Article review 1000 words

The Author(s) BMC Genomics 2016, 17(Suppl 9):750
DOI 10.1186/s12864-016-3087-2
RESEARCH
Open Access
More comprehensive forensic genetic
marker analyses for accurate human
remains identification using massively
parallel DNA sequencing
Angie D. Ambers1*, Jennifer D. Churchill1, Jonathan L. King1, Monika Stoljarova1,2, Harrell Gill-King3, Mourad Assidi4,
Muhammad Abu-Elmagd4, Abdelbaset Buhmeida4 and Bruce Budowle1,4*
From 3rd International Genomic Medicine Conference
Jeddah, Saudi Arabia. 30 November – 3 December 2015
Abstract
Background: Although the primary objective of forensic DNA analyses of unidentified human remains is positive
identification, cases involving historical or archaeological skeletal remains often lack reference samples for comparison.
Massively parallel sequencing (MPS) offers an opportunity to provide biometric data in such cases, and these cases
provide valuable data on the feasibility of applying MPS for characterization of modern forensic casework samples. In
this study, MPS was used to characterize 140-year-old human skeletal remains discovered at a historical site in
Deadwood, South Dakota, United States. The remains were in an unmarked grave and there were no records or other
metadata available regarding the identity of the individual. Due to the high throughput of MPS, a variety of biometric
markers could be typed using a single sample.
Results: Using MPS and suitable forensic genetic markers, more relevant information could be obtained from a limited
quantity and quality sample. Results were obtained for 25/26 Y-STRs, 34/34 Y SNPs, 166/166 ancestry-informative SNPs,
24/24 phenotype-informative SNPs, 102/102 human identity SNPs, 27/29 autosomal STRs (plus amelogenin), and 4/8
X-STRs (as well as ten regions of mtDNA). The Y-chromosome (Y-STR, Y-SNP) and mtDNA profiles of the unidentified
skeletal remains are consistent with the R1b and H1 haplogroups, respectively. Both of these haplogroups are the most
common haplogroups in Western Europe. Ancestry-informative SNP analysis also supported European ancestry. The
genetic results are consistent with anthropological findings that the remains belong to a male of European ancestry
(Caucasian). Phenotype-informative SNP data provided strong support that the individual had light red hair and brown
eyes.
Conclusions: This study is among the first to genetically characterize historical human remains with forensic genetic
marker kits specifically designed for MPS. The outcome demonstrates that substantially more genetic information can be
obtained from the same initial quantities of DNA as that of current CE-based analyses.
Keywords: Human skeletal remains, Massively parallel sequencing (MPS), Phenotype-informative SNPs, Y-STRs,
Mitochondrial DNA, Ancestry-informative markers (AIMS)
* Correspondence: angie.ambers2@unthsc.edu; bruce.budowle@unthsc.edu
1
Institute of Applied Genetics, Department of Molecular and Medical
Genetics, University of North, Texas Health Science Center, 3500 Camp Bowie
Boulevard, Fort Worth, TX, USA
Full list of author information is available at the end of the article
© 2016 The Author(s). Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
The Author(s) BMC Genomics 2016, 17(Suppl 9):750
Background
The paramount goal of forensic DNA testing of human
skeletal remains is identification of the unknown individual. A variety of genetic markers can be used to achieve
identification, including highly polymorphic autosomal
short tandem repeat (STR) loci and lineage markers [YSTRs, Y chromosome single nucleotide polymorphisms
(Y-SNPs), mitochondrial DNA (mtDNA)]. However, reference samples must be available for comparison for
these markers to be informative. In mass disasters, missing persons cases, or cases involving historical/archaeological remains, sometimes there are no clues as to the
person’s potential identity and/or there are no associations made with a reference sample or reference pedigree via a database search [1]. In such scenarios,
identification can be difficult or impossible using solely
autosomal STRs and lineage markers. However, there
are other genetic markers that can extend human identification capabilities, such as analysis of ancestryinformative markers [2–7] and phenotype-informative
SNPs [7–11].
Massively parallel sequencing (MPS) of ancestry- and
phenotype-informative SNPs, with its expanded capacity
for marker typing, offers the ability to develop investigative leads in such cases [12–16]. Thus, more genetic information can be gleaned from a sample without further
consumption of often very limited quantity and quality
samples. In this study, MPS was used in an effort to help
characterize 140-year-old human skeletal remains that
were buried in an unmarked grave in Deadwood, South
Dakota USA, a famous town of the American Old West.
