Algorithm 1. The referenceNof1 algorithm pseudocode to construct an optimized and unbiased reference standard.
	Variable	Description
Inputs	FC	A list of fold change thresholds
	Cutoffs	A list of expression thresholds
	Target	A minimum median Jaccard index to attain
Steps	For genes in region $R_i$ using Cutoffi and FCi, do
	For method $\in \left\{edgeR, DEGseq, DESeq2, NOISeq\right\}$ do
	Identify set of differentially expressed genes
	End for
	Calculate pairwise Jaccard Index (JI) for all (M,N) pairs of methods:
	$J{I}_{\left(M,N\right),R_i}=\frac{\left|M\cup N\right|}{\left|M\cap N\right|}$
	Calculate median Jaccard Index
	$J{I}_{R_{i,Med}}= median\left(J{I}_{\left(M,N\right),R_i}\right)$
	If ($J{I}_{R_{i,Med}}\ge$ Target)
	Return Cutoff* = Cutoffi, FC* = FCi
	Else
	Update FC, Cutoff parameters
	End for
	If (Target attained)
	Return Cutoff*, FC*
	Else
	Print No threshold achieved target Jaccard Index
The referenceNof1 algorithm requires a user to input the FC and expression cutoff filters for it to then identify the optimal region for producing the reference standard. For each pair of FC-region and expression cutoff combination, it calculates each method’s list of differentially expressed genes (DEGs), and then for each DEG list it calculates the Jaccard Index as a set-theoretic pairwise similarity measure. After calculating all pairwise Jaccard indices, it calculates the median for each region. If a parameter combination attains the desired median Jaccard Index, an early stopping rule is implemented, and the optimal parameter combination is returned. Otherwise, it continues the search until the target Jaccard Index is attained or the search through the parameter space is complete. (Legend: In the manuscript and figure, we represent FC for upregulated genes and 1/FC for downregulated genes.”)