Skip to main content
bioRxiv
  • Home
  • About
  • Submit
  • ALERTS / RSS
Advanced Search
New Results

Human Auditory Ossicles as an Alternative Optimal Source of Ancient DNA

Kendra Sirak, View ORCID ProfileDaniel 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, Dashtseveg Tumen, Sándor Évinger, Zdeněk Farkaš, Tamás Hajdu, Jamsranjav Bayarsaikhan, Lauren McIntyre, Vyacheslav Moiseyev, Ildikó Pap, Michael Pietrusewsky, Pál Raczky, Alena Šefčáková, Andrei Soficaru, Tamás Szeniczey, Béla Miklós Szőke, Tumurbaatar Tuvshinjargal, Dennis Van Gerven, Sergey Vasilyev, Lynne Bell, David Reich, Ron Pinhasi
doi: https://doi.org/10.1101/654749
Kendra Sirak
1Department of Genetics, Harvard Medical School, Boston, MA 02115, USA
2Earth Institute and School of Archaeology, University College Dublin, Dublin 4, Ireland
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • For correspondence: ron.pinhasi@univie.ac.at kendra_sirak@hms.harvard.edu daniel.fernandes@univie.ac.at
Daniel Fernandes
2Earth Institute and School of Archaeology, University College Dublin, Dublin 4, Ireland
3Department of Evolutionary Anthropology, University of Vienna, Vienna, 1090, Austria
4CIAS, Department of Life Sciences, University of Coimbra, 3000-456 Coimbra, Portugal
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • ORCID record for Daniel Fernandes
  • For correspondence: ron.pinhasi@univie.ac.at kendra_sirak@hms.harvard.edu daniel.fernandes@univie.ac.at
Olivia Cheronet
2Earth Institute and School of Archaeology, University College Dublin, Dublin 4, Ireland
3Department of Evolutionary Anthropology, University of Vienna, Vienna, 1090, Austria
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Eadaoin Harney
1Department of Genetics, Harvard Medical School, Boston, MA 02115, USA
5Dept. of Organismic and Evolutionary Biology, Harvard University, Cambridge, MA 02138, USA
6The Max Planck-Harvard Research Center for the Archaeoscience of the Ancient Mediterranean, Cambridge, MA 02138, USA and Jena, D-07745, Germany
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Matthew Mah
1Department of Genetics, Harvard Medical School, Boston, MA 02115, USA
7Broad Institute of Harvard and MIT, Cambridge, MA 02142, USA
8Howard Hughes Medical Institute, Harvard Medical School, Boston, MA 02115, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Swapan Mallick
1Department of Genetics, Harvard Medical School, Boston, MA 02115, USA
7Broad Institute of Harvard and MIT, Cambridge, MA 02142, USA
8Howard Hughes Medical Institute, Harvard Medical School, Boston, MA 02115, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Nadin Rohland
1Department of Genetics, Harvard Medical School, Boston, MA 02115, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Nicole Adamski
1Department of Genetics, Harvard Medical School, Boston, MA 02115, USA
8Howard Hughes Medical Institute, Harvard Medical School, Boston, MA 02115, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Nasreen Broomandkhoshbacht
1Department of Genetics, Harvard Medical School, Boston, MA 02115, USA
8Howard Hughes Medical Institute, Harvard Medical School, Boston, MA 02115, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Kimberly Callan
1Department of Genetics, Harvard Medical School, Boston, MA 02115, USA
8Howard Hughes Medical Institute, Harvard Medical School, Boston, MA 02115, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Francesca Candilio
3Department of Evolutionary Anthropology, University of Vienna, Vienna, 1090, Austria
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Ann Marie Lawson
1Department of Genetics, Harvard Medical School, Boston, MA 02115, USA
8Howard Hughes Medical Institute, Harvard Medical School, Boston, MA 02115, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Kirsten Mandl
3Department of Evolutionary Anthropology, University of Vienna, Vienna, 1090, Austria
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Jonas Oppenheimer
1Department of Genetics, Harvard Medical School, Boston, MA 02115, USA
8Howard Hughes Medical Institute, Harvard Medical School, Boston, MA 02115, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Kristin Stewardson
1Department of Genetics, Harvard Medical School, Boston, MA 02115, USA
8Howard Hughes Medical Institute, Harvard Medical School, Boston, MA 02115, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Fatma Zalzala
1Department of Genetics, Harvard Medical School, Boston, MA 02115, USA
8Howard Hughes Medical Institute, Harvard Medical School, Boston, MA 02115, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Alexandra Anders
9Institute of Archaeological Sciences, Eötvös Loránd University, 1088 Budapest, Múzeum körút 4/B, Hungary
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Juraj Bartík
10Slovak National Museum–Archaeological Museum, Žižkova 12, P.O. BOX 13, 810 06 Bratislava 16, Slovak Republi
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Alfredo Coppa
11Dipartimento di Biologia Ambientale, Sapienza Università di Roma, Rome 00185, Italy
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Dashtseveg Tumen
12Department of Anthropology and Archaeology, National University of Mongolia, Ulaanbaatar 14200, Mongolia
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Sándor Évinger
13Department of Anthropology, Hungarian Natural History Museum, H-1083 Budapest, Ludovika tér 2, Hungary
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Zdeněk Farkaš
10Slovak National Museum–Archaeological Museum, Žižkova 12, P.O. BOX 13, 810 06 Bratislava 16, Slovak Republi
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Tamás Hajdu
14Department of Biological Anthropology, Institute of Biology, Faculty of Science, Eötvös Loránd University Budapest, Pázmány Péter sétány 1/c. H-1117, Hungary
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Jamsranjav Bayarsaikhan
12Department of Anthropology and Archaeology, National University of Mongolia, Ulaanbaatar 14200, Mongolia
15National Museum of Mongolia, Juulchin Street -1, Ulaanbaatar 210146, Mongolia
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Lauren McIntyre
16Oxford Archaeology, Janus House, Osney Mead, Oxford OX2 0ES, UK
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Vyacheslav Moiseyev
17Peter the Great Museum of Anthropology and Ethnography (Kunstkamera), Russian Academy of Science, Univercity Emb. 3, St. Petersburg 199034, Russia
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Ildikó Pap
13Department of Anthropology, Hungarian Natural History Museum, H-1083 Budapest, Ludovika tér 2, Hungary
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Michael Pietrusewsky
18Department of Anthropology, University of Hawai’i at Mānoa, Honolulu, HI 96822, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Pál Raczky
9Institute of Archaeological Sciences, Eötvös Loránd University, 1088 Budapest, Múzeum körút 4/B, Hungary
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Alena Šefčáková
19Department of Anthropology, Slovak National Museum–Natural History Museum, Vajanského nábr. 2, P.O. BOX 13, 810 06 Bratislava 16, Slovak Republic
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Andrei Soficaru
20“Fr. J. Rainer” Institute of Anthropology, Romanian Academy, Eroii Sanitari 8, P. O. Box 35-13, Romania
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Tamás Szeniczey
14Department of Biological Anthropology, Institute of Biology, Faculty of Science, Eötvös Loránd University Budapest, Pázmány Péter sétány 1/c. H-1117, Hungary
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Béla Miklós Szőke
21Institute of Archaeology, Research Centre for the Humanities, Hungarian Academy of Sciences, Budapest, H-1097, Hungary
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Tumurbaatar Tuvshinjargal
15National Museum of Mongolia, Juulchin Street -1, Ulaanbaatar 210146, Mongolia
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Dennis Van Gerven
22Department of Anthropology, University of Colorado at Boulder, 1350 Pleasant Street, Boulder, CO 80309, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Sergey Vasilyev
23Institute of Ethnology and Anthropology, RAS, 32a Leninsky propsect, Moscow, 119991, Russia
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Lynne Bell
24School of Criminology, Forensic Research Centre, Simon Fraser University, 8888 University Drive, Burnaby,BC, V5A 1S6, Canada
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
David Reich
1Department of Genetics, Harvard Medical School, Boston, MA 02115, USA
7Broad Institute of Harvard and MIT, Cambridge, MA 02142, USA
8Howard Hughes Medical Institute, Harvard Medical School, Boston, MA 02115, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Ron Pinhasi
3Department of Evolutionary Anthropology, University of Vienna, Vienna, 1090, Austria
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • For correspondence: ron.pinhasi@univie.ac.at kendra_sirak@hms.harvard.edu daniel.fernandes@univie.ac.at
  • Abstract
  • Full Text
  • Info/History
  • Metrics
  • Supplementary material
  • Preview PDF
Loading

