Amplicon sequencing of LF microfilariae from blood smears
We designed three primer sets modified with Illumina adapters for library construction: cytochrome oxidase I (COI; 402 bp), ND4 (353 bp), and cytochrome b (cyt-b; 349 bp). PCR amplicons were purified with an ISOLATE II PCR and Gel kit (Bioline) following the manufacturers instructions. In total, 31 pools, each containing 5 mf from the same person, were prepared for three amplicons and sequenced on a MiSeq v3.0 2x300 at the La Trobe Institute for Molecular Science (LIMS). Reads were trimmed to remove any adapter and primer sequences, using Trimmomatic v0.32 in the paired-end, palindrome mode, with a minimum length of 200 bp and a minimum quality score of 30 (Bolger, Lohse et al. 2014). Reads were aligned against the W. bancrofti mitochondrial genome (GenBank accession HQ184469), using BWA v0.7.12 (Li and Durbin 2009) with the BWA-MEM algorithm. GATK v3.4_46 was used for local realignment around indels according to GATK Best Practices recommendations (van der Auwera, Arneiro et al. 2013).