Table 1. Summary of whole genome sequencing pipeline settings applied for each SNP pipeline.

Settings	RIVM SNP	Oxford University SNP	Research Center Borstel (MTBseq) SNP	SSI SNP
H37Rv reference genome version	3	2	3	3
Alignment software	Bowtie	Stampy	BWA	BWA
SNP calling software	Breseq	Samtools	Samtools	Samtools
Minimum mean sample coverage depth	≥ 20x	NA	≥ 30x	≥ 20x
Minimum sample coverage breadth	NA	> 88%	≥ 80% fulfilling thresholds for variant detection	≥ 95%
Genomic regions excluded	Repeats	Repeats	Repeats, resistance genes	Repeats
Minimum coverage depth to support a SNP	NA	5x (one forward, one reverse, < 10% alternative allele)	8x (four forward, four reverse, four with phred score ≥ 20)	8x (four forward, four reverse)
Excluding SNPs within 12bp	Yes	No	Yes	Yes
Allele frequency	≥ 80%	≥ 90%	≥ 75%	≥ 85%
Dealing with low coverage positions or positions not meeting variant call criteria when calculating the genetic distance	Report reference base	Report consensus base	Report consensus base or exclude position if data quality is below thresholds in >5% of samples	Complement with data from aligned reads if coverage is > 5x or exclude position if data quality is below threshold

BWA: Burrows-Wheeler Alignment; MTB: Mycobacterium tuberculosis; NA: not applicable; RIVM: National Institute for Public Health and the Environment; SNP: single nucleotide polymorphism; SSI: Statens Serum Institut.