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SUPPLEMENTARY DATA
Paleopathological analysis
The distinction between classical and atypical or early-stage TB cases is shown in the figures below.
Legend to Figure S1
Classical TB cases
a) Tuberculous spondylitis healed with gibbus formation (L1-L3) - Grave No. 65. (female,
mature)
b) Severe destruction of the 3rd vertebral body (inferior view) - Grave No. 65. (female,
mature)
c) Lumbosacral tuberculosis: severe erosion of the ventral sacral surface (traces of cold
abscess) - Grave No. 90. (male, elderly)
d) Coxitis tuberculosa: complete destruction and remodelling of the acetabulum - Grave
No. 90. (male, elderly)
1
a) b)
c) d)
2
Legend to Figure S2
Atypical or early-stage TB changes
a) Periosteal apposition on the visceral costal surface – Grave No. 88. (female, mature)
b) Maze like surface excavation (serpens endocrania symmetrica)- Grave No. 233.
(female, young adult)
c) Abnormal vertebral vascularisation - Grave No. 92. (male, young adult)
3
a) b)
c)
4
5
Details of DNA extraction
(a) Disaggregation of samples and DNA extraction
A small quantity (22–78mg) of each sample was crushed by a sterile pestle in a mortar and
added to 400μl of Proteinase K/EDTA. Samples were processed in batches of 7 plus a
negative extraction control. The slurry was incubated at 56ºC24, and mixed on a bead beater
daily. When the sample was solubilised, it was divided and one aliquot treated with 40μl of
0.1mol 1‒ of N-phenacylthiozolium bromide (PTB), to cleave any covalent cross-links thus
enabling DNA strand separation and amplification21. Sample tube contents were transferred
into separate 9ml tubes of NucliSens® (bioMérieux) lysis buffer containing 5mol 1‒ guanidium
thiocyanate and incubated for 1–3 days at 56ºC. To complete the disruption of bone and any
mycobacterial cell wall remnants, samples were boiled, then snap-frozen in liquid nitrogen
and thawed in a 65ºC waterbath. This procedure was repeated twice. Sample tubes were
centrifuged at 5000g for 15 min at 5ºC and the supernatants carefully removed into clean,
sterile tubes. DNA was captured by adding 40μl silica suspension (NucliSens®) and mixing
on a rotator wheel for 1 h. Tube contents were centrifuged and silica pellets washed once with
wash buffer (NucliSens®), twice with 70% (v/v) ethanol ( 20ºC) and once with acetone (‒ ‒
20ºC). After drying in a heated block, DNA was eluted using 60μl elution buffer
(NucliSens®), aliquoted and used immediately or stored at 20ºC. Silica supernates (500μl)‒
from PTB-negative samples were also collected from the 9ml tubes of lysis buffer, and the
2.0ml screw-capped Eppendorf tubes used to wash the silica. After chilling at 5ºC, supernates
were mixed vigorously for 20 s with 200μl of Protein Precipitation Solution (SLS Ltd., UK)
and centrifuged for 3min at 10,000g. Any pellet was discarded and 600μl isopropanol ( 20ºC)‒
added to 550μl of each supernate. Tubes were mixed by inversion 50 times and spun 3min.
Supernates were discarded and tubes washed once with 500μl 70% ethanol ( 20ºC). After‒
draining, tubes were dried in a heating block. Any precipitated DNA was re-hydrated with
60μl elution buffer (NucliSens®), aliquoted and used immediately or stored at 20ºC.‒
Negative extraction controls were processed in parallel with the test samples.
(b) DNA amplification and detection
Two specific regions of the MTBC were targeted in the repetitive elements IS6110 (1–25
copies/cell) and IS1081 (6 copies/cell). The IS6110 primers had a target region of 123 bp22
and the IS1081 primers designed by Taylor et al.23 produce an amplicon of 113 bp. Later,
6
specific M. tuberculosis primers and a fluorescent probe were used24 to enable shorter DNA
fragments to be detected in a real-time PCR reaction.
