Downloaded from genome.cshlp.org on September 30, 2021 - Published by Cold Spring Harbor Laboratory Press
Human Auditory Ossicles as an Alternative Optimal Source of Ancient DNA
Kendra Sirak1,2,*,†, Daniel Fernandes2,3,4,*,†, Olivia Cheronet2,3, Eadaoin Harney1,5,6, Matthew Mah1,7,8, Swapan Mallick1,7,8, Nadin Rohland1, Nicole Adamski1,8, Nasreen Broomandkhoshbacht1,8,‡, Kimberly Callan1,8, Francesca Candilio2,‡, Ann Marie Lawson1,8, Kirsten Mandl3, Jonas Oppenheimer1,8,‡, Kristin Stewardson1,8, Fatma Zalzala1,8,Alexandra Anders9, Juraj Bartík10, Alfredo Coppa11, Tumen Dashtseveg12, Sándor Évinger13, Zdeněk Farkaš10, Tamás Hajdu13,14, Jamsranjav Bayarsaikhan12,15, Lauren McIntyre16, Vyacheslav Moiseyev17, Mercedes Okumura18, Ildikó Pap13, Michael Pietrusewsky19, Pál Raczky9, Alena Šefčáková20, Andrei Soficaru21, Tamás Szeniczey13,14, Béla Miklós Szőke22, Dennis Van Gerven23, Sergey Vasilyev24, Lynne Bell25, David Reich1,7,8, Ron Pinhasi3,*
1 Department of Genetics, Harvard Medical School, Boston, MA 02115, USA 2 Earth Institute and School of Archaeology, University College Dublin, Dublin 4, Ireland 3 Department of Evolutionary Anthropology, University of Vienna, Vienna, 1090, Austria 4 CIAS, Department of Life Sciences, University of Coimbra, 3000-456 Coimbra, Portugal 5 Dept. of Organismic and Evolutionary Biology, Harvard University, Cambridge, MA 02138, USA 6 The Max Planck-Harvard Research Center for the Archaeoscience of the Ancient Mediterranean, Cambridge, MA 02138, USA and Jena, D-07745, Germany 7 Broad Institute of Harvard and MIT, Cambridge, MA 02142, USA 8 Howard Hughes Medical Institute, Harvard Medical School, Boston, MA 02115, USA 9 Institute of Archaeological Sciences, Eötvös Loránd University, H-1088 Budapest, Múzeum körút 4/B, Hungary 10 Slovak National Museum–Archaeological Museum, Žižkova 12, P.O. BOX 13, 810 06 Bratislava 16, Slovak Republic 11 Dipartimento di Biologia Ambientale, Sapienza Università di Roma, Rome 00185, Italy 12 Department of Anthropology and Archaeology, National University of Mongolia, Ulaanbaatar 14200, Mongolia 13 Department of Anthropology, Hungarian Natural History Museum, H-1083 Budapest, Ludovika tér 2, Hungary 14 Department of Biological Anthropology, Institute of Biology, Faculty of Science, Eötvös Loránd University Budapest, Pázmány Péter sétány 1/c. H-1117, Hungary 15 National Museum of Mongolia, Juulchin Street -1, Ulaanbaatar 210146, Mongolia 16 Oxford Archaeology, Janus House, Osney Mead, Oxford OX2 0ES, UK 17 Peter the Great Museum of Anthropology and Ethnography (Kunstkamera), Russian Academy of Science, Univercity Emb. 3, St. Petersburg 199034, Russia 18 Department of Genetics and Evolutionary Archaeology, Institute of Biosciences, University of São Paulo, Rua do Matão 277, Cidade Universitária 05508-090 São Paulo, Brazil 19 Department of Anthropology, University of Hawai’i at Mānoa, Honolulu, HI 96822, USA 20 Department of Anthropology, Slovak National Museum–Natural History Museum, Vajanského nábr. 2, P.O. BOX 13, 810 06 Bratislava 16, Slovak Republic 21 “Fr. J. Rainer” Institute of Anthropology, Romanian Academy, 13 Septembrie 13, P. O. Box 24, Bucharest, Romania 22 Institute of Archaeology, Research Centre for the Humanities, Hungarian Academy of Sciences, H-1097 Budapest, Tóth Kálmán utca 4, Hungary 23 Department of Anthropology, University of Colorado at Boulder, 1350 Pleasant Street, Boulder, CO 80309, USA
1 Downloaded from genome.cshlp.org on September 30, 2021 - Published by Cold Spring Harbor Laboratory Press
24 Institute of Ethnology and Anthropology, RAS, 32a Leninsky propsect, Moscow, 119991, Russia 25 Centre for Forensic Research, Simon Fraser University, 8888 University Drive, Burnaby,BC, V5A 1S6, Canada
†The first two authors (K.Si. and D.F.) should be regarded as joint-first authors.
* Correspondence to Ron Pinhasi (Tel: +43-664-60277-54721; [email protected]), Kendra Sirak ([email protected]), or Daniel Fernandes ([email protected]).
‡ Present addresses: Department of Anthropology, University of California, Santa Cruz, CA 95064, USA (N.B.); Department of Biomolecular Engineering, University of California, Santa Cruz, CA 95064, USA (J.O.); Soprintendenza Archeologia, Belle Arti e Paesaggio per la Città Metropolitana di Cagliari e le Province di Oristano e Sud Sardegna, Cagliari 09124, Italy (F.C.)
Running Title: Auditory ossicles as a source of ancient DNA
Keywords: Auditory ossicles, Ancient DNA, paleogenomic data, DNA optimization
2 Downloaded from genome.cshlp.org on September 30, 2021 - Published by Cold Spring Harbor Laboratory Press
1 ABSTRACT
2 DNA recovery from ancient human remains has revolutionized our ability to
3 reconstruct the genetic landscape of the past. Ancient DNA research has benefited from the
4 identification of skeletal elements, such as the cochlear part of the osseous inner ear, that
5 provide optimal contexts for DNA preservation; however, the rich genetic information
6 obtained from the cochlea must be counterbalanced against the loss of morphological
7 information caused by its sampling. Motivated by similarities in developmental processes
8 and histological properties between the cochlea and auditory ossicles, we evaluate the
9 ossicles as an alternative source of ancient DNA. We demonstrate that ossicles perform
10 comparably to the cochlea in terms of DNA recovery, finding no substantial reduction in data
11 quantity and minimal differences in data quality across preservation conditions. Ossicles can
12 be sampled from intact skulls or disarticulated petrous bones without damage to surrounding
13 bone, and we argue that they should be utilized when available to reduce damage to human
14 remains. Our results identify another optimal skeletal element for ancient DNA analysis and
15 add to a growing toolkit of sampling methods that help to better preserve skeletal remains for
16 future research while maximizing the likelihood that ancient DNA analysis will produce
17 useable results.
18
19 INTRODUCTION
20 Ancient DNA has become an important tool for addressing key questions about
21 human evolutionary and demographic history. Its rapid growth over the last decade has been
22 driven largely by advances in isolating (Dabney et al. 2013; Rohland et al. 2018), preparing
23 (Gansauge et al. 2017; Rohland et al. 2015), enriching (Fu et al. 2013, 2015; Haak et al.
24 2015; Mathieson et al. 2015), sequencing (Margulies et al. 2005), and analyzing (Briggs et al.
25 2007; Briggs et al. 2010; Ginolhac et al. 2011; Skoglund et al. 2014) small quantities of
26 degraded DNA. While these methodological advances have contributed to an improvement
27 in the quality and quantity of paleogenomic data obtained from ancient human remains, all
3 Downloaded from genome.cshlp.org on September 30, 2021 - Published by Cold Spring Harbor Laboratory Press
28 ancient DNA research fundamentally depends upon access to biological material that has
29 sufficient biomolecular preservation.
30 Skeletal tissue (i.e., bone or teeth) is the preferred biological material for human
31 ancient DNA analysis due to its ability to resist post-mortem degradation better than other
32 types of tissues, including skin and hair (Lindahl 1993; Smith et al. 2001, 2003; Collins et al.
33 2002). However, recent research has shown that not all bone elements are equally effective
34 in preserving DNA and has identified the dense bone encapsulating the cochlea within the
35 petrous pyramid of the temporal bone (referred to henceforth as the ‘cochlea’) (Gamba et al.
36 2014; Pinhasi et al. 2015), as well as the cementum layer in teeth roots (Damgaard et al.
37 2015; Hansen et al. 2017) as especially DNA-rich parts of the skeleton. The use of these
38 skeletal elements that act as repositories for the long-term survival of DNA has proven to be
39 particularly important for the analysis of biological samples recovered from regions where
40 high temperatures and/or humidity increase the rate of molecular degradation and result in
41 low concentrations of damaged DNA with reduced molecular complexity (e.g., Broushaki et
42 al. 2016; Lazaridis et al. 2016; Schuenemann et al. 2017; Skoglund et al. 2017; Fregel et al.
43 2018; Harney et al. 2018; van de Loosdrecht et al. 2018).
44 While use of the cochlea has contributed to the success of ancient DNA research
45 across a growing range of geographic and temporal contexts, it is important to balance
46 analytical goals with the irreparable damage to human skeletal remains that results from
47 destructive analyses (Prendergast and Sawchuk 2018; Sirak and Sedig 2019). Ancient DNA
48 is one of several such analyses that are now widely used in archaeology (others include
49 radiocarbon dating and stable isotope analysis) (Hublin et al. 2008; Mays et al. 2013;
50 Makarewicz et al. 2017; Pinhasi et al. 2019). To minimize damage to complete skulls from
51 ancient DNA sampling while still accessing the rich genetic data in the cochlea, we
52 developed a “Cranial Base Drilling” method to limit damage to surrounding bone areas when
53 a skull is intact (Sirak et al. 2017). However, even this method involves destructive sampling
54 of the cochlea. Recent work has highlighted the fact that the morphological analysis of the
55 inner ear part of the petrous pyramid (which includes the cochlea) can reveal population
4 Downloaded from genome.cshlp.org on September 30, 2021 - Published by Cold Spring Harbor Laboratory Press
56 relationships and thus that this part of the skeleton harbors some information about
57 population history (e.g., Spoor et al. 2003; Ponce de León et al. 2018). While genetic
58 comparisons of samples involve analysis of tens of thousands of independent markers
59 (single nucleotide polymorphisms, or SNPs) that provide far higher statistical resolution than
60 can be obtained by study of the smaller number of data points that can be extracted from
61 morphological analysis, not all cochlear bone yields sufficient amounts of ancient DNA. The
62 fact that there is morphological information in the petrous pyramid that will be destroyed
63 through sampling of ancient DNA highlights the importance of being a careful steward of
64 these elements.
