Applied Microbiology and Biotechnology A multiplex oligonucleotide ligation-PCR as a complementary tool for subtyping of Salmonella Typhimurium


0 0 NT 15 1 2-1-1 R D N C 3 -12-12-NA-211 S0059 1,4,[5],12:i:-Human 0 0 0 A1 15 1 2-1-1 R D N C 3 -13-

Fig. S1

 S1 Fig. S1 PFGE results of 53 isolates of the 3 most observed MOL-PCR profiles in the validation panel of 519 S. Typhimurium and S. 1,4,[5],12:i:-isolates. A horizontal line between two isolates indicates that these isolates were run on different gels (4 gels in total) 


Table S1

 S1 Primer pairs used in the development of the MOL-PCR subtyping assay 


	Target	Targeted	Primer	Sequence (5'→3') a	Annealing	Concn	Amplicon	Reference
		sequence			temp (°C)	(nM)	size (bp)	
	ST64B	immC cl-5'	SAL-1-F	ATGGTGGCCTTGTCGACGC	58	400	475	(Ross and
								Heuzenroeder 2005)
			SAL-1-R	GCTAACGTGAAGGATTTGTTCCG		400		(Ross and
								Heuzenroeder 2005)
	ST64B	immC cl-3'	SAL-2-F	CCATTACCGGCGCTTGCAC	58	400	453	(Ross and
								Heuzenroeder 2005)
			SAL-2-R	TAACGTATAACCATGCGATTTCCG		400		(Ross and
								Heuzenroeder 2005)
	ST64B	immC cro	SAL-3-F	GCGATATACGCAAAAGAAGGAGG	58	400	518	(Ross and
								Heuzenroeder 2005)
			SAL-3-R	TGGCTACTGAATGTGCCAGG		400		(Ross and
								Heuzenroeder 2005)
	ST64B	immC put c2	SAL-4-F	GCTGGTACTGCAACGTGCC	58	400	568	(Ross and
								Heuzenroeder 2005)
			SAL-4-R	CGAATGACATGGACATAAAGTCC		400		(Ross and
								Heuzenroeder 2005)
	ST64B	SB6	SAL-5-F	ACGACAAGCGCGTTGAGGC	58	400	280	(Fang et al. 2012)
			SAL-5-R	ACGGCATAACCACCATCTTCATC		400		(Fang et al. 2012)
	ST64B	SB26	SAL-6-F	GACACCATCAATGTATGGATCGC	58	400	276	(Fang et al. 2012)
			SAL-6-R	GGAAGTAATGCTTTTCTGGAGGC		400		(Fang et al. 2012)
	ST64B	SB28	SAL-7-F	CGTGGAAAGTGCAGTCAAGAGG	58	400	211	(Fang et al. 2012)
			SAL-7-R	CAAGGAAAAACCGGAGTTAAACAA		400		(Fang et al. 2012)
	ST64B	SB37	SAL-8-F	TGGTAGTGAATTGGTTAGCTGCG	58	400	279	(Fang et al. 2012)
			SAL-8-R	GCAATAAAGGCAAGGTAGTCCTCA		400		(Fang et al. 2012)
	ST64B	SB46	SAL-9-F	CATTGATGGTATCGAAGTTCGCC	58	400	490	(Ross and
								Heuzenroeder 2005)
			SAL-9-R	CCTGGAGTTTCTGGCACGC		400		(Ross and
	ST64T/P22	g9-5'	SAL-10-F b	GATTCCTTGCATCTGGAGCAC	58	400	217	Heuzenroeder 2005) (Fang et al. 2012)
			SAL-10-R SAL-10-F-nested c GTTGTTTCTAACCCTCGTCC GGCTGAGCAATCTGGACGTG		400 400	171	(Fang et al. 2012) This study
	ST64T/P22	g9-3'	SAL-11-F	TACCGTAGAAGATTGCGCTGG	58	400	276	(Fang et al. 2012)
			SAL-11-R	AACCCTTCTTCTGCCAAATTAGC		400		(Fang et al. 2012)
	ST64T/P22	g17	SAL-12-F	AGCGTTGCAGTAATCAGGTTTG	58	400	163	(Fang et al. 2012)
			SAL-12-R	AGGAAATATGAAATTACGTGTCTGG		400		(Fang et al. 2012)
	ST64T/P22	gtrA	SAL-13-F	TTGCCGAGAAAGTGATAAGGGTAA	58	400	179	(Fang et al. 2012)
			SAL-13-R	TGTTATCGCCGTATCGTTCAGC		400		(Fang et al. 2012)



  Used for initial PCR screening of the marker c Used for sequencing of PCR amplicons d Amplicon length depends on number of repeats (x) present in the locus: amplicon length = 311 + (6 × x) 