In 1874, the discovery of gold in the Black Hills of
South Dakota set off one of the last great gold rushes in
America. In 1876, miners moved to the area and formally established the city of Deadwood, now a U.S.
historical landmark. Deadwood’s original cemetery,
Ingleside, was located near the town’s core business district and contained approximately 100 burials (although
cemetery records are incomplete and some were buried
in unmarked graves). In 1878, the individuals interred
there were relocated to the hills above Deadwood, and
Mount Moriah Cemetery was established.
In 2012, a set of unidentified human skeletal remains
were unearthed by a construction crew in Deadwood’s
Presidential District, the original site of Ingleside Cemetery [17–19]. South Dakota State archaeologists and historic preservation officials for the City of Deadwood
recovered the skeleton from the site (with the exception
of one tooth and a few bones from the hands and feet).
Anthropological analyses indicated that the remains are
consistent with a male of European ancestry (Caucasian)
who was 18–24 years of age at the time of death and
65.7 − 70.7 inches tall. No indications of the cause of
death were evident in the skeletal samples [19–21].
Page 22 of 87
Forensic odontological analyses determined that this unknown individual was a habitual tobacco user and had
nine dental fillings (3 gold, 6 tin/amalgam). The latter observation is indicative of some level of affluence/wealth, as
most individuals in the late 19th century would simply
have had unhealthy teeth extracted [20, 21].
In June 2014, the City of Deadwood and the Deadwood
Historic Preservation Commission requested that the Institute of Applied Genetics (IAG) conduct DNA testing
on the remains to provide some level of identification
[18–21]. Given that the remains were in an unmarked
grave and no investigative leads existed regarding his identity, Deadwood city officials were interested in the analysis
of DNA markers that could help predict the individual’s
ancestry and external physical traits. Markers chosen for
analysis included Y-STRs, Y-SNPs, ancestry-informative
SNPs, phenotype-informative SNPs, and mitochondrial
DNA (mtDNA). To the best of our knowledge, this study
is among the first to genetically characterize historical human remains with forensic genetic marker kits specifically
designed for MPS.
Methods
The practices for minimizing contamination during the
analysis of the Deadwood remains were the same contamination controls recommended for archaeological
and ancient DNA specimens, including: (a) use of
protective suits, gloves, and masks; (b) bleach decontamination and UV-irradiation of work benches and
associated equipment; (c) physical removal and/or chemical destruction of contaminant/exogenous DNA on external bone surfaces; (d) extraction of bone samples in a
designated low-copy area; (e) PCR amplification in a location that is physically separated from the extraction area;
(f) use of appropriate negative controls, reagent blanks,
and positive controls; and (g) replicate testing [22–28].
Bone processing and DNA extraction
The right femur was provided to the IAG for DNA testing (Loan Accession No. 12–0051, South Dakota Archaeological Research Center) (Fig. 1). A portion of the
Fig. 1 Right femur of unidentified human skeletal remains discovered
in 2012 in Deadwood’s Presidential District
The Author(s) BMC Genomics 2016, 17(Suppl 9):750
femur diaphysis was surface-sanded with a Dremel®
4000 Rotary Tool and sterile grinding stone (Robert
Bosch Tool Corporation, Mount Prospect, Illinois USA)
followed by sectioning of eight adjacent regions with a
Stryker® autopsy saw (Mopec, Oak Park, Michigan
USA). DNA extractions were performed on six of the
eight bone sections in a designated low-copy number
(LCN) area of the laboratory, as described in Ambers
et al. [29].
DNA quantification
The quantity of DNA from seven bone powder fractions was determined using the Quantifiler® Human
DNA Quantification Kit and an ABI 7500 Real-Time
PCR System (Thermo Fisher Scientific, Waltham,
Massachusetts USA), according to manufacturers’ recommendations [30].
Traditional Y-STR typing via capillary electrophoresis
Human genomic DNA was amplified with reagents from
the AmpFlSTR® Yfiler™ PCR Amplification Kit and a
GeneAmp® PCR System 9700 (Thermo Fisher Scientific),
according to manufacturer’s recommendations [31].