65 As part of a search for alternative optimal sources for ancient DNA that can be used
66 in place of the cochlea, we noted that auditory ossicles have similar developmental
67 processes and histological properties as the osseous inner ear. We therefore tested whether
68 the ossicles – the smallest bones in the human body – might serve as alternative optimal
69 substrates for ancient DNA analysis.
70 71 Ossicle development and histology
72 The mechanism by which cochlear bone preserves endogenous DNA better than
73 other skeletal elements or other regions of the same petrous pyramid is not well understood;
74 however, it is likely related to the fact that human petrous bones are unique in being
75 characterized by a near-absence of growth or remodeling following the completion of
76 ossification by approximately 24 weeks in utero (Sølvsten Sørensen et al. 1992; Frisch et al.
77 1998; Hernandez et al. 2004). The inhibition of bone remodeling leads to the presence of a
78 larger number of mineralized osteocytes that reside in lacunae within the bone tissue
79 (Hernandez et al. 2004; Bell et al. 2008; Busse et al. 2010; Rask-Andersen et al. 2012). One
80 hypothesis (Pinhasi et al. 2019) is that ‘microniches’ created in the bone tissue by the
81 maintenance of mineralized osteocytes, combined with the protected location of the cochlea,
82 may act as repositories that encourage the long-term preservation of DNA (Bell et al. 2008;
5 Downloaded from genome.cshlp.org on September 30, 2021 - Published by Cold Spring Harbor Laboratory Press
83 Kontopoulos et al. 2019). Ossicles are similar to the cochlea in this respect (see below), and
84 we therefore hypothesized that they might also preserve high amounts of endogenous DNA.
85 In humans, the middle ear (the region of the ear located medial to the eardrum and
86 lateral to the oval window of the inner ear) is enclosed within the temporal bone and contains
87 the three auditory ossicles: the malleus, incus, and stapes (Figure 1). The ossicles
88 effectively allow humans to hear by transmitting sound-induced mechanical vibrations from
89 the outer to the inner ear. Though the ossicles do not experience high-strain biomechanical
90 loading, they are subject to unique vibrational patterns that impact their development and
91 characteristics over the course of an individual’s lifespan (Rolvien et al. 2018). In contrast to
92 the majority of the human skeleton, but similar to the cochlea, the auditory ossicles present
93 with their final size and morphology at birth following the onset of ossification of between 16
94 and 18 weeks in utero and the completion of ossification around 24 weeks gestational age
95 (Marotti et al. 1998; Yokoyama et al. 1999; Cunningham et al. 2000; Duboeuf et al. 2015;
96 Richard et al. 2017). The ossicles and cochlea appear to follow the same developmental
97 pattern of rapidly increasing bone volume through cortical thickening and densification, along
98 with mineralization of the bony matrix (Richard et al. 2017).
99
100
101 Figure 1: The three auditory ossicles. From left to right, the stapes, malleus, and incus.
6 Downloaded from genome.cshlp.org on September 30, 2021 - Published by Cold Spring Harbor Laboratory Press
102
103 Like the cochlea, ossicular bone tissue is rapidly modeled around the time of birth;
104 although it may undergo further postnatal maturation, there are no signs of bone remodeling
105 observed above the age of one year (Richard et al. 2017; Rolvien et al. 2018). The inhibition
106 of bone remodeling of the auditory ossicles is evident from features such as the presence of
107 a dense meshwork of collagenous fibers organized in an interlacing woven pattern, a smooth
108 fibrous appearance, and limited vascular channels and viable osteocytes (Marotti et al. 1998;
109 Chen et al. 2008). As in the case of the cochlea and in contrast to other skeletal elements,
110 mineralized osteocytes appear to accumulate in the ossicles throughout an individual’s life
111 without resulting in increased bone absorption (Marotti et al. 1998; Kanzaki et al. 2006;
112 Rolvien et al. 2018), likely conserving the overall architecture of the ossicles in order to
113 maintain optimal sound transmission (Kanzaki et al. 2006; Rolvien et al. 2018). While the
114 consequences of inhibited bone remodeling and the accumulation of mineralized osteocytes
115 have only been previously studied from a clinical perspective, we hypothesized that these
116 features might contribute to an optimized DNA preservation similar to that in the cochlea by
117 creating the ‘microniches’ that enable long-term DNA survival (Bell et al. 2008).
118
119 Use of ossicles in ancient DNA research
120 Due to their small size and tendency to become dislodged from the skull, ossicles are
121 only seldom recovered during excavation and are easily lost in collections excavated
122 decades ago. While ossicles are not recovered for every burial in every context, we have
123 found that these bones may remain lodged within the middle ear of intact skulls or can be
124 identified in the vicinity of a burial during excavation (Qvist et al. 2000). Given the value of
125 the ossicles as a substrate for ancient DNA analysis, as is demonstrated in this study, we
126 hope that more archaeologists, anthropologists, and museum curators will focus on
127 preserving these elements.
128 It is important to recognize that ossicles, just like the cochlea, are morphologically
129 informative. Indeed, there is a growing body of literature examining the comparative
7 Downloaded from genome.cshlp.org on September 30, 2021 - Published by Cold Spring Harbor Laboratory Press
130 morphology and pathology of the ossicles (e.g., Rak and Clarke 1979; Arensburg et al. 1981,
131 2005; Siori et al. 1995; Spoor et al. 2003; Crevecoeur et al. 2007; Quam and Rak 2008;
132 Quam et al. 2013a, 2013b; Stoessel et al. 2016). While differences in metric and non-metric
133 features of the auditory ossicles may be taxonomically informative for comparisons across
134 the genus Homo (e.g., Heim 1982; Spoor et al. 2003; Quam and Rak 2008; though see
135 Arensburg et al. 1981), it is unclear whether phylogenetic and population relationship
136 information can be retrieved from the auditory ossicles. In cases where ossicle morphology
137 may be a subject of future research, we encourage the anthropological study (including
138 description and measurement) and surface or micro-CT scanning of the ossicles to collect
139 metric and morphological information prior to ancient DNA analysis. Any ossicles that exhibit
140 visible pathologies should be avoided.
141 Though some anthropological attention has been given to the ossicles, we are not
142 aware of previous genetic analyses of these bones. Only a single study has specifically
143 attempted to analyze DNA from the ossicles, collecting the ossicles during medical autopsies
144 of recently deceased individuals and determining them to be a reliable DNA source from
145 bodies ranging from freshly deceased to highly putrefied (Schwark et al. 2015).
146
147 RESULTS
148 We carried out pilot work (described in Supplementary Note S1) to assess if the
149 quality and quantity of ancient DNA data recovered from the ossicles was approximately
150 similar to that recovered from the cochlea. The results of this pilot work (Supplementary
151 Table S1) suggested that ossicles perform comparably to the cochlea in metrics such as
152 amount of endogenous human DNA recovered and frequency of damage at the terminal
153 nucleotide of the DNA molecule (a commonly used measure of ancient DNA authenticity).
154 Based on these results, we selected an additional 16 archaeological samples for further
155 study from a wide range of geographic locations with varying climates and dated to between
156 ~6500–1720 years before present (yBP) and with both petrous bone and ossicles present
157 (Table 1, with detailed sample information in Supplementary Table S2). Skeletal samples for
8 Downloaded from genome.cshlp.org on September 30, 2021 - Published by Cold Spring Harbor Laboratory Press
158 both pilot work and the main study were excavated by professional archaeologists who held
159 all appropriate permits and permissions from the institutions managing archaeological
160 research in each region or country from which samples originate; these samples were
161 provided to us by the archaeologists for the purpose of paleogenomic analysis. No direct
162 descendants or stakeholder communities were identified for any sample included in this work.
163 Processing of all skeletal material was carried out following suggested best practices for
164 ancient DNA research, such as those outlinsed in Prendergast and Sawchuk (2018) and
165 Sirak and Sedig (2019). To be included in this study, each specimen was required to have at
166 least one ossicle as well as the cochlea of the petrous bone available for comparative
167 analysis. Whenever possible, a petrous bone that had an antimere was chosen (Prendergast
168 and Sawchuk 2018).
