Supplement Information (SI) - Online Material
Tracing Hepatitis B virus to the 16th century in a Korean mummy
Material
Sampling: An endoscopic examination of a mummified child from the 16th century AD excavated in
Yangju, Korea (1, 2) was performed in an operation room of Dankook University College hospital
using a sterilized fiber-optic endoscope (GIF XQ230, Olympus Co., Japan). Biopsies were taken from
the dry parenchymatous organ were subjected to microscopic and aDNA analysis. The liver biopsies
were immersed in sterile tubes to prevent contamination and were opened only in the ancient DNA
(aDNA) laboratories (3).
Tissue handling: Tissue handling and DNA extraction were performed in dedicated hoods which were
disinfected before and after use; disposables and all waste were autoclaved prior to discarding; nondisposable equipment was sterilized before and after use. Tissue samples surface was subjected to
sodium hypochchlorite 1,000ppm prior to the extraction to eliminate possible surface contamination.
Methods:
Identification of ancient HBV: The exploratory phase for HBV DNA detection was conducted
independently in three laboratories in Korea, Israel and the UK: In Korea, DNA was extracted using
phenol/chloroform/isoamyl alcohol (25:24:1) (4). Two gene regions the PreC/Core and the preS/S
were amplified cloned and sequenced using published primers (5, 6).
In Israel, specimens received from Korea were subjected to sodium hypochlorite 1000p.p.m in water
to destroy any contamination on the surface. DNA extractions were conducted using three methods:
commercial kits for DNA tissue extraction (Qiagen®, Germany and Machery-Nagel®, Germany) and
guanidinium thiocyanate (GuSCN) followed by silica capturing (7, 8). PCR amplification was
performed with HBV Monitor kit (Roche, USA) amplifying a PreC/C region of 245bp. In addition, a
short fragment of the PreS/S of 223bp was amplified using two published primer sets (9, 10). An HBV
Genotyping kit, INNO-LIPA HBV Genotyping Assay (Roche, USA) was used for amplification of a
fragment of the polymerase gene (11). Positive amplifications were directly sequenced and cloned.
Genetic profile of the mummy: Several attempts were carried out to amplify mitochondrial and
nuclear human DNA from liver and lung samples. The entire hypervariable region I (HVR-I) of the
human mitochondrial control region was amplified in two over-lapping fragments of 271-bp and
1
232-bp length following primers and PCR conditions described previously (12). The two
overlapping fragments (represented by 2-3 sequences obtained from different PCR attempts)
yielded a contiguous sequence with the following 2 substitutions relative to the CRS (13): 16223T
and 16362C. In addition the sequence of the lab technician (LH) differed from the mummy
sequence by 4 substitutions: 16223C — 16274A —16325C and 16362T supporting the authenticity
of the mummy sequence and lack of contamination. The obtained mummy HVR-I sequence
matches human haplogroup D, Asian origin, probably Japanese.
(http://www.bioanth.cam.ac.uk/mtDNA/toc.html).
DNA extracted from the same mummy lung and liver biopsies was further used to amplify nuclear
DNA using the commercial MiniFiler® kit of Short Tandem Repeat (STR) (Applied Biosystems).
Only a partial profile (7 out of 15 authosomal STR’s) was obtained due to degradation of the human
DNA. The determination of the Y chromosome STR (AMEL) confirms the initial morphologic
identification of the mummy as a male. Four alleles identified in the authosomal STR’s that were
successfully amplified, were not found in the positive control profile. The human genetic profile of
the mummy differed from that of the investigators (Table SI1). For three STR’s (D3S1358,
D1S1656, D21S11) only one allele was determined, causing difficulties to distinguish
homozygosity from heterozygosity in case of an allele drop. Moreover, the partial profile prevents
comparisons to other population profiles to determine the exact origin of the sample. Therefore, we
searched the Earth Human STR Allele Frequencies Database using the Most Probable Geographical
Origin model (http://www.ehstrafd.org/modules/MPGO) and found that the geographical origin
estimated for the partial profile is probably in Asia. The first 10 populations that were found with
high similarities are from India, China, Vietnam, South Korea and Japan. The identification of the
mummy as a male while all the investigators involved in the technical-laboratory work were
females together with the partial autosomal profile support the authenticity of the results and lack of
contamination.