Negative controls consisted of 10 μl low-TE buffer and
10 μl 9947A female DNA (0.1 ng/μl); 10 μl 007 Male
Control DNA (0.1 ng/μl) served as the positive control.
PCR products were separated via capillary electrophoresis (CE) on a 3500xl Genetic Analyzer, and analyzed
using GeneMapper® ID-X software (Thermo Fisher Scientific). DNA (elution #1 and elution #2) from seven
bone powder fractions was typed.
Massively Parallel Sequencing (MPS) with the Illumina
MiSeq®
DNA from four of the bone powder extracts (007.001
E1, 008.001 E1, 008.002 E1, 008.002 E2) that yielded partial to complete Yfiler™ Y-STR profiles was analyzed via
MPS. The beta version of the ForenSeq™ DNA Signature
Prep Kit (Illumina, San Diego, California USA) was used
to prepare libraries as described in [32]. For the Illumina® ForenSeq DNA Signature Prep Kit, the Y-STR
markers analyzed were: DYF387S1, DYS19, DYS385a/b,
DYS389I, DYS389II, DYS390, DYS391, DYS392,
DYS437, DYS438, DYS439, DYS448, DYS456, DYS460,
DYS481, DYS505, DYS522, DYS533, DYS549, DYS570,
DYS576, DYS612, DYS635, DYS643, and Y-GATA H4.
Input DNA was 0.20 ng, 1 ng, 1 ng, and 0.58 ng, respectively, for the first PCR. Ten microliters of pooled libraries were used for the proceeding “Denature and
Dilute Libraries” step. Subsequent sequencing on the
MiSeq® Desktop Sequencer (Illumina) and data analysis
were completed as detailed in [32].
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Massively Parallel Sequencing (MPS) with the Ion Torrent
PGM®
DNA from three of the same four bone extracts
(008.001 E1, 008.002 E1, 008.002 E2) was analyzed on
the Ion Torrent Personal Genome Machine® (PGM)
platform (Thermo Fisher Scientific). Library preparation, sequencing, and data analysis for three SNP
panels [HID-Ion AmpliSeq™ Identity Panel, HID-Ion
AmpliSeq™ Ancestry Panel, and an Externally Visible
Characteristics (EVC) prototype panel (Thermo Fisher
Scientific)] were completed as described in [33–36].
Input DNA was 1 ng, 1 ng, and 0.58 ng, respectively,
22 cycles were used in the initial PCR, and 25 μl of
pooled libraries were used for preparation of the Ion
OneTouch™ 2 (OT2) amplification solution. Mitochondrial DNA was amplified using an in-house PCR
multiplex assay [unpublished]. Eight positions of the
mtDNA coding region were sequenced: 4488–4656,
4727–4862, 8542–8707, 10674–10830, 13588–13745,
13809–14098, 14133–14301, and 14766–14923. The
noncoding hypervariable regions (HVI, HVII) also
were sequenced, as described in [37]. Library preparation, sequencing, and data analysis were completed
as outlined in [36] with one exception: 25 μl of
pooled libraries were used for preparation of the OT2
amplification solution.