169 A summary of sequencing results for the 16 individuals reported in this paper is
170 presented in Table 1 and Figure 2; for more detailed information, see Supplementary Table
171 S2 and Supplementary Figure S1.
172
173 Table 1: Sample information and summary of sequencing results. The 10 samples
174 considered to be ‘working’ for the purposes of this analysis are labeled with an asterisk. ID (%) Site Date [95CI] Match [Z-score] (Country) 1240K SNPs Deamination Initial Material 1240K ANGSD 1240K Coverage % Unique Reads Target Sequences mtDNA Consensus Contamination Mean Expected at 500K On- number of sequences) Base of Aligned Reads Endogenous DNA Yield (not normalized for total Frequencies on Terminal
Cochlear Powder; 0.996 0.008 7.53 622982 0.541 0.139 67.8 Katelai 1000-800 56 mg [0.990-0.999] [2.897]
614* (Pakistan) BCE Ossicle 0.994 0.005 30.55 776019 0.674 0.131 83.2 (2: I, M) [0.986-0.998] [2.593]
Cochlear Powder; 0.994 N/A 3.27 391574 0.340 0.239 50.4 Geoksiur 5000-2000 47 mg [0.984-0.998] (Female)
644* (Turkmenistan) BCE Ossicle 0.989 N/A 0.57 177819 0.155 0.225 31.5 (2: I, M) [0.974-0.996] (Female) Cochlear Powder; 0.999 0.002 70.15 829510 0.721 0.044 98.9 Fofonovo 6000-3000 47 mg [0.995-1.000] [2.053]
648* (Russia) BCE Ossicle 0.991 0.002 72.68 858204 0.746 0.045 98.8 (2: I, S) [0.983-0.996] [3.254]
9 Downloaded from genome.cshlp.org on September 30, 2021 - Published by Cold Spring Harbor Laboratory Press
Cochlear Powder; 0.995 0.005 Bayankhongor, 56.48 855679 0.744 0.139 98.7 Ulziit sum, 2500-400 55 mg [0.987-0.998] [5.841]
818* Maanit uul BCE Ossicle 0.965 0.040 71.80 883674 0.768 0.106 98.2 (Mongolia) (1: I) [0.953-0.974] [20.256] Cochlear Powder; 0.981 N/A Peugeot 46.79 843283 0.733 0.134 97.7 43-410 49 mg [0.972-0.988] (Female) Garage CE 1257* Ossicle 0.971 N/A (Great Britain) 55.83 829630 0.721 0.128 94.6 (2: I, M) [0.960-0.980] (Female) Cochlear Powder; N/A 0.01 266 0.000 0.064 - - Ban Chiang 900-300 48 mg (Female)
B26 (Thailand) BCE
BCES Ossicle 0.940 N/A 0.07 12438 0.008 0.013 - (3: I, M, S) [0.877-0.972] (Female) Cochlear Powder; 0.996 0.006 63.00 770152 3.909 0.091 98.6 Blatné 2200-2000 50 mg [0.990-0.999] [6.180]
33* (Slovakia) BCE BLAT Ossicle 0.998 0.005 68.25 725438 3.914 0.088 99.1 (2: I, M) [0.993-1.000] [5.184] Cochlear Powder; 0.995 0.005 73.88 779326 3.727 0.078 97.9 Glăvăneşti 3500-3000 47 mg [0.989-0.998] [4.856]
14* (Romania) BCE
GLAV Ossicle 0.991 0.004 59.40 749401 3.343 0.068 96.8 (2: I, M) [0.982-0.995] [4.574] Cochlear Powder; 0.002 253 0.000 0.014 - - - Al-Makhdarah 1190-800 53 mg
13H (Yemen) BCE Ossicle MKDH 0.001 531 0.000 0.025 - - - (2: I, M) Cochlear Powder; 0.004 115 0.000 0.024 - - - Kharibat al- 53 mg Ahjur 1000 BCE UA Ossicle KHRB (Yemen) 0.007 1244 0.001 0.098 - - - (2: I, M) Cochlear Powder; 0.983 N/A 0.99 134254 0.126 0.251 21.3 Laranjeiras I 2900-1800 50 mg [0.967-0.992] (Female)
3935 (Brazil) BCE Ossicle N/A 0.08 10743 0.009 0.244 - 2.4 (1: I) (Female) Cochlear Powder; 0.989 0.005 9.09 396963 0.478 0.213 50.6 Laranjeiras I 2900-1800 50 mg [0.977-0.995] [1.009]
3937 (Brazil) BCE Ossicle 0.08 7251 0.006 0.222 - - 1.6 (2: I, M) Cochlear Powder; 0.991 0.009 45.11 638507 1.712 0.150 92.4 Laranjeiras I 2900-1800 50 mg [0.982-0.996] [4.658] (Brazil) BCE 3938* Ossicle 0.992 0.010 30.58 696203 1.945 0.158 90.6 (2: I, M) [0.981-0.997] [5.142] Cochlear Powder; N/A 0.07 9791 0.008 0.207 - 2.2 Buccino 3000-2000 50 mg (Female)
9214 (Italy) BCE Ossicle N/A 0.13 37673 0.032 0.017 - 6.6 (2: I, M) (Female) Cochlear Powder; 0.961 0.006 57.47 689962 2.281 0.089 95.7 Olmo di Nogara 1400-1200 50 mg [0.972-0.994] [4.279] (Italy) BCE 9309* Ossicle 0.952 0.052 39.10 557972 0.833 0.089 68.8 (1: I) [0.932-0.967] [7.632] Cochlear Powder; 0.982 N/A 49.03 698186 2.466 0.103 95.5 Olmo di Nogara 1400-1200 50 mg [0.969-0.989] (Female) (Italy) BCE 9323* Ossicle 0.975 N/A 72.01 710183 2.719 0.118 96.7 (1: I) [0.961-0.984] (Female) 175
176 Out of 16 individuals included in this study, both the cochlea and ossicles produced
177 enough data to call mitochondrial DNA (mtDNA) haplogroups, assess damage patterns at
178 the terminal nucleotide of the molecule, and make estimations comparing contamination
179 between the cochlea and the ossicles for 10 individuals. These individuals are henceforth
180 referred to as the ‘working individuals.’ Any individuals that did not meet these three criteria
10 Downloaded from genome.cshlp.org on September 30, 2021 - Published by Cold Spring Harbor Laboratory Press
181 were considered to be ‘not working,’ as we were unable to effectively carry out inter-element
182 comparisons.
183 For four individuals, both the cochlea and ossicles were considered to have ‘failed’ our
184 analysis: one individual from Thailand produced marginal data for both skeletal elements
185 with a large error interval for the mtDNA contamination estimate – calculated as 1 minus the
186 rate of mitochondrial matches to the consensus sequence (Fu et al. 2013) – that was only
187 assessible when the ossicles were used; two individuals, both from Yemen, did not produce
188 enough data to allow for the determination of mtDNA haplogroup or contamination estimates;
189 and a Copper Age individual from Italy produced a very large interval for the mtDNA
190 contamination estimate and did not produce enough data to allow for the determination of
191 mtDNA haplogroup. For two individuals from Late Holocene Brazil, 3935 and 3937, mtDNA
192 haplogroups could not be determined and contamination error intervals were large for the
193 ossicles only.
194 We performed Wilcoxon Signed-Rank tests to compare the data generated using the
195 ossicles and cochlear samples for the 10 working individuals (Supplementary Table S3). For
196 completeness and confirmation, we present these results with and without including
197 individuals 3935 and 3937 for the six absolute metrics analyzed (out of eight total metrics,
198 see Supplementary Table S3), because with the exception of contamination estimates, these
199 individuals produced sufficient data to carry out statistical analysis.
200 With the Wilcoxon test, we obtained an average endogenous DNA yield of 47.83% for
201 the 10 working cochlea samples and 50.01% for the corresponding ossicles, a non-
202 significant difference (Table 1, Figure 2 Panel A) (p=0.557; Supplementary Table S3). The
203 average complexity for cochlea and ossicles was 89.36% and 85.83%, respectively (Table 1,
204 Figure 2 Panel B); this difference is also non-significant (p=0.275; Supplementary Table S3).
205 Complexity, defined here as the percentage of unique reads observed after down-sampling
206 to 500,000 sequences that align to the ~1.24 million targeted SNPs, is a potentially more
207 informative metric for comparing performance between the cochlea and ossicles because it
208 is directly related to the maximum amount of sequencing data the library can possibly yield
11 Downloaded from genome.cshlp.org on September 30, 2021 - Published by Cold Spring Harbor Laboratory Press
209 and is not biased by differences in sequencing depth across samples. Overall, these results
210 suggest that the quantity of data generated using ossicles is comparable to that generated
211 using the cochlea. Any minor differences are more likely due to chance than to a systematic
212 difference in DNA preservation between the cochlea and ossicles.
213
214
215 Figure 2: Comparative results between cochlea (yellow) and ossicle (green) samples from
216 the same individuals; bold font indicates the samples that were used in Wilcoxon Signed-
217 Rank test. A. Endogenous shotgun DNA ratios of the total reads. B. Complexity as
218 percentage of unique reads expected from 500,000 reads hitting targets. C. Deamination
219 frequencies on the terminal bases of sequences aligning to the human genome. D.
220 Contamination estimates calculated by subtracting the rate of mitochondrial matches to the
221 consensus sequence from 1 (smooth bars) and based on the heterozygosity of the X
12 Downloaded from genome.cshlp.org on September 30, 2021 - Published by Cold Spring Harbor Laboratory Press
222 Chromosome of male individuals (textured bars). Error bars indicate the 95% confidence
223 interval.
224
225 The average mtDNA coverage was ~423 (range ~61–1244 ) for the 10 working
226 cochlea samples and 388 for the corresponding ossicles (range ~31–1286 )
227 (Supplementary Table S2), this is also a non-significant difference (p=0.275; Supplementary
228 Table S3). The average coverage of the ~1.24 million targeted SNPs from across the
229 genome was 1.72 for the working cochlea samples, and 1.58 for the ossicles (Table 1);
230 on average, 711,916 SNPs were called when the cochlea was used and 696,454 were
231 called when the ossicles were used (without normalizing for total number of sequences;
232 Table 1). Consistent with other results, both of these differences were non-significant when
233 assessed with the Wilcoxon Signed-Rank test (p=1.000 and 0.770, respectively;
234 Supplementary Table S3).
235 For the 10 working samples, the average deamination frequency was slightly reduced
236 from 12.31% to 11.5% when the ossicles were used (Table 1, Figure 2 Panel C; see
237 Supplementary Figure S1), a decrease that, although small, was marginally statistically
238 significant (p=0.049; Supplementary Table S3). We note that if we include individuals 3935
239 and 3937, considered to be ‘not working’ by our previously described parameters, but for
240 which we were able to generate data using the cochlea, this statistic loses significance
241 (p=0.0923; Supplementary Table S3). This is the only metric for which there is a change in
242 significance when including individuals 3935 and 3937.
243 Mitochondrial contamination estimates (inferred by identifying mismatches to the
244 mtDNA consensus sequence (Fu et al. 2013)) increased from an average of 0.80% when the
245 cochlea was used to 1.80% when the ossicles were used, (Table 1, Figure 2 Panel D); this
246 difference has a significant p-value of 0.014 (Supplementary Table S3). Contamination
247 estimates based on the heterozygosity rate of the X Chromosome (a test only applicable to
248 males) (Korneliussen et al. 2014) averaged 0.58% for the cochlea and 1.70% for the
13 Downloaded from genome.cshlp.org on September 30, 2021 - Published by Cold Spring Harbor Laboratory Press
249 ossicles, a non-significant change (p=0.688) (Table 1, Figure 2, Supplementary Table S3).
