Amplicon sequencing of Malurus melanocephalus
Genomic DNA was extracted from each sample using DNeasy blood and tissue extraction kits (Qiagen, California, USA) and DNA concentrations were determined using a Qubit fluorometer (Life Technologies, California, USA). We amplified all the amplicons for each DNA sample in a single multiplex PCR by preparing a single primer mix containing all 240 primers each at a final concentration of 0.7uM. First round PCR was performed using the Qiagen Multiplex PCR Plus Kit (206152, Qiagen) with 0.3ul of the above primer mix and 20-40ng of DNA in a final volume of 11ul. Initial denaturation at 95 C for 5min was followed with 35 cycles of 95 C for 30 sec, 62 C for 90 seconds and 72 C for 30 sec and a final extension step of 68 C for 10min. A 10-fold dilution of these products was used as the template for a second round of PCR to add the Nextera i5 and i7 index primers. This second round of PCR was performed in a total volume of 11ul with 2ul of the diluted PCR product, 0.5U Platinum Taq DNA Polymerase (Thermofisher, 10966-034), 1 X PCR Buffer, 1.5 mM MgCl2, 0.2 mM dNTP and 0.5uM of i5 and i7 primer. Reaction conditions are as follows: initial denaturation at 95 C for 2min, followed by 7 cycles of 95 C for 30 sec, 55 C for 30 seconds and 72 C for 30 sec. This was followed by 95 C for 10sec, 55 C for 3min (with a 0.1deg/second ramp), 72 C for 30seconds and a final extension step of 72 C for 5min. PCR products were then pooled equally and cleaned using 1:1 ratio of SPRI beads (made using Sera-mag SpeedBeads (Fisher Scientific, 09-981-123) following methods in (Rohland and Reich 2012). This library was then sequenced by the Cornell Institute of Biotechnology Resource Center (BRC) on an Illumina MiSeq instrument (single end 150bp).