	Table S1	(continued)						
	Target		Targeted	Primer	Sequence (5'→3') a	Annealing	Concn	Amplicon	Reference
			sequence			temp (°C)	(nM)	size (bp)
	SNP TM1231	STM1269	SAL-59-F	TGAAGAAGAAGCGAAAAA	55	200	808	(Hu et al. 2006)
				SAL-59-R	CCAAGGTAGAGCAGATAA		200		(Hu et al. 2006)
	SNP TM973		STM1002	SAL-60-F	ATTTCCGCTGATGACTGT	55	200	732	(Hu et al. 2006)
				SAL-60-R	GCCACCTCTATTGATTTTA		200		(Hu et al. 2006)
	SNP 1880		otsA	SAL-61-F	TCTCCGCATGACGCGAAATA	60	200	306	This study
				SAL-61-R	GCTGGACGCCGCTCTATTAT		200		This study
	SNP 2199		napF	SAL-62-F	CCGGATAACGTGGGACGAAA	60	200	208	This study
				SAL-62-R	GCGTAGATTTCAAACGGGGC		200		This study
	SNP 101		araD	SAL-63-F	GTCATTTTGCGAGTGCAGGG	60	200	215	This study
				SAL-63-R	GAGAGCGGTGAAGTCGTTGA		200		This study
	SNP TM3124_1	intergenic	SAL-64-F	GAGGCGTAAAGCGACAG	55	200	776	(Hu et al. 2006)
				SAL-64-R	AATCCAGCGTAGGGTAG		200		(Hu et al. 2006)
	SNP TM3275_2	intergenic	SAL-65-F	TTGAAGAGGCGTGTTGG	55	200	590	(Hu et al. 2006)
				SAL-65-R	GCTGAAAGGCGAAGAAT		200		(Hu et al. 2006)
	Streptomycin/	aadA2	SAL-66-F	TGTTGGTTACTGTGGCCG	55	500	538	(Ng et al. 1999)
	spectinomycin resistance		SAL-66-R	TGCTTAGCTTCAAGTAAGACG		500		(Boyd et al. 2002)
	SNP 4213		yjeF	SAL-71-F	AGCCGGTCTGATTGAACAGG	60	200	616	This study
				SAL-71-R	ATCCGCATCCCACAGCATAG		200		This study
	SNP TM2079	intergenic	SAL-72-F	TCAGAAGGAGGGAGCAA	55	200	701	(Hu et al. 2006)
	MLVA locus STTR10	PSLT064	SAL-72-R SAL-74-F	GCGAAGTAGAGTAATGG CGGGCGCGGCTGGAGTATTTG	60	200 200	d	(Hu et al. 2006) (Lindstedt et al. 2004)
				SAL-74-R	GAAGGGGCCGGGCAGAGACAGC		200		(Lindstedt et al. 2004)

a W: A or T b 
Table S2

 S2 Probes used in MOL-PCR_1 


	Target	Targeted	Probe	Probe	Sequence (5'→3')	Bead	Accession	Reference of target-
		sequence		pre-		region	no.	specific sequence
				mix				
	All Salmonella	invA	invA-U	1.1	TAATACGACTCACTATAGGGgataagaaagtgaaatgtaaattgATAAACTT	51	AE006468	(Barbau-Piednoir et al.
	species				CATCGCACCGTCA		(McClelland	2013)
			invA-D	1.1	P-AAGGAACCGTAAAGCTGGCTTTCCCTTTAGTGAGGGTTAAT		et al. 2001)	
	Salmonella	rpoB	rpoB-U	1.2	TAATACGACTCACTATAGGGgtaattgaattgaaagataagtgtTTTCTCAG	18	AE006468	(Hernández Guijarro et
	enterica subsp.				CTGCACCGTAGC		(McClelland	al. 2012)
	enterica serovar		rpoB-D	1.2	P-CCTGGCGTCTTCTTTGACTCCTCCCTTTAGTGAGGGTTAAT		et al. 2001)	
	Typhimurium							
	ST64T/P22	g9-5'	SAL-10-U	-	TAATACGACTCACTATAGGGgtgtgttatttgtttgtaaagtatGGCTGAGCA	19	AY052766	(Fang et al. 2012)
					ATCTGGACGTG		(Mmolawa et	
			SAL-10-D	-	P-AGAGCCATCCTCATTTTCAATCCCTTTAGTGAGGGTTAAT		al. 2003)	
	ST64T/P22	g9-3'	SAL-11-U	1.2	TAATACGACTCACTATAGGGgtaagagtattgaaattagtaagaAACCCTTC	66	AY052766	(Fang et al. 2012)
					TTCTGCCAAATTAGC		(Mmolawa et	
			SAL-11-D	1.2	P-AACATTAATTCTAGAGGGGTCTACCTCCCTTTAGTGAGG		al. 2003)	
					GTTAAT			
	ST64T	mnt	SAL-16-U	1.2	TAATACGACTCACTATAGGGtgtatatgttaatgagatgttgtaTACAGGCGT	37	AY052766	(Fang et al. 2012)
					AGGCTTTTCAAGTG		(Mmolawa et	
			SAL-16-D	1.2	P-AATCAGCCACTATCTGCACTTCCCTTTAGTGAGGGTTAAT		al. 2003)	
	ST64T/P22	g8	SAL-23-U	1.2	TAATACGACTCACTATAGGGagtagaaagttgaaattgattatgTGCCAACG	12	NC_002371	(Fang et al. 2012)
					ACAATGCAGGTC		(Pedulla et	
			SAL-23-D	1.2	P-AGGAAGAGGGCTTTGAGATTGTCCCTTTAGTGAGGGTTAAT		al. 2003)	
	ST104	g9	SAL-25-U	-	TAATACGACTCACTATAGGGgaagatattgaaagaatttgatgtCGAAGTTG	55	AB102868	(Drahovská et al. 2007)
					GTCTGCACAATG		(Tanaka et	
			SAL-25-D	-	P-CAGCAGGGCGTCCGTCGCTTCTCCCTTTAGTGAGGGTTAAT		al. 2004)	
	Gifsy-1	gipA	SAL-29-U	1.1	TAATACGACTCACTATAGGGtttaagtgagttatagaagtagtaGGCAAGCT	29	AF246666	(Mikasová et al. 2005)
					GTACATGGCAAAG		(Stanley et	
			SAL-29-D	1.1	P-AACAAAATCCCCTTAGACGTCCCTTTAGTGAGGGTTAAT		al. 2000)	
	AFLP fragments	CA-1	SAL-33-U	-	TAATACGACTCACTATAGGGgttgagaattagaatttgataaagTTAACGAT	73	AF500153	(Fang et al. 2012)
					CAACGAAATTCAATCC		(Hu et al.	
			SAL-33-D	-	P-TAACCACAGCCGCTGAAAAGGTTCCCTTTAGTGAGGGTT		2002)	
					AAT			
	AFLP fragments	GA-1	SAL-45-U	1.2	TAATACGACTCACTATAGGGgttagttatgatgaatattgtgtaGCGTGGAAT	45	AF500166	(Lan et al. 2007)
					ATCGTTGATGG		(Hu et al.	
			SAL-45-D	1.2	P-GTTTGAAGCGGTGTCGAATCCCTTTAGTGAGGGTTAAT		2002)	