Whole genome analysis: Once HBV was identified in the liver biopsies, we proceeded to determine
the complete genome sequence. Analysis was performed in a dedicated ancient DNA laboratory at a
different campus of the Hebrew University and in Korea.
Primer design: Overlapping primer sets were designed using Primer 3 software
(http://primer3.sourceforge.net/) to amplify the entire genome based on published HBV genomes
(Table SI2). Sequences obtained from the liver samples were used to design new primer sets to
amplify and sequence the overlapping regions (Table SI2). Overall, 44 primer sets, were used to
2
amplify and sequence the entire HBV genome. To increase the overlapping of the sequences, we
amplified larger fragments, using combined primer sets (Table SI2).
DNA extraction and amplification: In Israel: Liver biopsies divided into small equal portions, ~10 mg
each, were used for separate extractions of DNA. Prior to DNA extraction, samples were incubated in
1,000ppm bleach for (5 min), followed by incubation in double distilled water (15 min) to eliminate
contemporary contamination during sampling. DNA extraction was conducted with guanidinium
thiocyanate (GuSCN) followed by silica capturing (7, 8). All PCR’s were performed in a volume of 25
l (10 PCR buffer, 0.2 mM of dNTPs, 2.5 mM of MgCl2, 0.4 µM of each primer and 0.5
Units/reaction of AmpliTaq Gold (Applied Biosystems, Inc. USA) using a touchdown PCR method
(14) consisting of an initial denaturation at 95°C for 10 min followed by a total of 45 cycles of 15 sec
denaturation at 94°C, 45 sec annealing for two cycles each at 60°C, 58°C, 56°C, 54°C, 52°C, and 35
cycles at 50°C or 48°C, and 45 sec elongation at 72°C, with a final elongation step of 10 min at 72°C.
The PCR’s were performed using high fidelity AmpliTaq Gold to minimize polymerase errors.
In Korea: Liver samples (0.1-0.2g) were incubated in 1 ml of lysis buffer (EDTA 50 mM, pH 8.0;
1mg/ml of proteinase K; SDS 1%; 0.1M DTT) at 56C for 24 hr. Total DNA was extracted using a
phenol/chloroform/isoamyl method (4). DNA isolation and purification was performed using a QIAmp
PCR purification kit (QIAGEN, Hilden, Germany). Purified DNA was eluted in 35 l of EB buffer
(QIAGEN, Germany). Quantity of extracted DNA was measured with Nano -Drop ND-1000
spectrophotometer (Thermo Fisher Scientific, USA). Overlapping primer sets (Table SI 2) and 40 ng
of the total DNA extract were subjected to PCR amplifications. The conditions of PCR were as
follows: pre-denaturation at 94C for 10 min; 40 or 45 cycles of denaturation at 94C for 45 sec;
annealing at 54 to 56C for 45 sec; extension at 72C for 45 sec; final extension at 72C for 10 min.
PCR amplification was performed using a PTC-200 DNA Engine (Bio-Rad. Laboratories, CA). PCR
products were separated on a 2.5% agarose gel electrophoresis and visualized under UV by staining
with ethidium bromide. PCR product electrophoresized was purified with QIAquick Gel Extraction
Kit (Qiagen, Germany).
Sequencing: In Israel: PCR products were analyzed using electrophoresis; positive amplifications
were purified using Exonuclease Shrimp Alkaline Phosphatase (Exo-Sap IT, HDV Pharmacia) or
Accura Kit (Bioneer® Country), with direct sequencing of the products. Both sense and anti-sense
strands were sequenced using the BigDye Terminator system (Applied Biosystems, USA) resolved on
an ABI PRISM 3700 (Applied Biosystems, USA) at the Center for Genomic Technologies, The
Hebrew University of Jerusalem.
3
Cloning: In Israel: Several of the purified PCR products of regions (PreS/S and PreC/C gene) were
cloned into a TOPO-TA vector and grown in One Shot TOP10 chemically competent E. coli cells
(Invitrogen, USA). At least 8-10 isolated colonies from each transformation were grown. DNA was
isolated using a QIAprep kit (Qiagen, Germany). Sequences were generated using the BigDye
Terminator Cycle Sequencing kit (ABI, USA) and resolved on an ABI PRISM 3700 DNA Analyzer.
For each positive clone, primers kz64 and kz77 (15) were used for sequencing the insert.