ABSTRACT

DNA recovery from ancient human remains has revolutionized our ability to reconstruct the genetic landscape of the past. Ancient DNA research has benefited from the identification of skeletal elements, such as the cochlear part of the osseous inner ear, that provide optimal contexts for DNA preservation; however, the rich genetic information obtained from the cochlea must be counterbalanced against the loss of valuable morphological information caused by its sampling. Motivated by similarities in developmental processes and histological properties between the cochlea and auditory ossicles, we evaluated the efficacy of ossicles as an alternative source of ancient DNA. We demonstrate that ossicles perform comparably to the cochlea in terms of DNA recovery, finding no substantial reduction in data quality, quantity, or authenticity across a range of preservation conditions. Ossicles can be sampled from intact skulls or disarticulated petrous bones without damage to surrounding bone, and we argue that, when available, they should be selected over the cochlea to reduce damage to skeletal integrity. These results identify a second optimal skeletal element for ancient DNA analysis and add to a growing toolkit of sampling methods that help to better preserve skeletal remains for future research while maximizing the likelihood that ancient DNA analysis will produce useable results.

INTRODUCTION

Ancient DNA has become an important tool for addressing key questions about human evolutionary and demographic history. Its rapid growth over the last decade has been driven largely by advances in isolating (Dabney et al. 2013; Rohland et al. 2018), preparing (Gansauge et al. 2017; Rohland et al. 2015), enriching (Fu et al. 2013, 2015; Haak et al. 2015; Mathieson et al. 2015), sequencing (Margulies et al. 2005), and analyzing (Briggs et al. 2007; Briggs et al. 2010; Ginolhac et al. 2011; Skoglund et al. 2014) small quantities of degraded DNA. While these methodological advances have contributed to an improvement in the quality and quantity of paleogenomic data obtained from ancient human remains, all ancient DNA research fundamentally depends upon access to biological material that has sufficient biomolecular preservation.

Skeletal tissue (i.e., bone or teeth) is the preferred biological material for human ancient DNA analysis due to its ability to resist post-mortem degradation better than other types of tissues, including skin and hair (Lindahl 1993; Smith et al. 2001, 2003; Collins et al. 2002). Recent research has shown that not all bone elements are equally effective in preserving DNA, however, and has identified the bone encapsulating the cochlea within the petrous pyramid of the temporal bone (referred to henceforth as the ‘cochlea’) (Gamba et al. 2014; Pinhasi et al. 2015), as well as the cementum layer in teeth roots (Damgaard et al. 2015; Hansen et al. 2017) as especially DNA-rich parts of the skeleton. The use of these skeletal elements that act as repositories for the long-term survival of DNA has proven to be particularly important for the analysis of biological samples recovered from regions where high temperatures and/or humidity increase the rate of molecular degradation and result in low concentrations of damaged DNA with reduced molecular complexity (e.g., Broushaki et al. 2016; Lazaridis et al. 2016; Schuenemann et al. 2017; Skoglund et al. 2017; Fregel et al. 2018; Harney et al. 2018; van de Loosdrecht et al. 2018).

While use of the cochlea has contributed to the application of ancient DNA research to a growing range of geographic and temporal contexts, it is important to balance analytical goals with the irreparable damage to human skeletal remains that results from destructive analyses (Prendergast and Sawchuk 2018; Sirak and Sedig in press). Ancient DNA is one of several such analyses that are now widely used in archaeology (others include radiocarbon dating and stable isotope analysis) (Hublin et al. 2008; Mays et al. 2013; Makarewicz et al. 2017; Pinhasi et al. 2019). To minimize damage to intact skulls from ancient DNA sampling while still accessing the rich genetic data in the cochlea, we developed a “Cranial Base Drilling” method to minimize damage to surrounding bone areas when a skull is intact (Sirak et al. 2017). However, even this method involves destructive sampling. Recent work has highlighted the fact that morphological analysis of the inner ear part of the petrous pyramid (including the cochlea) can reveal population relationships and thus harbors some information about population history (e.g., Spoor et al. 2003; Ponce de León et al. 2018). While genetic comparisons of samples involve analysis of tens of thousands of independent markers (single nucleotide polymorphisms, or SNPs) which provide far higher statistical resolution than can be obtained by study of the smaller number of data points that can be extracted from morphological analysis, not all cochlear bone yields sufficient amounts of ancient DNA. The fact that there is morphological information in the petrous pyramid that will be destroyed through sampling of ancient DNA highlights the importance of being a careful steward of these elements.

As part of a search for alternative optimal sources for ancient DNA that can be used in place of the cochlea, we noted that auditory ossicles have similar developmental processes and histological properties as the osseous inner ear. We therefore tested whether the ossicles – the smallest bones in the human body – might serve as alternative optimal substrates for ancient DNA analysis.

Ossicle development and histology

The mechanism by which cochlear bone preserves endogenous DNA better than other skeletal elements or other regions of the same petrous pyramid is not well understood; however, it is likely related to the fact that human petrous bones are unique in being characterized by a near-absence of growth or remodeling following the completion of ossification by approximately 24 weeks in utero (Sølvsten Sørensen et al. 1992; Frisch et al. 1998; Hernandez et al. 2004). The inhibition of bone remodeling leads to the presence of a larger number of mineralized osteocytes that reside in lacunae within the bone tissue (Hernandez et al. 2004; Bell et al. 2008; Busse et al. 2010; Rask-Andersen et al. 2012). One hypothesis (Pinhasi et al. 2019) is that ‘microniches’ created in the bone tissue by the maintenance of mineralized osteocytes, combined with the protected location of the cochlea, may act as repositories that encourage the long-term preservation of DNA (Bell et al. 2008; Kontopoulos et al. 2019). Ossicles are similar to the cochlea in this respect (see below), and we therefore hypothesized that they might also preserve high amounts of endogenous DNA.

In humans, the middle ear (the region of the ear located medial to the eardrum and lateral to the oval window of the inner ear) is enclosed within the temporal bone and contains the three auditory ossicles: the malleus, incus, and stapes (Figure 1). The ossicles effectively allow humans to hear by transmitting sound-induced mechanical vibrations from the outer to the inner ear. Though the ossicles do not experience high-strain biomechanical loading, they are subject to unique vibrational patterns that impact their development and characteristics over the course of an individual’s lifespan (Rolvien et al. 2018). In contrast to the majority of the human skeleton, but similar to the cochlea, the auditory ossicles present with their final size and morphology at birth following the onset of the ossification of between 16 and 18 weeks in utero and the completion of ossification around 24 weeks gestational age (Marotti et al. 1998; Yokoyama et al. 1999; Cunningham et al. 2000; Duboeuf et al. 2015; Richard et al. 2017). The ossicles and cochlea appear to follow the same developmental pattern of rapidly increasing bone volume through cortical thickening and densification, along with mineralization of the bony matrix (Richard et al. 2017).

Figure 1:
  • Download figure
  • Open in new tab
Figure 1: The three auditory ossicles. From left to right, the stapes, malleus, and incus.

Like the cochlea, ossicular bone tissue is rapidly modeled around the time of birth; although it may undergo further postnatal maturation, there are no signs of bone remodeling observed above the age of one year (Richard et al. 2017; Rolvien et al. 2018). The inhibition of bone remodeling of the auditory ossicles is evident from features such as the presence of a dense meshwork of collagenous fibers organized in an interlacing woven pattern, a smooth fibrous appearance, and limited vascular channels and viable osteocytes (Marotti et al. 1998; Chen et al. 2008). As in the case of the cochlea and in contrast to other skeletal elements, mineralized osteocytes appear to accumulate in the ossicles throughout an individual’s life without resulting in increased bone absorption (Marotti et al. 1998; Kanzaki et al. 2006; Rolvien et al. 2018), likely conserving the overall architecture of the ossicles in order to maintain optimal sound transmission (Kanzaki et al. 2006; Rolvien et al. 2018). While the consequences of inhibited bone remodeling and the accumulation of mineralized osteocytes have only been previously studied from a clinical perspective, we hypothesized that these features might contribute to optimized DNA preservation similar to that in the cochlea by creating the ‘microniches’ that enable long-term DNA survival (Bell et al. 2008).

