Table 4. Summary of the different levels of analysis performed to assess the effect of short-term parasite culturing on var and core gene expression, their rational, method, results, and interpretation.

Analysis level	Analysis	Rationale	Method	Results	Interpretation
var transcript	Per patient expression ranking	Relative quantification of var transcripts over consecutive generations of parasites originating from the same patient to reveal var gene switching events	Combine assembled var transcripts for each patient into a reference and quantify expression, validated with DBLα-tag analysis	46% of the patient samples had a change in the dominant or top 3 highest expressed var gene	Modest changes in most samples, but unpredictable var gene switching during culture in some samples
	Per patient var expression homogeneity (VEH)	Determine the overall diversity of var gene expression (number of different variants expressed and their abundance) to assess impact of culturing on the overall var gene expression pattern	Comparison of diversity curves based on per patient quantification of the var transcriptome	39% of ex vivo samples diversity curves distinct from in vitro samples	Some patient samples underwent a much greater var transcriptional change compared to others
	Conserved var variants	Assessing and comparing the expression levels of strain-transcendent var gene variants (var1, var2csa, var3) between samples	Reference of all assembled transcripts for each conserved var gene and quantify expression	var2csa expression increases in second in vitro generation	Parasites converge to var2csa during short-term in vitro culture
var-encoded PfEMP1 domains	Differential expression of PfEMP1 domains	Identification, quantification, and comparison of expression levels of different var gene-encoded PfEMP1 domains associated with different disease manifestations	Pool all assembled var transcripts into a reference and quantify expression of each domain	46% of the ex vivo samples cluster away from their in vitro samples in PCA plots, distinct clustering by in vitro generation was not observed; CIDRα2.5 significantly differentially expressed between ex vivo and generation 1	Transition to culture results in modest modulation of particular var domains
var group	Expression of NTS (NTSA vs NTSB) and DBLα (DBLα1 vs DBLα0+DBLα2)	Quantification and comparison of expression levels of different var gene groups (group A vs. groups B and C)	Create a reference of all assembled DBLα and NTS domains for each patient and quantify expression. Validated with DBLα-tag analysis	No significant changes	No preferential up- or downregulation of certain var groups during transition to culture
Global var expression	LARSFADIG coverage	Assessing the overall var gene expression level (excluding var2csa)	Assemble the LARSFADIG motif and map non-core reads to quantify coverage	Trend for decrease in global var expression during culture, but no significant changes	Subtle reduction in global var gene expression may reflect increase in parasite age during culture
Core genes	Differential gene expression (DGE)	Assessing the impact of cultivation on the parasite core gene transcriptome	Differential expression analysis of core genes (P. falciparum 3D7 used as reference)	19% of the core transcriptome significantly differentially expressed between paired ex vivo and generation 1 in vitro samples; distinct clustering by parasite generation observed; upregulation of invasion and replication-related genes in vitro	Parasites core gene expression changes substantially upon entering culture