  T7 and T3), anti-TAG, target-specific sequences and SNP positions are indicated by, respectively, italic, lower-case, underlined and boldface sequences. P: phosphate 


	Table S3 Target Salmonella enterica subsp. Probes used in MOL-PCR_2 Targeted sequence Probe enterica serovar Typhimurium rpoB rpoB-U rpoB-D ST64T/P22 g17 SAL-12-U SAL-12-D ST64T/P22 gtrA/gtrB SAL-14-U Table S3 (continued) Target Targeted sequence Probe AFLP fragments CA-3 SAL-35-U SAL-35-D AFLP fragments CA-7 SAL-36-U SAL-36-D AFLP fragments CA26.1 SAL-37-U SAL-37-D Table S3 (continued) Target Targeted sequence Probe Streptomycin/ spectinomycin resistance aadA2 SAL-66-U SAL-66-D Phase 2 flagellar gene fljB SAL-73-U SAL-73-D Probes used in MOL-PCR_SNP SNP Targeted sequence Probe Probe Probe pre-mix 2.2 2.2 --2.1 Probe pre-mix 2.1 2.1 2.2 2.2 2.2 2.2 Probe pre-mix ----pre-mix TM81 STM0080 SAL-56-U_SNP S.1 SAL-56-U_WT S.2 SAL-56-D S.1 TM3230_1 intergenic SAL-58-U_SNP -SAL-58-U_WT S.3 Primer (Table S4	Sequence (5'→3') TAATACGACTCACTATAGGGgtaattgaattgaaagataagtgtTTTCTCAG CTGCACCGTAGC P-CCTGGCGTCTTCTTTGACTCCTCCCTTTAGTGAGGGTTAAT TAATACGACTCACTATAGGGgtaagattagaagttaatgaagaaGCGTTGC AGTAATCAGGTTTG P-ATGTTGTTGTCATACTGAAATGCTCCCTTTAGTGAGGGTT AAT TAATACGACTCACTATAGGGatttgttatgataaatgtgtagtgCTTTCTCGG Sequence (5'→3') TAATACGACTCACTATAGGGaataagagaattgatatgaagatgAATGGCT GGCAGGGTCTGTTC P-GAACCTGACGGACAGGCGTCCCTTTAGTGAGGGTTAAT TAATACGACTCACTATAGGGattgtgaaagaaagagaagaaattCATCGGG GTGATCAGCA P-TGATTACCCTGTTTGCCCGTCCCTTTAGTGAGGGTTAAT TAATACGACTCACTATAGGgtatagtgtgattagatttgtaaaGCGGCGCGT ATTTCGTGC P-ATCGCATTGTCATTACCAGCATCCCTTTAGTGAGGGTTAAT Sequence (5'→3') TAATACGACTCACTATAGGGttgtgtagttaagagttgtttaatGGTATCTTC GAGCCAGCCAT P-GATCGACATTGATCTAGCTATCCCTTTAGTGAGGGTTAAT TAATACGACTCACTATAGGGtgaaatgtgtatttgtatgtttagCCAGCCGCA AGGGTTACTGTAC P-CGTCAGTAGCAACGTTAACTTCATAATCCCTTTAGTGAG GGTTAAT Sequence (5'→3') TAATACGACTCACTATAGGGaattagaagtaagtagagtttaagGGTCGATAAC GATACCGGTATGA TAATACGACTCACTATAGGGtttgatttaagagtgttgaatgtaGGTCGATAACG ATACCGGTATGG P-TGTCTTACAAAAACGCCGCCTCCCTTTAGTGAGGGTTAAT TAATACGACTCACTATAGGGttgtgatagtagttagatatttgtGGCTTTGACGAA GACTAAACCC TAATACGACTCACTATAGGgtgtgttatttgtttgtaaagtatGGCTTTGACGAAG ACTAAACCT	Bead region 18 52 42 Bead region 35 14 78 Bead region 36 62 Bead region	Accession no. AE006468 (McClelland et al. 2001) AY052766 (Mmolawa et al. 2003) AY052766 Accession no. AF500155 (Hu et al. 2002) AF500159 (Hu et al. 2002) DQ835569 (Lan et al. 2007) Accession no. AF261825 (Boyd et al. 2001) AE006468 (McClelland et al. 2001) Accession no. AE006468 (McClelland et al. 2001) AE006468 (McClelland et al. 2001)	Reference of target-specific sequence (Hernández Guijarro et al. 2012) (Fang et al. 2012) (Ross and Heuzenroeder Reference of target-specific sequence (Fang et al. 2012) (Fang et al. 2012; Lan et al. 2007) (Lan et al. 2007) Reference of target-specific sequence This study (Muñoz et al. 2010) Reference of SNP (Hu et al. 2006) (Hu et al. 2006)
	AFLP fragments	CA28.4 SAL-58-D SAL-38-U	S.3		2.2	CAATTAGCCTGGTATG TAATACGACTCACTATAGGGattaagtaagaattgagagtttgaTGATGCAC P-CCCCCTCTATACCCTATTTCTCCCTTTAGTGAGGGTTAAT	21	(Mmolawa et DQ835568	2005) (Lan et al. 2007)
	TM1231	SAL-14-D STM1269 SAL-59-U_SNP S.1	2.1	P-CGGCTTTATCTATTCCAGATTCCCTTTAGTGAGGGTTAAT TAATTCGTCG TAATACGACTCACTATAGGGtttgttgttaagtatgtgatttagCCAGCGCTTGAT		al. 2003) (Lan et al. AE006468	(Hu et al. 2006)
	ST64T/P22		gtrC	SAL-15-U SAL-38-D			2.1 2.2	TAATACGACTCACTATAGGGagagtattagtagttattgtaagtGCATTAAC P-CGAGGCTGCTGGATATATTCTCCCTTTAGTGAGGGTTAAT GGTCGGCT	57	AY052766 2007) (McClelland	(Ross and Heuzenroeder