250 The overall low levels of contamination are also supported by consistency in the estimation
251 of mtDNA haplogroups and molecular sex for all cochlea-ossicle pairs (Table 1, Figure 2,
252 Supplementary Table S2).
253
254 DISCUSSION
255 DNA recovery from the auditory ossicles
256 This study presents a direct comparison of DNA recovery from the ossicles and
257 corresponding cochlear bone using archaeological specimens that originate from varying
258 geographic and temporal contexts and offers several new insights. First, we demonstrate
259 that the ossicles perform comparably to the cochlea in terms of ancient DNA recovery
260 regardless of sample preservation. Focusing on 10 ‘working’ individuals from whom we were
261 able to generate enough ancient DNA data to call mtDNA haplogroups, assess damage
262 pattern, and make contamination estimates for both the cochlea and ossicles, we find that
263 the use of the cochlea or ossicles from each individual produces similar amounts of
264 endogenous DNA, mtDNA coverage, nuclear SNP coverage, and number of SNPs called.
265 We demonstrate that there is no substantial reduction in data quantity or complexity
266 associated with the analysis of the ossicles instead of the cochlea, despite a smaller
267 absolute quantity of bone material used. Second, although we find that the ossicles show a
268 marginal reduction in the frequency of deamination (a signal of ancient DNA authenticity)
269 compared to the corresponding cochlea, the magnitude of difference is not large enough to
270 affect whether we consider the DNA to be authentically ancient, and this change becomes
271 non-significant when we use a larger comparison pool of 12 individuals for which this metric
272 was available. This finding, which may be an artifact of sample size or possibly related to an
273 aspect of preservation that is currently unknown, should continue to be investigated using
274 additional samples in future research.
275 We observe a modest but significant increase in the amount of estimated
276 contamination between the cochlea and ossicles using mtDNA (from 0.80% to 1.80%), and a
14 Downloaded from genome.cshlp.org on September 30, 2021 - Published by Cold Spring Harbor Laboratory Press
277 non-significant increase using heterozygosity on the X Chromosome (from 0.58% to 1.70%).
278 These results suggest the possibility that ossicle extractions may be more susceptible to
279 contamination than petrous bones extractions, as might be expected by the greater difficulty
280 of cleaning the samples prior to ancient DNA analysis. However, we emphasize that the
281 average mtDNA contamination in the ossicles in our analysis remains well under the
282 accepted levels typically found in the ancient DNA literature (e.g. requiring a >95% match to
283 the consensus sequence (Fu et al. 2013)). Thus, our results support the conclusion that
284 ossicles are a useful and effective alternative to petrous samples in ancient DNA research.
285 Future cleaning methods for ossicles may be able to reduce contamination rates even
286 further.
287 While four of the six individuals classified as ‘not working’ did not produce sufficient
288 amounts of data for either the cochlea or ossicles, only the cochlea produced sufficient
289 amounts of authentic data for two first-degree relatives from the same site in Brazil (3935
290 and 3937). While it is possible that the 9-fold and 31-fold decrease in complexity between
291 the cochlea and ossicles of each individual (respectively) results from use of the ossicles,
292 other variables such as burial- or sample-specific diagenesis need to be considered as well.
293 In fact, the ossicles and cochlea from another individual (3938) from the same site produced
294 comparable data for the two skeletal elements. The opposite scenario, where the ossicles
295 out-perform the cochlea, has also been noted in the case of a previously published Pastoral
296 Neolithic individual from Tanzania (Prendergast et al. 2019), for which high quality data was
297 recovered only from the ossicles (for example, coverage of targeted SNPs increased from
298 0.233x using the cochlea to 1.58x using the ossicles).
299 Our data support no consistent differences between the ossicles and cochlea in
300 terms of ancient DNA data recovered, suggesting at most only minimal differences between
301 data quality associated with the analysis of the ossicles instead of the cochlea. We therefore
302 conclude that the auditory ossicles, when present, are an alternative optimal skeletal
303 element that can be used in ancient DNA research in place of the cochlea.
15 Downloaded from genome.cshlp.org on September 30, 2021 - Published by Cold Spring Harbor Laboratory Press
304 Though they are small, often isolated, and can be accessed without significant
305 impact to larger and more morphologically informative parts of the skeleton, the use of
306 ossicles for ancient DNA analysis still requires the destruction of human skeletal material
307 that may be anthropologically valuable. Ossicles have previously been used in studies of
308 comparative morphology; most notably, they have provided insight into morphological
309 differences and functional similarities in the middle ear of Neandertals and anatomically
310 modern humans, which has implications for understanding the auditory capacity of extinct
311 hominins (e.g., Stoessel et al. 2016). For this reason, we encourage all researchers
312 contemplating ancient DNA analysis to balance their analytical goals with the impact that
313 sampling for ancient DNA analysis will have on future availability of material.
314 In light of these findings, we suggest that archaeologists and curators attempt to
315 identify and preserve auditory ossicles whenever possible. Ideally, ossicles would be
316 identified and collected during archaeological recovery of human skeletal remains in a way
317 that minimizes the introduction of contamination, particularly given some evidence of this
318 contamination to have downstream effects during the analysis of the ossicles. Methods for
319 proactively reducing contamination include wearing disposable medical gloves that are
320 changed frequently when handling samples, avoiding washing skeletal material with water,
321 and storing samples in a cold, dry place as soon as possible (Llamas et al. 2017).
322 The use of ossicles for ancient DNA analysis will contribute to the successful analysis
323 of skeletal material that does not have a petrous bone present, or sets of remains that have
324 a petrous bone that cannot be processed in a destructive manner for ancient DNA research
325 (for example, those that may be morphologically-intact and displayed in museum collections).
326 On a broader level, the identification of the ossicles as an alternative optimal skeletal
327 element for ancient DNA analysis contributes to the reduction in the amount of damage
328 inflicted to human skeletal samples for the purposes of ancient DNA analysis. It is another
329 step toward the preservation of DNA-rich and anthropologically valuable skeletal material for
330 future studies that may benefit from methodological improvements unknown at present.
331
16 Downloaded from genome.cshlp.org on September 30, 2021 - Published by Cold Spring Harbor Laboratory Press
332 METHODS
333 Sample Selection and Preparation
334 The number of ossicles collected for each of the archaeological samples varied (see
335 Table 1), but the incus and malleus were identified and collected most frequently (n=10 and
336 n=8, respectively) while the stapes was identified and collected least frequently (n=2), likely
337 due to its diminutive size and fragility. In most cases, we recovered the ossicles while
338 following the standard cochlea sampling procedure (Pinhasi et al. 2019). In other cases, we
339 intentionally dislodged the ossicles from the skull for the purpose of this study; in most of
340 these instances, the ossicles were partially visible within the external auditory meatus. To
341 dislodge the ossicles, we cleaned a small engraving burr (described in Sirak et al. 2017) by
342 wiping it with a diluted bleach solution (~10% concentration). We placed the cleaned burr
343 inside the external auditory meatus and gently manipulated it within the inner ear canal. This
344 caused no apparent damage to the ossicles or to the cranium from which they were retrieved.
345 All ossicles were immediately placed into a sterile 2.0mL tube upon their removal from the
346 ear canal.
347 The preparation of all skeletal material for ancient DNA analysis was carried out in
348 dedicated cleanrooms at University College Dublin (UCD) or at the University of Vienna
349 following standard anti-contamination protocols (e.g., Hofreiter et al. 2001; Poinar 2003;
350 Llamas et al. 2017). All petrous bones were processed following a standard protocol (Pinhasi
351 et al. 2019). This protocol uses a dental sandblaster to systematically locate, isolate, and
352 clean the cochlea, which is then milled to homogeneous bone powder. Approximately 50 mg
353 of bone powder from the cochlea (range: 47–56 mg) was aliquoted for DNA extraction.
354 Complete auditory ossicles were decontaminated through exposure to UV irradiation for 10
355 minutes on each side. DNA clean room for DNA extraction and preparation of sequencing
356 libraries.
357
358 DNA Extraction
17 Downloaded from genome.cshlp.org on September 30, 2021 - Published by Cold Spring Harbor Laboratory Press
359 DNA was extracted from the cochlear bone powder and the whole auditory ossicles
360 in ancient DNA facilities at the University of Vienna following a standard ancient DNA
361 extraction protocol (Dabney et al. 2013) with a modification (Korlević et al. 2015) that uses
362 the tube assemblies from the High Pure Viral Nucleic Acid Large Volume kit (Roche,
363 05114403001). The intact ossicles were placed in the extraction buffer, and completely
364 dissolved during the incubation period in most cases. Lysates were mixed with Binding
365 Buffer and applied to the column assembly. DNA was bound to the silica matrix of the
366 columns by centrifugation and was washed twice with 650 µL of PE buffer (Qiagen) and
367 spun through the columns at 6000 rpm for 1 minute each time. After the column was put in a
368 fresh 1.5 mL collection tube, 25 µL of TET buffer was pipetted on the dry spun columns’
369 silica membrane. After a 10-minute incubation at room-temperature, the columns were spun
370 at maximum speed for 1 minute. The elution step was repeated to give a final volume of 50
371 µL of DNA extract. A negative control that contained no bone material was included with
372 each extraction batch.
373 374 Library Preparation
375 Next generation sequencing libraries were prepared in ancient DNA facilities at
376 Harvard Medical School from all extracts and controls included in the main study using a
377 library preparation method optimized for ancient DNA (Rohland et al. 2015). This protocol
378 uses a partial-UDG treatment that causes characteristic C-to-T ancient DNA damage to be
379 restricted to the terminal molecules while nearly eliminating it in the interior of the DNA
380 molecules so that the library can be used to test for ancient DNA authenticity. 10 μL of DNA
381 extract was used as input during library preparation. Libraries were enriched for ~1.2 million
382 nuclear sites across the genome (‘1240K capture’) in addition to sites on the human
383 mitochondrial genome (Fu et al. 2013, 2015; Haak et al. 2015; Mathieson et al. 2015).
