Author response table 1. Comparison of previous var assembly approaches based on DNA- and RNA-sequencing.

Study	Assembler	k-mer	Transcript or gene assembly	Validation on reference strain(s) (Yes/No)	Validation on field strain(s) (Yes/No)	Validation across different expression levels (Yes/No)	Read length (Short/Long)	Read correction (Yes/No)	Scaffolding (Yes/No)	Var transcript filter approach	Assumption	Other limitations
Duffy et al., 2016	Oases		Transcript	No	No	No	Short	Yes	Yes	Aligned to 399 var genes with BLAST (e-value< 10–5)		- No quantification of misassemblies - Unable to recover full length transcript assemblies
Dara et al., 2017	Sprai and Celera (no longer maintained)	71	Gene	Yes (strain NF54)	Yes (12 UM patient samples)	NA – only genome assemblies	Long and short	Method assumes combination of long and short-read sequencing will identify errors	No	>500 bp and aligned to VarDom database	Assumes a whole genome assembly is available	- Require prior filtering of human DNA - Need a combination of short-read and long-read sequencing
Tonkin-Hill et al., 2018*	SoapDeNovo-Trans	21, 31, 41, 52 & 61	Transcript	Yes (strain ITG)	No	No	Short	No	No	>500 bp and containing a sig. annotated var domain		- Unable to fully resolve N-terminus - No quantification of misassemblies
Otto et al., 2019	Masurca +post-assembly improvements	Default	Gene	Yes (clone 3D7)	Yes (15 Pf3k reference genomes)	No	Short	Yes	Yes		Whole genome dataset
Andrade et al., 2020	Velvet	41	Transcript	No	No	No	Short	No	No	Aligned to VarDom database		- No quantification of misassemblies
Stucke et al., 2021	rnaSPAdes	Default	Transcript	No	Yes (6 UM patient samples)	No – only the most expressed var gene	Short	Yes	Unclear	>500 bp and containing a sig. annotated var domain	Information about the true var annotation is available	- Performs de novo assembly on all non-human and P. falciparum mapping reads - Inconsistent results in 3 samples when comparing genomic and RNA-seq results for dominant var gene