	AFLP fragments	CG-1	SAL-39-U SAL-59-U_WT S.2		2.2	ACCTCTGACCACATC TAATACGACTCACTATAGGGaaattagttgaaagtatgagaaagCAGGGAA TAATACGACTCACTATAGGGtattagagtttgagaataagtagtCCAGCGCTTGA	20	(Mmolawa et AF500162 et al. 2001)	2005) (Lan et al. 2007)
				SAL-15-D			2.1	P-CAATTATTGTTAATAATGCGTGGTCCCTTTAGTGAGGGTT CCGTCTTGAG TGGTCGGCG		al. 2003) (Hu et al.
				SAL-39-D SAL-59-D	S.1		2.2	AAT P-CAAGTTCAGAGCGCAATGACTCCCTTTAGTGAGGGTTAAT P-CTTTCAGCCACGACGATATGGTGTCCCTTTAGTGAGGGTTAAT		2002)
	ST64T/P22 AFLP fragments TM973 STM1002 SAL-60-U_SNP S.1 int SAL-18-U CG-2 SAL-40-U	--	TAATACGACTCACTATAGGGaatgaaatagtgttaaatgagtgtCGGCAATC TAATACGACTCACTATAGGGtattagagagaaattgtagagattCGCTATAT TAATACGACTCACTATAGGGatgatgtgtttgatttgaattgaaGCTCATGTTCTG	74 61	NC_002371 AF500163 AE006468	(Fang et al. 2012) (Lan et al. 2007) (Hu et al. 2006)
								ATCACAAATGG CACGCTGAACAGAC CTTATTAGCACCGAAGGT		(Pedulla et (Hu et al. (McClelland
				SAL-18-D SAL-40-D SAL-60-U_WT S.2	--	P-GTGTTCGTCTACAAGGAAAGCTCCCTTTAGTGAGGGTTAAT P-CAGAACTAGGTGAAACAAAAGAGGTCCCTTTAGTGAGG TAATACGACTCACTATAGGGattgtgaaagaaagagaagaaattCTGCTTATTA		al. 2003) 2002) et al. 2001)
	P22		sieB	SAL-20-U			2.1	TAATACGACTCACTATAGGGtttgttagaatgagaagatttatgACAACTCA GTTAAT GCACCGAAGGC	75	NC_002371	(Ross and Heuzenroeder
	AFLP fragments	CT-1	SAL-42-U SAL-60-D	S.1		-	TGGTGGCAGGAG TAATACGACTCACTATAGGGtattgttgaatgtgtttaaagagaCATCTGCTG P-GATACGTCCCCAGATATTTATGAAGATATAGTCCCTTTAGT	47	(Pedulla et AF500164	2005) (Lan et al. 2007)
				SAL-20-D			2.1	P-CTAATGCGTTTTTTCCTGCATCCCTTTAGTGAGGGTTAAT ATAGCTTAGTTGTC GAGGGTTAAT		al. 2003) (Hu et al.
	ST64T/P22 1880	otsA	eac	SAL-21-U SAL-42-D SAL-61-U_SNP S.1		2.1 -	TAATACGACTCACTATAGGGaaagaattagtatgatagatgagaCAGCTCTT P-GATAATGCCAACGACAATGCAGGTCCCTTTAGTGAGGGT TAATACGACTCACTATAGGGtttgtgtgttattgtaattgagatCACGATTGAGCG	76	NC_002371 2002) AE006468	(Fang et al. 2012) (Pang et al. 2012)
								TGTTGTATGCGC TAAT CCGCA		(Pedulla et (McClelland
	AFLP fragments	CT-2	SAL-21-D SAL-43-U SAL-61-U_WT S.2		2.1 2.2	P-GGCCTTCCTTTGTGTTTCCCTCCCTTTAGTGAGGGTTAAT TAATACGACTCACTATAGGGgtatgttgtaatgtattaagaaagCCCAGGTA TAATACGACTCACTATAGGGtatgaatgttattgtgtgttgattCACGATTGAGCG	25	al. 2003) AF500165 et al. 2001)	(Fang et al. 2012)
	P22		g13	SAL-24-U			2.1	TAATACGACTCACTATAGGGtattagagtttgagaataagtagtCAGCGAAG AACAGGAAATCCA CCGCC	33	NC_002371 (Hu et al.	(Mikasová et al. 2005)
				SAL-43-D SAL-61-D	S.1		2.2	CGTTTGATTAG P-TTCCGGCACTGAAAATACTGCTCCCTTTAGTGAGGGTTAAT P-GCCACGTCATCCCGATCGTCCCTTTAGTGAGGGTTAAT		(Pedulla et 2002)
	AFLP fragments 2199 napF	SAL-24-D SAL-47-U SAL-62-U_SNP S.1 GA27.1		2.1 2.2	P-CGAACCAATCGAGTCTGTGTCCCTTTAGTGAGGGTTAAT TAATACGACTCACTATAGGGaagatgatagttaagtgtaagttaGGTTTGT TAATACGACTCACTATAGGGagtgaatgtaagattatgtatttgCGGAAGAAAAA	27	al. 2003) AY052766 AE006468	(Fang et al. 2012) (Pang et al. 2012)
	ST104		g44	SAL-26-U			2.1	TAATACGACTCACTATAGGGtttgatttaagagtgttgaatgtaCAACGCCCC CCTACGACCCC GCGATTCA	26	AB102868 (Mmolawa et (McClelland	(Fang et al. 2012)
				SAL-47-D SAL-62-U_WT S.2		2.2	ACACACCA P-GGAATCCTTCCATCGGAAATGATCCCTTTAGTGAGGGTT TAATACGACTCACTATAGGGagagtattagtagttattgtaagtGAAGAAAAAGC		(Tanaka et al. 2003) et al. 2001)
				SAL-26-D			2.1	P-GGTTCGGTACCACCTTTAATGTCCCTTTAGTGAGGGTTAAT AAT GATTCG		al. 2004)
	ST104 AFLP fragments	g61 GC-1	SAL-27-U SAL-48-U SAL-62-D	S.1		2.2 2.2	TAATACGACTCACTATAGGGaattgagaaagagataaatgatagCGCTACA TAATACGACTCACTATAGGGtgagtaagtttgtatgtttaagtaTGGAAGAA P-GTACAGGCCTGCGCACAGTCCCTTTAGTGAGGGTTAAT	72 65	AB102868 AF500167	(Drahovská et al. 2007) (Fang et al. 2012)
								GCAACAAAAACG CAAGCAAACAAGATTC		(Tanaka et (Hu et al.
				SAL-27-D SAL-48-D		2.2 2.2	P-TATGCTCCAGATGGAAGAGAGGTCCCTTTAGTGAGGGTT P-TCGTAGAACTACTGCAAAAAGCCAGTCCCTTTAGTGAGG		al. 2004) 2002)
								AAT GTTAAT	



