Validation

Validation

spoligotyper was checked on public reference genomes and on read sets of known species, lineage and spoligotype. Everything below is reproducible with validation/run_validation.sh, which downloads the data, simulates the read sets (BBTools randomreads.sh, fixed seeds) and checks each result.

Result: 45 of 45 checks passed.

Reference genomes

• M. tuberculosis, M. bovis, BCG, M. africanum GM041182: the expected lineages are the predictions of Coll et al. 2014 (Supplementary Table 4) for the same genomes; spoligotyper may report a more specific sublineage on the same branch (EAI5: 1.1.2 within 1.1). Documented spoligotypes: H37Rv 777777477760771 (SIT451), AF2122/97 SB0140, BCG SB0120, and the Beijing signature (spacers 1-34 absent, 35-43 present) for CCDC5079.
• Other members of the complex (M. caprae, M. orygis, M. microti, M. pinnipedii, M. mungi, M. africanum, M. canettii): the expected species is the NCBI taxonomy of the genome, with the RD profile of the RD PCR scheme (see Species and lineage), and the lineage the clade of the barcode (BOV: M. bovis clade; BOV_AFRI: animal lineages and lineage 6). These genomes were not used to choose the RD segments: they are an independent test. The M. africanum RB30001 genome has the spoligotype of GM041182 (AFRI_1, lineage 6); the lineage of RB30065 is not documented.
• The RD profile is shown after the species: RD1, RD4, RD7, RD9 and RD12, + present, - deleted, p partially deleted (see Species and lineage), r present at reduced depth.
• SIT and SITVIT2 family: from the SITVIT2 patterns published with SpolLineages (spoligotyper-download-sit). The documented SITs are checked: H37Rv SIT451, BCG SIT482, and SIT1 for the Beijing strain CCDC5079.