Use of ossicles in ancient DNA research

Due to their small size and tendency to become dislodged from the skull, ossicles are only seldom recovered during excavation and are easily lost in collections excavated decades ago. While ossicles are not recovered for every burial in every context, we have empirically found that these bones may remain lodged within the middle ear of intact skulls or can be identified in the vicinity of a burial during excavation (Qvist et al. 2000). Given the value of the ossicles as a substrate for ancient DNA analysis, demonstrated in this study, we hope that more archaeologists and anthropologists and museum curators will focus on preserving these elements.

It is important to recognize that ossicles, just like the cochlea, are morphologically informative. Indeed, there is a growing body of literature examining the comparative morphology and pathology of the ossicles (e.g., Rak and Clarke 1979; Arensburg et al. 1981, 2005; Siori et al. 1995; Spoor et al. 2003; Crevecoeur et al. 2007; Quam and Rak 2008; Quam et al. 2013a, 2013b; Stoessel et al. 2016). While differences in metric and non-metric features of the auditory ossicles may be taxonomically informative for comparisons across the genus Homo (e.g., Heim 1982; Spoor et al. 2003; Quam and Rak 2008; though see Arensburg et al. 1981), it is unclear whether phylogenetic and population relationship information can be retrieved from the auditory ossicles. In cases where ossicle morphology may be a subject of future research, we encourage that anthropological study (including description, measurement, and evaluation of any apparent pathologies) and surface or micro-CT scanning to collect metric and morphological information prior to ancient DNA analysis. Any ossicles that exhibit visible pathologies should be avoided.

Though some anthropological attention has been given to the ossicles, we are not aware of previous genetic analyses of these bones. Only a single study has attempted to analyze DNA from the ossicles, collecting the ossicles during medical autopsies of recently-deceased individuals and determining them to be a reliable DNA source from bodies ranging from freshly deceased to highly putrefied (Schwark et al. 2015).

RESULTS

We carried out pilot work to assess if the quality and quantity of ancient DNA data recovered from the ossicles was approximately similar to that recovered from the cochlea (described in Supplemental Material). The results of this pilot work (Supplementary Table 1) suggested that ossicles perform comparably to the cochlea in metrics such as amount of endogenous human DNA recovered and frequency of damage at the terminal nucleotide of the DNA molecule (a commonly used measure of ancient DNA authenticity). Based on these results we selected 10 ossicles from archaeological samples from a wide range of geographic locations with varying climates and dated to between ∼6500–1720 years before present (yBP) (Table 1, with detailed sample information in Supplementary Table 2). To be included in this study, each specimen was required to have at least one ossicle as well as the cochlea of the petrous bone available for comparative analysis. Whenever possible, a petrous bone that had an antimere was chosen (Prendergast and Sawchuk 2018); we did not sample the antimeres in order to preserve them for future analyses.

View this table:
  • View inline
  • View popup
  • Download powerpoint
Table 1: Sample information and summary of sequencing results.

A summary of sequencing results for the 10 individuals reported in this paper is presented in Table 1 and Figure 2; for more detailed information, see Supplementary Table 2.

Figure 2:
  • Download figure
  • Open in new tab
Figure 2:

Comparative results between cochlea (yellow) and ossicle (green) samples from the same individuals. Panel a. Endogenous shotgun DNA ratios of the total reads. Panel b. Complexity as percentage of unique reads expected from 500,000 reads hitting targets. Panel c. Deamination frequencies on the terminal bases of the 1240K capture sequences. Panel d. Contamination estimates calculated by subtracting the rate of mitochondrial matches to the consensus sequence from 1 (smooth bars) and based the heterozygosity of the X-chromosome of male individuals (textured bars). Error bars indicate the 95% confidence interval.

Out of 10 individuals included in this study, both the cochlea and ossicles produced enough data to call mitochondrial DNA (mtDNA) haplogroups, assess damage patterns at the terminal nucleotide of the molecule, and make contamination estimations for seven individuals; these individuals are henceforth referred to as the ‘working individuals.’ One individual from Thailand produced marginal data that allowed the same analyses, but produced a larger error interval for the mtDNA contamination estimate – calculated as 1 minus the rate of mitochondrial matches to the consensus sequence (Fu et al. 2013) – only when the ossicles were used; two individuals, both from Yemen, did not produce enough data to allow for the determination of the mtDNA haplogroup or contamination estimates. Both the cochlea and ossicles were therefore considered to have ‘failed’ our analysis for these latter individuals. We performed Wilcoxon Signed-Rank tests to compare the data generated using the ossicles and cochlear samples.

We obtained an average endogenous DNA yield of 45.87% for the seven working cochlea samples and 51.30% for the corresponding ossicles (Table 1, Figure 2 Panel A) (p=0.2969 for the difference; Supplementary Table 3). Complexity, defined here as the percentage of unique reads expected out after down-sampling to 500,000 sequences that align to the ∼1.2 million targeted SNPs, is a potentially more informative metric for comparing performance between the cochlea and ossicles because it is directly related to the maximum amount of sequencing data the extract or library can possibly yield and is not biased by differences in sequencing depth across samples. The average complexity for cochlea and ossicles was 87.1% and 86.0%, respectively (Table 1, Figure 2 Panel B); this difference is also non-significant (p=0.4688; Supplementary Table 3). Overall, these results suggest that the data generated using ossicles is comparable to that generated using the cochlea. Any minor differences are likely due to chance rather than a systematic difference in DNA preservation between the cochlea and ossicles.

The average mtDNA coverage was 525× for the seven working petrous samples and 486× for the corresponding ossicles (Supplementary Table 2), which were not significantly different (p=0.6875; Supplementary Table 3). The average coverage of the ∼1.2 million targeted SNPs from across the genome was 1.53× for the seven working petrous samples, and 1.47× for the ossicles (Table 1); on average, 727,500 SNPs were called when the cochlea was used and 714,312 were called when the ossicles were used (Table 1). Both of these differences were non-significant (p=0.9375 and 0.6875, respectively; Supplementary Table 3).

For a sample from burial phase Middle Period VII at Ban Chiang, northeast Thailand (BCB 26), the cochlea failed to produce enough data even for estimating contamination, with only 266 nuclear SNPs covered; however, we observe a ∼46-fold increase in SNPs hit associated with the use of the ossicles (12,438 SNPs) (Table 1). In addition, the mitochondrial coverage was seen to increase from 0.08x with the cochlea to 5.15x with the ossicles, an increase of ∼63-fold (Table 1, Supplementary Table 2). Looking further into this data increase, we note a ∼4-fold decrease in frequency of deamination at the terminal base (from 6.40% to 1.30%) for the nuclear data as well as a high mitochondrial contamination estimate (point estimate, 6.0%; 95% confidence interval: 2.8–12.3%), which may indicate the presence of DNA contamination (Table 1, Figure 2). Because of this, we are unable to equate the increase in data to the use of the ossicle.

For the seven working samples, the average deamination frequency was slightly reduced from 12.32% to 11.28% when the ossicles were used, a decrease (Table 1, Figure 2 Panel C) that, although small, was statistically significant (p=0.0313; Supplementary Table 3). Mitochondrial contamination estimates (inferred by identifying mismatches to the mtDNA consensus sequence (Fu et al. 2013)) increased from an average of 0.63% to 1.44%, (Table 1, Figure 2 Panel D) with a significant p-value of 0.0469 (Supplementary Table 3). This change was driven by a single individual (818), which exhibited increased contamination in the ossicle relative to the cochlea (Table 1, Figure 2, Supplementary Table 3). Contamination based on the heterozygosity rate of the X-chromosome (a test only applicable to males) (Korneliussen et al. 2014) averaged 0.52% for the cochlea and 1.12% for the ossicles (or excluding individual 818, 0.53% and 0.40%, respectively), a non-significant change (p=0.625 for the full test and 0.125 without individual 818) (Table 1, Figure 2, Supplementary Table 3). The overall low levels of contamination are also supported by consistency in the estimation of mtDNA haplogroups and molecular sex for all cochlea-ossicles pairs (Table 1, Figure 2, Supplementary Table 2).

