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Protocols used for MLST of Moraxella catarrhalis

Genes

The M. catarrhalis MLST scheme uses internal fragments of the following eight house-keeping genes:
glyRS (glycyl-tRNA synthetase beta subunit)
ppa (Pyrophosphate phospho-hydrolase)
efp (elongation factor P)
fumC (fumarate hydratase)
trpE (anthranilate synthase component I)
mutY (adenine glycosylase)
adk (Adenylate kinase)
abcZ (ATP-binding protein)

PCR Amplification

The primer pairs we use for the PCR amplification of internal fragments of these genes are:

		Product length:
glyRS:	F 5'-GCACCGAAGAGTTGCCACCA-3'	762 bp
glyRS:	R 5'-ACGCAACGGGCAAATCCACC-3'

ppa:	F 5'-AATAAAATTCTAGATGCTGGC-3'	523 bp
ppa:	R 5'-ACTTATTGCTCTGTCCAGCG-3'

efp:	F 5'-CTCTGATTGACAACTGGCAGG-3'	582 bp
efp:	R 5'-GATATTCGCCAGTACGCG-3'

fumC:	F 5'-GGGCGGTACAGCAGTCGGCAC-3'	675 bp
fumC:	R 5'-CTCATCAAATTCAGCTTCAG-3'

trpE:	F 5'-TTATCCCGCATCGAAAATGG-3'	545 bp
trpE:	R 5'-GGTTTCATCCCATTCAGCC-3'

mutY:	F 5'-GGCAATACCATCATCAGCCG-3'	609 bp
mutY:	R 5'-GGTAACTGACTTTGAACGCC-3'

adk:	F 5'-GGCATTCCTCAAATCTCAAC-3'	631 bp
adk:	R 5'-GATGGGCTTTATTGTCAAATG-3'

abcZ:	F 5'-ACATGCTGATGATGGTGAG-3'	610 bp
abcZ:	R 5'-CACTGGCAAGTTCAAGCGC-3'

An annealing temperature of 52° C is fine for all genes, except GlyRS (58° C) and adk (54° C).

Sequencing

We use the amplification primer pairs for the sequencing step except for the following genes.

glyRS: F 5'-GCACCGAAGAGTTGCCACCA-3'
glyRS: R 5'-ATATCGGCTTGACGCTGATC-3'

fumC: F 5'-GCTGTCAAAGTCGCTAAAG-3'
fumC: R 5'-CTCATCAAATTCAGCTTCAG-

mutY: F 5'-TATGCTGTGTGGGTATCTG-3'
mutY: R 5'-GGTAACTGACTTTGAACGCC-3'

Allele template

Allelic profile of M. catarrhalis MC216

glyRS (537 bp):
GTTGGCACTTTACTAAATGATCAAAATATCAGCTTTGATGATATTCATGCCTTTGCCGCACCACGCCGTTTGGCACTTACTATTAC
AGGTGTGGGCGAGTTTCAGCCTGACCGTATCGAAACTAAAAAAGGCCCAAGTGTTAAGGCAGCGTTTGATGACGCAGGTAATTTAA
CGCGTGCAGGGCAAGGATTTTTGCAAGGTTTAAATGCCACTGGGCGAAATCTTAGTGTCAAAGATTTGGGGCGTATTGCTGATAAA
AAAGGCGAGTACATCGCTTATGATTTGACCATTAAAGGCGAAAAAATCACCGATTTAATGCCAAATATCTTACAAAAAGCGCTTGA
TGATTTGCCAATTGCTAAGCGTATGCGTTCTGGCATTGATCGCCATGAGTTCATACGGCCTGTTCATTGGGTGGTTTTGATGTCAG
ATGATGTTGTAATTGAGGCGACCATTCAAGGCCATAAAACTGGCAACCAAAGCCGTGGTCATCGCTATCATTCACCTGATTTTTTT
GCCATTGATCATGCCGATCAT

ppa (393 bp):
TCAAATCATAAAATTGAGTGGAATCGTCATTTGGCATGCTTTGAGCTTGATCGTGTTGAGCCAATTGCCTTTGCCAAACCATGCAA
CTATGGCTTTATTCCCCAAACCTTAGATGAAGATGGTGATGAATTAGACGCCTTAATCATCACAGAACAGCCTTTGACGACGGGTA
TTTTTCTTAAAGCCAAAGTCATCGGGGTAATGAAATTTGTGGATGATGGCGAGGTCGACGATAAAGTCATCGTTGTTCCTGCCGAT
GATCGTAATAATGGCAACGCCTATAATAGTCTTGACGACCTACCACCACAGTTAATAAAGCAACTTGAATTTCATTTTAGTCATTA
TAAAGACTTAAAAAAACCAGGCTCAACTGTGGTTGAGGGCTTTTTTGAT

efp (414 bp):
AATGAATATGTGAAACCAGGTAAAGGTCAAGCTTTTAACCGTGTTAAGTTGCGTAATCTACGCACAGGCAAAGTGCTTGAGCAGAC
TTTTAAGTCAGGCGATTCACTTGAAGGTGCTGATGTGGTGGATGTGGAAATGAACTATCTATACAACGATGGCGAATTTTGGCATT
TTATGCACCCTGAGACTTTTGAGCAATTGCAAGCAGATAAAACCGCAATGGGTGATGCTGCACAGTGGCTAAAAGAAAATTCAAAT
GCGTTATGCACTTTGACGCTATTTAATGGTGCGCCGCTGTCGGTCACACCACCAAACTTTGTAGAGCTTGAAATCGTTGAAACTGA
CCCAGGTGTTCGCGGTGATACCTCAGGTGGTGGCGGTAAGCCTGCACGCCTTGAAACAGGTGCAACCGTG