Final data analysis
30X and 10X coverage were set as minimum detection
thresholds for the autosomal markers and mtDNA
typed by MPS in this study, respectively. The Y
Table 1 DNA concentrations (ng/μl) obtained from the right
femur of Deadwood’s unidentified human skeletal remains
(E1 = elution #1; E2 = elution #2; total elution volume = 30 μl)
Sample ID
ng/μl
Total DNA (ng)
Femur 001.001 E1
0.0327
0.98
Femur 001.001 E2
0.0082
0.24
Femur 002.002 E1
0.0232
0.70
Femur 002.002 E2
0.0029
0.09
Femur 003.001 E1
0.0147
0.44
Femur 003.001 E2
undetermined
undetermined
Femur 006.002 E1
0.1730
5.19
Femur 006.002 E2
0.0147
0.44
Femur 007.001 E1
0.0383
1.15
Femur 007.001 E2
0.0014
0.04
Femur 008.001 E1
0.3080
9.24
Femur 008.001 E2
0.0347
1.04
Femur 008.002 E1
0.3350
10.05
Femur 008.002 E2
0.0579
1.74
30
DYS389 II
19
DYS458
14
DYS19
11,14
DYS385 a/b
13
DYS393
11
DYS391
11
DYS439
23
DYS635
13
DYS392
12
Y GATA H4
DYS437
14
24
30
19
19
19
14
14
14
11,14
14
13
13
13
16
24
30
Consensus Y-STR Haplotype:
14
14
24
16
11
11
11
11
16
13
FEMUR 008.002 E2
11
FEMUR 008.002 E1
19
19
11
14
16
14
16
FEMUR 008.001 E2
11
FEMUR 008.001 E1
13
11
19
16
FEMUR 007.001 E1
11
FEMUR 007.001 E2
14
13
13
16
FEMUR 006.002 E1
FEMUR 006.002 E2
FEMUR 003.001 E2
FEMUR 003.001 E1
11
11
11
11
23
23
23
23
13
12
12
12
12
15
15
15
15
15
15
15
15
15
24
DYS390
15
14
DYS389 I
FEMUR 002.002 E2
DYS456
FEMUR 002.002 E1
FEMUR 001.001 E2
FEMUR 001.001 E1
Sample Name
12
12
12
12
12
DYS438
19
19
19
19
19
DYS448
Table 2 Y-STR typing allele results for 17 loci in seven different bone powder fractions using the AmpFlSTR® Yfiler™ PCR Amplification Kit and CE (E1 = elution #1; E2 = elution #2)
The Author(s) BMC Genomics 2016, 17(Suppl 9):750
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The Author(s) BMC Genomics 2016, 17(Suppl 9):750
Page 25 of 87
haplogroup was determined using the ancestry feature
and metapopulation tool of the Y-STR haplotype reference database YHRD (www.yhrd.org). A PCA plot of
ancestry-informative SNP data was generated with the
Illumina® ForenSeq™ Universal Analysis Software.
Mitochondrial DNA sequence alignment was performed with the mitoSAVE workbook [38], and haplogroup determination was made using HaploGrep
software (http://haplogrep.uibk.ac.at/) [39]. Phenotypic SNP data were analyzed with the Illumina® ForenSeq™ Universal Analysis Software as well as with the
HIrisplex hair/eye color prediction tool (http://hirisplex.erasmusmc.nl) [9, 10].
Results and discussion
DNA concentrations recovered from the right femur
powder fractions ranged from 0.0147–0.3350 ng/μl for
elution #1 and 0–0.0579 ng/μl for elution #2, respectively. The elution volume for each DNA extract was
30 μl, and the total DNA recovered per elution is reported in Table 1.
A variety of STR and SNP markers were analyzed via
CE and MPS. No DNA was detected in any of the negative controls and reagent blanks, and positive controls
yielded the correct type for all analyses.
Y-chromosome (Y-STR and Y-SNP) DNA Analysis: CE and
MPS
Y-STR typing results varied among the samples (Tables 2 and 3). Allele calls among all extracts were concordant. Individual sample results and the complete 17locus Yfiler™ consensus profile are shown in Table 2.
Fifteen of the twenty-six Y-STR markers analyzed with
the Illumina® ForenSeq™ DNA Signature Prep Kit overlap with the AmpFlSTR® Yfiler™ PCR Amplification Kit.
The Y-STR alleles recovered from all bone samples
among the common markers between MPS and CE
were concordant. Y-STR typing results were obtained
for 17 of the 26 markers assayed with MPS (Table 3);
coverage ranged from 31x to 620x [148 ± 137 (Avg ±
SD)]. The total number of Y-STR loci that yielded results for both methods was 25.
The composite 17-locus Y-STR profile generated
with AmpFlSTR® Yfiler™ and the additional Y STR
loci from the Illumina® ForenSeq™ DNA Signature
Prep Kit is consistent with the R1b haplogroup. R1b
is the most common Y haplogroup in Western Europe, spanning 80 % of the population in Ireland,
western Wales, the Scottish Highlands, the Atlantic
fringe of France, Catalonia, and the Basque country.
It also is common around the Caucasus and in Anatolia, in parts of Russia, and in Central and South
Asia [40–45].
In addition to Y-STR data, a consensus Y-SNP profile was compiled using data from three different
bone powder fractions from the Deadwood unidentified skeletal remains. All 34 upper clade Y-SNPs in
the HID-Ion AmpliSeq™ Identity Panel provided typing results [Additional file 1], and these haplogroupinformative Y-SNP results also supported an R1b haplogroup assignment.