DISCUSSION

DNA recovery from the auditory ossicles

This study presents a direct comparison of DNA recovery from the ossicles and corresponding cochlear bone using archaeological specimens that originate from varying geographic and temporal contexts and offers several new insights. First, we demonstrate that the ossicles perform comparably to the cochlea in terms of ancient DNA recovery regardless of sample preservation. Focusing on seven individuals from whom we were able to generate enough working ancient DNA data to call mtDNA haplogroups, assess damage pattern, and make contamination estimates, we find that the use of the cochlea or ossicles from each individual produces similar amounts of endogenous DNA, mtDNA coverage, nuclear SNP coverage, and number of SNPs called. We demonstrate that there is no substantial reduction in data quantity or complexity associated with the analysis of the ossicles instead of the cochlea. Second, although we find that the ossicles show a slight reduction in the frequency of deamination (a signal of ancient DNA authenticity) compared to the corresponding cochlea, the amounts of contamination estimated using both mtDNA and heterozygosity on the X chromosome are comparable. Considered together, our data suggest that there is little reduction in data quality associated with the analysis of the ossicles instead of the cochlea. We conclude that the auditory ossicles, when present, are an alternative optimal skeletal element that can be used in ancient DNA research in place of the cochlea

Though they are small, often isolated, and can be accessed without significant impact to larger, morphologically-informative parts of the skeleton, the use of ossicles for ancient DNA analysis still requires the destruction of human skeletal material that may be anthropologically valuable. Ossicles have previously been used in studies of comparative morphology; most notably, they have provided insight into morphological differences and functional similarities in the middle ear of Neandertals and anatomically modern humans, which has implications for understanding the auditory capacity of extinct hominins (e.g., Stoessel et al. 2016). For this reason, we encourage all researchers contemplating ancient DNA analysis to balance their analytical goals with the impact that sampling for ancient DNA analysis will have on future availability of material.

In light of these findings, we suggest that archaeologists and curators attempt to identify and preserve auditory ossicles whenever possible. Ideally, ossicles would be identified and collected during archaeological recovery of human skeletal remains in a way that minimizes the introduction of contamination. This includes wearing disposable medical gloves that are changed frequently when handling samples, avoiding washing skeletal material with water, and storing samples in a cold, dry place as soon as possible (Llamas et al. 2017).

The use of ossicles for ancient DNA analysis will contribute to the successful analysis of skeletal material that does not have a petrous bone present, or sets of remains that have a petrous bone that cannot be processed in a destructive manner for ancient DNA research (for example, those that may be morphologically-intact and displayed in museum collections). On a broader level, the identification of the ossicles as an alternative optimal skeletal element for ancient DNA analysis contributes to the reduction in the amount of damage inflicted to human skeletal samples for the purposes of ancient DNA analysis. It is another step toward the preservation of DNA-rich and anthropologically-valuable skeletal material for future studies that may benefit from methodological improvements that are unknown at present.

METHODS

Sample Selection and Preparation

The number of ossicles collected for each of the 10 archaeological samples varied (see Table 1), but the incus and malleus were identified and collected most frequently (n=10 and n=8, respectively) while the stapes was identified and collected least frequently (n=2), likely due to its diminutive size and fragility. In most cases, we recovered the ossicles while following the standard cochlea sampling procedure (Pinhasi et al. 2019). In other cases, we intentionally dislodged the ossicles from the skull for the purpose of this study; in most of these instances, the ossicles were partially visible within the external auditory meatus. To dislodge the ossicles, we cleaned a small engraving burr (described in Sirak et al. 2017) by wiping it with a diluted bleach solution (∼10% concentration). We placed the cleaned burr inside the external auditory meatus and gently manipulated it within the inner ear canal. This caused no apparent damage to the ossicles or to the cranium from which they were retrieved. All ossicles were immediately placed into a sterile 2.0mL tube upon their removal from the ear canal.

The preparation of all skeletal material for ancient DNA analysis was carried out in dedicated cleanrooms at University College Dublin (UCD) or at the University of Vienna following standard anti-contamination protocols (e.g., Hofreiter et al. 2001; Poinar 2003; Llamas et al. 2017). All petrous bones were processed following a standard protocol (Pinhasi et al. 2019). This protocol uses a dental sandblaster to systematically locate, isolate, and clean the cochlea, which is then milled to homogeneous bone powder. Approximately 50 mg of bone powder from the cochlea (range: 47–56 mg) was aliquoted for DNA extraction. Complete auditory ossicles were decontaminated through exposure to UV irradiation for 10 minutes on each side; after noting a substantial reduction in amount of bone powder associated with the milling of complete ossicles to bone powder during pilot work, we chose not to grind the ossicles to a fine powder, instead placing them inside a new sterile 2.0mL tube following decontamination with UV irradiation. The tubes that included the whole ossicles or petrous bone powder were then taken to a separate ancient DNA clean room for DNA extraction and preparation of sequencing libraries.

DNA Extraction

DNA was extracted from the cochlear bone powder and the whole auditory ossicles in ancient DNA facilities at the University of Vienna following a standard ancient DNA extravtion protocol (Dabney et al. 2013) with a modification (Korlevic et al. 2015) that uses the tube assemblies from the High Pure Viral Nucleic Acid Large Volume kit (Roche, 05114403001). The intact ossicles were placed in the extraction buffer, and completely dissolved during the incubation period in most cases. Lysates were washed twice with 650 µL of PE buffer (Qiagen) and spun through the columns at 6000 rpm for 1 minute. After being put in a fresh 1.5mL collection tube, 25µL of TET buffer was pipetted on the dry spun MinElute columns’ silica membrane. After a 10-minute incubation at room-temperature, the columns were spun at maximum speed for 1 minute. The elution step was repeated to give a final volume of 50µL of DNA extract. A negative control that contained no bone material was included with each extraction batch.

Library Preparation

Next generation sequencing libraries were prepared in ancient DNA facilities at Harvard Medical School from all extracts and controls using a library preparation method optimized for ancient DNA (Rohland et al. 2015). This protocol uses a partial-UDG treatment that causes characteristic C-to-T ancient DNA damage to be restricted to the terminal molecules while nearly eliminating it in the interior of the DNA molecules so that the library can be used to test for ancient DNA authenticity. 10μL of DNA extract was used as input during library preparation. Libraries were enriched for ∼1.2 million nuclear sites across the genome (‘1240K capture’) in addition to sites on the human mitochondrial genome (Fu et al. 2013, 2015; Haak et al. 2015; Mathieson et al. 2015). Enriched libraries were sequenced on an Illumina NextSeq500 instrument, with 2×76 cycles and an additional 2×7 cycles used for identification of indices. In addition, a small proportion of reads were generated from unenriched versions of each library. This unenriched (‘shotgun’) data was used to estimate the proportion of endogenous molecules in each library.

Data Processing

Following sequencing, we trimmed molecular adapters and barcodes from sequenced reads prior to merging forward and reverse reads using custom software (https://github.com/DReichLab/ADNA-Tools). We allowed up to three mismatches of low base quality (<20) and up to one mismatch at higher base quality (≥20), ensuring that the highest base quality in the overlap region was regained. We aligned reads to the mitochondrial RSRS genome (Behar et al. 2012) and to the hg19 human reference sequence with the samse command in bwa (v0.6.1) (Li and Durbin 2009).

We used the tool ContamMix (Fu et al. 2014) to determine the rate of matching between the consensus RSRS sequence and reads which aligned to the mitochondrial genome. We determined the rate of C-to-T substitution at the terminal ends of each molecule using PMDtools (https://github.com/pontussk/PMDtools; Skoglund et al. 2014). We used the tool ANGSD (Korneliussen et al. 2014) to determine the amount of contamination in the X-chromosome of individuals identified as genetically male. The complexity of the sample was assessed by quantifying the number of unique reads expected from a pre-determined number of reads hitting target.

DATA ACCESS

Data are available at the European Nucleotide Archive under accession number PRJEB32751.

DISCLOSURE DECLARATION

The authors declare no conflict of interest.

