Kochakarn, T. | bioRxiv Science

bioRxiv2022

Generation of a mutator parasite to drive resistome discovery in Plasmodium falciparum

In vitro evolution of drug resistance is a powerful approach for identifying antimalarial targets, however key obstacles to eliciting resistance are the parasite inoculum size and mutation rate. Here we sought to increase parasite genetic diversity to potentiate resistance selections by editing catalytic residues of Plasmodium falciparum DNA polymerase {delta}. Mutation accumulation assays revealed a [~]5-8 fold elevation in the mutation rate, with an increase of 13-28 fold in drug-pressured lines. When challenged with KAE609, high-level resistance was obtained more rapidly and at lower inoculum than wild-type parasites. Selections were also successful with an "irresistible" compound, MMV665794 that failed to yield resistance with other strains. Mutations in a previously uncharacterized gene, PF3D7_1359900, which we term quinoxaline resistance protein (QRP1), were validated as causal for resistance to MMV665794 and an analog, MMV007224. The increased genetic repertoire available to this "mutator" parasite can be leveraged to drive P. falciparum resistome discovery.

microbiology↗

bioRxiv2020

Comparing library preparation methods for SARS-CoV-2 multiplex amplicon sequencing on the Illumina MiSeq platform

Genomic surveillance has a key role in tracking the ongoing COVID-19 pandemic, but information on how different sequencing library preparation approaches affect the data produced are lacking. We compared three library preparation methods using both tagmentation (Nextera XT and Nextera Flex) and ligation-based (KAPA HyperPrep) approaches on both positive and negative samples to provide insights into any methodological differences between the methods, and validate their use in SARS-CoV-2 amplicon sequencing. We show that all three library preparation methods allow us to recover near-complete SARS-CoV-2 genomes with identical SNP calls. The Nextera Flex and KAPA library preparation methods gave better coverage than libraries prepared with Nextera XT, which required more reads to call the same number of genomic positions. The KAPA ligation-based approach shows the lowest levels of human contamination, but contaminating reads had no effect on the downstream analysis. We found some examples of library preparation-specific differences in minority variant calling. Overall our data shows that the choice of Illumina library preparation method has minimal effects on consensus base calling and downstream phylogenetic analysis, and suggests that all methods would be suitable for use if specific reagents are difficult to obtain.

genomics↗