Single cell sequencing of the small and AT-skewed genome of malaria parasites

In this study, we developed a single cell sequencing pipeline for Plasmodium falciparum parasites, which included efficient isolation of single infected erythrocytes, an optimized WGA method inspired by MALBAC, and a sensitive method of assessing sample quality prior to sequencing. We tested our pipeline on erythrocytes infected with laboratory-reared parasites as well as patient-isolated parasites with heavy human genome contamination. We sequenced single-cell laboratory samples amplified by optimized MALBAC (13 early-stage parasite samples and 10 late-stage parasite samples), non-optimized MALBAC (1 early-stage parasite sample and 1 late-stage parasite sample), 2 patient-isolated parasite samples amplified by optimized MALBAC, alongside bulk samples (1 Dd2 bulk DNA sample and 1 clinical bulk DNA sample). Genome amplification using our optimized protocol showed increased genome coverage, better coverage uniformity, and strong amplification reproducibility. These improvements will enable the detection of parasite-to-parasite heterogeneity to clarify the role of genetic variations, such as CNVs, in the adaptation of P. falciparum. This study also provides a framework for the optimization of single cell amplification in other organisms with challenging genomes.