(c) The PCR conditions
The PCR mix included 2mM bovine serum albumin (BSA) to reduce PCR inhibition25 and
2.0mM MgCl2. PCR assays were initially run at an annealing temperature of 58ºC and
amplified DNA was examined by agarose gel electrophoresis.26 Subsequently, amplification
was performed in a final volume of 25μl using a RotorGene© 3000 (Qiagen) real-time
platform27, to enable the detection of DNA using SYBR Green and melt analysis or specific
primers with fluorescent probe. Annealing was at 60ºC. A hot-start Taq polymerase was used
to minimize non-specific primer and template binding. Negative DNA extraction and PCR
controls were processed alongside the test samples.
(d) Results
Single-stage PCRs with outer primer pairs
Gel with IS6110 PCR products Gel with IS1081 PCR products
7
Key to abbreviations:
EC = negative extraction control; s = silica supernate (fluid left in 2 ml tubes after silica spun
down, normally short aDNA fragments); LVs = large volume silica supernate (fluid left in 9
ml lysis buffer tubes after silica spun down, short aDNA fragments); wb = water blank
negative control in PCR.
Lanes (left to right): 1: Phi X-174 HaeIII markers; 11: 20bp and 100bp molecular markers
Top row: 2: +ve control; 3: BC-12s; 4: BC-12 LVs; 5: wb1; 6: BC-17s; 7: BC-17 LVs; 8: BC-
22s; 9: wb2; 10: BC-22 LVs;
Bottom row: 2: +ve control; 3: BC-33s; 4: BC-33 LVs; 5: BC-38s; 6: ECs; 7: BC-38 LVs; 8:
BC-48s; 9: BC-48 LVs; 10: EC LVs;
Conclusions:
IS6110: possible weak positives with BC-12 LVs and BC-17 LVs. Positive with BC-33s. Non-
specific bands from BC-12s, BC-22s, BC-33 LVs, and BC-48 LVs. Others negative.
IS1081: positive with BC-12 LVs. All other samples (except positive controls) were negative
Single-stage IS 1081 PCRs using inner primers (113 bp)
Lanes (left to right): 11: 20bp and 100bp molecular markers
Top row: Lane 1: +ve control; 2: BC-12; 3: wb5; 4: BC-12+; 5: BC-17; 6: wb6; 7: BC-17+; 8:
BC-22; 9: BC22+; 10: wb7.
Bottom row: Lane 1: BC-33; 2: BC-33+; 3: BC-48; 4: wb8; 5: BC-48+; 6: EC; (lanes 7–10:
different samples and another PCR)
Conclusions:
Positives from samples BC-12, BC-12+, BC-17, BC-17+, BC-48.
Doubtful results from BC-22, BC-22+ (very faint trace) and BC-48+.
Negatives from BC-33 and 33+, and all water blanks.
BC-51 was examined separately for MTB IS1081 but was negative.
Nested IS 6110 PCRs using inner primer pair
Samples loaded in the same order as above, using the stage 1 PCR products that were re-
amplified for a further 25 cycles.
Conclusions:
Positive and negative controls were satisfactory. Only BC-33s was positive.
Real-time experiments were also carried out with the same primers and melt analysis. Results
are summarized at the end of the document.
Real-time PCR with IS 1081 primers and probe
The lower the cycle threshold (Ct) the greater the quantity of target aDNA in the sample. In
this image, the positive samples in order of their Ct was as follows:
Positive control (a 1/10 dilution of extract from a Vác mummy) Ct 32 cycles
BC-189+ (+ indicates the DNA was extracted using PTB) Ct 39 cycles
Negatives were obtained from BC-12, BC-22, BC-116+, BC-116, BC-134+, BC-134, BC-
134s, BC-188+, BC-188, BC-189, BC-215, wb1, wb2, wb3, wb4, EC, EC1, EC+, ECs
In this experiment a nested PCR was performed on the PCR product from BC-189+ which
explains the high level of signal at the start of the reaction. The positive control had a Ct of 28
and sample BC-92 had a Ct of 41.
Negatives were obtained from BC-22, BC-65+, BC-65, BC-66+, BC-66, BC86+, BC-86, BC-
88+, BC-88, BC-90+, BC-90, BC-92+, BC-154+, BC-154, wb1, wb2, wb3, wb4, EC+, EC.
