Vertebrate amplification
Following extractions, we amplified each pooled sample of sandflies in
two separate reactions using a modification of the primer pair
12SV5F/12SV5R (Riaz et al., 2011). We used the reverse primer
(TTAGATACCCCACTATGC) as Riaz et al. (2011) and a modified version of the
forward primer to allow for broader binding of vertebrate targets
(YAGAACAGGCTCCTCTAG). These primers target approximately 100 base pairs
in the 12S rRNA gene region of the vertebrate mitochondrial genome.
Additionally, we used twin-tags to better detect tag jumping. Briefly,
we carried out PCR reactions in a volume of 20 ul using 10 ul AmpliTaq
Gold 360 Master Mix (final concentration of 1x), 5 ul of forward and
reverse primers (final concentration of 0.25 uM), 3 ul of water, and 2
ul of DNA template. PCR cycling was as follows: initial denaturing at
95°C for 10 min; 40 cycles: 95°C for 30 sec, 58°C for 30 sec, 72°C for
30 sec; and a final extension at 72°C for 7 min.