384 Enriched libraries were sequenced on an Illumina NextSeq 500 instrument, with 2x76 cycles
385 and an additional 2x7 cycles used for identification of indices. In addition, a small proportion
386 of reads were generated from unenriched versions of each library. This unenriched
18 Downloaded from genome.cshlp.org on September 30, 2021 - Published by Cold Spring Harbor Laboratory Press
387 (‘shotgun’) data was used to estimate the proportion of endogenous molecules in each
388 library.
389
390 Data Processing
391 Following sequencing, we trimmed molecular adapters and barcodes from
392 sequenced reads prior to merging forward and reverse reads using publicly available
393 software and pipelines found at https://github.com/DReichLab/ADNA-Tools. We allowed up
394 to three mismatches at positions of low base quality (<20) and up to one mismatch at higher
395 base quality (≥20); in cases of base matching, we kept the highest base quality in the
396 overlap region, while in cases where bases differed, we retained the higher quality base with
397 reduced base quality of the difference of the two base qualities. We aligned reads to the
398 mitochondrial RSRS genome (Behar et al. 2012) and to the hg19 human reference
399 sequence with the samse command in BWA (v0.7.15) (Li and Durbin 2009). We use the
400 hg19 build of the human genome to avoid potential reference bias possible when performing
401 population genetics analysis with other samples previously aligned to hg19, which is the
402 historical norm for ancient DNA studies. Other versions of the human reference are expected
403 to have nearly identical numbers of SNPs in aligned reads, but those SNPs may be biased
404 relative to hg19.
405 We used the tool ContamMix (Fu et al. 2014) to determine the rate of matching
406 between the consensus RSRS sequence and reads which aligned to the mitochondrial
407 genome. We determined the rate of C-to-T substitution at the terminal ends of each
408 molecule using PMDtools (https://github.com/pontussk/PMDtools; Skoglund et al. 2014). We
409 used the tool ANGSD (Korneliussen et al. 2014) to determine the amount of contamination in
410 the X Chromosome of individuals identified as genetically male. The complexity of the
411 sample was assessed by quantifying the number of unique reads expected from a pre-
412 determined number of reads hitting target.
413
414 DATA ACCESS
19 Downloaded from genome.cshlp.org on September 30, 2021 - Published by Cold Spring Harbor Laboratory Press
415 All sequencing data generated in this study have been submitted to the European
416 Nucleotide Archive. (ENA https://www.ebi.ac.uk/ena) under accession number PRJEB32751.
417
418 ACKNOWLEDGEMENTS
419 This work was partially supported by a European Research Council starting grant
420 ADNABIOARC 263441 to R.P, a National Science Foundation (NSF) Doctoral Dissertation
421 Research Improvement Grant BCS-1613577 to K.Si, an Irish Research Council grant
422 GOIPG/2013/36 to D.F., a graduate student fellowship from the Max Planck-Harvard
423 Research Center for the Archaeoscience of the Ancient Mediterranean (MHAAM) to E.H,
424 and Russian Foundation for Basic Research grants 18-00-00360, 18-09-00349 to V.M. T.H.
425 and T.S. were supported by grants from the Hungarian Research, Development and
426 Innovation Office, project numbers FK128013 and TÉT_16-1-2016-0020. D.R. is an
427 investigator of the Howard Hughes Medical Institute. The authors would like to thank
428 Canterbury Archaeological Trust for permission to analyze sample 1257 (I4491); full details
429 about this sample can be found in the CAT PXA report (Helm et al. 2017). We also would
430 like to thank Museu do Homem do Sambaqui “Pe. João Alfredo Rohr” for permission to
431 analyse the samples from Brazil included in this analysis.
432
433 DISCLOSURE DECLARATION
434 The authors declare no conflict of interest.
435
436 REFERENCES
437 Anders, A. 2017. Emberek az újkőkorban. Polgár környéki neolitikus temetkezések 438 bioszociális feldolgozásának első eredményei (People from the Neolithic. First results of the 439 biosocialarchaeological investigations on Neolithic burials of the Polgár micro region). Thesis 440 submitted to Eötvös Loránd University, Budapest. 441 442 Anders A, Nagy EG. 2007. Late Neolithic burial rites at the site of Polgár-Csőszhalom-dűlő. 443 In The Lengyel, Polgár and related cultures in the Middle/Late Neolithic in Central Europe. 444 (eds. Kozłowski JK, Raczky P), pp.83-96. Polish Academy of Arts and Sciences, Kraków. 445 446 Arensburg B, Belkin V, Wolf M. 2005. Middle ear pathology in ancient and modern 447 populations: incudal osteoma. Acta oto-laryngologica 125, 1164-1167.
20 Downloaded from genome.cshlp.org on September 30, 2021 - Published by Cold Spring Harbor Laboratory Press
448 449 Arensburg B, Harell M, Nathan H. 1981. The human middle ear ossicles: Morphometry, and 450 taxonomic implications. Journal of Human Evolution 10, 199-205. 451 452 Bartík J, Šefčáková A. 2004. Hrob so šálkou protoúnětickej kultúry z Blatného, okr. Bratislava- 453 vidiek, Slovensko (A grave with a cup of Proto Únětice Culture from Blatné, Greater city of 454 Bratislava, Slovakia). In Zborník K poctě Vladimíru Podborskému (Proceedings to Honor 455 Vladimír Podborský). (eds. Kazdová E, Měřínský Z, Šabatová K), pp.235-244. FF Masaryk 456 University in Brno, Brno. 457 458 Behar DM, van Oven M, Rosset S, Metspalu M, Loogväli E-L, Silva NM, Kivisild T, Torroni A, 459 Villems R. 2012. A “Copernican” Reassessment of the Human Mitochondrial DNA Tree from 460 its Root. The American Journal of Human Genetics 90, 675-684. 461 462 Bell LS, Kayser M, Jones C. 2008. The mineralized osteocyte: A living fossil. American 463 Journal of Physical Anthropology 137, 449-456. 464 465 Briggs AW, Stenzel U, Johnson PL, Green RE, Kelso J, Prüfer K, Meyer M, Krause J, Ronan 466 MT, Lachmann M, Pääbo S. 2007. Patterns of damage in genomic DNA sequences from a 467 Neandertal. Proceedings of the National Academy of Sciences 104, 14616-14621. 468 469 Broushaki F, Thomas MG, Link V, López S, van Dorp L, Kirsanow K, Hofmanová Z, 470 Diekmann Y, Cassidy LM, Díez-del-Molino D, et al. 2016. Early Neolithic genomes from the 471 eastern Fertile Crescent. Science 353, 499-503. 472 473 Busse B, Djonic D, Milovanovic P, Hahn M, Püschel K, Ritchie RO, Djuric M, Amling M. 2010. 474 Decrease in the osteocyte lacunar density accompanied by hypermineralized lacunar 475 occlusion reveals failure and delay of remodeling in aged human bone. Aging Cell 9, 1065- 476 1075. 477 478 Canci A, Contursi D, Fornaciari G. 2005. La necropoli dell'età del bronzo di Olmo di Nogara 479 (Verona): primi risultati dello studio paleopatologico. La necropoli dell’età del Bronzo 480 all’Olmo di Nogara, in Memorie del Museo Civico di Storia Naturale di Verona 8, 495-501. 481 482 Chen H, Okumura T, Emura S, Shoumura S. 2008. Scanning electron microscopic study of 483 the human auditory ossicles. Annals of Anatomy - Anatomischer Anzeiger 190, 53-58. 484 485 Collins MJ, Nielsen–Marsh CM, Hiller J, Smith CI, Roberts JP, Prigodich RV, Wess TJ, 486 Csapò J, Millard AR, Turner–Walker G. 2002. The survival of organic matter in bone: a 487 review. Archaeometry 44, 383-394. 488 489 Coppa A, Damadio S. 2005. Palaeobiology of the populations of Yemen. In South Arabian 490 Necropolises. Italian Excavations at Al- Makhdarah and Kharabit al-Ahjur (Republic of 491 Yemen). (eds. de Maigret A, Antonini S), pp. 91-146. Istituto Italiano per l’Africa e l’Oriente, 492 Centro Studi e Scavi Archeologici, Rome. 493 494 Corrain C, Capitanio M, Pesarin F. 1975. Confronto statistico tra le serie osteologiche delle 495 stazioni eneolitiche di Buccino e di Madonna della Catena (Salerno). Atti della XVII Riunione 496 Scientifica dell'Istituto Italiano di Preistoria e Protostoria, Firenze, 187-197. 497 498 Crevecoeur I. 2007. New discovery of an Upper Paleolithic auditory ossicle: the right malleus 499 of Nazlet Khater 2. Journal of Human Evolution 52, 341-345. 500 501 Cunningham C, Scheuer L, Black S. 2000. Developmental Juvenile Osteology. Academic 502 Press, San Diego, CA.