SUPPLEMENTARY MATERIAL

Pilot Work

Five archaeological samples representing a range of geographic locations were selected for a pilot project aimed at obtaining initial insight into use of the ossicles for ancient DNA analysis. We chose samples based on their age and depositional contexts to represent a range of molecular preservation (sample information provided in Supplementary Table 1). All specimens had at least two ossicles, and one petrous pyramid from the same individual was selected for comparative analysis. Skeletal material was processed in dedicated ancient DNA clean rooms at University College Dublin following standard anti-contamination protocols (Hofreiter et al. 2001; Poinar 2003; Llamas et al. 2017). Petrous bones were processed as described in Pinhasi et al. (2019) to create bone powder, and complete auditory ossicles were decontaminated through exposure to UV irradiation for 10 minutes on each side and milled to fine powder. DNA extraction and library preparation followed standard ancient DNA protocols, described in the following section. All extraction and library preparation took place in a separate clean room from that used for processing bones and also followed standard anti-contamination protocols.

We generated raw sequencing data for this pilot work using low-coverage whole-genome shotgun sequencing on the Illumina MiSeq and NextSeq platforms. Data were processed using a custom bioinformatics pipeline to enable a basic comparison of endogenous DNA yield from the cochlea and from the auditory ossicles (Supplementary Table 1). Our results suggested that the auditory ossicles were approximately equivalent to the cochlea for endogenous DNA preservation, with the difference in endogenous DNA content ranging between a 0.17-fold decrease and a 0.3-fold increase (Supplementary Table 1). The endogenous DNA yields ranged from 0.16 to 68.19%, with a median of 54.68%, and no substantial difference between the ossicles and cochlea detected (Supplementary Table 1). We identified damage patterns consistent with expectations for ancient DNA in the sequencing data generated using both the ossicle and cochlea samples, with an average substitution frequency on the 5’-end of the DNA molecule of 14.50% for the ossicle samples and 14.40% for the petrous bone samples (Supplementary Table 1). Like endogenous yield, this difference is not substantial. Overall similarity in endogenous yield and damage frequencies between the auditory ossicle and cochlea samples from the same individual supported our hypothesis that auditory ossicles may also be an effective substrate for ancient DNA analysis.

ACKNOWLEDGEMENTS

This work was partially supported by a European Research Council starting grant ADNABIOARC 263441 to R.P, a National Science Foundation (NSF) Doctoral Dissertation Research Improvement Grant BCS-1613577 to K.Si, an Irish Research Council grant GOIPG/2013/36 to D.F., a graduate student fellowship from the Max Planck-Harvard Research Center for the Archaeoscience of the Ancient Mediterranean (MHAAM) to E.H, and Russian Foundation for Basic Research grants 18-00-00360, 18-09-00349 to V.M. T.H. and T.S. were supported by grants from the Hungarian Research, Development and Innovation Office, project numbers FK128013 and TÉT_16-1-2016-0020. D.R. is an investigator of the Howard Hughes Medical Institute. The authors would like to thank Canterbury Archaeological Trust for permission to analyze sample 1257 (I4491); full details about this sample can be found in the CAT PXA report (Helm et al. 2017).

Footnotes

  • ↵† The first two authors (K.Si. and D.F.) should be regarded as joint-first authors.

