Table 1.

Comparison of BISCUIT, Bismark, BSBolt, bwa-meth and gemBS. Tools in parentheses are third party tools used to perform the stated functionality

	BISCUIT	Bismark	BSBolt	bwa-meth	gemBS
General
Language	C	Perl	Python/C	Python/C	Rust/C
Multi-threaded	Yes	Yes	Yes	Yes	Yes
Availability	GitHub, Bioconda, Docker	GitHub, Bioconda	GitHub, Bioconda, Pip	GitHub, Bioconda	GitHub, Bioconda, Docker
Supported Libraries (WGBS, EM-seq, RRBS, PBAT, NOME-seq)
Mode	Directional, Non-Directional	Directional, Non-Directional	Directional, Non-Directional	Directional	Directional, Non-Directional
Index / Algorithm
Reference	4-base	3-base	3-base	3-base	3-base
Seed Creation	3-base	3-base	3-base	3-base	3-base
Asymmetric Scoring	Yes	No	No	No	No
Alignment Algorithm	BWA-MEM-based	Bowtie2 / HiSAT2	BWA-MEM-based	BWA-MEM	GEM3
Global or Local Alignment?	Local	Global	Local	Local	Both
Functionality
Handle Spike-ins	Yes	Yes	Yes	Yes	Yes
UMI Support	Yes	Yes	No	No	No
Handle Cell Barcodes	Yes	Yes	No	No	No
5′/3′ Trimming	Yes	(TrimGalore!)	(TrimGalore!)	(TrimGalore!)	BScall
Duplicate Marking	(dupsifter)	Yes	(samtools)	(Picard)	BScall
Collapse Overlapping PE Reads	Yes	Yes	Yes	No	BScall
Variant Calling	Yes	No	Yes	(BISCUIT)	Yes
Methylation Extraction	Yes	Yes	Yes	(MethylDackel)	Yes
Accessibility Extraction	Yes	Yes	No	No	No
Companion R Tool	Yes	Yes	No	No	No
Visualize WGBS BAM	Yes	No	No	No	No
Input / Output
FASTA / FASTQ	Yes	Yes	Yes	Yes	Yes
Standard Input	Yes	No	No	Yes	Yes
Write Directly to SAM/BAM	No	Yes	Yes	No	Yes
Write directly to Standard Output	Yes	No	Yes	Yes	No
CpG and CpH	Yes	Yes	Yes	No	Yes
Quality Control
MultiQC Support?	Yes	Yes	No	No	No
Non-CpG Stats	CpA / CpC / CpT	CpH	CpH	No	No
M-Bias Plot	CpG / CpH	CpG / CpH	No	No	CpH