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

Shiwei Liu,Jennifer L Güler,Jennifer L Güler,Michael J Mcconnell,Michael J Mcconnell,Adam C Huckaby,Ian Burbulis,Ian Burbulis,Audrey C Brown,Christopher C Moore

doi:10.1186/s13073-021-00889-9

Shiwei Liu, Jennifer L Güler + Show 8 more

Open Access

https://doi.org/10.1186/s13073-021-00889-9

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Abstract

Single-cell genomics is a rapidly advancing field; however, most techniques are designed for mammalian cells. We present a single-cell sequencing pipeline for an intracellular parasite, Plasmodium falciparum, with a small genome of extreme base content. Through optimization of a quasi-linear amplification method, we target the parasite genome over contaminants and generate coverage levels allowing detection of minor genetic variants. This work, as well as efforts that build on these findings, will enable detection of parasite heterogeneity contributing to P. falciparum adaptation. Furthermore, this study provides a framework for optimizing single-cell amplification and variant analysis in challenging genomes.

Highlights

Malaria is a life-threatening disease caused by protozoan Plasmodium parasites
Plasmodium falciparum genomes from single-infected erythrocytes are amplified by Multiple annealing and looping-based amplification cycles (MALBAC) Our single-cell sequencing pipeline for P. falciparum parasites included stage-specific parasite enrichment, isolation of single infected erythrocytes, cell lysis, whole genome amplification, pre-sequencing quality control, whole genome sequencing, and analysis steps (Fig. 1a)
We applied a version of MALBAC that we optimized for the small AT-rich P. falciparum genome to 42 early- (EOM) and 20 late-stage (LOM) laboratory Dd2 parasite samples as well as 4 clinical samples (COM) (Additional file 2: Table S8)

Summary

Introduction

Malaria is a life-threatening disease caused by protozoan Plasmodium parasites. P. falciparum causes the greatest number of human malaria deaths [1]. The clinical symptoms of malaria occur when parasites invade human erythrocytes and undergo rounds of asexual reproduction by maturing from early-stage into late-stage forms and bursting from erythrocytes to begin the cycle again [2]. Drug efficacy is mitigated by the frequent emergence of resistant populations [3] Both single-nucleotide polymorphisms (SNPs) and copy number variations (CNVs; the amplification or Recent studies have begun to overcome these limitations for SNP analysis; methods including leukocyte depletion [13], selective whole-genome amplification (WGA) of parasite DNA [14], hybrid selection with RNA baits [15], and single-cell sequencing of P. falciparum parasites [16, 17] help enrich parasite DNA, determine genetic diversity, and understand the accumulation of SNPs in long-term culture. The study of genetic diversity in early stage parasites on a single-cell level remains challenging [16]; the lack of alternative single-cell approaches for P. falciparum parasites impedes the validation of SNP results by parallel investigations [18]

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