REFERENCES

  1. Anders, A. 2017. Emberek az újkökorban. Polgár környéki neolitikus temetkezések bioszociális feldolgozásának elsö eredményei (People from the Neolithic. First results of the biosocialarchaeological investigations on Neolithic burials of the Polgár micro region). Thesis submitted to Eötvös Loránd University, Budapest.
    1. Kozlowski JK,
    2. Raczky P
    Anders A, Nagy EG. 2007. Late Neolithic burial rites at the site of Polgár-Csöszhalom-dulö. In The Lengyel, Polgár and related cultures in the Middle/Late Neolithic in Central Europe. (eds. Kozlowski JK, Raczky P), pp. 83–96. Polish Academy of Arts and Sciences, Kraków.
  2. ↵
    Arensburg B, Belkin V, Wolf M. 2005. Middle ear pathology in ancient and modern populations: incudal osteoma. Acta oto-laryngologica 125, 1164–1167.
    OpenUrlPubMed
  3. ↵
    Arensburg B, Harell M, Nathan H. 1981. The human middle ear ossicles: Morphometry, and taxonomic implications. Journal of Human Evolution 10, 199–205.
    OpenUrl
    1. Kazdová E,
    2. Měřínský Z,
    3. Šabatová K
    Bartík J, Šefčáková A. 2004. Hrob so šálkou protoúnětickej kultúry z Blatného, okr. Bratislavavidiek, Slovensko (A grave with a cup of Proto Únětice Culture from Blatné, Greater city of Bratislava, Slovakia). In Zborník K poctě Vladimíru Podborskému (Proceedings to Honor Vladimír Podborský). (eds. Kazdová E, Měřínský Z, Šabatová K), pp.235–244. FF Masaryk University in Brno, Brno.
  4. ↵
    Behar DM, van Oven M, Rosset S, Metspalu M, Loogväli E-L, Silva NM, Kivisild T, Torroni A, Villems R. 2012. A “Copernican” Reassessment of the Human Mitochondrial DNA Tree from its Root. The American Journal of Human Genetics 90, 675–684.
    OpenUrlCrossRefPubMed
  5. ↵
    Bell LS, Kayser M, Jones C. 2008. The mineralized osteocyte: A living fossil. American Journal of Physical Anthropology 137, 449–456.
    OpenUrlCrossRefPubMedWeb of Science
  6. ↵
    Briggs AW, Stenzel U, Johnson PL, Green RE, Kelso J, Prüfer K, Meyer M, Krause J, Ronan MT, Lachmann M, Pääbo S. 2007. Patterns of damage in genomic DNA sequences from a Neandertal. Proceedings of the National Academy of Sciences 104, 14616–14621.
    OpenUrlAbstract/FREE Full Text
  7. ↵
    Broushaki F, Thomas MG, Link V, López S, van Dorp L, Kirsanow K, Hofmanová Z, Diekmann Y, Cassidy LM, Díez-del-Molino D, et al. 2016. Early Neolithic genomes from the eastern Fertile Crescent. Science 353, 499–503.
    OpenUrlAbstract/FREE Full Text
  8. ↵
    Busse B, Djonic D, Milovanovic P, Hahn M, Püschel K, Ritchie RO, Djuric M, Amling M. 2010. Decrease in the osteocyte lacunar density accompanied by hypermineralized lacunar occlusion reveals failure and delay of remodeling in aged human bone. Aging Cell 9, 1065–1075.
    OpenUrlCrossRefPubMedWeb of Science
  9. ↵
    Chen H, Okumura T, Emura S, Shoumura S. 2008. Scanning electron microscopic study of the human auditory ossicles. Annals of Anatomy - Anatomischer Anzeiger 190, 53–58.
    OpenUrl
  10. ↵
    Collins MJ, Nielsen–Marsh CM, Hiller J, Smith CI, Roberts JP, Prigodich RV, Wess TJ, Csapò J, Millard AR, Turner–Walker G. 2002. The survival of organic matter in bone: a review. Archaeometry 44, 383–394.
    OpenUrlCrossRef
    1. de Maigret A,
    2. Antonini S
    Coppa A, Damadio S. 2005. Palaeobiology of the populations of Yemen. In South Arabian Necropolises. Italian Excavations at Al-Makhdarah and Kharabit al-Ahjur (Republic of Yemen). (eds. de Maigret A, Antonini S), pp. 91–146. Istituto Italiano per l’Africa e l’Oriente, Centro Studi e Scavi Archeologici, Rome.
  11. ↵
    Crevecoeur I. 2007. New discovery of an Upper Paleolithic auditory ossicle: the right malleus of Nazlet Khater 2. Journal of Human Evolution 52, 341–345.
    OpenUrlPubMed
  12. ↵
    Cunningham C, Scheuer L, Black S. 2000. Developmental Juvenile Osteology. Academic Press, San Diego, CA.
  13. ↵
    Dabney J, Knapp M, Glocke I, Gansauge M-T, Weihmann A, Nickel B, Valdiosera C, García N, Pääbo S, Arsuaga J-L, Meyer M. 2013. Complete mitochondrial genome sequence of a Middle Pleistocene cave bear reconstructed from ultrashort DNA fragments. Proceedings of the National Academy of Sciences 110, 15758–15763.
    OpenUrlAbstract/FREE Full Text
  14. Daley T, Smith AD. 2013. Predicting the molecular complexity of sequencing libraries. Nature Methods 10, 325–327.
    OpenUrl
  15. ↵
    Damgaard PB, Margaryan A, Schroeder H, Orlando L, Willerslev E, Allentoft ME. 2015. Improving access to endogenous DNA in ancient bone and teeth. Scientific Reports 5, 11184.
    OpenUrl
  16. ↵
    Duboeuf F, Burt-Pichat B, Farlay D, Suy P, Truy E, Boivin G. 2015. Bone quality and biomechanical function: A lesson from human ossicles. Bone 73, 105–110.
    OpenUrlCrossRef
  17. Farkaš, Z., 2002. Nálezy ludských pozostatkov v prostredí kultúry ludu s lineárnou keramikou na Slovensku (Human remains from the Linear Pottery Culture area in Slovakia). Archeologické rozhledy 54, 23–43.
    OpenUrl
  18. ↵
    Fregel R, Mendez FL, Bokbot Y, Martin-Socas D, Camalich-Massieu MD, Santana J, Morales J, Avila-Arcos MC, Underhill PA, Shapiro B, et al. 2018. Ancient genomes from North Africa evidence prehistoric migrations to the Maghreb from both the Levant and Europe. Proceedings of the National Academy of Sciences 115, 6774–6779.
    OpenUrlAbstract/FREE Full Text
  19. ↵
    Frisch T, Sølvsten Sørensen M, Overgaard S, Lind M, Bretlau P. 1998. Volume-referent bone turnover estimated from the interlabel area fraction after sequential labeling. Bone 22, 677–682.
    OpenUrlCrossRef
  20. ↵
    Fu Q, Hajdinjak M, Moldovan OT, Constantin S, Mallick S, Skoglund P, Patterson N, Rohland N, Lazaridis I, Nickel B, et al. 2015. An early modern human from Romania with a recent Neanderthal ancestor. Nature 524, 216–219.
    OpenUrlCrossRefGeoRefPubMed
  21. ↵
    Fu Q, Li H, Moorjani P, Jay F, Slepchenko SM, Bondarev AA, Johnson PLF, Aximu-Petri A, Prüfer K, de Filippo C, et al. 2014. Genome sequence of a 45,000-year-old modern human from western Siberia. Nature 514, 445–449.
    OpenUrlCrossRefPubMedWeb of Science
  22. ↵
    Fu Q, Meyer M, Gao X, Stenzel U, Burbano HA, Kelso J, Pääbo S. 2013. DNA analysis of an early modern human from Tianyuan Cave, China. Proceedings of the National Academy of Sciences 110, 2223–2227.
    OpenUrlAbstract/FREE Full Text
  23. ↵
    Gamba C, Jones ER, Teasdale MD, McLaughlin RL, Gonzalez-Fortes G, Mattiangeli V, Domboroczki L, Kovari I, Pap I, Anders A, Whittle A, Dani J, Raczky P, Higham TFG, Hofreiter M, Bradley DG, Pinhasi R. 2014. Genome flux and stasis in a five millennium transect of European prehistory. Nature Communications 5, 5257.
    OpenUrl
  24. ↵
    Gansauge M-T, Gerber T, Glocke I, Korlević P, Lippik L, Nagel S, Riehl L, Schmidt A, Meyer (F) 2017. Single-stranded DNA library preparation from highly degraded DNA using T4 DNA ligase. Nucleic Acids Research 45, e79.
    OpenUrlCrossRefPubMed
  25. ↵
    Ginolhac A, Rasmussen M, Gilbert MTP, Willerslev E, Orlando L. 2011. mapDamage: testing for damage patterns in ancient DNA sequences. Bioinformatics 27, 2153–2155.
    OpenUrlCrossRefPubMedWeb of Science
  26. Green RE, Krause J, Briggs AW, Maricic T, Stenzel U, Kircher M, Patterson N, Li H, Zhai W, Fritz MH-Y, et al. 2010. A draft sequence of the Neandertal genome. Science 328, 710–722.
    OpenUrlAbstract/FREE Full Text
    1. Gokhman II
    Gromov AV. 1995. Bronze Age population of the Southern Khakassia and problem of the origin of the Karasuk culture, vol. 1. In Antropologia segodnya. (ed. Gokhman II), pp. 130-150. MAE RAS, Saint-Petersburg.
  27. Haas N, Maximilian K. 1958. Anthropological study of the human bones from graves with ochre from Glăvăneştii Vechi, Corlăteni and Stoicani Cetăţuie. Soviet Anthropology 4, 133–146.
    OpenUrl
  28. ↵
    Haak W, Lazaridis I, Patterson N, Rohland N, Mallick S, Llamas B, Brandt G, Nordenfelt S, Harney E, Stewardson K, et al. 2015. Massive migration from the steppe is a source for Indo-European languages in Europe. Nature 522, 207–211.
    OpenUrlCrossRefPubMed
  29. ↵
    Hansen HB, Damgaard PB, Margaryan A, Stenderup J, Lynnerup N, Willerslev E, Allentoft ME. 2017. Comparing Ancient DNA Preservation in Petrous Bone and Tooth Cementum. PLoS ONE 12, e0170940.
    OpenUrlCrossRefPubMed
  30. ↵
    Harney E, May H, Shalem D, Rohland N, Mallick S, Lazaridis I, Sarig E, Stewardson K, Nordenfelt S, Patterson N, et al. 2018. Ancient DNA from Chalcolithic Israel reveals the role of population mixture in cultural transformation. Nature Communications 9, 3336.
    OpenUrl
  31. ↵
    Heim JL. 1982. Les Enfants Néandertaliens de La Ferrassie. Publié sous les auspices de la Fondation Singer-Polignac. Bulletins et Mémoires de la Société d’Anthropologie de Paris 9, 347.
    OpenUrl
  32. ↵
    Helm R, Allison E, Anderson I, Barber L, Broadley R, Carruthers W, Locker A, Lyne M, McIntyre L, McNee B, Richardson A, Smith I, Wilson T. 2017. Former Peugeot Garage, Rhodaus Town (A28), Canterbury, Kent CT1 2RH. Post-excavation assessment, client report no. 2017/107, Canterbury Archaeological Trust.
  33. ↵
    Hernandez CJ, Majeska RJ, Schaffler MB. 2004. Osteocyte density in woven bone. Bone 35, 1095–1099.
    OpenUrlCrossRefPubMed
  34. ↵
    Hofreiter M, Jaenicke V, Serre D, von Haeseler A, Pääbo S. 2001. DNA sequences from multiple amplifications reveal artifacts induced by cytosine deamination in ancient DNA. Nucleic Acids Research 29, 4793–4799.
    OpenUrlCrossRefPubMedWeb of Science
  35. ↵
    Hublin JJ, Pääbo S, Derevianko AP, Doronichev VB, Golovanova LV, Freiss M, Froment A, Hoffmann A, Jillani Kachache NE, Kullmer O, et al. 2008. Suggested guidelines for invasive sampling of hominid remains. Journal of Human Evolution 55, 756–757.
    OpenUrlCrossRefPubMed
  36. ↵
    Kanzaki S, Ito M, Takada Y, Ogawa K, Matsuo K. 2006. Resorption of auditory ossicles and hearing loss in mice lacking osteoprotegerin. Bone 39, 414–419.
    OpenUrlCrossRefPubMed
  37. ↵
    Kontopoulos I, Penkman K, McAllister GD, Lynnerup N, Damgaard PB, Hansen HB, Allentoft ME, Collins MJ. 2019. Petrous bone diagenesis: a multi-analytical approach. Palaeogeography, Palaeoclimatology, Palaeoecology 518, 143–154.
    OpenUrl
  38. Korlević P, Gerber T, Gansauge M-T, Hajdinjak M, Nagel S, Aximu-Petri A, Meyer M. 2015. Reducing microbial and human contamintion in DNA extractions from ancient bones and teeth. Biotechniques 58, 87–93.
    OpenUrl
  39. ↵
    Korneliussen TS, Albrechtsen A, Nielsen R. 2014. ANGSD: Analysis of Next Generation Sequencing Data. BMC Bioinformatics 15, 356.
    OpenUrlCrossRefPubMed
  40. ↵