fumC (465 bp):
GAGGCTTTGGCCGCACGAGATGCCGAAGTATTTGCTTCAGGTGCACTAAAAACACTGGCAGCAAGCCTAAATAAAATCGCCAATGA
TGTGCGTTGGCTGGCTTCAGGCCCACGCTGCGGTCTAGGTGAGCTTAGCATTCCTGAAAATGAGCCTGGTTCATCCATCATGCCTG
GTAAAGTCAATCCAACTCAGTGTGAGGCGATGACCATGGTGGTCACCCAAGTCTTTGGTAATGATACGACCATTTCTATGGCAGGG
GCTTCTGGTAATTTTGAATTAAATGTTTATATGCCTGTGATTGGATATAACTTATTGCAGTCGATTCGTCTGCTAACGGATGCGAT
CAATAGCTTTAATGAGCATTGTGCGGTAGGGATCGAACCAATTTACGAAAAAATTGACCATTATTTACATCATTCTTTGATGTTGG
TCACCGCACTCAATCGCCATATTGGCTATGAAAAT

trpE (372 bp):
TTAAATGACCAAAAAGAGATCGCAGAACATCTGATGCTCATTGATCTTGGGCGTAATGATATTGGGCGTGTCTGTGAGATTGGTTC
GGTTAAAGTGACAGATCAGATGTTCATTGAACGCTACTCGCAAGTGATGCATATCGCTAGTAATGTTGAGGGGCGTATACGCCAAG
ATGTCGATGCTTTAGATGTGTTTTGTGCGACCTTTCCAGCGGGTACTTTGTCTGGTGCGCCAAAAATCCGTGCCATGCAAATTATT
GATGAAGTTGAGCCAGTGCGTCGCACCGTTTTTGGCGGGGCGATCGGATATCTTGGCTGGCATGGCAATATGGATACTGCCATCGC
AATACGCACAGCGGTCATGAAAGATGGT

mutY (426 bp):
TTTTTTGAGCCGTTTTTGGCACGATTTGCAACCGTACAAGAGCTTGCTGTAGCAGATTGGCAAGAAGTAGCGTCTTTTTGGGCAGG
GCTTGGGTATTATGCTCGGGCAAGAAATTTACACGCAGGCGCACAGCAAGTGGCTGATTTTATTGATACTCATGGCAGGTTTCCTG
AAACGGTCAATGAGTGGCAAGCAGTCAAAGGGGTGGGGCGATCGACAGCTGGCGCAATCGTTGCGATGGGCGTTAAGAAGTTTGGC
GTCATCTGCGATGGCAATGTCAAGCGTGTACTTGCTCGCCATCGCGCGGTTTTTGGTGATGTGACAAAAAGCGCAACTGACAAAAG
GCTTTGGGAAATTGCCACCGCACTAACCCCGAAAGAGTATAGTGGACACTATGCCCAAGCGATGATGGATTTGGGTGCAACA

adk (471 bp):
CAAGCAAAAAGTGTCATGGATGCAGGTCAATTGGTTTCTGATGAGCTGATTATTAATTTGGTTAAAGAACGCTTAAAACAGCCAGA
TTGTGACAATGGTTGTATTTTTGATGGATTCCCACGCACAATCCCACAAGCCAAAGCACTGGCAGATGCTGGCATTGCCATTGACC
ATGTGATTGAAATCAGCGCCCCTGATGATGAAATCGTAAGCCGTATGTCGGGTCGCCGTGCACATTTGGCATCAGGTCGTACTTAT
CATGTGATCTATAATCCCCCAAAAACTGACGGTATTGATGATGTCACAGGCGAGGCACTGGTGCAGCGTGATGATGATAAAGAAGA
AGTGGTGCGTGATCGCTTATCGGTATATCACGCCCAAACCGCTACATTGATTAGTCATTATCAAGAAGTTGCCAAATCGGGCGAAA
ATGCCCCTGAATATCATGACTTTGATGGAACAAAATCCATC

abcZ (429 bp):
CTTGATGTGGTGATGGAGGGTGATGACAAAGTTGCCCAAGCCTTAAAAAACTATCATCAAGCCAGTGCAAAATGCGCCATGGGCGA
CAATGAAGCTTGTGATGCCATGAGCGGTCTTCAGCAAACACTCGATGAGTTAAATGGTTGGGACTTGGAGCGGCGTGCGCGTGCAT
TGATTGACAGCATGGGGTTTGACGCTGATGCTTGCTTATCAACATTATCAGGGGGTCGTAAACGCCGTGTTCTGTTAGCACAGGCT
TTGGTAACAAATCCTGATGTGCTACTTTTAGACGAGCCAACCAACCATTTAGATATTGAAAGCATCACTTGGCTTGAAAAATTTTT
ATTAAATGAACGATTAACCGTTCTTTTCATCTCACATGATCGGAAGTTTGTTGATACACTTGCCACTTATATCATCGAGCTTGAC