21 Downloaded from genome.cshlp.org on September 30, 2021 - Published by Cold Spring Harbor Laboratory Press
503 504 Dabney J, Knapp M, Glocke I, Gansauge M-T, Weihmann A, Nickel B, Valdiosera C, García 505 N, Pääbo S, Arsuaga J-L, Meyer M. 2013. Complete mitochondrial genome sequence of a 506 Middle Pleistocene cave bear reconstructed from ultrashort DNA fragments. Proceedings of 507 the National Academy of Sciences 110, 15758-15763. 508 509 Daley T, Smith AD. 2013. Predicting the molecular complexity of sequencing libraries. 510 Nature Methods 10, 325-327. 511 512 Damgaard PB, Margaryan A, Schroeder H, Orlando L, Willerslev E, Allentoft ME. 2015. 513 Improving access to endogenous DNA in ancient bone and teeth. Scientific Reports 5, 514 11184. 515 516 Duboeuf F, Burt-Pichat B, Farlay D, Suy P, Truy E, Boivin G. 2015. Bone quality and 517 biomechanical function: A lesson from human ossicles. Bone 73, 105-110. 518 519 Farkaš Z., 2002. Nálezy ľudských pozostatkov v prostredí kultúry ľudu s lineárnou keramikou 520 na Slovensku (Human remains from the Linear Pottery Culture area in Slovakia). 521 Archeologické rozhledy 54, 23–43. 522 523 Fregel R, Mendez FL, Bokbot Y, Martin-Socas D, Camalich-Massieu MD, Santana J, 524 Morales J, Avila-Arcos MC, Underhill PA, Shapiro B, et al. 2018. Ancient genomes from 525 North Africa evidence prehistoric migrations to the Maghreb from both the Levant and 526 Europe. Proceedings of the National Academy of Sciences 115, 6774-6779. 527 528 Frisch T, Sølvsten Sørensen M, Overgaard S, Lind M, Bretlau P. 1998. Volume-referent 529 bone turnover estimated from the interlabel area fraction after sequential labeling. Bone 22, 530 677-682. 531 532 Fu Q, Hajdinjak M, Moldovan OT, Constantin S, Mallick S, Skoglund P, Patterson N, 533 Rohland N, Lazaridis I, Nickel B, et al. 2015. An early modern human from Romania with a 534 recent Neanderthal ancestor. Nature 524, 216-219. 535 536 Fu Q, Li H, Moorjani P, Jay F, Slepchenko SM, Bondarev AA, Johnson PLF, Aximu-Petri A, 537 Prüfer K, de Filippo C, et al. 2014. Genome sequence of a 45,000-year-old modern human 538 from western Siberia. Nature 514, 445-449. 539 540 Fu Q, Meyer M, Gao X, Stenzel U, Burbano HA, Kelso J, Pääbo S. 2013. DNA analysis of an 541 early modern human from Tianyuan Cave, China. Proceedings of the National Academy of 542 Sciences 110, 2223-2227. 543 544 Gamba C, Jones ER, Teasdale MD, McLaughlin RL, Gonzalez-Fortes G, Mattiangeli V, 545 Domboróczki L, Kővari I, Pap I, Anders A, Whittle A, Dani J, Raczky P, Higham TFG, 546 Hofreiter M, Bradley DG, Pinhasi R. 2014. Genome flux and stasis in a five millennium 547 transect of European prehistory. Nature Communications 5, 5257. 548 549 Gansauge M-T, Gerber T, Glocke I, Korlević P, Lippik L, Nagel S, Riehl L, Schmidt A, Meyer 550 M. 2017. Single-stranded DNA library preparation from highly degraded DNA using T4 DNA 551 ligase. Nucleic Acids Research 45, e79. 552 553 Ginolhac A, Rasmussen M, Gilbert MTP, Willerslev E, Orlando L. 2011. mapDamage: testing 554 for damage patterns in ancient DNA sequences. Bioinformatics 27, 2153-2155. 555 556 Green RE, Krause J, Briggs AW, Maricic T, Stenzel U, Kircher M, Patterson N, Li H, Zhai W, 557 Fritz MH-Y, et al. 2010. A draft sequence of the Neandertal genome. Science 328, 710-722.
22 Downloaded from genome.cshlp.org on September 30, 2021 - Published by Cold Spring Harbor Laboratory Press
558 559 Gromov AV. 1995. Bronze Age population of the Southern Khakassia and problem of the 560 origin of the Karasuk culture, vol. 1. In Antropologia segodnya. (ed. Gokhman II), pp.130-150. 561 MAE RAS, Saint-Petersburg. 562 563 Haas N, Maximilian K. 1958. Anthropological study of the human bones from graves with 564 ochre from Glăvăneștii Vechi, Corlăteni and Stoicani Cetățuie. Soviet Anthropology 4, 133- 565 146. 566 567 Haak W, Lazaridis I, Patterson N, Rohland N, Mallick S, Llamas B, Brandt G, Nordenfelt S, 568 Harney E, Stewardson K, et al. 2015. Massive migration from the steppe is a source for 569 Indo-European languages in Europe. Nature 522, 207-211. 570 571 Hansen HB, Damgaard PB, Margaryan A, Stenderup J, Lynnerup N, Willerslev E, Allentoft 572 ME. 2017. Comparing Ancient DNA Preservation in Petrous Bone and Tooth Cementum. 573 PLoS ONE 12, e0170940. 574 575 Harney E, May H, Shalem D, Rohland N, Mallick S, Lazaridis I, Sarig E, Stewardson K, 576 Nordenfelt S, Patterson N, et al. 2018. Ancient DNA from Chalcolithic Israel reveals the role 577 of population mixture in cultural transformation. Nature Communications 9, 3336. 578 579 Heim JL. 1982. Les Enfants Néandertaliens de La Ferrassie. Publié sous les auspices de la 580 Fondation Singer-Polignac. Bulletins et Mémoires de la Société d’Anthropologie de Paris 9, 581 347. 582 583 Helm R, Allison E, Anderson I, Barber L, Broadley R, Carruthers W, Locker A, Lyne M, 584 McIntyre L, McNee B, Richardson A, Smith I, Wilson T. 2017. Former Peugeot Garage, 585 Rhodaus Town (A28), Canterbury, Kent CT1 2RH. Post-excavation assessment, client 586 report no. 2017/107, Canterbury Archaeological Trust. 587 588 Hernandez CJ, Majeska RJ, Schaffler MB. 2004. Osteocyte density in woven bone. Bone 35, 589 1095-1099. 590 591 Hofreiter M, Jaenicke V, Serre D, von Haeseler A, Pääbo S. 2001. DNA sequences from 592 multiple amplifications reveal artifacts induced by cytosine deamination in ancient DNA. 593 Nucleic Acids Research 29, 4793-4799. 594 595 Hublin JJ, Pääbo S, Derevianko AP, Doronichev VB, Golovanova LV, Freiss M, Froment A, 596 Hoffmann A, Jillani Kachache NE, Kullmer O, et al. 2008. Suggested guidelines for invasive 597 sampling of hominid remains. Journal of Human Evolution 55, 756-757. 598 599 Kanzaki S, Ito M, Takada Y, Ogawa K, Matsuo K. 2006. Resorption of auditory ossicles and 600 hearing loss in mice lacking osteoprotegerin. Bone 39, 414-419. 601 602 Kontopoulos I, Penkman K, McAllister GD, Lynnerup N, Damgaard PB, Hansen HB, Allentoft 603 ME, Collins MJ. 2019. Petrous bone diagenesis: a multi-analytical approach. 604 Palaeogeography, Palaeoclimatology, Palaeoecology 518, 143-154. 605 606 Korlević P, Gerber T, Gansauge M-T, Hajdinjak M, Nagel S, Aximu-Petri A, Meyer M. 2015. 607 Reducing microbial and human contamintion in DNA extractions from ancient bones and 608 teeth. Biotechniques 58, 87-93. 609 610 Korneliussen TS, Albrechtsen A, Nielsen R. 2014. ANGSD: Analysis of Next Generation 611 Sequencing Data. BMC Bioinformatics 15, 356. 612
23 Downloaded from genome.cshlp.org on September 30, 2021 - Published by Cold Spring Harbor Laboratory Press
613 Lazaridis I, Nadel D, Rollefson G, Merrett DC, Rohland N, Mallick S, Fernandes D, Novak M, 614 Gamarra B, Sirak K, et al. 2016. Genomic insights into the origin of farming in the ancient 615 Near East. Nature 536, 419-424. 616 617 Li H, Durbin R. 2009. Fast and accurate short read alignment with Burrows-Wheeler 618 transform. Bioinformatics 14, 1754-1760. 619 620 Lindahl T. 1993. Instability and decay of the primary structure of DNA. Nature 362, 709-715. 621 622 Llamas B, Valverde G, Fehren-Schmitz L, Weyrich LS, Cooper A, Haak W. 2017. From the 623 field to the laboratory: Controlling DNA contamination in human ancient DNA resarch in the 624 high-throughput sequencing era. STAR: Science & Technology of Archaeological Research 625 3, 1-14. 626 627 Makarewicz C, Marom N, Bar-Oz G. 2017. Palaeobiology: Ensure equal access to ancient 628 DNA. Nature 548, 158. 629 630 Margulies M, Egholm M, Altman WE, Attiya S, Bader JS, Bemben LA, Berka J, Braverman 631 MS, Chen YJ, Chen Z, et al. 2005. Genome sequencing in microfabricated high-density 632 picolitre reactors. Nature 437, 376-380. 633 634 Marotti G, Farneti D, Remaggi F, Tartari F. 1998. Morphometric investigation on osteocytes 635 in human auditory ossicles. Annals of Anatomy-Anatomischer Anzeiger 180, 449-453. 636 637 Mathieson I, Lazaridis I, Rohland N, Mallick S, Patterson N, Alpaslan-Roodenberg S, Harney 638 E, Stewardson K, Fernandes D, Novak M, et al. 2015. Genome-wide patterns of selection in 639 230 ancient Eurasians. Nature 528, 499-503. 640 641 Mays S, Elders J, Humphrey L, White W, Marshall P. 2013. Science and the Dead: A 642 guideline for the destructive sampling of archaeological human remains for scientific analysis. 643 Advisory Panel on the Archaeology of Burials in England, English Heritage Publishing, 644 Swindon, England. 645 646 Okumura M. 2007. Diversidade morfológica craniana, micro-evolução e ocupação pré- 647 histórica da costa brasileira. PhD Thesis. Institute of Biosciences, University of São Paulo. 648 649 Passarello P, Macchiarelli R. 1987. La necropoli protostorica di Kātelai (Swāt, Pakistan). 650 Analisi antropologica del materiale scheletrico con riferimento al contesto paleobiologico 651 umano dell’area medio-orientale. Rivista di Antropologia 65, 5-104. 652 653 Pietrusewsky M, Douglas MT. 2002. Ban Chiang, a Prehistoric Village Site in Northeast 654 Thailand. Volume I: The Human Skeletal Remains. University Museum Monograph 111. 655 Philadelphia: University of Pennsylvania Museum of Archaeology and Anthropology. 656 657 Pinhasi R, Fernandes DM, Sirak K, Cheronet O. 2019. Isolating the human cochlea to 658 generate bone powder for ancient DNA analysis. Nature Protocols 14, 1194-1205. 659 660 Pinhasi R, Fernandes DM, Sirak K, Novak M, Connell S, Alpaslan-Roodenberg S, Gerritsen 661 F, Moiseyev V, Gromov A, Raczky P, Anders A, Pietrusewsky M, Rollefson G, Jovanovic M, 662 Trinhhoang H, Bar-Oz G, Oxenham M, Matsumura H, Hofreiter M. 2015. Optimal Ancient 663 DNA Yields from the Inner Ear Part of the Human Petrous Bone. PLoS ONE 10, e0129102. 664 665 Poinar HN. 2003. The top 10 list: criteria of authenticity for DNA from ancient and forensic 666 samples. International Congress Series 1239, 575-579. 667
