Text S1
1
Distinct clones of Yersinia pestis caused the Black Death
Haensch S, Bianucci R, Signoli M, Rajerison M, Schultz M, Kacki S, Vermunt M, Weston DA, Hurst D,
Achtman M, Carniel E, Bramanti B.*
*To whom correspondence should be addressed at the Institute of Anthropology,
Colonel Kleinmann Weg 2, SBII 02, Johannes Gutenberg University,
D-55128 Mainz, Germany. E-mail: bramanti@uni-mainz.de
Archaeological information
The rural cemetery of Saint-Laurent-de-la-Cabrerisse is located mid-way between Narbonne and
Carcassonne in south-eastern France. During the 2007 excavations, an early medieval church and graveyard
used during the 8th-14th centuries were discovered and 149 graves were identified. Three of the graves,
located close to the southern wall of the church, were mass graves belonging to a funerary phase dating
between the 11th and 14th centuries. Two of these interments contained two bodies each, while the third
contained the remains of five individuals, making a total sum of six adults and three juveniles. As all the
bodies were buried simultaneously and there were no signs of violent trauma, the three multiple graves
were suspected to be plague pits. Historical accounts suggest that the Black Death reached Marseilles (in
south-eastern France) by November 1347 and spread to the west of France by land and sea, reaching
Narbonne and Carcassonne at the beginning of 1348 [S1, S2]. Unfortunately, no additional written records
exist for the rural cemetery of Saint-Laurent-de-la-Cabrerisse. A copper and iron buckle found in
association with one skeleton (SLC 1083) from grave SLC 160, together with soil stratigraphy dated the
graves to between the early 13th and late 14th centuries, indicating that the plague epidemic associated with
these graves could be either the outbreak of 1348 or the resurgence of 1374. Radiocarbon measurements
have been carried out on three samples from the site, among these one from grave SLC 141, the grave of
the PCR-positive sample SLC 1006 (sample SLC 1010; OxA-21213). For this sample, calibration indicates
calAD 1279-1389 (95.4 % probability) as the most likely range, i.e. calAD 1279-1315 (49.6 %) and 13551389 (46.4 %). Individuals from the two other pits, which were positive only to the RDT analyses, were
also dated: SLC 1013 (OxA 21214) - calAD 1286-1325 (39.7 %), 1345-1394 (55.7 %); SLC 1081 (OxA
21215) - cal AD 1303-1366 (70.7 %), 1383-1409 (24.7 %).
Text S1
2
From the English cemetery site of Hereford, a town near the Welsh border, 188 individuals were found in
three mass graves (pits) associated with the cathedral [S3]. The pit sample, a relatively small proportion of
the 1194 skeletons excavated from the cemetery, had a large number of juveniles, particularly children aged
between 5 and 14 years. Adults from the mass pits were mainly aged between 26-35 years, and the sexes
were evenly represented. The three pits were of similar dimensions and were found in a row, each built up
of layers containing bodies separated by thin deposits of clay-rich soil. It is likely that many more
individuals (ca. 300-400) were buried in the mass graves, as only the end of each burial pit was excavated.
All archaeological evidence points to the Black Death as the cause of death for the individuals in the mass
graves. Plague is thought to have been exported from France to England via shipping and trade in the
summer of 1348 [S4]. Most of the Hereford samples used here were derived from individuals from Plague
Pit 2, which has been directly AMS radiocarbon dated to between calAD 1281-1389 (2 sigma range,
probability 95.4 %, KIA23704), and fits into either the time of the first plague outbreak in 1349, the second
outbreak in 1361, or the third outbreak in 1369 [S5].
The skeletal collection from Bergen op Zoom (The Netherlands) was excavated from the site of an ancient
hospital, where all the available ground was used for mass burial. The samples were taken from eleven
mass graves containing between 10 and 25 individuals, most of them buried in wooden coffins, some in
linen shrouds. The mass graves, which can be dated to the middle of the 14th century on the basis of soil
stratigraphy, artefacts and coins, were located very close to one another, did not intersect and were all
contained within the same stratigraphic level. Moreover, 30 graves were found surrounding the altar inside
the hospital chapel and an additional 150 graves were found in the same level as the central hospital
building itself and in a small structure adjacent to the hospital [S6]. Out of an estimated population of
2,400-3,200 medieval inhabitants, approximately 800 were buried in the mass graves, indicating a sudden,
high mortality due to a major catastrophe. The administrative records for Bergen op Zoom, once a thriving
port city, were destroyed by fire in 1397 and there are no written reports describing plague for the years
1349-1351 or later. However, historical data indicate that plague ravaged the Low Countries at least four
times between 1349 and 1390.
Text S1
3
Plague epidemics have been well-documented in the southern German town of Augsburg since 1380,
repeatedly afflicting the town until 1634/1635. During the 15th and 16th centuries, plague episodes were
recorded once a year, and in the years 1627/1628 a total of 12,103 people died [S7]. The excavated site
(now factory premises at Heinrich-von-Buz-Straße 28) was dated to the 16th/17th century by ceramics and
was associated with either the Thirty Years War or a disease epidemic [S8]. This mass grave was found
roughly 300m away from a site excavated in 1936 near an earlier plague repository. The mass grave
consisted of 4-5 layers of skeletons, which were difficult to separate. The individuals were all buried
together in an anonymous fashion and all but 9 individuals were placed in a prone position, alternating eastwest, reflecting the typical rapid burial of plague victims.
The Italian archaeological site of Ex-Gelmini in Parma was situated in the Oltretorrente area of the city,
where several lazarettos (quarantine stations) were organized during the plague epidemic of 1630 [S9].
During the excavation, numerous pits were discovered and estimated to contain up to 5,000 individuals in
several stratified layers. Associated ceramics date the pits to the end of the 16th or beginning of 17th century,
indicating that the skeletons might belong to victims of the 1630 plague wave that killed 75 % of the
population (ca. 16,000-18,000 individuals [S10]).
The samples used as negative controls for the analyses (Table 1) originated from Germany, France and the
Netherlands. Those indicated as ‘Bösfeld’ (Bös) were bones derived from skeletons excavated from the
Bösfeld site, near Mannheim, a Frankish cemetery dating roughly to the 7th century. The ‘SLC’ samples (all
teeth) were unearthed from the oldest part of the cemetery of Saint-Laurent-de-la-Cabrerisse (8th-10th
centuries), whereas the teeth indicated as ‘BNK’ originated from sepultures in an ordinary cemetery of the
16th and 17th century in Bergen op Zoom. All samples used as negative controls were exhumed from sites
without archaeological evidence of catastrophic events, like an epidemic. A soil sample for aDNA analysis
was obtained from the femur of individual Man 30 from the Augsburg site.