Sample	Organism	SB	SIT (family)	Octal	Species (RD1 RD4 RD7 RD9 RD12)	Lineage	Expected lineage	Result
H37Rv	M. tuberculosis H37Rv	Not in Mbovis.org	SIT451 (T-H37Rv)	777777477760771	M. tuberculosis (+ + + + +)	4.9	4.9	OK
CDC1551	M. tuberculosis CDC1551	Not in Mbovis.org	SIT549 (X3)	700076757760771	M. tuberculosis (+ + + + +)	4.1.1.3	4.1.1.3	OK
Erdman	M. tuberculosis Erdman ATCC 35801	Not in Mbovis.org	SIT1230 (H1)	777757774020771	M. tuberculosis (+ + + + +)	4.1.2.1	4.1.2.1	OK
F11	M. tuberculosis F11	Not in Mbovis.org	SIT33 (LAM3)	776177607760771	M. tuberculosis (+ + + + +)	4.3.2.1	4.3.2.1	OK
KZN1435	M. tuberculosis KZN 1435	Not in Mbovis.org	SIT60 (LAM4)	777777607760731	M. tuberculosis (+ + + + +)	4.3.3	4.3.3	OK
CCDC5079	M. tuberculosis CCDC5079 (Beijing)	Not in Mbovis.org	SIT1 (Beijing)	000000000003771	M. tuberculosis (+ + + + +)	2.2.1	2.2.1	OK
CAS_NITR204	M. tuberculosis CAS/NITR204	Not in Mbovis.org	Not in SITVIT2 list	677777441741771	M. tuberculosis (+ + + + +)	3	3	OK
EAI5_NITR206	M. tuberculosis EAI5/NITR206	Not in Mbovis.org	Not in SITVIT2 list	667777467740071	M. tuberculosis (+ + + + +)	1.1.2	1.1	OK
RGTB423	M. tuberculosis RGTB423	Not in Mbovis.org	Not in SITVIT2 list	777736033740711	M. tuberculosis (+ + + + +)	1.2.2	1.2.2	OK
GM041182	M. africanum GM041182 (lineage 6)	SB0147	SIT181 (AFRI_1)	770777777777671	M. africanum (lineage 6) (+ + - - +)	6	6	OK
AF2122_97	M. bovis AF2122/97	SB0140	SIT683 (BOV_2)	664073777777600	M. bovis (+ - - - -)	BOV	BOV	OK
BCG_Pasteur	M. bovis BCG Pasteur 1173P2	SB0120	SIT482 (BOV_1)	676773777777600	M. bovis BCG (- - - - -)	BOV	BOV	OK
M_canettii	M. canettii CIPT 140010059	SB2277	SIT2669 (ATYPIC)	000000000000000	M. canettii (+ + + + -)	-	-	OK
M_marinum	M. marinum M	SB2277	SIT2669 (ATYPIC)	000000000000000	MTBC not detected (? ? ? ? ?)	-	-	OK
M_kansasii	M. kansasii ATCC 12478	SB2277	SIT2669 (ATYPIC)	000000000000000	MTBC not detected (? ? ? ? ?)	-	-	OK
M_avium	M. avium 104	SB2277	SIT2669 (ATYPIC)	000000000000000	MTBC not detected (? ? ? ? ?)	-	-	OK
M_africanum_RB30001	M. africanum RB30001 (same spoligotype as GM041182, AFRI_1)	SB0147	SIT181 (AFRI_1)	770777777777671	M. africanum (lineage 6) (+ + - - +)	6	6	OK
M_africanum_RB30065	M. africanum RB30065 (lineage not documented)	Not in Mbovis.org	Orphan (AFRI_2)	474077607177071	M. africanum (lineage 5) (+ + p - +)	5	not documented	OK
M_caprae_Allgaeu	M. caprae Allgaeu	SB0418	SIT647 (BOV_4-CAPRAE)	200003777377600	M. orygis or M. caprae (+ + - - -)	BOV	BOV	OK
M_caprae_SY-1	M. caprae SY-1	SB0418	SIT647 (BOV_4-CAPRAE)	200003777377600	M. orygis or M. caprae (+ + - - -)	BOV	BOV	OK
M_orygis_51145	M. orygis 51145	Not in Mbovis.org	Not in SITVIT2 list	600000000000271	M. orygis or M. caprae (+ + - - -)	BOV	BOV	OK
M_orygis_NIAB	M. orygis NIAB_BDWBCSHFL_1	Not in Mbovis.org	Not in SITVIT2 list	600740007774671	M. orygis or M. caprae (+ + - - -)	BOV	BOV	OK
M_microti_OV254	M. microti OV254	SB0118	SIT539 (microti)	000000000000600	M. microti (p + - - +)	BOV_AFRI	BOV_AFRI	OK
M_microti_MausIV	M. microti Maus IV	SB0118	SIT539 (microti)	000000000000600	M. microti (p + - - +)	BOV_AFRI	BOV_AFRI	OK
M_microti_94-2272	M. microti 94-2272	SB0118	SIT539 (microti)	000000000000600	M. microti (p + - - +)	BOV_AFRI	BOV_AFRI	OK
M_pinnipedii_MP1	M. pinnipedii MP1 (draft)	SB0155	SIT593 (PINI1)	074000037777600	M. microti, M. pinnipedii or M. mungi (+ + - - +)	BOV_AFRI	BOV_AFRI	OK
M_pinnipedii_BAA-688	M. pinnipedii ATCC BAA-688 (draft)	Not in Mbovis.org	Not in SITVIT2 list	074000033747400	M. microti, M. pinnipedii or M. mungi (+ + - - +)	BOV_AFRI	BOV_AFRI	OK
M_mungi_BM22813	M. mungi BM22813 (draft)	SB1960	SIT3151 (mungi)	672600000000671	M. microti, M. pinnipedii or M. mungi (p + - - +)	BOV_AFRI	BOV_AFRI	OK
M_canettii_ET1291	M. canettii ET1291	SB2277	SIT2669 (ATYPIC)	000000000000000	M. canettii (+ p + + +)	-	-	OK
M_canettii_CIPT140070010	M. canettii CIPT 140070010	SB2277	SIT2669 (ATYPIC)	000000000000000	M. canettii (+ + + + +)	-	-	OK
M_canettii_CIPT140070017	M. canettii CIPT 140070017	SB2277	SIT2669 (ATYPIC)	000000000000000	M. canettii (+ + + + +)	4	not documented	OK

The non-tuberculous mycobacteria and the M. canettii genomes have none of the 43 standard spacers: their pattern is SB2277, the pattern with no spacer, flagged with a warning (see the FAQ). M. canettii CIPT 140070017 carries the lineage 4 SNP (the H37Rv allele); it is identified as M. canettii from its missing spacers, with a warning that the lineage SNPs are not reliable for M. canettii.

Reads

Public reads (ENA), of strains of known spoligotype, species and lineage, most of them the strains of the reference genomes above:

• ERR1744454: M. bovis AF2122/97, Illumina single-end.
• SRR12006063: M. tuberculosis H37Rv, Illumina HiSeq 4000 paired-end. This lab stock is not quite the reference genome: in most reads, spacer 39 is followed directly by the direct repeat and the sequence that follows spacer 43 in the reference, so most cells lost spacers 40 to 43, and a minority kept spacers 41 to 43 (recombination between direct repeats during passage). Another H37Rv run (ERR15989112) also lacks spacer 40. The resulting pattern, 777777477760731, is SIT1647 of the T-H37Rv family in SITVIT2. spoligotyper flags the minority as a mixed sample.
• ERR027297: M. microti Maus IV, Illumina GAII paired-end (2010, first 1.5 million pairs). With the strong GC bias of these reads, one RD1^mic segment gets a few stray reads and one GC-rich RD1 segment none: M. microti is still recognised from its RD1^mic deletion, which tolerates one error on each side.
• SRR16643349: M. orygis 51145, Illumina MiniSeq paired-end. 8 reads (median of the present spacers: 73) carry a variant of spacer 3 with one SNP, flanked by direct repeats, absent from the PacBio assembly of the strain: a minority population or cross-contamination. Spacer 3 is called present (octal 700000000000271, instead of 600000000000271 for the assembly), and flagged; the octal code is not checked.
• ERR2383628: M. africanum RB30001 (lineage 6), Illumina HiSeq 2500 paired-end (first million pairs).
• SRR18636082, SRR23035463: M. canettii ET1291, Illumina NextSeq paired-end, and nanopore (first 30,000 reads, mean length 4.3 kb): the nanopore reads get the long-read warning.