24 Downloaded from genome.cshlp.org on September 30, 2021 - Published by Cold Spring Harbor Laboratory Press
668 Ponce de León MS, Koesbardiati T, Weissmann JD, Milella M, Reyna-Blanco CS, Suwa G, 669 Kondo O, Malaspinas A-S, White TD, Zollikofer CPE. 2018. Human bony labyrinth is an 670 indicator of population history and dispersal from Africa. Proceedings of the National 671 Academy of Sciences 115, 4128-4133. 672 673 Prendergast ME, Sawchuk E. 2018. Boots on the ground in Africa's ancient DNA ‘revolution’: 674 archaeological perspectives on ethics and best practices. Antiquity 92, 803-815. 675 676 Pulcini ML. 2014. La necropoli di Olmo di Nogara (Verona). Studio paleobiologico dei resti 677 umani per la ricostruzione dell'organizzazione di una comunità dell'Età del bronzo padana. 678 PhD Thesis. Dipartimento dei Beni Culturali: Archeologia, Storia dell'Arte, del Cinema e della 679 Musica, Università Degli Studi di Padova. 680 681 Quam R, Martínez I, Arsuaga JL. 2013a Reassessment of the La Ferrassie 3 Neandertal 682 ossicular chain. Journal of Human Evolution 64, 250-262. 683 684 Quam R, Rak Y. 2008. Auditory ossicles from southwest Asian Mousterian sites. Journal of 685 Human Evolution 54, 414-433. 686 687 Quam R, de Ruiter DJ, Masali M, Arsuaga J-L, Martínez I, Moggi-Cecchi J. 2013b. Early 688 hominin auditory ossicles from South Africa. Proceedings of the National Academy of 689 Sciences 110, 8847-8851. 690 691 Qvist M. 2000. Auditory ossicles in archaeological skeletal material from medieval Denmark. 692 Acta Oto-Laryngologica 120, 82-85. 693 694 Raczky P. 2018. A complex monument in the making at the Late Neolithic site of Polgár- 695 Csőszhalom. In Across the Mediterranean – along the Nile. Studies in Egyptology, Nubiology 696 and Late Antiquity Dedicated to László Török on the Occasion of His 75th Birthday. (eds. 697 Bács TA, Bollók Á, Vida T), pp. 15-60. Institute of Archaeology, Research Centre for the 698 Humanities Hungarian Academy of Sciences, Budapest. 699 700 Rak Y, Clarke RJ. 1979. Ear ossicle of Australopithecus robustus. Nature 279, 62-63. 701 702 Rask Andersen H, Liu W, Erixon E, Kinnefors A, Pfaller K, Schrott Fischer A, Gluckert R. 703 2012. Human cochlea: anatomical characteristics and their relevance for cochlear 704 implantation.‐ The Anatomical Record: Advances in Integrative Anatomy‐ and Evolutionary 705 Biology 295, 1791-1811. 706 707 Richard C, Courbon G, Laroche N, Prades JM, Vico L, Malaval L. 2017. Inner ear 708 ossification and mineralization kinetics in human embryonic development-microtomographic 709 and histomorphological study. Scientific Reports 7, 4825. 710 711 Rohland N, Glocke I, Aximu-Petri A, Meyer M. 2018. Extraction of highly degraded DNA from 712 ancient bones, teeth and sediments for high-throughput sequencing. Nature Protocols 713 13, 2447–2461. 714 715 Rohland N, Harney E, Mallick S, Nordenfelt S, Reich D. 2015. Partial uracil–DNA– 716 glycosylase treatment for screening of ancient DNA. Philosophical Transactions of the Royal 717 Society of London B: Biological Sciences 370, 20130624. 718 719 Rolvien T, Schmidt FN, Milovanovic P, Jähn K, Riedel C, Butscheidt S, Püschel K, Jeschke 720 A, Amling M, Busse B. 2018. Early bone tissue aging in human auditory ossicles is 721 accompanied by excessive hypermineralization, osteocyte death and micropetrosis. 722 Scientific Reports 8, 1920.
25 Downloaded from genome.cshlp.org on September 30, 2021 - Published by Cold Spring Harbor Laboratory Press
723 724 Schmitz PI, Bittencourt ALV. 1996. O sítio arqueológico de Laranjeiras I, S.C. Pesquisas 725 (Antropologia) 53, 13-76. 726 727 Schuenemann VJ, Peltzer A, Welte B, van Pelt WP, Molak M, Wang C-C, Furtwängler A, 728 Urban C, Reiter E, Nieselt K, et al. 2017. Ancient Egyptian mummy genomes suggest an 729 increase of Sub-Saharan African ancestry in post-Roman periods. Nature Communications 8, 730 15694. 731 732 Schwark T, Modrow JH, Steinmeier E, Poetsch M, Hasse J, Fischer H, von Wurmb-Schwark 733 N. 2015. The auditory ossicles as a DNA source for genetic identification of highly putrefied 734 cadavers. International Journal of Legal Medicine 129, 457-462. 735 736 Siori MS, Monchietto MJ, Masali M. 1995. Morphometrics of human auditory ossicles from 737 Antinoe Necropolis (Egypt). International Journal of Anthropology 10, 29. 738 739 Sirak K, Fernandes DM, Cheronet O, Novak M, Gamarra B, Balassa T, Cséki A, Dani J, 740 Gallina JZ, Kocsis-Buruzs G, László O, Pap I, Patay R, Petkes Z, Szenthe G, Szeniczey T, 741 Hajdu T, Pinhasi R. 2017. A minimally-invasive method for sampling human petrous bones 742 from the cranial base for ancient DNA analysis. BioTechniques 62, 283-289. 743 744 Sirak K, Sedig JW. In press. Balancing Analytical Goals and Anthropological Stewardship in 745 the Midst of the Paleogenomics Revolution.” Accepted at World Archaeology. 746 747 Skoglund P, Northoff BH, Shunkov MV, Derevianko AP, Pääbo S, Krause J, Jakobsson M. 748 2014. Separating endogenous ancient DNA from modern day contamination in a Siberian 749 Neandertal. Proceedings of the National Academy of Sciences 111, 2229-2234. 750 751 Skoglund P, Thompson JC, Prendergast ME, Mittnik A, Sirak K, Hajdinjak M, Salie T, 752 Rohland N, Mallick S, Peltzer A, et al. 2017. Reconstructing prehistoric African population 753 structure. Cell 171, 59-71. 754 755 Smith CI, Chamberlain AT, Riley MS, Cooper A, Stringer CB, Collins MJ. 2001. Neanderthal 756 DNA: Not just old but old and cold? Nature 410, 771-772. 757 758 Smith CI, Chamberlain AT, Riley MS, Cooper A, Stringer CB, Collins, MJ. 2003. The thermal 759 history of human fossils and the likelihood of successful DNA amplification. Journal of 760 Human Evolution 45, 203-217. 761 762 Sølvsten Sørensen M, Bretlau P, Balslev Jørgensen M. 1992. Quantum type bone 763 remodeling in the human otic capsule: morphometric findings. Acta Oto-Laryngologica 112, 764 4-10. 765 766 Spoor F, Hublin JJ, Braun M, Zonneveld F. 2003. The bony labyrinth of Neanderthals. 767 Journal of Human Evolution 44, 141-165. 768 769 Stoessel A, Gunz P, David R, Spoor F. 2016. Comparative anatomy of the middle ear 770 ossicles of extant hominids–Introducing a geometric morphometric protocol. Journal of 771 Human Evolution 91, 1-25. 772 773 Szőke BM. 2008. Pannonien in der Karolingerzeit Bemerkungen zur Chronologie des 774 frühmittelalterlichen Fundmaterials in Westungarn. Schild von Steier Beiheft 4, 41-56. 775 776 Szőke BM. 2010. Mosaburg/Zalavár und Pannonien in der Karolingerzeit. Antæus: 777 Communications ex Instituto Archaeological Academiae Scientarum Hungaricae 31, 9-52.
26 Downloaded from genome.cshlp.org on September 30, 2021 - Published by Cold Spring Harbor Laboratory Press
778 779 Szőke BM. 2014. The Carolingian Age in the Carpathian Basin: Permanent Exhibition of the 780 Hungarian National Museum. Magyar Nemzeti Múzeum, Budapest. 781 782 van de Loosdrecht M, Bouzouggar A, Humphrey L, Posth C, Barton N, Aximu-Petri A, Nickel 783 B, Nagel S, Talbi EH, El Hajraoui MA, et al. 2018. Pleistocene North African genomes link 784 Near Eastern and sub-Saharan African human populations. Science 360, 548-552. 785 786 Vasilyev SV, Gerasimova MM, Borutskaya SB, Frizen SY, Zamballarova ED. 2010. 787 Anthropological study of the Fofonovo burial (Transbaikalia) Neolithic and early bronze age. 788 Herald of anthropology 18, 113-127. 789 790 Yokoyama T, Iino Y, Kakizaki K, Murakami Y. 1999. Human temporal bone study on the 791 postnatal ossification process of auditory ossicles. The Laryngoscope 109, 927-930.
27 Downloaded from genome.cshlp.org on September 30, 2021 - Published by Cold Spring Harbor Laboratory Press
Figure 1: The three auditory ossicles. From left to right, the stapes, malleus, and incus.
Downloaded from genome.cshlp.org on September 30, 2021 - Published by Cold Spring Harbor Laboratory Press Downloaded from genome.cshlp.org on September 30, 2021 - Published by Cold Spring Harbor Laboratory Press
Table 1: Sample information and summary of sequencing results. The 10 samples considered to be ‘working’ for the purposes of this analysis are labeled with an asterisk.
