mHVR amplicon  sequencing of T.cruzi: TeDa2cl4
In order to amplify  the minicircles hypervariable region, kDNA specific primers 121  (5-ACACTCTTTCCCTACACGACGCTCTTCCGATCTAAATAATGTACGGG(T/G)GAGATGCATGA-3) and 122  (5-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGGTTCGATTGGGGTTGGTGTAATATA-3) were  modified by adding an oligo adapter to be used in an Illumina platform. The  mHVR libraries were generated by a one-step PCR performed in 5 l reaction  volumes containing 5 ng of DNA, 250 nM of each primer, 2 M of barcode  primers, 5 U of Fast Start High Fidelity Enzyme Blend (Roche), 0.50 l of 10X  buffer (supplied with the Fast Start High Fidelity Enzyme Blend), 25 nM of  MgCl2 (Roche), 0.25 l of DMSO (Roche), 10 mM of PCR grade nucleotide mix  (Roche) and 0.25 l of 20X access array loading reagent (Fluidigm ). The PCR  reaction was carried out on a Veriti Thermal Cycler (Life Technologies) and  ran as follow: an initial denaturation step (10 min at 95C), 10 cycles (95C  for 15 seconds, 60C 30 seconds, 72C 1 min), 2 cycles (95C for 15 seconds, 80C  30 seconds, 60C 30 seconds, 72C 1 min), 8 cycles (95C for 15 seconds, 60C 30  seconds, 72C 1 min), 2 cycles (95C for 15 seconds, 80C 30 seconds, 60C 30  seconds, 72C 1 min), 8 cycles (95C for 15 seconds, 60C 30 seconds, 72C 1 min)  and 5 cycles (95C for 15 seconds, 80C 30 seconds, 60C 30 seconds, 72C 1 min).  Amplicons were then purified using the magnetic beads Agencourt AMPure XP-PCR  Purification (Beckman Genomics, USA). The concentration of the purified  amplicons was controlled using Qubit Fluorometer 2.0 (Invitrogen, USA). All  libraries were validated using the Fragment Analyzer system (Advanced  Analytical Technologies, USA). The average size of the mHVR amplicons was  480bp. All samples were then pooled  and prepared according to the manufacturer's recommendations (Illumina  Protocols: Sequencing Library Preparation) and sequenced on an Illumina MiSeq  using a 500 cycle v2 kit (Illumina, San Diego, USA) to produce amplicons of  approximately 480 bp in length (250 bp paired-end reads).