    Lazaridis I, Nadel D, Rollefson G, Merrett DC, Rohland N, Mallick S, Fernandes D, Novak M, Gamarra B, Sirak K, et al. 2016. Genomic insights into the origin of farming in the ancient Near East. Nature 536, 419–424.
    OpenUrlCrossRefPubMed
  41. ↵
    Li H, Durbin R. 2009. Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics 14, 1754–1760.
    OpenUrl
  42. ↵
    Lindahl T. 1993. Instability and decay of the primary structure of DNA. Nature 362, 709–715.
    OpenUrlCrossRefPubMedWeb of Science
  43. ↵
    Llamas B, Valverde G, Fehren-Schmitz L, Weyrich LS, Cooper A, Haak W. 2017. From the field to the laboratory: Controlling DNA contamination in human ancient DNA resarch in the high-throughput sequencing era. STAR: Science & Technology of Archaeological Research 3, 1–14.
    OpenUrlCrossRef
  44. ↵
    Makarewicz C, Marom N, Bar-Oz G. 2017. Palaeobiology: Ensure equal access to ancient DNA. Nature 548, 158.
    OpenUrl
  45. ↵
    Margulies M, Egholm M, Altman WE, Attiya S, Bader JS, Bemben LA, Berka J, Braverman MS, Chen YJ, Chen Z, et al. 2005. Genome sequencing in microfabricated high-density picolitre reactors. Nature 437, 376–380.
    OpenUrlCrossRefPubMedWeb of Science
  46. ↵
    Marotti G, Farneti D, Remaggi F, Tartari F. 1998. Morphometric investigation on osteocytes in human auditory ossicles. Annals of Anatomy-Anatomischer Anzeiger 180, 449–453.
    OpenUrlCrossRefPubMedWeb of Science
  47. ↵
    Mathieson I, Lazaridis I, Rohland N, Mallick S, Patterson N, Alpaslan-Roodenberg S, Harney E, Stewardson K, Fernandes D, Novak M, et al. 2015. Genome-wide patterns of selection in 230 ancient Eurasians. Nature 528, 499–503.
    OpenUrlCrossRefPubMed
  48. ↵
    Mays S, Elders J, Humphrey L, White W, Marshall P. 2013. Science and the Dead: A guideline for the destructive sampling of archaeological human remains for scientific analysis. Advisory Panel on the Archaeology of Burials in England, English Heritage Publishing, Swindon, England.
  49. Passarello P, Macchiarelli R. 1987. La necropoli protostorica di Katelai (Swat, Pakistan). Analisi antropologica del materiale scheletrico con riferimento al contesto paleobiologico umano dell’area medio-orientale. Rivista di Antropologia 65, 5–104.
    OpenUrl
  50. Pietrusewsky M, Douglas MT. 2002. Ban Chiang, a prehistoric village site in northeast Thailand. Volume One: the human skeletal remains. University Museum Monograph 111. Philadelphia: University of Pennsylvania Museum of Archaeology and Anthropology.
  51. ↵
    Pinhasi R, Fernandes DM, Sirak K, Cheronet O. 2019. Isolating the human cochlea to generate bone powder for ancient DNA analysis. Nature Protocols 14, 1194–1205.
    OpenUrl
  52. ↵
    Pinhasi R, Fernandes DM, Sirak K, Novak M, Connell S, Alpaslan-Roodenberg S, Gerritsen F, Moiseyev V, Gromov A, Raczky P, Anders A, Pietrusewsky M, Rollefson G, Jovanovic M, Trinhhoang H, Bar-Oz G, Oxenham M, Matsumura H, Hofreiter M. 2015. Optimal Ancient DNA Yields from the Inner Ear Part of the Human Petrous Bone. PLoS ONE 10, e0129102.
    OpenUrlCrossRefPubMed
  53. ↵
    Poinar HN. 2003. The top 10 list: criteria of authenticity for DNA from ancient and forensic samples. International Congress Series 1239, 575–579.
    OpenUrlCrossRef
  54. ↵
    Ponce de León MS, Koesbardiati T, Weissmann JD, Milella M, Reyna-Blanco CS, Suwa G, Kondo O, Malaspinas A-S, White TD, Zollikofer CPE. 2018. Human bony labyrinth is an indicator of population history and dispersal from Africa. Proceedings of the National Academy of Sciences 115, 4128–4133.
    OpenUrlAbstract/FREE Full Text
  55. ↵
    Prendergast ME, Sawchuk E. 2018. Boots on the ground in Africa’s ancient DNA ‘revolution’: archaeological perspectives on ethics and best practices. Antiquity 92, 803–815.
    OpenUrlCrossRef
  56. ↵
    Quam R, Martínez I, Arsuaga JL. 2013a Reassessment of the La Ferrassie 3 Neandertal ossicular chain. Journal of Human Evolution 64, 250–262.
    OpenUrlCrossRefPubMed
  57. ↵
    Quam R, Rak Y. 2008. Auditory ossicles from southwest Asian Mousterian sites. Journal of Human Evolution 54, 414–433.
    OpenUrlCrossRefPubMedWeb of Science
  58. ↵
    Quam R, de Ruiter DJ, Masali M, Arsuaga J-L, Martínez I, Moggi-Cecchi J. 2013b. Early hominin auditory ossicles from South Africa. Proceedings of the National Academy of Sciences 110, 8847–8851.
    OpenUrlAbstract/FREE Full Text
  59. ↵
    Qvist M. 2000. Auditory ossicles in archaeological skeletal material from medieval Denmark. Acta Oto-Laryngologica 120, 82–85.
    OpenUrl
    1. Bács TA,
    2. Bollók Á,
    3. Vida T
    Raczky P. 2018. A complex monument in the making at the Late Neolithic site of Polgár-Csöszhalom. In Across the Mediterranean – along the Nile. Studies in Egyptology, Nubiology and Late Antiquity Dedicated to László Török on the Occasion of His 75th Birthday. (eds. Bács TA, Bollók Á, Vida T), pp. 15–60. Institute of Archaeology, Research Centre for the Humanities Hungarian Academy of Sciences, Budapest.
  60. ↵
    Rak Y, Clarke RJ. 1979. Ear ossicle of Australopithecus robustus. Nature 279, 62–63.
    OpenUrlCrossRefGeoRefPubMedWeb of Science
  61. ↵
    Rask-Andersen H, Liu W, Erixon E, Kinnefors A, Pfaller K, Schrott-Fischer A, Gluckert R. 2012. Human cochlea: anatomical characteristics and their relevance for cochlear implantation. The Anatomical Record: Advances in Integrative Anatomy and Evolutionary Biology 295, 1791–1811.
    OpenUrl
  62. ↵
    Richard C, Courbon G, Laroche N, Prades JM, Vico L, Malaval L. 2017. Inner ear ossification and mineralization kinetics in human embryonic development-microtomographic and histomorphological study. Scientific Reports 7, 4825.
    OpenUrl
  63. ↵
    Rohland N, Glocke I, Aximu-Petri A, Meyer M. 2018. Extraction of highly degraded DNA from ancient bones, teeth and sediments for high-throughput sequencing. Nature Protocols 13, 2447–2461.
    OpenUrl
  64. ↵
    Rohland N, Harney E, Mallick S, Nordenfelt S, Reich D. 2015. Partial uracil–DNA– glycosylase treatment for screening of ancient DNA. Philosophical Transactions of the Royal Society of London B: Biological Sciences 370, 20130624.
    OpenUrlCrossRefPubMed
  65. ↵
    Rolvien T, Schmidt FN, Milovanovic P, Jähn K, Riedel C, Butscheidt S, Püschel K, Jeschke A, Amling M, Busse B. 2018. Early bone tissue aging in human auditory ossicles is accompanied by excessive hypermineralization, osteocyte death and micropetrosis. Scientific Reports 8, 1920.
    OpenUrl
  66. ↵
    Schuenemann VJ, Peltzer A, Welte B, van Pelt WP, Molak M, Wang C-C, Furtwängler A, Urban C, Reiter E, Nieselt K, et al. 2017. Ancient Egyptian mummy genomes suggest an increase of Sub-Saharan African ancestry in post-Roman periods. Nature Communications 8, 15694.
    OpenUrl
  67. ↵
    Schwark T, Modrow JH, Steinmeier E, Poetsch M, Hasse J, Fischer H, von Wurmb-Schwark (G) 2015. The auditory ossicles as a DNA source for genetic identification of highly putrefied cadavers. International Journal of Legal Medicine 129, 457–462.
    OpenUrl
  68. ↵
    Siori MS, Monchietto MJ, Masali M. 1995. Morphometrics of human auditory ossicles from Antinoe Necropolis (Egypt). International Journal of Anthropology 10, 29.
    OpenUrl
  69. ↵
    Sirak K, Fernandes DM, Cheronet O, Novak M, Gamarra B, Balassa T, Cséki A, Dani J, Gallina JZ, Kocsis-Buruzs G, László O, Pap I, Patay R, Petkes Z, Szenthe G, Szeniczey T, Hajdu T, and Pinhasi R. 2017. A minimally-invasive method for sampling human petrous bones from the cranial base for ancient DNA analysis. BioTechniques 62, 283–289.
    OpenUrlCrossRefPubMed
  70. Sirak K, Sedig JW. In press. Balancing Analytical Goals and Anthropological Stewardship in the Midst of the Paleogenomics Revolution.” Accepted at World Archaeology.
  71. ↵
    Skoglund P, Northoff BH, Shunkov MV, Derevianko AP, Pääbo S, Krause J, Jakobsson M. 2014. Separating endogenous ancient DNA from modern day contamination in a Siberian Neandertal. Proceedings of the National Academy of Sciences 111, 2229–2234.
    OpenUrlAbstract/FREE Full Text
  72. ↵
    Skoglund P, Thompson JC, Prendergast ME, Mittnik A, Sirak K, Hajdinjak M, Salie T, Rohland N, Mallick S, Peltzer A, et al. 2017. Reconstructing Prehistoric African Population Structure. Cell 171, 59–71.
    OpenUrlCrossRefPubMed
  73. ↵
    Smith CI, Chamberlain AT, Riley MS, Cooper A, Stringer CB, Collins MJ. 2001. Neanderthal DNA: Not just old but old and cold? Nature 410, 771–772.
    OpenUrlGeoRefPubMed
  74. ↵
    Smith CI, Chamberlain AT, Riley MS, Cooper A, Stringer CB, Collins, MJ. 2003. The thermal history of human fossils and the likelihood of successful DNA amplification. Journal of Human Evolution 45, 203–217.
    OpenUrlCrossRefPubMedWeb of Science
  75. ↵
    Sølvsten Sørensen M, Bretlau P, Balslev Jørgensen M. 1992. Quantum type bone remodeling in the human otic capsule: morphometric findings. Acta Oto-Laryngologica 112, 4–10.
    OpenUrl
  76. ↵
    Spoor F, Hublin JJ, Braun M, Zonneveld F. 2003. The bony labyrinth of Neanderthals. Journal of Human Evolution 44, 141–165.
    OpenUrlCrossRefPubMedWeb of Science
  77. ↵
    Stoessel A, Gunz P, David R, Spoor F. 2016. Comparative anatomy of the middle ear ossicles of extant hominids–Introducing a geometric morphometric protocol. Journal of Human Evolution 91, 1–25.
    OpenUrlCrossRefPubMed
  78. Szöke BM. 2008. Pannonien in der Karolingerzeit Bemerkungen zur Chronologie des frühmittelalterlichen Fundmaterials in Westungarn. Schild von Steierschen Beiheft 4, 41–56.
    OpenUrl
  79. Szöke BM. 2010. Mosaburg/Zalavár und Pannonien in der Karolingerze. Antæus: Communications ex Instituto Archaeological Academiae Scientarum Hungaricae 31, 9–52.
    OpenUrl
  80. Szöke BM. 2014. The Carolingian Age in the Carpathian Basin: Permanent Exhibition of the Hungarian National Museum. Magyar Nemzeti Múzeu, Budapest.
  81. ↵
    van de Loosdrecht M, Bouzouggar A, Humphrey L, Posth C, Barton N, Aximu-Petri A, Nickel B, Nagel S, Talbi EH, El Hajraoui MA, et al. 2018. Pleistocene North African genomes link Near Eastern and sub-Saharan African human populations. Science 360, 548–552.
    OpenUrlAbstract/FREE Full Text
  82. Vasilyev SV, Gerasimova MM, Borutskaya SB, Frizen SY, Zamballarova ED. 2010. Anthropological study of the Fofonovo burial (Transbaikalia) Neolithic and early bronze age. Herald of anthropology 18, 113–127.
    OpenUrl
  83. ↵
    Yokoyama T, Iino Y, Kakizaki K, Murakami Y. 1999. Human temporal bone study on the postnatal ossification process of auditory ossicles. The Laryngoscope 109, 927–930.
    OpenUrlCrossRefPubMed
Back to top
PreviousNext
Posted May 31, 2019.
Download PDF