In Korea: The positive amplifications were cloned using pGEM-T Easy Vector (Promega, US) and
competent cells (ECOS-101, Yeastern Biotech, Taiwan). Plasmid isolation was performed using
QIAprep spin miniprep kit (Qiagen, Germany). Sequencing was performed on ABI 3730xl Genetic
Analyzer (Applied Biosystems, USA).
Cytosine deamination: Ancient DNA is frequently extensively damaged as manifested by cytosine
deamination converting into uracil residues. Such reactions can cause nucleotide misincorporations
during PCR, generating so-called type II errors (ie. GC>AT mismatches) (16). The nucleotide
misincorporations principle could have provided a means of establishing that DNA is ancient but
this approach was found to be limited, therefore best used as a quantitative rather then a qualitative
difference (17, 18). In our study, treatment of extracted DNA from the liver biopsies with Uracil Nglycosylase, the enzyme which removes deaminated cytosine from DNA, prevents DNA
amplification, confirmed that the ancient DNA was damaged and nucleotide misincorporations, (C
<> T and G <> A) which are often seen in ancient DNA were expected. Therefore, our consensus
sequence was determined from multiple overlaping sequences that were obtained from different
PCR reactions using different extracted DNA from different tissue biopsies (Table SI4, Figure SI1).
The consensus sequence represents either identical or the majority identical regions among the
overlapping sequences. Ambiguity of one nucleotide in one sequence among the multiple
overlapping sequences was treated as type II errors (ie. GC>AT mismatches) and was ignored. The
four different consensus sequences represent four substitutions that were found in several sequences
among the overlapping sequences. These substitutions may be authentic, representing different viral
strains. The authenticity is supported by previous reports that identified natural mutations occurring
in the same liver cell during replication, enabling more than one variant per cell (19, 20). The
representation of four aHBV sequences in the analysis is taking into account possible variation
among the HBV sequences, which represent the diversity existing in the different cells. As
indicated in the manuscript (P.10 L.219-220), the sequences were obtained from 24 liver samples.
4
Phylogenetic and allele sharing analysis: A total of 161 sequences, both sense and
antisense, were found to be of high quality and used to determine the entire HBV genome.
The number of sequences of each region in the genome varied from two to 16 sequences, with
21bp up to 250bp overlap between them (Table SI4). The phylogenetic relationships inferred
for the aHBV DNA sequences using alignment of each gene separately, representing all four
ORF’s, distinguished genotype C from the other genotypes supported by high bootstrap
values. The focus of the analysis was to determine the phylogenetic relationship of the aHBV
sequences with other genotype C sequences, in contrast to a number of representative of other
genotypes which were used as an outgroup. The genotype C sequences are separated into two
clusters, HBV- C1 and HBV- C2 with high bootstrap support. Among the HBV- C2 clade
there are two groups that can be identified but this separation is not supported by high
bootstrap values. In all gene phylogenetic analysis the aHBV sequences establish a distinct
cluster within the HBV/C2 clade, which is supported by high bootstrap values (Figure 4 and
Table SI 3). The clustering of the aHBV consensus sequences distinguishes the aHBV DNA
C2 from the other contemporary sequences studied.
Estimation of tMRCA: tMRCA was estimated under relaxed molecular clocks model using
the BEAST software. The aHBV sequences from the Korean mummy were found to cluster
together. The aHBV cluster has a posterior distribution to some of the contemporary
HBV/C2 sequences indicating that the aHBV sequences are ancestral (Figure SI3).
If the HBV virus co-diverged with its host, then our estimate of the mummy tMRCA, should
have a similar age to the origin of the virus. As we do not have a specific calibration we used
the estimated divergence time available in the literature. Different theories have been
proposed by investigators on HBV origin. The common theory proposed that the
evolutionary history of HBV corresponds to the spread of anatomically modern humans as
they migrated from Africa ~100, 000 years ago and different genotypes infecting humans
evolved since this dispersal (Norder et al., 2004). Alternatively, Gunther and colleagues
suggested that the HBV genotypes might have evolved later than, and independent of, human
migration (Gunther et al., 1999). Based on 22 years of observations of nucleotide
substitutions among HBV sequences Orito and colleagues estimated that the origin of the
HBV is ~3000 years (Orito et al., 1989; Zhou et al., 2007 and Jazayeri et al., 2010). The
results of the relaxed molecular clock models indicates tMRCA of the aHBV is similar to the
outgroup tMRCA (Figure SI3b). Therefore, the aHBV sequence represents and ancient
sequence probably one of the first viruses migrated to Asia from Africa.