  TAG, target-specific sequences and SNP positions are indicated by, respectively, italic, lower-case, underlined and boldface sequences. P: phosphate 


	Table S4 SNP 101 TM3124_1 intergenic SAL-64-U_SNP S.1 (continued) Targeted sequence Probe Probe pre-mix araD SAL-63-U_SNP S.1 SAL-63-U_WT S.2 SAL-63-D S.1 SAL-64-U_WT S.2 SAL-64-D S.1 TM3275_2 intergenic SAL-65-U_SNP S.1 SAL-65-U_WT S.2 SAL-65-D S.1 4213 yjeF SAL-71-U_SNP S.1 SAL-71-U_WT S.2 SAL-71-D S.1 TM2079 intergenic SAL-72-U_SNP S.3 SAL-72-U_WT S.3 SAL-72-D S.3 Specificity of the internal positive control probes Sequence (5'→3') TAATACGACTCACTATAGGGattaagtaagaattgagagtttgaGGCGCGAATG GGTGTGTACA TAATACGACTCACTATAGGGaattgagaaagagataaatgatagCGAATGGGTG TGTACG P-ATGCCGCCGATAGTCGGTCCCTTTAGTGAGGGTTAAT TAATACGACTCACTATAGGGgatagatttagaatgaattaagtgTCACAACTTCA AAATAAAACGTTATAAATTAATAT TAATACGACTCACTATAGGGaatgaaatagtgttaaatgagtgtTCACAACTTCA AAATAAAACGTTATAAATTAATAG P-ATTATATCAACAATCGCTTTTATCCTTGCTCCCTTTAGTGAGG GTTAAT TAATACGACTCACTATAGGGagtagaaagttgaaattgattatgCCTGCAAATAC TCGTACGGGTCGCGC TAATACGACTCACTATAGGGtttgttagaatgagaagatttatgCCTGCAAATAC TCGTACGGGTCGCGG P-TCTTTTACATCATTACGACGTCAAACTCCCTTTAGTGAGGGT TAAT TAATACGACTCACTATAGGgtatgttgtaatgtattaagaaagTCTGGCGATTAT TGGCGGT TAATACGACTCACTATAGGgtttgtgtttgtataagttgttaaTCTGGCGATTATT GGCGGC P-GACCAGGGAACAGCGGGCGCTCCCTTTAGTGAGGGTTAAT TAATACGACTCACTATAGGGaatgtaaagtaaagaaagtgatgaTACATCAGGC AACGGTACGT TAATACGACTCACTATAGGGatttgttatgataaatgtgtagtgATACATCAGGC AACGGTACGA P-CTATAGGACACCGCGCTAAGTCCCTTTAGTGAGGGTTAAT Genus Species Subspecies Serovar Reference a invA rpoB Escherichia coli NA NA ATCC 25922 --Listeria monocytogenes NA NA ATCC 51772 --Shigella sonneï NA NA 10-03865 --Salmonella enterica enterica Agona NH.II.18.12 + -Salmonella enterica enterica Derby H.II.34.34 + -Salmonella enterica enterica Enteritidis H.VI.6.32 + -Salmonella enterica enterica Infantis NH.III.66.30 + -Salmonella enterica enterica Kentucky H.I.70.26 + -Salmonella enterica enterica Newport H.V.70.42 + -Salmonella enterica enterica Paratyphi B H.VII.64.65 + -Salmonella enterica enterica Typhi H.III.28.24 + -Salmonella enterica enterica Typhimurium H.II.32.32 + + Salmonella enterica enterica Typhimurium var. Copenhagen H.II.13.13 + + Primer (T7 and T3), anti-Table S5 + positive reaction in MOL-PCR; -negative reaction in MOL-PCR	Bead region 21 72 28 74 12 75 25 53 44 42	Accession no. AE006468 (McClelland et al. 2001) AE006468 (McClelland et al. 2001) AE006468 (McClelland et al. 2001) AE006468 (McClelland et al. 2001) AE006468 (McClelland et al. 2001)	Reference of SNP (Pang et al. 2012) (Hu et al. 2006) (Hu et al. 2006) (Pang et al. 2012) (Hu et al. 2006)