Additional considerations on the aDNA analysis
Text S1
4
For aDNA amplification we used oligonucleotide primers that were designed to amplify amplicons of 120
bp on average, a length that is compatible with degraded DNA (Table S2). Nevertheless, we failed to
amplify the aDNA of Y. pestis from plague skeletons in 87 % of all individuals, similar to previous results
obtained by other groups [S11, S12]. The recovery of amplifiable Y. pestis DNA from ancient samples is
problematic even when analyses are repeated several times and the skeletons are from known plague
victims. In some cases, only a few regions of the bacterial DNA could be amplified (Fig. 3; Table S3),
probably because the DNA molecules were severely damaged and fragmented. For some individuals, even
extracts obtained from different teeth from the same jaw showed differences in the success rate (e.g. Ber10,
Table S3). Moreover, individuals who were positive for the RDT analysis often failed to amplify the caf1
gene (Table 1), confirming published results by others [S12]. We did not consider two samples (Ber3 and
Ber9) among the pla-positives, as although both amplified once, the amplification could not be positively
replicated, even when the same extract was used. Most likely the DNA content in the extracts was too low
and thus the possibility for amplification was strongly influenced by chance. However, Ber3 yielded
positive results for the RDT analysis.
With regards to the series that failed to amplify Y. pestis specific DNA but were positive for the RDT
reaction (Parma and Augsburg), we stress that for these two series bone samples were principally analyzed.
While trabecular bone from the femoral heads revealed positive results for the RDT test, the genetically
analyzed cortical bone may have possessed too few surfaces for bacteria to infiltrate in the last phase of
infection. Moreover, unlike teeth, bone is more readily subjected to environmental degradation processes
that destroy DNA.
For those samples that were positively amplified, all the sequences obtained were determined by up to three
independent extractions and several independent amplifications, depending on the availability of biological
material and extract (Table S3). When necessary, amplifications were repeated, changing the amount of
template to exclude artifacts like stochastic substitution due to degradation processes, especially if those
were observed at the SNP positions.
Text S1
5
Each obtained sequence was tested by performing BLAST searches to find matches from Yersinia
sequences sharing a MaxIdent score over 90 % (identity scores lower than 100 % were due to deaminations
since the largest scores still remained Y. pestis ssp.). Deaminations are the principal changes that occur due
to the degradation of ancient DNA [S13], thus suggesting an archaic origin for the molecules.
Blanks and Negative controls
No milling, extraction and PCR blanks amplified with Y. pestis specific primers yielded amplification
results. An exception was a PCR control amplified for the SNP s19. This yielded a sequence that could be
identified as Escherichia coli DNA (Fig. S6 in Text S2). The presence of traces of E. coli DNA in the
laboratory environment is not unexpected since chemicals employed for the PCR reaction can be obtained
from the modified bacterium. Blank controls amplified with unspecific primers (rpoB and 16S primers) in
some cases gave reaction products that could always be unambiguously identified with primer dimers or
artifacts.
Soil samples used for the RDT analyses always yielded negative results. Soil from one Augsburg bone
sample (Man30) was used for genetic analyses. While rpoB-primers yielded no amplification, DNA
amplified with the 16S-primers yielded only DNA from environmental and soil bacteria and no Y. pestisspecific sequence could be obtained.
No reaction products were obtained from the negative controls used for the RDT and PCR analyses. This
cannot be attributed to inhibition effects since from all negative controls (but Bös 844) human mtDNA
sequences were amplified (data not shown).
The high number of likely false negatives among the samples from the plague pits both for RDT and PCR
analyses required a large number of negative controls to ensure that the sequences and the protein did not
stem from other unknown bacteria. The main goal of our aDNA investigation was to genotype ancient Y.
pestis DNA, not to genetically prove the existence of the infection in each skeleton. Thus we concentrated
our efforts on obtaining reproductions of up to 20 specific markers from some individuals (Table S3)
instead of performing a clinical case-control experiment by repeating the analysis a fixed number of times.
Text S1
6
Conversely, in several cases we abandoned the search after the first attempts, conscious that in these cases
we would have had more false negatives among the plague individuals. Nevertheless, to establish whether
we used an adequate number of negative controls we performed a Fisher's exact test on a contingency table
(Table S4) under the hypothesis that ‘case’ and ‘controls’ were individuals originating from the same
population. This gave a high significant p-value for both the RDT (P 0.01 %) and PCR (P 2.25 %) analyses.
This indicates that it is extremely unlikely that the negative results obtained from the negative controls
(non-plague individuals) were due to false negative reactions.
References
S1. Biraben J-N (1975) Les hommes et la peste en France et dans les pays Européens et Méditerranéens, 2 vols. Paris: Mouton.
S2. Benedictow OJ (2004) The Black Death 1346-1353: the complete history. Woodbridge: Boydell Press.
S3. Weston DA, Boylston AE, Ogden AR, Hurst D (2007) The palaeodemography of the Black Death: the Hereford Cathedral
Close cemetery. Am J Phys Anthropol S44: 247-248.
S4. Shrewsbury JFD (1970) A history of bubonic plague in the British Isles. Cambridge: Cambridge Univ. Press.
S5. Hatcher J (1977) Plague, population and the English economy 1348-1530. London: Macmillan.
S6. Vermunt M (2007). In: Degraeve A, Demeter S, De Meulemeester J, editors. Hospitalen van de Middeleeuwen en de
moderne tijden. Brussels: Ministerie van het Brussels Hoodstedelijk Gewest, pp. 127-142.
S7. Grünsteudel G, editor (1998) Augsburger Stadtlexikon. Augsburg: Perlach-Verlag.
S8. Bakker L (1988) Zeitschrift des Historischen Vereins für Schwaben 81: 7.
S9. Banzola MO (1980) L’Ospedale vecchio di Parma. Notizie storiche e vicende costruttive precedute da una sintesi della
formazione urbana di Parma, cenni sulle origini e sulla storia degli ospedali nell’Occidente elementi di storia dell’ospitalità a
Parma. Parma: Palatina.