Ancestry informative SNPs
Ancestry-informative SNP results were obtained for 51
of the 54 SNP markers amplified via the Illumina® ForenSeq™ DNA Signature Prep Kit, and for all 165 markers
tested using the HID-Ion AmpliSeq™ Ancestry Panel.
Depth of coverage ranged from 31x to 2240x (170 ±
107) and 53x to 1190x (379 ± 243), respectively. Fiftythree of the ancestry-informative SNPs in the Illumina®
ForenSeq™ kit are included in the HID-Ion AmpliSeq™
Ancestry Panel, and 51 of these SNPs yielded results
with both panels. The results were concordant, and a
composite profile was generated [Additional file 2].
Using the ancestry-informative SNP data, the major
population bio-ancestry was determined to be European (Fig. 2).
Mitochondrial DNA (mtDNA) Analysis
An in-house PCR multiplex assay comprised of short
amplicons (~200 bp in length) at targeted sites on the
coding and non-coding regions (HVI and HVII) of the
mitochondrial DNA (mtDNA) genome was used to
characterize the maternal lineage of the Deadwood
Table 3 Y-STR typing allele results for four different bone powder fractions using the Illumina® ForenSeq™ DNA Signature Prep Kit and
MPS (E1 = elution #1; E2 = elution #2; markers in common with AmpFlSTR® Yfiler™ are shown in bold)
Sample Name
DYF387S1 DYS19 DYS385a-b DYS389I DYS389II DYS390 DYS391 DYS392 DYS437 DYS438 DYS439 DYS448 DYS456
FEMUR 007.001 E1
12
FEMUR 008.001 E1
14
11
15
15
FEMUR 008.002 E1
36
14
11
FEMUR 008.002 E2
36
14
14
11
Consensus Y-STR Haplotype 36
14
14
11
15
12
11
12
11
12
11
19
12
11
19
The Author(s) BMC Genomics 2016, 17(Suppl 9):750
Page 26 of 87
Table 3 Y-STR typing allele results for four different bone powder fractions using the Illumina® ForenSeq™ DNA Signature Prep Kit and
MPS (E1 = elution #1; E2 = elution #2; markers in common with AmpFlSTR® Yfiler™ are shown in bold) (Continued)
Sample Name
DYS460
DYS481
FEMUR 007.001 E1
22
FEMUR 008.001 E1
22
FEMUR 008.002 E1
22
FEMUR 008.002 E2
22
Consensus Y-STR Haplotype
22
DYS505
DYS570
DYS576
12
17
17
12
17
17
39
17
17
39
12
DYS522
DYS533
10
12
10
12
skeletal remains. A total of 10 regions of the mtDNA
genome were assayed (HVI, HVII, and eight coding regions covering 2120 bases). The mtDNA regions targeted by the in-house multiplex assay increase
discrimination power by 17.4 % beyond sequencing of
HV1 and HVII alone (unpublished data). Quality sequencing results were obtained for all 10 regions, with
a range in coverage of 19x to 23,987x for 95.3 % of the
targeted regions. Nine of the 10 regions sequenced
had greater than 1000x coverage (14133–14301 had an
average coverage of ~100x) (Additional file 3). No inference of heteroplasmy was observed.
The reportable mtDNA data from these ten regions
(2020 bp; 30–305, 4488–4656, 4707–4880, 8520–8726,
10657–10847, 13570–13760, 13791–13990, 14133–
14301, 14749–14941, 15980–16407) allowed for the
following haplotype to be constructed for the reported region (146C, 263G, 4769G, 16181G, 16183C,
16189C). These data provided sufficient genetic
DYS549
DYS612
DYS635
DYS643
Y-GATA H4
10
10
23
10
23
10
information to determine that the most likely mtDNA
haplogroup is H1 with numerous subgroups of H1
(e.g., H1 + 16189, H1f, H1y, etc.) giving 83.2 % quality
score via HaploGrep [39]. Mitochondrial haplogroup
H1 is the most common in Western Europe and is
found throughout Europe, northern Africa, the Levant, the Caucasus, Anatolia, and as far as Central
Asia and Siberia [46–52]. Hence, the biogeographic
ancestry determined by the Y-STR, Y-SNP, ancestryinformative SNP, and mtDNA data are all consistent
with that obtained by anthropological analyses of a
European ancestry.