5
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dry bone to a genetic portrait: a case study of sickle cell anemia. Am J Phys Anthropol
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6
Table SI1: Summery of the authosomal STR alleles characterizing the Korean mummy.
Sample/STR Amel
HBV12
(Liver)
HBV14
(Liver)
HBV 15
(Lung)
HBV 15a
(Lung)
D3
S1358
D19
S433
D2
D22 D16
S1338 S1045 S539
D18
D1
D10
S51 S1656 S1248
D2
S441
TH01 vWA
D21
S11
D12
D8
FGA
S391 S1179
16
23, ?
?, 29
Y, ?
?, 12
?, 29
16, ?
9, 12
14,
16
aHBV
?, Y 16, ?
Consensus
9, 12
14,
16
?, 16
9, 13
16,
18
1, 13
Positive
control
X,Y
17,18
13,14
22,25
Investigator
1
X,X
15,18
13,13
20,23
Investigator
2
X,X
16,18 12,15.2 19,24
16
?, 29
13, 15
15,19 10,11 13,13 11, 13 14, 15
15,16
8, 9
15,17
12,
18.3
12, 13 19,?
13, 15
19,
23
29,
31.2
18,
23
14, 15
20,
23
6, 6
17,19 29,30
17,
20
13,16
20,
25
8, 9
16,18 29,30 18,23 12, 14
24,
25
10, 14
6, 9.3
11.3, 11.3
10, 14
16,
19
12,
13
?= unknown allele
7
Table SI2: Summary of primer sets designed and used in the study
Primer
L1
L2
L3
L7
F1R1
F2R2
F3R3
F7R7
F8R8
F11R11
S1
S2
X2
X3
Hep1
Hep2
Hep3
Hep4
Hep5
Hep6
Hep7
Hep8
Hep9
Position
750
914
850
1057
981
1222
1629
1821
135
339
280
F
R
F
R
F
R
F
R
F
R
F
Sequence (5' - 3')
GGTATTGGGGGCCAAGTCTG
TGCGGTAAAGTACCCCAAC
AAACGTTGGGGCTACTCCCT
GCATCAAGGCAGGATAGCCA
GAAAGTATGTCAAAGAATTGTGG
CTATGGCCAAGCCCCATC
CACCAGGTCTTGCCCAAGGT
AGTTGCATGGTGCTGGTGA
CTGGGGACCCTGCACCGAAC
GGTGAGTGATTGGAGGTTGG
AGGGGGAGCACCCACGTGTC
467
567
770
1770
1977
2098
2306
2457
2657
3195
202
422
611
2910
3085
3042
39
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
GCAACATACCTTGGTAGTCC
GCTGTACAAAACCTTCGGACG
ACAGACTTGGCCCCCAATAC
GTACTAGGAGGCTGTAGGCA
GAAGGAAAGAAGTCAGAAGG
GAATCTGGCCACCTGGGTGGGA
TTGGTGGTCTGTAAGCGGGAGG
CCTTGGACTCATAAGGTGGG
AGGATAGAACCTAGCAGGCA
TCCTCAGGCCATGCAGTGGA
CTGTAACACGAGAAGGGGTCCTAG
TGCCTCATCTTCTTGTTGGT
GGATGGGAATACAAGTGCAG
TCTGGGATTCTTTCCCGATCACC
GTTGTCAATATGCCCTGAGCCTG
AGGGTTCACCCCACCACACG
ACTCTGGGATCTAGCAGAGCTTG
90
271
249
420
337
478
459
608
573
734
710
900
876
1033
1013
1132
1153
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
CTGTTCCGACTACTGCCTCAC
GAGAGAAGTCCACCACGAGTCTA
TAGACTCGTGGTGGACTTCTCTC
AGCAGCAGGATGAAGAGGAA
ACCAACCTCTTGTCCTCCAA
AGGACAAACGGGCAACATAC
GTATGTTGCCCGTTTGTCCT
TGGGAATACAAGTGCAGTTTC
AAAACCTTCGGACGGAAACT
CTGAAAGCCAAACAGTGGGGGAAAG
CTTTCCCCCACTGTTTGGCTTTCAG
CCAACTTCCAATTACATATCCCATG
CATGGGATATGTAATTGGAAGTTGG
GTGTAAAAGGGGCAGCAAAGC
GCTTTGCTGCCCCTTTTACAC
CAACGGGGTAAAGGTTCAGATA
TATCTGAACCTTTACCCCGTTG
Product
164
207
241
192
204
187
Annealing
Temp.