S10. Del Panta L, Livi Bacci M, Pinto G, Sonnino E editors (1996) La popolazione Italiana dal Medioevo a Oggi. Bari: Laterza.
S11. Gilbert MT, Cuccui J, White W, Lynnerup N, Titball RW et al. (2004) Absence of Yersinia pestis-specific DNA in human
teeth from five European excavations of putative plague victims. Microbiology 150: 341-354.
S12. Pusch CM, Rahalison L, Blin N, Graeme JN, Czarnetzki A (2004) Yersinial F1 antigen and the cause of Black Death.
Lancet Infect Dis 4: 484-485.
S13. Hofreiter M, Jaenicke V, Serre D, von Haeseler A, Pääbo S (2001) DNA sequences from multiple amplifications reveal
artifacts induced by cytosine deamination in ancient DNA. Nucleic Acids Res. 29: 4793-4799.
Text S1
7
Figure S1: Alignment of the pla-nucleotide sequences from ancient samples. As reference the sequence from Y. pestis CO92
was used (AL10996; 1.ORI). Sample Ber16 is not considered in the alignment because it was not sequenced.
Text S1
480
Yp_CO92_AL109969
Ber3a_XXBB.47.1_YP10R
Ber3a_XXBB.47.1_YP11D
Ber9a_XXBB.47.4_YP11D
Ber9a_XXBB.47.4_YP10R
Ber10a_XXBB.47.5_YP11D
Ber10a_XXBB.47.5_YP11D
Ber10a_XIV.59.10_YP11D
Ber10a_XIV.59.10_YP10R
Ber10a_XIV.60.11_YP10R
Ber10a_XIV.60.11_YP11D
Ber10a_XIV.68.13_YP11D
Ber10a_XIV.68.13_YP10R
Ber12a_XVI.68.5_YP10R
Ber13b_XVI.68.6_YP11D
Ber13b_XVI.68.6_YP10R
Ber13a_XXIII.93.14_YP11D
Ber36b_XVI.68.10_YP11D
Ber36b_XVI.68.10_YP10R
Ber37b_XXIV.95.5_YP11D
Ber37a_XXV.100.1_YP10R
Ber37a_XXV.100.1_YP11D
Ber45a_XVI.68.12_YP11D
Ber45a_XVI.68.12_YP10R
Her7a_XVIIIBB.63.3_YP12D
Her7a_XV.63.15_YP11R
Her7a_XV.63.15_YP12D
Her7a_XVIIIBB.62.3_YP11D
Her7a_XVIIIBB.62.3_YP10R
Her7a_XXI.86.7_YP11D
Her7a_XXI.86.7_YP10R
Her21a_XV.63.16_YP12D
Her21a_XXIVBB.63.7_YP12D
Her21a_XXIVBB.62.7_YP11D
Her21a_XXIVBB.62.7_YP10R
SLC1006a_XXIX.105.1_YP10R
SLC1006a_XXIX.105.1_YP11D
SLC1006a_XXIX.105.1.2_YP10R
SLC1006a_XXIX.105.1.2_YP11D
SLC1006a_XXIX.106.8_YP11D
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Yp_CO92_AL109969
Ber3a_XXBB.47.1_YP10R
Ber3a_XXBB.47.1_YP11D
Ber9a_XXBB.47.4_YP11D
Ber9a_XXBB.47.4_YP10R
Ber10a_XXBB.47.5_YP11D
Ber10a_XXBB.47.5_YP11D
Ber10a_XIV.59.10_YP11D
Ber10a_XIV.59.10_YP10R
Ber10a_XIV.60.11_YP10R
Ber10a_XIV.60.11_YP11D
Ber10a_XIV.68.13_YP11D
Ber10a_XIV.68.13_YP10R
Ber12a_XVI.68.5_YP10R
Ber13b_XVI.68.6_YP11D
Ber13b_XVI.68.6_YP10R
Ber13a_XXIII.93.14_YP11D
Ber36b_XVI.68.10_YP11D
Ber36b_XVI.68.10_YP10R
Ber37b_XXIV.95.5_YP11D
Ber37a_XXV.100.1_YP10R
Ber37a_XXV.100.1_YP11D
Ber45a_XVI.68.12_YP11D
Ber45a_XVI.68.12_YP10R
Her7a_XVIIIBB.63.3_YP12D
Her7a_XV.63.15_YP11R
Her7a_XV.63.15_YP12D
Her7a_XVIIIBB.62.3_YP11D
Her7a_XVIIIBB.62.3_YP10R
Her7a_XXI.86.7_YP11D
Her7a_XXI.86.7_YP10R
Her21a_XV.63.16_YP12D
Her21a_XXIVBB.63.7_YP12D
Her21a_XXIVBB.62.7_YP11D
Her21a_XXIVBB.62.7_YP10R
SLC1006a_XXIX.105.1_YP10R
SLC1006a_XXIX.105.1_YP11D
SLC1006a_XXIX.105.1.2_YP10R
SLC1006a_XXIX.105.1.2_YP11D
SLC1006a_XXIX.106.8_YP11D
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Yp_CO92_AL109969
Ber3a_XXBB.47.1_YP10R
Ber3a_XXBB.47.1_YP11D
Ber9a_XXBB.47.4_YP11D
Ber9a_XXBB.47.4_YP10R
Ber10a_XXBB.47.5_YP11D
Ber10a_XXBB.47.5_YP11D
Ber10a_XIV.59.10_YP11D
Ber10a_XIV.59.10_YP10R
Ber10a_XIV.60.11_YP10R
Ber10a_XIV.60.11_YP11D
Ber10a_XIV.68.13_YP11D
Ber10a_XIV.68.13_YP10R
Ber12a_XVI.68.5_YP10R
Ber13b_XVI.68.6_YP11D
Ber13b_XVI.68.6_YP10R
Ber13a_XXIII.93.14_YP11D
Ber36b_XVI.68.10_YP11D
Ber36b_XVI.68.10_YP10R
Ber37b_XXIV.95.5_YP11D
Ber37a_XXV.100.1_YP10R
Ber37a_XXV.100.1_YP11D
Ber45a_XVI.68.12_YP11D
Ber45a_XVI.68.12_YP10R
Her7a_XVIIIBB.63.3_YP12D
Her7a_XV.63.15_YP11R
Her7a_XV.63.15_YP12D
Her7a_XVIIIBB.62.3_YP11D
Her7a_XVIIIBB.62.3_YP10R
Her7a_XXI.86.7_YP11D
Her7a_XXI.86.7_YP10R
Her21a_XV.63.16_YP12D
Her21a_XXIVBB.63.7_YP12D
Her21a_XXIVBB.62.7_YP11D
Her21a_XXIVBB.62.7_YP10R
SLC1006a_XXIX.105.1_YP10R
SLC1006a_XXIX.105.1_YP11D
SLC1006a_XXIX.105.1.2_YP10R
SLC1006a_XXIX.105.1.2_YP11D
SLC1006a_XXIX.106.8_YP11D
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Text S1
9
Figure S2: Alignment of the (A-B) caf1 and (C-D) rpoB sequences from ancient samples. The corresponding gene sequences
of Y. pestis CO92 served as reference sequence. Different primer sets were used for each amplification fragment (Tab. S2): for
caf 1 (A and B) caf1 F1/R1or U2 / L2 and rpoB F1/ R1 or rpoB F2 / R2 for rpoB.
