Pfs25 PCR amplicon sequencing of Plasmodium falciparum: human blood
DNA was extracted from dried blood spots (DBS) using QIAmp DNA Blood Mini Kit. Pfs25  was PCR amplified using high fidelity polymerase. PCR amplicons were bead-purified (Omega) and quantified by Qubit and adjusted to equivalent concentration. Library prep was performed with Nextera XT using unique dual indexes (UDIs). After library prep, samples were again bead-purified and quantified by qPCR using Roche KAPA Library Quantification Kit. All samples were normalized to a concentration of 4nM. After this normalization, quantified and nornalized libraries were pooled into 8-sub pools. These 8 sub-pools were bead-purified and quantity was measured by KAPA qPCR. The 8 sub-pools were normalized and combined in equal parts to form one final pool. This final pool was sent to the Yale Center for Genome Analysis (YCGA) for sequencing on an Illumina NovaSeq 6000 platform with targeted coverage of 500,000 reads per sample.