Comparison of whole-genome amplification methods for next-generation sequencing of individual Onchocerca volvulus microfilariae to obtain better quality nuclear genome
Multiple displacement whole genome amplification (WGA) was performed using 1l of lysate from microfilariae of Onchcoerca volvulus according to the manufacturer's instructions (GenomiPhi v2, Cytiva, Marlborough, Massachusetts, USA): WGA reactions were performed at 30C for 1.5 hrs and heat inactivated at 65C for 10 min. Concentrations of the WGA DNA were determined using a Qubit 2.0 fluorometer (Thermo Fisher Scientific, Waltham, Massachussets, USA). Sequencing libraries were constructed on samples whose DNA concentrations were above 1.0 ng/l, using the Nextera DNA Flex/Illumina DNA prep library construction kits and barcoded using unique dual indexes, according to the manufacturers instructions (Illumina, San Diego, California, USA). An Agilent TapeStation analysis was performed on libraries using D1000 ScreenTapes (Agilent Technologies, Santa Clara, California, USA) to ensure that the libraries were within the selected size range of 400  650bp. The Qubit fluorometer was used to quantify libraries for pooling. The final 10nM pooled library was  run on a NovaSeq SP, 300 cycles.