Genomic Study of Plasmodium falciparum in Sierra Leone

Blood samples were collected by the 23rd batch of China Medical Team from clinical malaria patients at the Sierra Leone China Friendship Hospital Freetown between December 2022 and March 2023. Approval for this study was obtained from the Ethics Committee of the Chinese Center for Disease Control and Prevention. All participants were fully informed about the study's protocol, potential risks, and benefits, and they provided written informed consent. Blood samples were collected from patients who tested RDT and microscopically positive for P. falciparum infection, which was further confirmed through PCR as single-species infections. To ensure high-quality genomic data, only samples with a high parasite density were selected. DNA was extracted from frozen blood samples using the QIAGEN DNeasy Blood & Tissue Kit. Per the requirement of the Sierra Leone Ministry of Health, the first batch of 50 samples was sequenced locally at the Sierra Leone-China Biological Laboratory, which housed a MGISEQ-200 sequencer donated by BGI. Due to the lack of automated equipment, sequencing libraries were manually constructed. DNA was fragmented using enzymatic digestion to control fragment size, with the MGIEasy Universal DNA Library Prep Kit used for this process. After end-repair, A-tailing, and adapter ligation, DNA fragments of approximately 430 bp were selected. Circularized libraries were prepared using the MGISEQ-200RS sequencing kit, and DNA nanoballs were loaded for sequencing. The sequencing process on the MGISEQ-200 machine generated an average of 39.6 million paired-end reads per sample, each measuring 150 bp in length. Adapter sequences and low-quality reads were removed using Trimmomatic, and reads were mapped to the 3D7 reference genome using BWA. Variant calling followed the GATK4 workflow, generating 165,000 high-quality SNPs by filtering out those with over 5% missing calls. Given the requirement for 90% genome coverage, only 19 samples from the first sequencing round met the criteria. Following approval from the Ministry of Health, the remaining samples were sent to China for sequencing on the Illumina platform. DNA was fragmented into 500 bp segments using the Covaris S2 system, and libraries were prepared and sequenced on an Illumina X-10 platform, generating an average of 136 million reads. In the second round 16 samples met the desired sequencing coverage threshold.