-
Mean
DNA Yield DNA ID (%) score] Site Date - [95CI] Match Aligned Reads Aligned [Z (Country) 1240K SNPs1240K Deamination Initial Material 1240K ANGSD1240K 1240K Coverage 1240K % Unique Reads Reads Unique % Target Sequences Target mtDNA Consensus Consensus mtDNA Contamination Contamination Expected at 500K On 500K at Expected number of sequences) of number Base of of Base Endogenous Endogenous (notnormalized for total Frequencies on Terminal Terminal on Frequencies
Cochlear Powder; 0.996 0.008
7.53 622982 0.541 0.139 67.8 * Katelai 1000-800 56 mg [0.990-0.999] [2.897]
614 (Pakistan) BCE Ossicle 0.994 0.005 30.55 776019 0.674 0.131 83.2 (2: I, M) [0.986-0.998] [2.593]
Cochlear Powder; 0.994 N/A 3.27 391574 0.340 0.239 50.4 * Geoksiur 5000-2000 47 mg [0.984-0.998] (Female)
644 (Turkmenistan) BCE Ossicle 0.989 N/A 0.57 177819 0.155 0.225 31.5 (2: I, M) [0.974-0.996] (Female) Cochlear Powder; 0.999 0.002 70.15 829510 0.721 0.044 98.9 * Fofonovo 6000-3000 47 mg [0.995-1.000] [2.053]
648 (Russia) BCE Ossicle 0.991 0.002 72.68 858204 0.746 0.045 98.8 (2: I, S) [0.983-0.996] [3.254] Bayankhongor, Cochlear Powder; 0.995 0.005 56.48 855679 0.744 0.139 98.7 * Ulziit sum, 2500-400 55 mg [0.987-0.998] [5.841]
818 Maanit uul BCE Ossicle 0.965 0.040 71.80 883674 0.768 0.106 98.2 (Mongolia) (1: I) [0.953-0.974] [20.256]
Cochlear Powder; 0.981 N/A
* Peugeot 46.79 843283 0.733 0.134 97.7 43-410 49 mg [0.972-0.988] (Female) Garage CE Ossicle 0.971 N/A 1257 (Great Britain) 55.83 829630 0.721 0.128 94.6 (2: I, M) [0.960-0.980] (Female) Cochlear Powder; N/A
0.01 266 0.000 0.064 - -
6 48 mg (Female)
ES Ban Chiang 900-300
B2 (Thailand) BCE Ossicle 0.940 N/A BC 0.07 12438 0.008 0.013 - (3: I, M, S) [0.877-0.972] (Female)
Cochlear Powder; 0.996 0.006
63.00 770152 3.909 0.091 98.6
* Blatné 2200-2000 50 mg [0.990-0.999] [6.180] AT
33 (Slovakia) BCE Ossicle 0.998 0.005 BL 68.25 725438 3.914 0.088 99.1 (2: I, M) [0.993-1.000] [5.184]
Cochlear Powder; 0.995 0.005
73.88 779326 3.727 0.078 97.9
* Glăvăneşti 3500-3000 47 mg [0.989-0.998] [4.856] AV
14 (Romania) BCE Ossicle 0.991 0.004 GL 59.40 749401 3.343 0.068 96.8 (2: I, M) [0.982-0.995] [4.574]
Cochlear Powder;
H 0.002 253 0.000 0.014 - - - 53 mg D Al-Makhdarah 1190-800
13H (Yemen) BCE Ossicle MK 0.001 531 0.000 0.025 - - - (2: I, M) Cochlear Powder; 0.004 115 0.000 0.024 - - -
Kharibat al- 53 mg Ahjur 1000 BCE UA Ossicle KHRB (Yemen) 0.007 1244 0.001 0.098 - - - (2: I, M) Cochlear Powder; 0.983 N/A 0.99 134254 0.126 0.251 21.3 Laranjeiras I 2900-1800 50 mg [0.967-0.992] (Female)
3935 (Brazil) BCE Ossicle N/A 0.08 10743 0.009 0.244 - 2.4 (1: I) (Female)
9323* 9309* 9214 3938* 3937 Downloaded from Ol Ol Lar Laranjeiras I m m Bu o o (B (Brazil) anj (I (I (I
d d t t t ra c a a a i i c
ei l l l No No z y y y i n i r ) ) ) l as o )
g g
a a
I r r
a a genome.cshlp.org
1400 1400 3000 2900 2900 BC BC BC BC BCE - - - - - 1800 1200 1200 2000 1800 E E E E
Co Co Co Co Cochlear c c c c h h h h onSeptember30,2021-Publishedby (2 (2 (2 l l l l Os Os Os Os Os e e e e 50 50 50 50 50 mg (1 (1 a a a a : : :
s s s s s r r r r I I I : : , , , m m m m
i i i i i
Powder Po Po Po Po c c c c c I I M M M ) ) g g g g l l l l l
e e e e e ) ) )
w w w w
d d d d e e e e r r r r ; ; ; ; ;
72. 49. 39. 57. 30. 45. 0. 0. 0. 9.09 0 13 07
10 0 03 47 5 1 8 1 8 1
710183 698186 557972 689962 696203 638507 396963 37673 9791 7251
2. 2. 0. 2. 0. 0. 1. 1. 0. 0.478 719 466 833 281 032 008 945 712 006 Cold SpringHarborLaboratoryPress
0. 0. 0. 0. 0. 0. 0. 0. 0. 0.213 118 103 089 089 017 207 158 150 222
[0 [0 [0 [0 [0 [0 [0.977 .9 .9 .9 .9 .9 .9 6 6 3 7 8 8 0. 0. 0. 0. 0. 0. 0.989 1 9 2 2 1 2 975 982 952 961 992 991 ------0. 0. 0. 0. 0. 0. 0.995] 984] 989] 967] 994] 997] 996]
(F (F (F (F [7 [4 [5 [4 [1.009] 0. 0. 0. 0. 0.005 e e e e N/ N/ N/ N/ .6 .2 .1 .6 m m m m 052 006 010 009 - 3 7 4 5 A A A A a a a a 2 9 2 8 l l l l e e e e ] ] ] ]
) ) ) )
96. 95. 68. 95. 90. 92. 50.6 6. 2. 1. 6 2 6 7 5 8 7 6 4
Corrigenda
Genome Research 30: 427–436 (2020)
Corrigendum: Human auditory ossicles as an alternative optimal source of ancient DNA Kendra Sirak, Daniel Fernandes, Olivia Cheronet, Eadaoin Harney, Matthew Mah, Swapan Mallick, Nadin Rohland, Nicole Adamski, Nasreen Broomandkhoshbacht, Kimberly Callan, Francesca Candilio, Ann Marie Lawson, Kirsten Mandl, Jonas Oppenheimer, Kristin Stewardson, Fatma Zalzala, Alexandra Anders, Juraj Bartík, Alfredo Coppa, Tumen Dashtseveg, Sándor Évinger, Zdeněk Farkaš, Tamás Hajdu, Jamsranjav Bayarsaikhan, Lauren McIntyre, Vyacheslav Moiseyev, Mercedes Okumura, Ildikó Pap, Michael Pietrusewsky, Pál Raczky, Alena Šefčáková, Andrei Soficaru, Tamás Szeniczey, Béla Miklós Szőke, Dennis Van Gerven, Sergey Vasilyev, Lynne Bell, David Reich, and Ron Pinhasi
The authors would like to correct the affiliation of coauthor Mercedes Okumura to the following:
Department of Genetics and Evolutionary Biology, Institute of Biosciences, University of São Paulo, Cidade Universitária 05508-090 São Paulo, Brazil
The correction has been made in the revised article online.
doi: 10.1101/gr.266098.120
Genome Research 29: 1152–1163 (2019)
Corrigendum: OMA standalone: orthology inference among public and custom genomes and transcriptomes Adrian M. Altenhoff, Jeremy Levy, Magdalena Zarowiecki, Bartłomiej Tomiczek, Alex Warwick Vesztrocy, Daniel A. Dalquen, Steven Müller, Maximilian J. Telford, Natasha M. Glover, David Dylus, and Christophe Dessimoz
The authors would like to correct the omission of additional Supplemental Data files, which were inadvertent- ly excluded from the initial publication of this article. Please note that the complete Supplemental Data file has now been published online as Revised Supplemental Material. The authors apologize for the inconvenience.
doi: 10.1101/gr.266809.120
938 Genome Research www.genome.org Downloaded from genome.cshlp.org on September 30, 2021 - Published by Cold Spring Harbor Laboratory Press
Human auditory ossicles as an alternative optimal source of ancient DNA
Kendra Ann Sirak, Daniel Magalhaes Fernandes, Olivia Cheronet, et al.
Genome Res. published online February 25, 2020 Access the most recent version at doi:10.1101/gr.260141.119
Supplemental http://genome.cshlp.org/content/suppl/2020/03/12/gr.260141.119.DC1 Material
Related Content Corrigendum: Human auditory ossicles as an alternative optimal source of ancient DNA Kendra Sirak, Daniel Fernandes, Olivia Cheronet, et al. Genome Res. June , 2020 30: 938_1
P
Accepted Peer-reviewed and accepted for publication but not copyedited or typeset; Manuscript accepted manuscript is likely to differ from the final, published version.
Creative This article is distributed exclusively by Cold Spring Harbor Laboratory Press for Commons the first six months after the full-issue publication date (see License http://genome.cshlp.org/site/misc/terms.xhtml). After six months, it is available under a Creative Commons License (Attribution-NonCommercial 4.0 International), as described at http://creativecommons.org/licenses/by-nc/4.0/.
Email Alerting Receive free email alerts when new articles cite this article - sign up in the box at Service the top right corner of the article or click here.
Advance online articles have been peer reviewed and accepted for publication but have not yet appeared in the paper journal (edited, typeset versions may be posted when available prior to final publication). Advance online articles are citable and establish publication priority; they are indexed by PubMed from initial publication. Citations to Advance online articles must include the digital object identifier (DOIs) and date of initial publication.
To subscribe to Genome Research go to: https://genome.cshlp.org/subscriptions
Published by Cold Spring Harbor Laboratory Press