54
54
54
54
54
54
54
54
56
56
56
56
56
56
56
56
56
56
56
56
56
56
56
56
56
56
56
56
203
207
208
200
222
189
175
210
181
61.8
62.4
62.4
57.3
57.3
57.3
57.3
55.9
55.3
64.6
64.6
59.7
59.7
59.8
59.8
58.4
58.4
171
141
149
161
190
157
119
128
8
Hep10
Hep11
Hep12
Hep13
Hep14
Hep15
Hep16
Hep17
Hep18
Hep19
Hep20
Hep21
Hep22
Hep23
OHep1
OHep2
OHep3
OHep4
OHep5
OHep6
1281
1261
1436
1417
1546
1527
1680
1660
1816
1787
1942
1922
2078
2054
2275
2256
2370
2345
2543
2524
2656
2635
2803
2784
2914
2895
3102
3084
3205
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
F
R
GAGTTCCGCAGTATGGATCG
CGATCCATACTGCGGAACTC
GACGGGACGTAGACAAAGGA
TCCTTTGTCTACGTCCCGTC
AGACCGCGTAAAGAGAGGTG
CACCTCTCTTTACGCGGTCT
TTGCTGAGAGTCCAAGAGTCC
GGACTCTTGGACTCTCAGCAA
GGCAGAGGTGAAAAAGTTGC
GCATAAATTGGTCTGTTCACCAG
CTCCACAGAAGCTCCAAATTC
GAATTTGGAGCTTCTGTGGAG
TCATCAACTCACCCCAACACAG
CATACAGCACTCAGGCAAGC
CCACACTCCAAAAGACACCA
TGGTGTCTTTTGGAGTGTGG
GAGGCGAGGGAGTTCTTCTT
GTTAGACGACGAGGCAGGTC
AAAGGAGGGAGTTTGCCACT
AGTGGCAAACTCCCTCCTTT
GGATAGAACCTAGCAGGCATAA
TTATGCCTGCTAGGTTCTATCC
GCGCTGCGTGTAGTTTCTCT
AGAGAAACTACACGCAGCGC
CCAGAGGATTGGGAACAGAA
TTCTGTTCCCAATCCTCTGG
CAATATGCCCTGAGCCTGA
TCAGGCTCAGGGCATATTG
AGACAGTCATCCTCAGGCCA
659
875
942
966
1044
1235
1212
1436
1861
2588
2771
F
R
F
R
F
R
F
R
F
R
F
CGTTTCTCCTGGCTCAGTTTAC
AAGTTAAGGGAGTAGCCCCAAC
TTTTCGGAAACTGCCTGTAAAT
ATGTTTTCGGAAACTGCCTGTA
ACCTGCCTTGATGCCTTTAT
ATGCGCTGATGGCCTATG
CTTGGCCATAGGCCATCAG
GACGGGACGTAGACAAAGGA
TGTTCAAGCCTCCAAGCTGT
ATATGTGGGCCCTCTCACAG
CAGCCTTGCCCACAAAGTAT
59.4
59.4
59.4
59.4
59.4
59.4
59.8
59.8
57.3
58.9
57.9
57.9
60.3
59.4
57.3
57.3
59.4
61.4
57.3
57.3
58.4
58.4
59.4
59.4
57.3
57.3
56.7
56.7
59.4
175
129
153
156
155
156
221
114
198
132
168
130
207
121
216
58.8
59.4
59.7
60.1
61.2
60.3
59.8
59.6
60.1
60.1
60.9
191
224
183
9
Table SI3: Published contemporary HBV sequences used in the analyses of the aHBV
DNA genome.
Accession no.