(Barbau-Piednoir et al. 2013)in our laboratory(Barbau-Piednoir et al. 2013) 
Table S6

 S6 Minimum and maximum of observed signal-to-noise ratios, calculated cut-off values and assigned prime numbers for the markers in MOL-PCR_1 


	Marker	Negative samples	Positive samples	Calculated	Assigned prime
		Minimum SN	Maximum SN	Minimum SN	Maximum SN	cut-off value	number
	invA	NA	NA	8.15	27.66	8	2
	rpoB	NA	NA	10.45	47.59	10	2
	SAL-10	0.64	2.83	3.82	30.31	3.5	7
	SAL-11	0.60	2.54	7.66	28.62	5	13
	SAL-16	0.39	2.51	4.05	28.12	3.5	17
	SAL-23	0.64	2.66	8.90	33.98	6	11
	SAL-25	0.76	2.93	8.82	29.40	6	31
	SAL-29	0.72	2.77	3.87	31.78	3.5	5
	SAL-33	0.42	2.19	4.67	18.69	3.5	67
	SAL-45	0.51	2.64	5.13	28.60	4	19
	SAL-49	0.72	2.79	6.93	30.38	5	3
	SAL-50	0.63	2.78	6.17	24.37	4	43
	SAL-51	0.72	2.67	5.82	20.00	4	47
	SAL-53	0.23	2.49	6.63	31.33	5	37
	SAL-55	0.72	2.81	4.31	21.29	3.5	41
	SAL-67	0.57	2.33	10.01	39.68	6	53
	SAL-68	0.34	2.39	4.54	18.55	3.5	61
	SAL-69	0.37	2.25	7.30	21.88	5	59
	SAL-70	0.51	2.45	12.37	33.85	7	29
	SAL-74	0.67	2.55	10.15	31.22	6	23
	SN: signal-to-noise ratio; NA: not applicable				

Table S7

 S7 Minimum and maximum of observed signal-to-noise ratios, calculated cut-off values and assigned prime numbers for the markers in MOL-PCR_2 


	Marker	Negative samples	Positive samples	Calculated	Assigned prime
		Minimum SN	Maximum SN	Minimum SN	Maximum SN	cut-off value	number
	rpoB	NA	NA	4.85	22.02	4	2
	SAL-12	0.70	2.26	5.34	15.10	4	41
	SAL-14	0.67	2.23	4.10	17.46	3	23
	SAL-15	0.55	1.91	3.35	22.65	3	7
	SAL-18	0.70	2.33	3.20	17.03	3	11
	SAL-20	0.52	2.01	4.48	16.80	3	17
	SAL-21	0.54	2.10	3.93	15.77	3	53
	SAL-24	0.29	1.78	3.27	13.22	3	59
	SAL-26	0.70	2.45	3.62	14.82	3	29
	SAL-27	0.69	2.21	3.13	13.77	3	13
	SAL-35	0.55	1.99	3.14	12.18	3	19
	SAL-36	0.60	1.96	13.23	26.13	8	71
	SAL-37	0.48	2.94	5.28	10.58	4	61
	SAL-38	0.61	2.15	4.72	12.31	3.5	67
	SAL-39	0.61	1.96	7.11	22.12	5	37
	SAL-40	0.41	1.96	3.14	11.55	3	73
	SAL-42	0.55	2.23	3.02	9.88	3	5
	SAL-43	0.43	2.16	7.36	15.93	5	79
	SAL-47	0.36	1.65	3.21	16.40	3	47
	SAL-48	0.49	1.81	5.05	18.16	3.5	31
	SAL-66	0.33	2.44	7.79	28.81	5	43
	SAL-73	0.77	2.05	10.88	25.10	6	3
	SN: signal-to-noise ratio; NA: not applicable				

Table S8

 S8 Minimum and maximum of median fluorescence intensity, calculated cut-off values and assigned prime numbers for the markers in MOL-PCR_SNP 


