amplification of the libraries and the Dynamag-96 side (Invitrogen) for the clean-up of the libraries.