The positive control had a Ct of 28.6, BC-66s: 36.6, BC-86s: 36.9, BC-92: 36.0, BC-92s:
36.3, and BC-154: 36.1. Individual screenshots are available for each positive sample.
Negative results were obtained from BC-65s, BC-86s, BC-90s, wb, EC and ECs.
These results were confirmed by agarose gel electrophoresis.
Overall findings for M. tuberculosis complex in these samples
Positives with one or both target regions:
BC-12, BC-17, BC-33, BC-48, BC-66, BC-88, BC-92, BC-154, BC-189
Negative (but cannot exclude poor preservation):
BC-22, BC-38, BC-65, BC-86, BC-90, BC-116, BC-134, BC-188, BC-212, BC-215, BC-233
Analysis of mycolipenate and mycocerosates
The initial analyses were performed at Bristol University, using a ThermoFinnigan
MAT95 XP-Trap mass spectrometer, fitted with a Phenomenex Zebron ZB-5 (5%
phenyl, 95% dimethylpolysiloxane) capillary column (30 m × 0.25 mm i.d. × 0.25 µm
film thickness) using He as carrier gas (constant flow mode 1ml min-1) and ammonia
as the CI reagent gas. A GC oven temperature gradient from 200 to 300ºC at 6.7ºC
min-1 was used, the final temperature being held for 20 min. The ion source
temperature was 250ºC, the injector 300ºC and the transfer line 300ºC. Selected ion
monitoring (SIM) was used for mycocerosate ions at m/z 367.6311 (C24), 395.6844
(C26), 409.7111 (C27), 437.7645 (C29), 451.7911 (C30), 479.8445 (C32), 493.8712 (C33)
and 507.8978 (C34); additionally, m/z 407.6952 was monitored for the presence of C27
mycolipenic acid. Later studies were carried out at Swansea University with the same
Phenonemex Zebron ZB-5 column, using He as carrier gas. PFB esters, on NICI-
GCMS, fragment to produce negative carboxylate [M – H]- ions, which can be
detected at high sensitivity. Selected ion monitoring (SIM) was used to search for
mycocerosate carboxylate ions at m/z 367.6311 (C24), 395.6844 (C26), 409.7111 (C27),
437.7645 (C29), 451.7911 (C30), 479.8445 (C32), 493.8712 (C33) and 507.8978 (C34).29
Additionally, m/z 407.6952 was monitored for the presence of the C27 mycolipenate
carboxylate ion.29 Partial racemisation of mycocerosates during the alkaline
hydrolysis leads to the formation of diasteroisomers, which resolve on gas
chromatography to give characteristic doublets; in contrast, mycolipenates are singlets
as they cannot racemise.29
Legend to Figure S3
Selected ion monitoring (SIM) negative ion chemical ionisation (NI-CI) gas
chromatography mass spectrometry (GC-MS) of pentafluorobenzyl esters (Swansea
University). A. Standard M. leprae (Swansea University); B-H. Samples extracted
from graves (BC) 22, 12, 17, 33, 38, 65, 66, respectively. Intensities (bold in
brackets) are normalised to the major component (100).