	Marker	Negative samples	Positive samples	Calculated	Assigned prime	Assigned prime
		Minimum MFI	Maximum MFI	Minimum MFI	Maximum MFI	cut-off value	number -presence of SNP	number -absence of locus
	SAL-56_SNP SAL-56_WT	106 189	357 485	771 737.5	2063 2342	600 600	5 1	47
	SAL-58_SNP SAL-58_WT	103 290	422.5 590	624.5 885	1423 2319.5	500 700	29 1	41
	SAL-59_SNP SAL-59_WT	83 93	311.5 240.5	407 440	1607 2154.5	350 350	11 1	53
	SAL-60_SNP SAL-60_WT	179 108	769.5 259	1243 463	2407.5 1556	1000 350	3 1	59
	SAL-61_SNP SAL-61_WT	71 163	230.5 558	251.5 1106	726.5 2994	250 800	13 1	61
	SAL-62_SNP SAL-62_WT	71 99.5	302 182.5	385 217.5	1106 1110	350 200	19 1	67
	SAL-63_SNP SAL-63_WT	90 118	500 360	985 409	2091 1955.5	700 400	17 1	71
	SAL-64_SNP SAL-64_WT	99 118	540.5 168	803 217	1193 1543	700 200	31 1	73
	SAL-65_SNP SAL-65_WT	102 97	383 242	1153 600	2072 1699	800 400	7 1	79
	SAL-71_SNP SAL-71_WT	204 117	810.5 227	1565 431	2411 1558	1200 350	23 1	43
	SAL-72_SNP SAL-72_WT	114 158	443 165	1908 319	2027.5 1914	1200 250	37 1	83
	MFI: median fluorescence intensity						

Table S9

 S9 In-house code for MOL-PCR_1 


	Unique GPP In-house code
	3	1
	15	2
	69	3
	87	4
	123	5
	345	6
	435	7
	795	8
	851	9
	10005	10
	15015	11
	19095	12
	23055	13
	34891	14
	248235	15
	255255	16
	345345	17
	1312311	18
	5870865	19
	6561555	20
	19114095	21
	23138115	22
	43444401	23
	150345195	24
	324939615	25
	574893165	26
	3457939485	27
	1.0028E+11	28
	2.04963E+16	29
	1.20928E+18	30
	9.54E+19	31
	1.06E+20	32
	3.91E+21	

Table S10

 S10 In-house code for MOL-PCR_2 


	Unique GPP In-house code
	1	1
	3	2
	43	3
	57	4
	129	5
	82203	6
	97055	7
	291165	8
	465465	9
	680295	10
	1228371	11
	8843835	12
	17606651	13
	23339049	14
	224989611	15
	430422405	16
	8178025695	17
	10874497865	18
	32623493595	19
	66371935245	20
	1.54007E+11	21
	1.69216E+11	22
	2.58277E+11	23
	9.23053E+11	24
	1.40281E+12	25
	3.62117E+12	26
	4.90726E+12	27
	1.11059E+13	28
	1.76239E+14	29
	4.50435E+17	30



  Salmonella enterica subsp. enterica serovar Typhimurium and 1,4,[5],12:i:-isolates used in the development and validation of the multiplex oligonucleotide ligation-PCR (MOL-PCR) and their subtyping data. 