Forensic DNA phenotyping
Twenty-four phenotype-informative SNPs were assayed
using the Ion Torrent PGM® and HID-Ion AmpliSeq™
Externally Visible Characteristics (EVC) Prototype Panel.
Results were obtained for 23 of the 24 phenotype-
Fig. 2 PCA plot of ancestry-informative SNPs, displaying the best fit population assignment. The population assignment for the 140-year-old
unidentified Deadwood skeletal remains is circled in the scatter plot
The Author(s) BMC Genomics 2016, 17(Suppl 9):750
Page 27 of 87
amelogenin), and 4/8 X-STRs. Range in coverage for
the human identity SNPs, autosomal STRs, and X-STRs
were 32x-1085x (217 ± 213), 31x-2838x (297 ± 485), and
31x-361x (170 ± 107), respectively. With the HID-Ion
AmpliSeq™ Identity Panel, results were obtained for 90/
90 human identity SNPs [Additional files 5, 6 and 7],
with a depth of coverage of 33x-1419x (282 ± 205).
There are 80 human identity SNPs in common between
the two kits, and 75 of these common markers yielded
results with both panels. Results were concordant between the two identity SNP panels. These results further support the potential of MPS to enable typing of a
much larger number of genetic markers from the same
amount of DNA than would have been possible with
current CE-based systems.
informative SNPs assayed, with a depth of coverage of
33x to 1419x (282 ± 205) (Table 4).
Additional testing was performed on the skeletal
samples using the Illumina® ForenSeq™ DNA Signature
Prep Kit and MiSeq® platform. Results were obtained
for all 24 phenotype-informative SNP markers assayed,
with a depth of coverage of 32x to 1187x (288 ± 407).
Typing results were concordant between assays and between the two MPS platforms. A composite phenotypeinformative SNP profile was generated and is shown in
Additional file 4. Phenotypic SNP analysis was performed using the HIrisPlex hair/eye color prediction
tool (http://hirisplex.erasmusmc.nl), which generates
individual probabilities for four hair color categories
(red, blonde, brown, black), two hair color shades
(light, dark), and three eye color categories (blue, intermediate, brown) [9, 10]. The 24 predictive DNA variants (23 SNPs and 1 INDEL) of the HIrisPlex assay
are included in the Illumina® ForenSeq™and HID-Ion
AmpliSeq™ Externally Visible Characteristics (EVC)
Prototype Panel, and the system was designed to cope
with low template and degraded DNA. All 24 DNA
variants have small amplicon sizes (< 160 bp). In terms of specificity, HIrisPlex variants provide blue and brown human eye color predictions with over 90 % precision [9] and average hair color prediction accuracies of 0.70, 0.79, 0.80, and 0.88 for red, blonde, brown, and black hair, respectively [10]. Analysis of the Deadwood skeletal remains indicated that this individual likely had light red hair and light brown eyes. Probabilities for hair color, hair color shade, and eye color were 0.69, 0.71, and 0.72, respectively (Table 5). Conclusion In an effort to learn more about the late-19th-century human skeletal remains discovered at the site of Deadwood’s first cemetery, historic preservation officials enlisted a number of forensic specialists to conduct analyses on the remains that could assist in his identification [17–21, 53]. Since the individual was buried in an unmarked grave and no investigative leads existed regarding his identity, lineage testing and forensic DNA phenotyping were performed to predict ancestry and external physical traits. The Y-chromosome (Y-STR, Y-SNP) and mitochondrial DNA (mtDNA) profiles of the unidentified skeletal remains are consistent with the R1b and H1 haplogroups, respectively. Both of these haplogroups are the most common ones in Western Europe. The ancestry-informative SNPs also indicated a European background. These genetic results are consistent with the findings of a previous anthropological report which determined that the Deadwood unidentified skeletal remains belong to a male of European ancestry (Caucasian). The phenotype-informative SNPs provided strong support that the individual had light red hair and brown eyes. This study is among the first known historical remains case that has been characterized with genetic panels designed specifically Other markers assayed with MPS panels The Illumina® ForenSeq™ DNA