10 15 20 Minutes
10 15 20 Minutes
0
50
0
50
15.41
15.53
0
10
23.07
20.43
1
3
12.02
2
50
100
13.20
10.06
50
100
0
50
100
0
17.20
(95)
(100)
(5)
(12)
(3)
Figure S3
13.20
C27 m/z 407
C27 m/z 409
C29 m/z 437
C30 m/z 451
C32 m/z 479
C33 m/z 493
C34 m/z 507
Relative Abundance
21.37
0
0
(0)
(0)
0
15.41
0
20
(29)
0
1
12.02
(28)
0
50
10.06
2
4
6
11.95
50
100
13.20
(100)
(0)
(32)
19.87
17.20
13.20
C27 m/z 407
C27 m/z 409
C29 m/z 437
C30 m/z 451
C32 m/z 479
10 15 20 Minutes
0
Relative Abundance
10 15 20 Minutes
C27 m/z 407
C27 m/z 409
C29 m/z 437
C30 m/z 451
C32 m/z 479
0
50
100
0
50
100
13.22
17.22
19.91
10
20
10.06
(0)
Relative Abundance
13.22
0
(0)
0.0
0.1
0.2
12.02
0
20
40
15.43
(100)
(54)
(5)
0
50
100
0
50
100
0
50
100
0
50
100
13.20
10.06
17.20
20.83
0
1
12.02
(100)
(78)
19.87
13.20
C27 m/z 407
C27 m/z 409
C29 m/z 437
C30 m/z 451
C32 m/z 479
Relative Abundance
(0)
(0)
(0)
0
50
100
13.22
10.07
13.22
17.22
15.43
C27 m/z 407
C27 m/z 409
C29 m/z 437
C30 m/z 451
C32 m/z 479
0.0
0.5
12.02
(100)
(13)
(34)
10 15 20 Minutes
0
50
100
0
50
100
0
50
100
Relative Abundance
(0)
(0)
0
50
100
0
50
100
17.18
19.86
20.81
15.39
10 15 20 Minutes
0.0
0.5
12.00
10
20
10.04
100)
(16)
(44)
C27 m/z 407
C27 m/z 409
C29 m/z 437
C30 m/z 451
C32 m/z 479
13.06
0
Relative Abundance
(0)
0
50
100
13.18
17.18
20.81
(0)
19.84
C27 m/z 407
C27 m/z 409
C29 m/z 437
C30 m/z 451
C32 m/z 479
13.20
Relative Abundance
10 15 20 Minutes
(0)
(0)
0
50
100
0
50
100
0
50
100
0
50
100
0
100
9.67
C27 m/z 407
C27 m/z 409
C29 m/z 437
C30 m/z 451
C32 m/z 479
C33 m/z 493
C34 m/z 507
10 15 20 Minutes
Relative Abundance
(0)
(2)
11.92
(4)
12.00
(62)
15.39
(50)
15.51
(8)
20.41
(100)
0
100
0
100
0
100
0
100
0
100
0
100
A. M. leprae (Swansea)
B. BC 22
C. BC 12
D. BC 17
E. BC 33
F. BC 38
G. BC 65
H. BC 66
50
100
13.18
0
(0)
(0)
(0)
Legend to Figure S4
Selected ion monitoring (SIM) negative ion chemical ionisation (NI-CI) gas
chromatography mass spectrometry (GC-MS) of pentafluorobenzyl esters (Swansea
University). A. Standard M. tuberculosis (Strain C) (Swansea University); B-H.
Samples extracted from graves (BC) 116, 134, 154, 188, 189, 212, 215, respectively.
Intensities (bold in brackets) are normalised to the major component (100).
10 15 20 Minutes
10 15 20 Minutes
C27 m/z 407
C27 m/z 409
C29 m/z 437
C30 m/z 451
Figure S4
C32 m/z 479
17.18
10
20
10.06
0
C27 m/z 407
C27 m/z 409
C29 m/z 437
C30 m/z 451
C32 m/z 479
13.18
0
20
40
15.39
0.0
0.1
12.00
(100)
(23)
(32)
10 15 20 Minutes
0.0
0.5
11.72
(98)
19.86
0
10 15 20 Minutes
0
50
100
0
50
100
13.22
17.22
50
15.43
0
20
10.07
0.1
0.2
0.3
12.02
(94)
(42)
(100)
19.91
13.22
C27 m/z 407
C27 m/z 409
C29 m/z 437
C30 m/z 451
C32 m/z 479
Relative Abundance
0.0
(0)
(0)
0
50
100
13.20
17.20
19.86
10
20
10.06
(100)
C27 m/z 407
C27 m/z 409
C29 m/z 437
C30 m/z 451
C32 m/z 479
10 15 20 Minutes