Text S1
10
Figure S3: Sequence comparison of glpD (A) and napA613 (B) of Y. pestis CO92 and KIM6 with sequences from ancient
samples. Ancient samples lack both, the 93bp-deletion of 1.ORI and the G/T mutation of 2.Med (indicated with red boxes in
S3.A and S3.B, respectively).
Text S1
11
Figure S4: Alignment of the sequences encompassing five SNPs defining branch 0. The relevant codons are marked with red
rectangles. A: s29 defining branch 0.PE2b (GCA), B: s31 defining branch 0.PE2a (GGA), C: s81excluding 0.PE4 and Y.
pseudotuberculosis (AAA) and D-E: s82/s87 defining branch 0.PE (shortly before splitting into branches 1and 2).
A
98 0
Yp stIP32 953 _BX 936 39 8s2 9
Be r1 0a_ XIV.72.8 s29 L1 a
Be r1 0a_ XIV.72.8 s29 U1 a
Be r3 6b_ XIV.72.1 0s2 9U1a
Be r4 5a_ XIV.72.1 1s2 9U1a
He r7 a_X V.72.12 s2 9U1 a
He r2 1a_ XV.72 .1 3s29 U1a
99 0
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YpstIP32953_BX936398s31
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Ber36b_XVI.73.10s31L
Ber36b_XVI.73.10s31U
Ber45a_XVI.73.11s31L
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Her7a_XV.73.12s31L
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.
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.
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T
.
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G
.
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390
C
.
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T
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.
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A
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1610
1600
G C G A C C A T C T G A C C A C G A T A A G C C A T
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T
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T
.
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.
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1940
A
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.
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.
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1620
A
.
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1180
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.
380
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1630
T
.
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1950
A
.
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10 20
T
.
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.
370
T
.
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1640
C
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1190
C
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360
G
R
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1960
T
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10 10
A
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1650
A
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1200
A
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C
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350
T
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1970
A
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C
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340
A
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1210
T
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10 00
A
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1660
E
YpstIP_BX936398s87
Ber10a_XIV.92.13s87U1
Ber10a_XIV.92.13s87L1
Ber45b_XVII.84.7s87L1
Ber45b_XVII.84.7s87U1
G
.
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1670
D
YpstIP32953_BX936398s82
Ber10a_XIV.72.1s82L1
Ber10a_XIV.72.1s82U1
Ber36b_XVI.72.3s82L1
Ber36b_XVI.72.3s82U1
Ber45a_XVI.72.4s82L1
Ber45a_XVI.72.4s82U1
Her7a_XV.72.5s82L1
Her7a_XV.72.5s82U1
Her21a_XV.72.6s82L1
Her21a_XV.72.6s82U1
SLC1006b_XXIX.109.9s82L1
SLC1006b_XXIX.109.9s82U1
T
.
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.
330
A
.
.
.
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C
Yp stIP32 953_BX 936398s81
Be r10a_XVI.74.1s81U5
Be r13b_XVI.77.3s81U7
Be r16a_XVII.77.1s81U7
Be r36b_XVI.74.3s81U5
Be r45a_XVI.74.4s81U5
Be r45a_XVI.73.4s81U5
Be r45a_XVI.80.4s81L1b
Be r45a_XVI.80.4s81U7
Be r45b_XVII.77.2s81U7
A
.
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1930
A
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C
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G A T
. . .
. . .
.
.
.
.
Text S1
12
Figure S5: Alignment of the sequences encompassing five SNPs defining branch 1. The relevant codons are marked with red
rectangles. A and B: 1.ORI specific SNPs s2 (CGA) and s7 (ACC). C-F: branch 1 specific SNPs s11 (CAA), s12 (CGT), s13
(GCT) and s14 (ACG).
A
100
YpCO92_NC_003143_s2
Ber10a_XIV.63.21s2L2.
Ber10a_XIV.63.21s2U2
Ber10a_XVI.69.23s2U2
Ber10a_XVI.69.23s2L2
Ber36b_XVI.69.21s2U2
Ber36b_XVI.69.21s2L2
Ber36b_XVI.70.10s2L2
Ber36b_XVI.70.10s2U2
Ber45a_XVI.69.22s2U2
Ber45a_XVI.69.22s2L2
Ber45a_XVI.70.11s2U2
Ber45a_XVI.70.11s2L2
Ber13b_XVI.70.9s2U2
Ber13b_XVI.70.9s2L2
Her7a_XV.70.12s2L2
Her7a_XV.70.12s2U2
Her21a_XV.70.13s2L2
SLC1006b_XXIX.114.8s2L2
T
.
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.
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.
-
B
YpCO92_NC_003143_s7
Ber10a_XVI.94.9_s7U4
Ber10a_XVI.94.9_s7L4
Ber45b_XVIII.84.9s7L4
Ber45b_XVIII.84.9s7U4
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C
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360
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YpCO92_NC_003143_s11
Ber10a_XIV.79.1s11l
Ber10a_XIV.79.1s11u
Ber36b_XVI.79.4s11l
Ber36b_XVI.79.4s11u
Ber45a_XVI.79.5s11l
Ber45a_XVI.79.5s11u
Ber45b_XVII.79.6s11l
Ber45b_XVII.79.6s11u
Her21a_XV.79.8s11l
Her21a_XV.79.8s11u
SLC1006b_XXIX.109.21s11u
SLC1006b_XXIX.109.21s11l
SLC1006b_XXIX.113.11s11l
SLC1006b_XXIX.113.11s11u
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900
T
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Y
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C
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Y
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F
Yp CO92_NC_003143_s14
G
Ber10a_XIV.66.1s14L1
.