DQ536410
DQ536412
AY247030
AY247031
X14193
Genotype
C2
C2
C2
C2
C2
C2
Location
Korea
Korea
Korea
Korea
Korea
Korea
Y18857
EU916241
AF182805
EU579443
FJ032361
EU589345
AF533983
C2
C2
C2
C2
C2
C2
C2
China
China
China
China
China
China
China
Guo and Hou (1999)
Fang and Gu Direct submission
Lin et al. (2001)
Liu et al. Direct submission
Liu et al. Direct submission
Liu et al. Direct submission
Dong, and He Direct submission
D50520
D23681
AB298721
AB113879
AB033553
AB368297
X04615
C2
C2
C2
C2
C2
C2
C2
Japan
Japan
Japan
Japan
Japan
Japan
Japan
Asahina et al. (1996)
Horikita et al. (1994)
Inoue et al. (2008)
Michitaka and Tran, Direct submission
Okamoto et al. (1987)
Nakajima and Abe, Direct submission
Okamoto et al. (1986)
AB111946
AB112065
AB031262
C1
C1
C1
Vietnam
Vietnam
Vietnam
Huy et al. (2004)
Huy et al. (2004)
Yuasa et al. (2000)
AB112066
AB112348
AB112408
C1
C1
C1
Myanmar
Myanmar
Myanmar
Huy et al. (2004)
Huy et al. (2004)
Huy et al. (2004)
AF068756
AB112472
AB112471
AB074755
C1
C1
C1
C1
Thailand
Thailand
Thailand
Thailand
Monkongdee et al. (1998)
Huy et al. (2004)
Huy et al. (2004)
Sugauchi et al. (2002)
AB205118
AY128092
AB205126
AB120308
AM422939
AB205191
AB205010
A
A
D
D
D
E
H
Japan
Canada
Japan
Japan
France
Ghana
Japan
Nakajima et al. (2005)
Osiowy and Giles (2003)
Nakajima et al. (2005)
Michitaka et al. (2006)
Mrani et al. Direct submission
Huy et al. (2006)
Nakajima et al. (2005)
AY641563
Reference
Kim et al. (2007)
Kim et al. (2007)
Song et al. (2005)
Odgerel et al. (2003)
Odgerel et al. (2003)
Rho et al. (1989)
10
Table SI4: Summary of sequences obtained for each primer set and the overlap coverage.
Nucleotide
Position
1
90
135
249
272
337
347
280
422
574
567
710
750
874
850
981
1013
1132
1262
1418
1528
1629
1661
1770
1820
1923
2049
2098
2256
2346
2457
2525
2,689
2785
2841
2894
2910
3042
3195
Primer
KPS/S
Hep1
F1R1
Hep2
SAT-KZ
Hep3
Hep4
Hep3-Hep4
F2R2
S2
Hep5
F3R3
Hep6
L1
Hep7
Hep6-Hep7
L2
L3
Hep8
Hep9
Hep8-Hep9
Hep10
Hep11
Hep12
L7
Hep13
F7R7
Hep14
Hep15
Hep16
F8R8
Hep17
Hep18
F11R11
Hep19
Hep20
Hep19-Hep20
Hep21
PS+M13
Hep22
X2
X3
S1
Product
size
178
183
204
173
438
142
150
263
187
189
161
203
192
164
161
304
207
241
142
151
268
175
129
153
192
176
207
124
176
222
208
136
199
200
133
121
234
131
436
147
175
210
222
No.
Sequences
9
6
1
4
3
4
3
6
1
12
4
1
3
1
4
2
1
1
4
4
4
2
2
8
1
4
1
2
6
5
1
6
5
1
6
4
4
3
12
4
3
2
1
Overlap (bp)
60; 89
24
165
150, 85
272, 263, 35
35
148
24
27
22
23
20
19
20
20
17
21
43
21
46
20
20, 76, 21
210
144
149
Direct/
Cloned
C
D
D
D
C
D
D
D
D
C
D
D
D
C
D
D
C
C
D
D
D
D
D
D
C
D
D
D
D
D
D
D
D
D
D
D
D
D
C
D
C
C
C
No
PCR’s
2
3
1
2
2
2
3
3
1
3
2
1
2
1
2
1
1
1
3
3
2
1
1
4
1
2
1
2
3
3
1
3
3
1
3
4
4
2
3
2
1
1
1
Gene in annotation
Pre-S2/S
S gene
S gene
S gene
GRE
S gene
S gene
S gene
S gene
S gene
S gene
S gene
S gene
S gene
S gene
S gene
Enhancer
Enhancer
Enhancer
X gene
X gene
DR1, P gene
P gene
PreC
P gene, PreC
DR2, X, C , Poly A
Poly A, C gene
C gene
P gene
C gene
P gene
TATA box
PreS1
PreS1
PreS1/2
PreS1/2
PreS/S
D = direct sequencing; C = cloned sequencing
11