Simulated reads:

• sim_...: 150 bp reads with sequencing errors, simulated from the reference genomes above at 10x or 30x.
• sim_mixed_H37Rv70_AF2122_30: 70% H37Rv and 30% M. bovis reads.
• sim_contaminated_H37Rv15x_marinum15x: H37Rv and M. marinum reads at the same depth. The M. marinum genome is larger (6.6 Mb), so 40% of the reads are MTBC: the estimated MTBC fraction, 0.40, is right.

Sample	SB	Octal	Species	Lineage	MTBC fraction	Warnings	Result
ERR1744454	SB0140	664073777777600	M. bovis	BOV	1.00	-	OK
SRR12006063	Not in Mbovis.org	777777477760731	M. tuberculosis	4.9	0.69	spacers with few reads, mixed sample	OK
ERR027297	SB0118	000000000000600	M. microti	BOV_AFRI	0.70	-	OK
SRR16643349	Not in Mbovis.org	700000000000271	M. orygis or M. caprae	BOV	0.87	mixed sample	OK
ERR2383628	SB0147	770777777777671	M. africanum (lineage 6)	6	1.00	-	OK
SRR18636082	SB2277	000000000000000	M. canettii	-	0.86	RD4 partially deleted, no spacer (M. canettii)	OK
SRR23035463	SB2277	000000000000000	M. canettii	-	0.95	long reads, RD4 partially deleted, no spacer (M. canettii)	OK
sim_H37Rv_30x	Not in Mbovis.org	777777477760771	M. tuberculosis	4.9	1.00	-	OK
sim_H37Rv_30x_PE	Not in Mbovis.org	777777477760771	M. tuberculosis	4.9	0.97	-	OK
sim_H37Rv_10x	Not in Mbovis.org	777677475760761	M. tuberculosis	4.9	1.00	low depth, spacers with few reads	OK
sim_Beijing_30x	Not in Mbovis.org	000000000003771	M. tuberculosis	2.2.1	1.00	-	OK
sim_AF2122_97_30x	SB0140	664073777777600	M. bovis	BOV	1.00	-	OK
sim_mixed_H37Rv70_AF2122_30	Not in Mbovis.org	777777777767771	MTBC, mixed sample?	mixed: 4 68%, BOV 35%, BOV_AFRI 26%	1.00	mixed sample, spacers with few reads	OK
sim_contaminated_H37Rv15x_marinum15x	Not in Mbovis.org	777777477760771	M. tuberculosis	4.9	0.40	contamination, low MTBC depth	OK

• At 10x, three present spacers get fewer than 5 reads and are called absent: the pattern is wrong, but flagged ("low depth", "spacers with few reads"). Use --min-count 2 or 3 for low depth data, see How it works.
• The mixed sample is detected from both alleles of the lineage SNPs; its spoligotype is the union of the two strains' patterns.
• The contaminated sample keeps its spoligotype, species and lineage, with a contamination warning.

Comparison with SpoTyping

SpoTyping 2.1 (Xia et al. 2016, BLAST-based), an independent in silico spoligotyping tool, was run on the 13 original MTBC genomes (--seq): the spoligotypes are identical for 12 of 13 genomes.

Sample	spoligotyper	SpoTyping	Difference
H37Rv	777777477760771	777777477760771	identical
CDC1551	700076757760771	700076757760771	identical
Erdman	777757774020771	777757774020771	identical
F11	776177607760771	776177607760771	identical
KZN1435	777777607760731	777777607760731	identical
CCDC5079	000000000003771	000000000003771	identical
CAS_NITR204	677777441741771	677777441741771	identical
EAI5_NITR206	667777467740071	667777477740671	spacers 24, 37, 38
RGTB423	777736033740711	777736033740711	identical
GM041182	770777777777671	770777777777671	identical
AF2122_97	664073777777600	664073777777600	identical
BCG_Pasteur	676773777777600	676773777777600	identical
M_canettii	000000000000000	000000000000000	identical

SpoTyping's EAI5/NITR206 pattern has spacers 24, 37 and 38 present. In this assembly, the closest sequences to these spacers have 6, 4 and 5 mismatches out of 25 bp, so spoligotyper, which allows 1 mismatch, calls them absent.