	Data set S1 Sample_ID Serovar	Source Outbreak_related PCR_screening Stability_experiment PFGE_cluster a GPP_MOL-PCR_1 GPP_MOL-PCR_2 GPP_MOL-PCR_SNP MOL-PCR_code Phage_type b MLVA
	S0041	Typhimurium Human	0	0	0 NT	34891	3	14-2-10	DT1	3-11-16-23-311
	S0042	Typhimurium Human	0	0	0 NT	34891	3	14-2-10	DT1	3-11-16-23-311
	Table S11 Unique GPP In-house code In-house code for MOL-PCR_SNP 1 1 29 2 37 3 41 4 69 5 105 6 221 7 1155 8 2139 9 4199 10 Source Outbreak_related PCR_screening Stability_experiment PFGE_cluster a GPP_MOL-PCR_1 GPP_MOL-PCR_2 GPP_MOL-PCR_SNP MOL-PCR_code Phage_type b MLVA S0001 Typhimurium Human 0 1 1 NT 3.91E+21 1.11059E+13 33-28-10 DT104 3-15-10-23-311 S0002 Typhimurium Human 0 1 1 NT 248235 224989611 15-15-1 DT120 3-13-10-NA-211 S0003 Typhimurium Human 0 1 0 NT 19114095 32623493595 21-19-1 DT120 3-13-10-NA-211 S0004 Typhimurium Human 0 1 0 NT 15 3 2-2-1 DT120 3-12-10-NA-211 S0005 Typhimurium Human 0 1 0 B3 255255 8843835 16-12-8 D T 3 5 3 -15-11-NA-311 S0006 Typhimurium Human 0 1 1 NT 3.91E+21 1.11059E+13 33-28-10 DT104 3-15-10-23-311 S0007 Typhimurium Human 0 1 1 NT 345345 8178025695 17-17-9 U302 4-14-18-7-211 S0008 1,4,[5],12:i:-Human 0 1 0 NT 15 1 2-1-1 DT193 3-13-10-NA-211 S0009 Typhimurium Human 0 1 1 NT 15 3 2-2-1 DT120 3-12-11-NA-211 S0010 Typhimurium Human 0 1 0 NT 3.91E+21 1.11059E+13 33-28-10 DT12 3-17-16-11-NA S0011 1,4,[5],12:i:-Human 0 1 1 NT 15 1 2-1-1 DT193 3-13-9-NA-211 S0012 Typhimurium Human 0 1 0 NT 3.91E+21 1.11059E+13 33-28-10 DT104 3-13-12-22-311 S0013 Typhimurium Human 0 1 0 NT 15 3 2-2-1 N T 3 -12-11-NA-211 S0014 1,4,[5],12:i:-Human 0 1 1 NT 15 1 2-1-1 DT120 3-13-10-NA-211 S0016 Typhimurium Human 0 1 1 NT 255255 8843835 16-12-8 DT195 3-15-13-NA-311 S0017 Typhimurium Human 0 1 1 NT 255255 8843835 16-12-8 U302 3-16-10-NA-311 S0018 1,4,[5],12:i:-Human 0 1 0 NT 15 1 2-1-1 R D N C 3 -12-11-NA-211 S0019 Typhimurium Human 0 1 1 NT 255255 8843835 16-12-8 D T 1 2 3 -14-10-NA-311 S0020 1,4,[5],12:i:-Human 0 1 1 NT 15 1 2-1-1 DT195 3-13-10-NA-211 S0021 Typhimurium Human 0 1 0 NT 255255 8843835 16-12-8 DT195 3-16-10-NA-311 S0022 1,4,[5],12:i:-Human 0 1 1 A10 15 1 2-1-1 DT195 3-13-10-NA-211 S0023 Typhimurium Human 0 1 0 NT 345345 430422405 17-16-5 U302 4-8-10-9-211 S0024 Typhimurium Human 0 1 1 B1 255255 8843835 16-12-8 U302 3-15-11-NA-311 S0025 Typhimurium Human 0 1 1 NT 345345 8178025695 17-17-9 D T 1 2 5 -14-11-8-211 S0026 1,4,[5],12:i:-Human 0 1 0 NT 15 1 2-1-1 DT193 3-12-10-NA-211 S0027 Typhimurium Human 0 1 1 NT 3.91E+21 1.11059E+13 33-28-10 DT104 3-14-11-21-311 S0028 Typhimurium Human 0 1 1 NT 255255 8843835 16-12-8 DT193 3-15-13-NA-311 S0029 Typhimurium Human 0 1 1 NT 255255 8843835 16-12-8 DT193 3-15-13-NA-311 S0030 Typhimurium Human 0 1 1 NT 345345 8178025695 17-17-9 D T 1 2 5 -13-15-8-211 S0031 Typhimurium Human 0 0 0 NT 851 3 9-2-7 D T 1 3 -14-10-15-NA S0032 Typhimurium Human 0 0 0 NT 851 3 9-2-7 D T 1 3 -14-10-15-311 S0033 Typhimurium Human 0 0 0 NT 3.91E+21 1.11059E+13 33-28-10 U302 3-14-18-14-311 S0034 Typhimurium Human 0 0 0 NT 255255 8843835 16-12-8 D T 3 5 3 -14-10-NA-311 S0035 Typhimurium Human 0 0 1 NT 3.91E+21 1.11059E+13 33-28-10 DT12 3-14-18-14-311 S0036 Typhimurium Human 0 0 0 NT 3 1228371 1-11-6 D T 1 3 -19-5-NA-311 S0037 Typhimurium Human 0 0 1 NT 3.91E+21 1.11059E+13 33-28-10 U302 3-18-16-17-311 S0038 Typhimurium Human 0 0 1 NT 255255 8843835 16-12-8 DT195 3-14-10-NA-311 S0039 Typhimurium Human 0 0 1 NT 15 3 2-2-1 DT120 3-12-9-NA-211 S0040 Typhimurium Human 0 0 1 A3 15 3 2-2-1 DT195 3-12-11-NA-211 S0043 Typhimurium Human 0 0 0 NT 34891 3 14-2-10 DT1 3-11-16-23-311 S0044 Typhimurium Human 0 0 0 NT 255255 3.62117E+12 16-26-8 DT208 3-15-13-NA-311 S0045 1,4,[5],12:i:-Human 0 0 0 A5 15 1 2-1-1 U311 3-15-13-NA-311 S0046 Typhimurium Human 0 0 0 NT 255255 8843835 16-12-8 DT193 3-15-13-NA-311 S0047 Typhimurium Human 0 0 0 NT 255255 3.62117E+12 16-26-8 DT195 3-15-13-NA-311 S0048 Typhimurium Human 0 0 0 NT 255255 8843835 16-12-8 DT195 3-15-13-NA-311 S0049 Typhimurium Human 0 0 0 NT 34891 3 14-2-10 DT1 3-11-16-23-NA S0050 Typhimurium Human 0 0 0 NT 34891 1228371 14-11-10 DT1 3-11-15-22-NA S0051 Typhimurium Human 0 0 0 NT 255255 8843835 16-12-8 DT193 3-15-12-NA-311 S0052 1,4,[5],12:i:-Human 0 0 0 A10 15 1 2-1-1 DT193 3-12-9-NA-211 S0053 Typhimurium Human 0 0 0 NT 3.91E+21 1.11059E+13 33-28-10 DT104 3-14-13-20-311 S0054 1,4,[5],12:i:-Human 0 0 0 NT 15 1 2-1-1 DT110 3-12-8-NA-211 S0055 1,4,[5],12:i:-Human 0 0 0 NT 15 1 2-1-1 DT110 3-11-12-NA-211 S0056 Typhimurium Human 0 0 0 B2 255255 8843835 16-12-8 DT208 3-10-7-NA-311 S0057 Typhimurium Human 0 0 0 NT 19114095 1.40281E+12 21-25-1 DT193 3-15-9-NA-211 180557 Sample_ID Serovar S0015 Typhimurium Human 0 1 0 A3 15 3 2-2-1 DT193 3-12-11-NA-211 S0058 1,4,[5],12:i:-Human 0

 4,[5],12:i
Véronique Wuyts, Wesley Mattheus, Nancy H C Roosens, Kathleen Marchal, Sophie Bertrand, Sigrid C J De Keersmaecker
Applied Microbiology and Biotechnology A multiplex oligonucleotide ligation-PCR as a complementary tool for subtyping of Salmonella Typhimurium