Relative Abundance
n-27
13.22
20.81
0
50
100
0
50
100
0
50
100
0
(0)
(0)
(0)
(0)
0
50
100
20.83
15.41
(100)
10 15 20 Minutes
0
2
11.93
5
10
10.06
0.0
0.5
12.00
0
(92)
(0)
(60)
(32)
C27 m/z 407
C27 m/z 409
C29 m/z 437
C30 m/z 451
C32 m/z 479
Relative Abundance
19.87
17.20
13.20
13.08
0
0
20
15.41
0.0
0.1
0.2
12.02
0.2
0.4
0.6
11.93
0
50
10.06
0
50
100
13.20
(100)
(13)
(29)
(19)
13.20
17.20
19.87
(0)
0.0
Relative Abundance
0
50
100
13.20
17.18
19.87
22.74
C27 m/z 407
C27 m/z 409
C29 m/z 437
C30 m/z 451
C32 m/z 479
10 15 20 Minutes
Relative Abundance
50
100
0
50
100
0
50
100
0
(0)
(0)
(0)
(0)
0
10
20
0
10
20
10.06
15.41
0.0
0.4
0.6
12.02
19.87
(100)
(45)
(15)
22.76
100
0
50
100
17.20
13.20
13.20
50
0
0.2
C27 m/z 407
C27 m/z 409
C29 m/z 437
C30 m/z 451
C32 m/z 479
Relative Abundance
(0)
(0)
0
50
100
10.04
9.66
C27 m/z 407
C27 m/z 409
C29 m/z 437
C30 m/z 451
C32 m/z 479
(70)
10 15 20 Minutes
Relative Abundance
(3)
11.92
(33)
12.00
(20)
15.39
(100)
0
50
100
0
50
100
0
50
100
0
50
100
A. M. tuberculosis Strain C (Swansea)
B. BC 116
Relative Abundance
D. BC 154
C. BC 134
E. BC 188
F. BC 189
G. BC 212
H. BC 215
0
50
100
13.20
50
100
13.18
n-27
0
(0)
50
100
(0)
Legend to Figure S5
Selected ion monitoring (SIM) negative ion chemical ionisation (NI-CI) gas
chromatography mass spectrometry (GC-MS) of pentafluorobenzyl esters (Bristol
University). A-F. Samples extracted from graves (BC) 48, 86, 88, 90, 92, 233,
respectively. Intensities (bold in brackets) are normalised to the major component
(100).
0
50
100
Figure S5
0
50
100
0
26.31
24.14
15 20 25 30 Minutes
0.0
0.5
19.96
20
40
17.51
(100)
(0)
(84)
(30)
C27 m/z 407
C27 m/z 409
C29 m/z 437
C30 m/z 451
C32 m/z 479
0
Relative Abundance
(0)
0
50
100
17.52
21.47
26.33
29.55
16.56
24.16
27.83
33.35
2
16.97
1
2
3
19.97
(100)
(55)
(0)
(47)
(93)
15 20 25 30 Minutes
0
50
100
0
50
100
0
0
Relative Abundance
C27 m/z 407
C27 m/z 409
C29 m/z 437
C30 m/z 451
C32 m/z 479
0
0
0
5
0
5
0
19.85
19.97
50
100
21.45
17.50
50
100
24.12
15 20 25 30 Minutes
Relative Abundance
(49)
C27 m/z 407
C27 m/z 409
C29 m/z 437
C30 m/z 451
C32 m/z 479
(68)
(93)
(100)
26.30
29.52
100
100
100
Relative Abundance
50
50
50
17.51
26.32
24.12
0.0
0.5
1.0
1.5
19.98
C27 m/z 407
C27 m/z 409
C29 m/z 437
C30 m/z 451
C32 m/z 479
0
0
0
15 20 25 30 Minutes
(100)
(0)
(83)
(23)
0
0
50
100
17.52
26.33
29.55
24.14
1.5
19.98
(89)
(0)
(78)
(100)
(0)
15 20 25 30 Minutes
Relative Abundance
0
50
100
0
50
100
1.0
0.5
0.0
C27 m/z 407
C27 m/z 409
C29 m/z 437
C30 m/z 451
C32 m/z 479
0
50
17.50
100
24.13
0.0
0.5
1.0
19.96
100
26.30
C27 m/z 407
C27 m/z 409
C29 m/z 437
C30 m/z 451
C32 m/z 479
21.45
29.53
(85)
(100)
(15)
15 20 25 30 Minutes
Relative Abundance
(0)
0
0
A. BC 48
C. BC 88
E. BC 92
B. BC 86
D. BC 90
F. BC 233
50
100
21.46
50
21.45
100
(0)
50
100
21.47
50
100
21.46
0
100
21.45
(0)
(0)