Ber10a_XIV.66.1s14U1
.
Ber10a_XVI.69.5s14U1
.
Ber10a_XVI.69.5s14L1
.
Ber13b_XVI.69.2s14L1
.
Ber36b_XVI.69.3s14U1
.
Ber36b_XVI.69.3s14L1
.
Ber36b_XVI.71.3s14L1
.
Ber36b_XVI.71.3s14U1
.
Ber45a_XVI.69.4s14U1
.
Ber45a_XVI.71.4s14L1
.
Ber45a_XVI.71.4s14U1
.
Ber45a_XVI.114.12s14U1 .
Ber45a_XVI.114.12s14L1 .
Her21a_XV.67.11s14U1
.
Her21a_XV.67.11s14L1
.
Her21a_XV.71.6s14U1
.
Her21a_XV.71.6s14L1
.
Her21a_XV.114.13s14L1
.
SLC1006b_XXIX.109.5s14L1 .
SLC1006b_XXIX.109.5s14U1 .
T
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2120
400
390
380
370 2130
360
YpCO92_NC_003143_s12
G A A G A T T C A C A T C T G G G T G A T T
CA GA A GA GT T GGCGGA A CGGA T GT T GA T GCCGGA A GA T A A GA T CCGT A A A GT G
Ber10a_XIV.79.10s12l
. . . . . . . . . . . . . . . . . . . . . .
. .
Ber10a_XIV.79.10s12u
. . . . . . . . . . . . . . . . . . . . . .
Ber13b_XVI.79.11s12l
. . . . . . . . . . . . . . . . . . . . . .
. .
Ber13b_XVI.79.11s12u
. . . . . . . . . . . . . . . . . . . . . .
. .
Ber36b_XVI.79.13s12l
. . . . . . . . . . . . . . . . . . . . . .
. .
Ber36b_XVI.79.13s12u
. . . . . . . . . . . . . . . . . . . . . .
. . Ber37b_XXV.116.3s12u
. . . . . K . . . . . . . . . . . . . . . .
. . Ber37b_XXV.116.3s12l
. . . . . Y . . . . . . . . . . . . . . C .
. . Ber45a_XVI.79.14s12l
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. R Ber45b_XVIII.79.15s12l
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. . Ber45a_XVI.81.2s12l
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. . Her7a_XV.79.16s12l
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SLC1006b_XXIX.109.15s12l
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T
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.
.
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A
.
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.
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.
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G
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A
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A
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C
.
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A
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Y
T
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.
Text S1
13
Figure S6: Alignment of the sequences encompassing five SNPs defining branch 2. The relevant codons are marked
with red rectangles. A-D: branch 2 specific SNPs s15 (CAT), s17 (ACT), s18 (AAA) and s19 (CTA). E: s20 (GGT) defining
2.MED.
Text S1
14
Table S1. Results of the RDT analysis. Code numbers for the samples, the results of the RDT for plague and the AgF1
concentrations are indicated. For the samples from Parma, the corresponding code used for genetic analysis is reported in
parentheses.
Archaeological site
Parma (Italy)
Augsburg (Germany)
Bergen op Zoom
(The Netherlands)
Hereford
(England)
Saint-Laurent-de-laCabrerisse (France)
Negative controls
Code number
PR pit 1US 18
PR pit 1US 19 (PAR 119)
PR pit 1US 20 (PAR 120)
PR pit 2 US 202 ind. 01
PR pit 2 US 204 ind. 04
PR pit 3 US 5 ind. 15 (PAR 315)
PR pit 3 US 19 ind.02
PR pit 3 US 20 ind. 11 (PAR 311)
PR pit 3 US 32 ind.17 (PAR 317)
PR pit 3 US 53 ind. 5
PR pit 3 US 55 ind. 9
PR pit 3 US 64 ind. 08 (PAR 308)
PR pit 3 US 67 ind .06
PR pit 3 US 68 ind. 13
PR pit 3 US 69 ind. 07
PR pit 3 US 78 ind. 10
PR pit 3 US 93 ind. 03 (PAR 303)
PR pit 3 US 97 ind. 16 (PAR 316)
PR pit 3 US 99 ind. 14
Man 3
Man 37
Man 9
Man 38
Man 30
Man 2
Man 41
BER 1
BER 2
BER 3
BER 6
BER 8
Her 7a
Her 23a
Her 24a
Her 27a
Her 28a
Her 29a
Her 30a
SLC 1006
SLC 1010
SLC 1013
SLC 1014
SLC 1080
SLC 1081
SLC 1082
SLC 1083
SLC 1084
Bös 844
Bös 842
SLC 128
SLC 136
SLC 156
SLC 370
SLC 144
SLC 367
BNK1
BNK2
BNK3
BNK4
RDT bone
positive
positive
negative
positive
positive
negative
negative
positive
negative
negative
negative
negative
negative
negative
negative
negative
negative
positive
negative
positive
positive
positive
positive
negative
negative
negative
positive
negative
positive
negative
positive
negative
positive
negative
negative
positive
positive
positive
positive
positive
positive
positive
positive
negative
negative
positive
positive
negative
negative
negative
negative
negative
negative
negative
negative
negative
negative
negative
negative
[AgF1] bone
>1.25-0.625 ng/ml
>1.25-0.625 ng/ml
absent
>0.625 ng/ml
>0.625 ng/ml
absent
absent
>0.625 ng/ml
absent
absent
absent
absent
absent
absent
absent
absent
absent
>0.625 ng/ml
absent
>0.625 ng/ml
>2.5-1.25 ng/ml
>1.25-0.625 ng/ml
>1.25-0.625 ng/ml
absent
absent
absent
>0.625 ng/ml
absent
>0.625 ng/ml
absent
>0.625 ng/ml
absent
>0.625 ng/ml
absent
absent
>1.25 ng/ml
>1.25-0.625 ng/ml
>1.25 ng/ml
>2.5-1.25 ng/ml
>0.625 ng/ml
>0.625 ng/ml
>0.625 ng/ml
>1.25-0.625 ng/ml
absent
absent
>1.25-0.625 ng/ml
>0.625 ng/ml
absent
absent
absent
absent
absent
absent
absent
absent
absent
absent
absent
absent
Text S1
BNK5
BNK6
BNK7
BNK8
BNK9
BNK10
BNK11
BNK12
BNK13
BNK14
BNK15
BNK16
BNK17
BNK18
BNK19
BNK20
negative
negative
negative
negative
negative
negative
negative
negative
negative
negative
negative
negative
negative
negative
negative
negative
absent
absent
absent
absent
absent
absent
absent
absent
absent
absent
absent
absent
absent
absent
absent
absent
15
Text S1
16
Table S2. Primers used in this study. Sequences are given in 5’3’. PCR product length is given with and without primers (in
parentheses). Note: primers for glpD by Drancourt et al. (2007; reference [14] in Text) are designed on Y. pestis Orientalis with
the 93bp deletion, while primers gU3/gL2 and gU4/gL4 were designed on the glpD-gene of Y. pestis Medievalis lacking the
deletion. *: 16S rDNA primers were used for amplification of the soil sample only.