Signature Prep Kit and the HID-Ion AmpliSeq™ Identity Panel also contain markers that do not contribute to the characterization of bioancestry or phenotype, but nonetheless were able to be typed. With the Illumina® ForenSeq™ DNA Signature Prep Kit, results were obtained for 88/95 human identity SNPs, 27/29 autosomal STRs (plus Table 4 Phenotype-informative SNP analysis results for three Deadwood skeletal samples using the Ion Torrent PGM® and HID-Ion AmpliSeq™ Externally Visible Characteristics (EVC) Prototype Panel Sample Name rs12896399 rs28777 rs1393350 rs16891982 rs12203592 N29insA rs1042602 rs2378249 rs12821256 rs1800407 rs12913832 rs683 Femur G/G 008.001 E1 A/A G/G G/G C/C C/C G/A T/T G/G A/G C/A Femur G/G 008.002 E1 A/A G/G G/G C/C C/C G/A T/T G/G A/G C/A Femur 008.002 E2 A/A G/G G/G C/C G/A T/T G/G A/G C/A The Author(s) BMC Genomics 2016, 17(Suppl 9):750 Page 28 of 87 Table 4 Phenotype-informative SNP analysis results for three Deadwood skeletal samples using the Ion Torrent PGM® and HID-Ion AmpliSeq™ Externally Visible Characteristics (EVC) Prototype Panel (Continued) Sample Name rs2402130 rs4959270 rs1805005 rs1805006 rs2228479 rs1110400 rs11547464 rs1805007 rs1805008 rs1805009 Y152OCH rs2228479 Femur 008.001 E1 A/A C/A G/G C/C G/G T/T G/G C/C T/T G/G C/C G/G Femur 008.002 E1 A/A C/A G/G C/C G/G T/T G/G C/C T/T G/G C/C G/G Femur 008.002 E2 A/A C/A G/G C/C G/G T/T G/G C/C T/T C/C G/G for forensic human identification purposes. The results were highly informative. The study demonstrates the potential of MPS to analyze unidentified human skeletal remains and to provide substantially more genetic information from the same initial quantities of DNA sample as that of CE-based analyses. Using the Illumina® ForenSeq™ DNA Signature Prep Kit, results were obtained for 25/26 Y-STRs, 88/95 human identity SNPs, 51/54 ancestry-informative SNPs, 24/24 phenotype-informative SNPs, 27/29 autosomal STRs (plus amelogenin), and 4/8 X-STRs. With the HID-Ion AmpliSeq™ Identity Panel, results were obtained for 34/34 Y-SNPs and 90/90 human identity SNPs. The HID-Ion AmpliSeq™ Ancestry Panel yielded data for 165/165 ancestry-informative SNP markers assayed. Combined results for all MPS panels included genetic data for 25/26 Y-STRs, 34/ 34 Y SNPs, 166/166 ancestry-informative SNPs, 24/ 24 phenotype-informative SNPs, 102/102 human identity SNPs, 27/29 autosomal STRs (plus amelogenin), and 4/8 X-STRs (as well as ten regions of mtDNA). An important point about DNA testing of historical or archaeological skeletal remains should be emphasized. Six bone sections/cuttings were taken, and bone powder fractions from each were analyzed. Adjacent bone sections yielded vastly different results in terms of DNA quantity and number of allele calls; some regions of Table 5 Prediction of Deadwood undentified skeletal remains’ potential hair and eye color using phenotype-informative SNP data and the HIrisplex hair/eye color prediction tool (http://hirisplex. erasmusmc.nl/) [10] HAIR COLOR HAIR SHADE EYE COLOR Brown 0.19 Light 0.71 Brown 0.72 Red 0.69 Dark 0.29 Intermediate 0.19 Blue 0.09 Black 0.04 Blonde 0.09 bone did not yield any DNA, while other areas yielded complete profiles. These findings are consistent with a previous study performed on the 120-year-old skeletal remains of an American Civil War soldier [29], which required testing of multiple bone sections and a consensus testing approach to obtain a complete Y-STR haplotype. With its capacity for simultaneous analysis of a multitude of different types of DNA markers, MPS technology holds promise for use in the characterization of historical and archaeological remains, and in missing persons cases. In addition, in mass disasters or other types of cases where reference samples are not available/known, genetic markers such as ancestry-informative and phenotype-informative SNPs can provide data for craniofacial reconstructions that could be useful for positive identification. Additional files Additional file 1: Y-SNP data for three different bone powder fractions using the HID-Ion AmpliSeq™ Identity Panel and the Ion Torrent PGM® MPS platform (E1 = elution #1; E2 = elution #2). (XLSX 12 kb) Additional file 2: Ancestry-informative SNP results for 140-year-old skeletal remains from Deadwood, Dakota