Supplementary Material

Email

Thank you for your interest in spreading the word about bioRxiv.

NOTE: Your email address is requested solely to identify you as the sender of this article.

Enter multiple addresses on separate lines or separate them with commas.
Human Auditory Ossicles as an Alternative Optimal Source of Ancient DNA
(Your Name) has forwarded a page to you from bioRxiv
(Your Name) thought you would like to see this page from the bioRxiv website.
CAPTCHA
This question is for testing whether or not you are a human visitor and to prevent automated spam submissions.
Share
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, Dashtseveg Tumen, Sándor Évinger, Zdeněk Farkaš, Tamás Hajdu, Jamsranjav Bayarsaikhan, Lauren McIntyre, Vyacheslav Moiseyev, Ildikó Pap, Michael Pietrusewsky, Pál Raczky, Alena Šefčáková, Andrei Soficaru, Tamás Szeniczey, Béla Miklós Szőke, Tumurbaatar Tuvshinjargal, Dennis Van Gerven, Sergey Vasilyev, Lynne Bell, David Reich, Ron Pinhasi
bioRxiv 654749; doi: https://doi.org/10.1101/654749
Digg logo Reddit logo Twitter logo Facebook logo Google logo LinkedIn logo Mendeley logo
Citation Tools
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, Dashtseveg Tumen, Sándor Évinger, Zdeněk Farkaš, Tamás Hajdu, Jamsranjav Bayarsaikhan, Lauren McIntyre, Vyacheslav Moiseyev, Ildikó Pap, Michael Pietrusewsky, Pál Raczky, Alena Šefčáková, Andrei Soficaru, Tamás Szeniczey, Béla Miklós Szőke, Tumurbaatar Tuvshinjargal, Dennis Van Gerven, Sergey Vasilyev, Lynne Bell, David Reich, Ron Pinhasi
bioRxiv 654749; doi: https://doi.org/10.1101/654749

Citation Manager Formats

  • BibTeX
  • Bookends
  • EasyBib
  • EndNote (tagged)
  • EndNote 8 (xml)
  • Medlars
  • Mendeley
  • Papers
  • RefWorks Tagged
  • Ref Manager
  • RIS
  • Zotero
  • Tweet Widget
  • Facebook Like
  • Google Plus One

Subject Area

  • Genomics
Subject Areas
All Articles
  • Animal Behavior and Cognition (3597)
  • Biochemistry (7563)
  • Bioengineering (5517)
  • Bioinformatics (20777)
  • Biophysics (10316)
  • Cancer Biology (7973)
  • Cell Biology (11629)
  • Clinical Trials (138)
  • Developmental Biology (6602)
  • Ecology (10197)
  • Epidemiology (2065)
  • Evolutionary Biology (13605)
  • Genetics (9537)
  • Genomics (12842)
  • Immunology (7919)
  • Microbiology (19536)
  • Molecular Biology (7653)
  • Neuroscience (42050)
  • Paleontology (307)
  • Pathology (1257)
  • Pharmacology and Toxicology (2199)
  • Physiology (3266)
  • Plant Biology (7036)
  • Scientific Communication and Education (1294)
  • Synthetic Biology (1951)
  • Systems Biology (5426)
  • Zoology (1115)