Locus or
branch
pla
caf1
16S rDNA*
rpoB
glpD
napA
1.ORI
Primer pairs
YP12D
cagcaggatatcaggaaaca
YP11R
gcaagtccaatatatggcatag
YP11D
ctatgccatatattggacttgc
YP10R
gagccggatgtcttctcacg
caf1 F1
aaccagcccgcatcactctta
caf1 R1
atcacccgcggcatctgta
U2
aaataaccaccaattcactacaaaag
L2
tgagcgaacaaagaaatcctg
16S F3
acacggtccagactcctacg
16S R3
aaactcaaccccttcctcctc
rpoB F1
ttgacagcgataagggtaaaac
rpoB R1
caatgcacgcagaatgatg
rpoB F2
cgtaacggtagaggcgaagtg
rpoB R2
tgtggcatcagagtatcaaggtc
glpD-F3
cgctgtttcgaacattcaga
glpD-R3
ggccaaggcttcacttacca
gU3
tgtttcgaacattcagaggaaggtaa
gL2
tttggaactggcggaagacg
gU4
ggaaggtaacatggaaaccaaagact
gL4
cttccagcagcagtaccgacag
aggcggcggctggtcatac
U60
L60
s2 U2
gagccgatggggtgctacga
s2 L2
ctgcgcctaccaagactcgtt
s7 U4
aatctggagcgttggtttgagaag
s7 L4
s11 U1
aggcatgtgggttggcaatg
s11 L1
ccattaacaaactcaaccgtatctca
s12 U1
agagtccagcggtagctccagagt
s12 L1
ggaaaccccgattggtgatgac
s13 U4
ctggcctgttgcgtcatcagta
s13 L4
s14 U1
cgaaaatgggtgtaaaagcgaaat
s14 L1
ccgtaatgttcccttcttggatag
s15 U2
tgcacgagcatcactttgtaa
s15 L2
s17 U1
acaggctaaagcagagcacc
s17 U2
acaagccgcaatcaatgagtc
s18 U4
aggtgtgggaatagctcaaaatgtt
s18 L5
s19 L3
ttacggttaatgggattgctgtta
s19 U3
ggatgtggatcgggactttc
s20 L1
ggcgcgtcacattgaatggtatt
s20 U1
s29U1
ccgggaactcagagcacaga
ttcccttgatcaacggtcatacc
s29L1
s31U1
ggtttatcgagtgggatcttcagttc
Branch 1
Branch 2
2.MED
0.PE2.b
0.PE2
Sequence
5’ - 3’
ggatggcaccttggaacagat
atccgttccgccaactcttc
ggtgaggtagtcgtcgtttgtg
aatacgcgcaaaattgttctga
atcttgtggcagattggcatc
gaagccgtacagatcgtgtttttc
Annealing-temperature
Product length
Source
55°C
148bp (106bp)
Raoult
et al. 2000
(ref. [5] in
Text)
56°C
161bp (121bp)
this study
55°C
139bp (92bp)
this study
58°C
138bp (97bp)
this study
55-56°C
170bp (129bp)
this study
55-57°C
164bp (120bp)
this study
54, 56, 58°C
144 bp (104bp)
on 1.ORI
Drancourt
et al. 2007
55°C
167bp (122bp)
on 2.MED excluding deletion
this study
59°C
110bp (62bp)
on 2.MED excluding deletion
this study
60-62°C
113bp (74bp)
this study
55-62°C
92bp (50bp)
this study
57°C
130bp (86bp)
this study
56/62°C
85bp (39bp)
this study
59-60°C
82bp (36bp)
this study
59°C
104bp (58bp)
this study
57°C
100bp (56bp)
this study
53°C
130bp (89bp)
this study
55°C
102bp (59bp)
this study
57°C
103bp (54bp)
this study
55°C
80bp (39bp)
this study
60°C
92bp (49bp)
this study
57-58°C
97bp (48bp)
this study
58°C
118bp (70bp)
this study
Text S1
Branch 0
Branch 0
just before
the split of
branches
1 and 2
s31L1
tgaggggtttgagctaggtgatag
s81U5
gatcgtggtcagatggtcgc
cgttgagcggtgtacggatt
aagtgaaggagcgcactctgg
s81U7
s81L1
s82U1
acagcttggggatgcttatcttc
s82L1
tgagtagcaggctttgcgagag
s87U1
tatgggtaaataccgcctgaat
s87L1
aagcgattgtatttgcctatcat
U5/L1: 58°C
U7/L1: 57-60°C
U5/L1: 89bp (48bp)
this study
U7/L1: 117bp (75bp)
57°C
87bp (42bp)
this study
52-54°C
93bp (48bp)
this study
17
Text S1
18
Table S3. Amplified products obtained from the various samples analysed, using different sets of primers. Amplification
success rate is given as relative frequency based on the number of attempts. Samples from the same locality are listed together.