using the A) Illumina® ForenSeq™ panel and B) HID-Ion AmpliSeq™ Ancestry Panel. Concordant results for the 53 ancestry-informative SNPs that were common between the two assays were used to generate a composite AIMS profile (C). (XLSX 20 kb) Additional file 3: Sequence results for ten regions of the mtDNA genome using an in-house mtDNA panel (unpublished). The overall coverage ranged from 0-23987x. The lowest coverage (~100x) region (14133–14301) is indicated with black box and arrow. A) 140-year-old skeletal remains, B) reagent blank, and C) positive control. (JPG 136 kb) Additional file 4: Phenotype-informative SNP analysis results for 140-year-old skeletal remains from Deadwood, Dakota using the: A) Illumina® ForenSeq™ DNA Signature Prep Kit and B) HID-Ion AmpliSeq™ Externally Visible Characteristics (EVC) Prototype Panel (E1 = elution #1; E2 = elution #2). Concordant results were used to generate a composite profile (C). (XLSX 12 kb) Additional file 5: Human identity SNP results for 140-year-old skeletal remains from Deadwood, Dakota using the: A) Illumina® ForenSeq™ DNA Signature Prep Kit and B) HID-Ion AmpliSeq™ Identity Panel (E1 = elution #1; E2 = elution #2). Eighty of the human identity SNPs were common The Author(s) BMC Genomics 2016, 17(Suppl 9):750 between the two panels (shown in bold). Concordant results were used to generate a composite profile (C). (XLSX 17 kb) Additional file 6: Autosomal STR typing results for 140-year-old skeletal remains from Deadwood, Dakota using the Illumina® ForenSeq™ DNA Signature Prep Kit and MiSeq® MPS platform (E1 = elution #1; E2 = elution #2). (XLSX 12 kb) Additional file 7: X-STR typing results for 140-year-old skeletal remains from Deadwood, South Dakota using the Illumina® ForenSeq™ DNA Signature Prep Kit and MiSeq® MPS platform (E1 = elution #1; E2 = elution #2). (XLSX 11 kb) Acknowledgements This collaborative project was funded by the Institute of Applied Genetics (IAG) and the City of Deadwood Office of Historic Preservation. The skeletal remains were loaned to the IAG by the South Dakota State Historical Society-Archaeological Research Center (Loan #218, Accession #12–0051) for DNA testing. We would like to express our thanks to Deadwood City Archivist Michael Runge and the City of Deadwood’s Historic Preservation Officer Kevin Kuchenbecker for their invaluable insights into the historical aspects of the case; South Dakota archaeologist and repository manager Katie Lamie for providing information regarding burial excavation and handling of the remains prior to submission for DNA analyses; and Maiko Takahashi for technical assistance in the laboratory. Reagents for MPS were kindly provided by Illumina and Thermo Fisher Scientific. The opinions, findings, and conclusions or recommendations expressed in this publication are those of the authors and do not necessarily reflect those of the City of Deadwood, the South Dakota State Historical Society-Archaeological Research Center, Illumina, or Thermo Fisher Scientific. Publication charges were paid by the Center of Excellence in Genomic Medicine Research, King Abdulaziz University, Jeddah, Saudi Arabia. Declarations This article has been published as part of BMC Genomics Volume 17 Supplement 9, 2016: Proceedings of the 3rd International Genomic Medicine Conference: genomics. The full contents of the supplement are available online at http://bmcgenomics.biomedcentral.com/articles/supplements/ volume-17-supplement-9. Availability of data and materials All datasets supporting the conclusions of this article are included within the article and its additional files. Authors’ contributions ADA was the lead scientist who processed the bones, typed samples, analyzed the data, and prepared the manuscript; JDC and MK generated sequencing data; JLK performed data analyses; H G-K facilitated acquisition of the samples and contributed to the design of the study; MA, MA-E, AB, and MA-Q contributed to review of the data and preparation of the manuscript; and BB designed the study, performed data analyses, and contributed to manuscript preparation. All authors read and approved the final manuscript. Competing interests The authors declare that they have no competing interests. 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