A
Tooth
XXIBB
XII
Ber1
B
Tooth
Ber2
B
Tooth
Ber3
Ber4
Ber5
B
Tooth
A
Tooth
Ber6
Ber7
B
Tooth
A
Tooth
XXBB
1/1
B
Tooth
A
Tooth
Ber9
B
Tooth
C
Tooth
0/1
XII
0/2
XVI
0/1
XXIBB
0/1
0/3
XXIBB
0/1
XII
0/1
XIV
0/3
XXIBB
0/1
Ber10
Ber11
Ber12
Ber13
B
Tooth
C
Tooth
A
Tooth
B
Tooth
A
Tooth
A
Tooth
B
Tooth
s87
s82
s81
s31
s29
s20
s19
s18
s17
s15
s14
s13
s12
s11
s7
s2
glpD-F3/R3
glpD gU3/gL2
glpD gU4/gL4
napA 613
aspA
0/1
0/1
0/2
0/2
0/1
0/1
0/1
0/1
0/2
0/2
1/1
1/1
1/1
1/1
0/2
0/2
1/2
0/2
0/1
XIV
0/3
XXBB
0/1
XIII
0/1
XIV
0/3
XXBB
0/1
0/2
XIV
0/3
XXBB
1/1
XV
0/1
XII
0/2
XIV
0/3
XVI
0/1
XXBB
1/1
1/1
XVI
XII
0/1
0/2
XIV
XIV
A
Tooth
0/1
0/1
XV
XII
Ber8
rpoB2
0/2
0/3
XII
B
Tooth
A
Tooth
rpoB1
0/3
XIV
XII
B
Tooth
A
Tooth
caf1 U2/L2
0/2
XIII
A
Tooth
B
Tooth
C
Tooth
A
Tooth
caf1 F1/R1
0/1
XIV
XII
YP11D/YP10R
YP12D/YP11R
Sample
Extraction
(BK) indicates negative controls.
3/3
1/1
0/1
2/2
1/2
2/3
1/1
1/1
1/1
1/1
1/1
1/1
1/1
1/1
1/1
1/1
0/1
1/1
1/1
1/1
1/1
1/1
0/1
1/1
1/1
1/1
1/1
0/1
1/1
1/3
0/1
0/2
XIV
0/3
XVI
0/1
XVI
0/1
XXII
0/1
XVI
1/1
XXIII
1/1
XVI
1/1
0/1
0/1
0/1
0/2
1/2
0/1
0/1
1/1
0/2
1/2
1/2
0/1
1/1
1/2
1/2
0/1
2/2
1/1
Text S1
Ber14
Ber15
Ber16
Ber17
Ber18
Ber19
Ber20
Ber21
Ber22
Ber24
Ber25
Ber26
Ber27
Ber28
Ber29
Ber30
Ber31
Ber32
Ber33
Ber34
Ber35
Ber36
Ber37
Ber38
Ber39
Ber40
Ber41
Ber42
Ber43
Ber45
BNK1
(BK)
BNK2
(BK)
BNK3
(BK)
BNK4
(BK)
A
Tooth
A
Tooth
B
Tooth
A
Tooth
B
Tooth
A
Tooth
A
Tooth
A
Tooth
A
Tooth
B
Tooth
A
Tooth
A
Tooth
B
Tooth
A
Tooth
A
Tooth
B
Tooth
A
Tooth
A
Tooth
B
Tooth
B
Tooth
A
Tooth
A
Tooth
B
Tooth
A
Tooth
A
Tooth
B
Tooth
A
Tooth
B
Tooth
A
Tooth
B
Tooth
B
Tooth
A
Tooth
A
Tooth
B
Tooth
B
Tooth
B
Tooth
A
Tooth
A
Tooth
A
Tooth
B
Tooth
A
Tooth
B
Tooth
A
Tooth
B
Tooth
A
Tooth
B
Tooth
A
Tooth
B
Tooth
XVI
0/1
XXIV
0/1
XVII
0/1
XXIII
XVII
0/1
1/1
XXIV
0/1
0/1
XVIII
0/1
XVII
0/1
XVII
0/1
XXII
0/1
XVII
0/1
XVIII
0/1
XXIV
0/1
XVI
0/1
0/1
XXIV
0/1
XXIII
0/1
XVIII
0/1
XXIII
0/1
XXIV
0/1
0/1
XXIII
0/1
XVI
0/1
XXIII
0/1
0/1
XXIII
0/1
XVI
1/1
XXIV
1/1
XXV
1/1
XXII
0/1
XVIII
0/1
XVII
0/1
XVI
1/1
1/1
0/1
0/2
0/1
2/2
1/1
2/2
1/1
2/2
1/1
2/2
3/3
1/1
1/1
1/3
1/1
2/2
2/2
1/1
1/1
3/3
1/1
1/1
0/1
0/1
XXV
0/1
XVI
XVII
1/2
1/1
0/1
XVIII
XVII
1/1
0/1
0/1
XXIV
XVIII
0/1
0/1
XXIV
XVII
0/1
0/1
XVII
XVII
19
1/1
1/1
1/1
1/1
1/1
2/2
2/2
1/1
2/2
1/1
1/1
1/1
1/1
1/1
1/1
1/1
XLI
0/2
0/1
0/1
XLII
0/2
0/1
0/1
XLI
0/3
0/1
0/1
XLII
0/2
0/1
0/1
XLI
0/2
0/1
0/1
XLII
0/2
0/1
0/1
XLI
0/2
0/1
0/1
XLII
0/2
0/1
0/1
1/1
1/1
Text S1
BNK5
(BK)
BNK6
(BK)
BNK7
(BK)
BNK8
(BK)
BNK9
(BK)
BNK10
(BK)
BNK11
(BK)
BNK12
(BK)
BNK13
(BK)
BNK14
(BK)
BNK15
(BK)
BNK16
(BK)
BNK17
(BK)
BNK18
(BK)
BNK19
(BK)
BNK20
(BK)
Her2
Her5
Her7
Her8
Her17
Her18
Her21
A
Tooth
B
Tooth
A
Tooth
B
Tooth
A
Tooth
B
Tooth
A
Tooth
B
Tooth
A
Tooth
B
Tooth
A
Tooth
B
Tooth
A
Tooth
B
Tooth
A
Tooth
B
Tooth
A
Tooth
B
Tooth
A
Tooth
B
Tooth
A
Tooth
B
Tooth
A
Tooth
B
Tooth
A
Tooth
B
Tooth
A
Tooth
B
Tooth
A
Tooth
B
Tooth
A
Tooth
B
Tooth
A
Tooth
A
Tooth
A
Tooth
A
Tooth
A
Tooth
A
Tooth
A
Tooth
B
Tooth
Her24
A
Tooth
A
Tooth
A
Tooth
Her25
A
Tooth
Her22
Her23
XLI
0/2
0/1
0/1
XLII
0/2
0/1
0/1
XLI
0/2
0/1
0/1
XLII
0/2
0/1
0/1
XLI
0/2
0/1
0/1
XLII
0/2
0/1
0/1
XLI
0/2
0/1
0/1
XLII
0/2
0/1
0/1
XLI
0/2
0/1
0/2
XLII
0/2
0/1
0/1
XLI
0/2
0/1
0/2
XLII
0/3
0/1
0/1
XLI
0/2
0/1
0/1
XLII
0/2
0/1
0/1
XLI
0/2
0/1
0/1
XLII
0/2
0/1
0/1
XLI
0/2
0/1
0/1
XLII
0/2
0/1
0/1
XLI
0/2
0/1
0/1
XLII
0/2
0/1
0/1
XLI
0/2
0/1
0/1
XLII
0/2
0/1
0/1
XLI
0/2
0/1
0/1
XLII
0/2
0/1
0/1
XLII
0/2
0/1
0/1
XLII
0/2
0/1
0/1
XLII
0/2
0/1
0/1
XLII
0/2
0/1
0/1
0/2
0/1
0/1
XLII
0/2
0/1
0/1
XLII
0/2
0/1
0/1
XLII
0/2
0/1
0/1
XLII
0/1
0/1
0/1
0/1
0/1
XVIIBB
0/1
0/1
0/1
XVIIBB
0/1
0/1
0/1
XVIIIBB
1/1
1/1
0/1
XV
1/1
XXI
1/2
2/3
XVIIIBB
0/1
0/1
0/1
XVIIBB
0/1
0/1
0/1
XVIIBB
0/1
0/1
0/1
XXIVBB
1/1
1/1
0/1
XV
1/1
1/1
1/3
XXI
0/1
0/4
0/2
XXIVBB
0/1
0/1
XXI
0/1
0/4
XXIVBB
0/1
0/1
0/1
XXIVBB
0/1
0/1
0/1
XXIVBB
0/1
0/1
0/1
XXI
0/1
0/4
0/1
0/1
0/1
0/2
4/4
0/3
1/1
1/2
1/1
0/3
0/2
0/3
20
2/2
1/1
2/2
1/1
1/2
0/1
0/1
0/1
2/2
1/2
1/1
1/1
0/3
1/1
0/1
2/2
1/1
1/2
1/1
2/2
0/1
0/2
1/1
2/2
2/2
1/1
1/1
0/3
1/1
1/1
0/2
0/1
0/1
0/1
1/1
0/3
0/1
Text S1
Her26
SLC
1006
SLC
1010
SLC
1013
SLC
1080
SLC
1081
SLC
1083
SLC
128
(BK)
SLC
136
(BK)
SLC
156
(BK)
SCL
370
(BK)
SLC
144
(BK)
SLC
367
(BK)
PAR
311
PAR
316
PAR
119
PAR
120
PAR
308
PAR
317
PAR
303
PAR
315
A
Tooth
A
Tooth
B
Tooth
A
Tooth
B
Tooth
A
Tooth
B
Tooth
A
Tooth
A
Tooth
B
Tooth
A
Tooth
A
Tooth
B
Tooth
A
Tooth
B
Tooth
A
Tooth
B
Tooth
A
Tooth
B
Tooth
A
Tooth
B
Tooth
A
Tooth
B
Tooth
A
Bone
A
Bone
A
Bone
A
Bone
A
Bone
A
Bone
A
Bone
A
Bone
A
Tooth
XXIVBB
0/1
0/1
XXI
0/1
0/4
XXVIII
0/1
XXIX
2/2
XXVIII
0/1
XXIX
0/1
XXVIII
0/1
XXIX
0/1
XXVIII
0/1
XXXI
0/1
XXIX
0/1
XXIX
0/1
XXXI
0/1
XXIX
0/1
XXVIII
0/1
XXIX
0/1
XXVIII
0/1
XXIX
0/1
XXVIII
0/1
XXIX
0/1
XXXI
0/1
XXIX
0/1
XXVIII
0/1
XXIX
0/1
VIII
1/1
0/2
0/3
0/1
0/4
0/1
0/1
0/1
0/2
0/3
0/1
0/4
0/1
0/1
0/1
0/2
0/3
0/1
0/4
0/1
0/2
0/3
0/1
0/4
0/1
0/2
0/3
0/1
0/4
VIII
0/1
0/2
0/3
0/1
0/4
VIII
0/1
0/2
0/3
0/1
0/4
VIII
0/1
0/1
VIII
0/1
0/1
0/1
0/2
0/3
0/1
0/4
VIII
0/1
0/1
0/1
0/2
0/2
0/4
0/3
0/3
VII
0/1
0/1
0/4
0/7
0/7
0/1
0/5
0/1
II
0/3
III
Man3
A
Bone
VII
0/4
0/1
0/1
I
0/4
IV
A
Bone
VI
0/1
Man30
A
Bone
VII
0/4
0/1
0/1
II
III
0/5
0/3
0/7
0/6
III
Man9
0/7
0/6
0/1
0/1
I
A
Bone
0/7
0/3
III
Man37
1/4
VIII
III
Man38
2/2
0/1
II
B
Bone
0/4
VIII
VIII
21
0/1
0/3
0/3
0/7
0/7
0/5
0/3
0/3
0/4
0/7
0/7
1/2
2/2
2/2
2/2
1/1
2/2
1/1
1/2
1/1
Text S1
VI
0/1
0/1
0/5
II
0/3
III
Man2
A
Bone
VII
0/4
0/1
0/1
II
A
Bone
VII
0/7
0/6
0/5
0/3
0/3
V
Man41
22
0/3
0/4
0/1
0/1
I
0/8
0/7
0/1
0/1
0/3
II
Bös
844
(BK)
III
BK1
Bone
VII
0/4
0/1
0/1
I
VII
0/3
0/4
IV
BK2
Bone
0/7
0/3
0/3
III
Bös
842
(BK)
0/7
0/1
0/1
0/7
0/6
0/3
0/3
Table S4: Summary of the results and test of the hypothesis of false negatives among the negative
controls. The significant tests were calculated on contingency tables obtained from the number of
individuals investigated for the pla-gene (see Table S3). Since bone samples did not appear to contain
enough bacterial DNA, for the PCR analyses only results from tooth samples were considered.
RDTanalysis
PCRanalysis
F1-positive
F1-negative
pla-positive
pla-negative
Cases
Controls
24
23
10
51
0
28
0
26
Fisher’s exact
test
P 0.0001
P 0.0225