Alu SINE analyses of 3,000-year-old human skeletal remains: a pilot study
© Kothe et al. 2016
Received: 23 December 2015
Accepted: 31 March 2016
Published: 18 April 2016
As Short Interspersed Elements (SINEs), human-specific Alu elements can be used for population genetic studies. Very recent inserts are polymorphic within and between human populations. In a sample of 30 elements originating from three different Alu subfamilies, we investigated whether they are preserved in prehistorical skeletal human remains from the Bronze Age Lichtenstein cave in Lower Saxony, Germany. In the present study, we examined a prehistoric triad of father, mother and daughter.
For 26 of the 30 Alu loci investigated, definite results were obtained. We were able to demonstrate that presence/absence analyses of Alu elements can be conducted on individuals who lived 3,000 years ago. The preservation of the ancient DNA (aDNA) is good enough in two out of three ancient individuals to routinely allow the amplification of 500 bp fragments. The third individual revealed less well-preserved DNA, which results in allelic dropout or complete amplification failures. We here present an alternative molecular approach to deal with these degradation phenomena by using internal Alu subfamily specific primers producing short fragments of approximately 150 bp.
Our data clearly show the possibility of presence/absence analyses of Alu elements in individuals from the Lichtenstein cave. Thus, we demonstrate that our method is reliably applicable for aDNA samples with good or moderate DNA preservation. This method will be very useful for further investigations with more Alu loci and larger datasets. Human population genetic studies and other large-scale investigations would provide insight into Alu SINE-based microevolutionary processes in humans during the last few thousand years and help us comprehend the evolutionary dynamics of our genome.
KeywordsTransposable elements Alu SINE retrotransposon aDNA ancientDNA DNA preservation Lichtenstein cave Bronze Age
After the discoveries of Barbara McClintock on Zea Mays [1, 2], much research has been conducted in the field of transposable elements (TEs). It is now known that TEs, long classified as junk DNA , have major effects on the genomes of all organisms. For example, they may affect gene functions or alternate transcription rates [4–9]. In eukaryotes, TEs are mostly inherited vertically from generation to generation and in rare cases horizontally, e.g. via a viral vector . In humans, TEs make up a great part of the total genome. Estimates vary from ~45 %  to ~69 % . Thousands of new TE loci have recently been identified in the human 1,000 Genome Project [13, 14]. The transposition mechanism of TEs can generally be divided into two classes: class I retrotransposons and class II DNA transposons. While DNA transposons move via a “cut-and-paste” mechanism, the retrotransposons move by a “copy-and-paste” mechanism. As class l elements, the non-autonomous Short Interspersed Elements (SINEs) present the largest group of TEs in eukaryotic genomes in terms of copy numbers . More than 200 SINE families have been identified so far . Their sequence information can be retrieved at SINEBase  and RepBase . The retrotransposition mechanism of a SINE requires a Long Interspersed Element (LINE)-encoded protein from a LINE partner with reverse transcriptase and endonuclease activity .
The absence of an element at a specific locus can be described as the ancestral state, whereas presence is the derived state [18, 19]. Due to the irreversibility of an insertion and its homoplasy-free character, SINE insertions are a powerful tool for phylogenetic analyses [20, 21]. The most abundant SINEs in humans are the primate-specific Alu elements, reaching a copy number of about 1.1 million [11, 22]. Their partner LINEs are L1 elements that represent a family of mammalian retrotransposons that have been replicating and evolving for more than 100 Myr . Alu elements usually have a length of approximately 300 base pairs. They began to expand with the primate radiation 65 Mya and peaked in activity 40 Mya. It is believed that only a few “Master Genes” are retropositionally competent . Due to accumulations of new mutations, over evolutionary time, new Alu subfamilies are created. The 7SL RNA-derived Alu elements can be classified in three subfamilies J, S and Y, with AluJ being the oldest, followed by AluS and AluY as the youngest and only active subfamily . Within the AluY elements, the subfamilies AluYa5 and AluYb8 are the groups with the largest numbers of copies. Some of these elements retrotransposed so recently that they are absent in other primate lineages and are even polymorphic between and within human populations [25–28]. These polymorphic elements are perfectly suited for population genetic and phylogenetic studies. In cases of rapid radiation of taxa or simultaneous lineage divergence, some TEs may not show the real phylogenetic state. This phenomenon is called incomplete lineage sorting [29–32]. Nonetheless, polymorphic Alu elements are excellent ancestry markers for resolving relationships within and between human populations . In a genome-wide study of polymorphic TEs in 2,504 individuals across 26 human populations, Rishishwar et al.  recently showed that the genetic diversity represented by TE polymorphisms, mainly by Alu elements, reflects known patterns of human evolution. Alu elements and TEs in general insert nearly randomly into the genome, exist in large copy numbers, and are mostly non-autonomous [34, 35]. Our genome is constantly in evolutionary change . Normally, the long-term effects of gene evolution and function alternation become visible [37, 38]. The effects of short-term or microevolutionary processes can be detected by analyzing the presence/absence situations of human specific Alu elements.
For such analyses, human remains with well-preserved DNA are required. Usually, DNA degradation in bones is too advanced for analyses of fragments that exceed 200–300 base pairs [39, 40], but it was proven, for example, that larger fragments of 397 bp from bone samples of the Lichtenstein cave can be amplified, too . The main causes of DNA loss in remains are autolysis directly after death, hydrolisis, and oxidation [42, 43]. The degree of post-mortem DNA degradation depends on environmental factors such as acid conditions, microbial activity, and high mean temperatures . On the other hand, constant low temperatures and neutral or slightly alkaline pH-values provide optimal conditions for DNA preservation [40, 42]. These conditions are found in the Lichtenstein cave near Osterode in Lower Saxony, Germany. For thousands of years, the cave has had a constant temperature of 6–8 °C. Additionally, the skeletal remains were coated with a gypsum layer, which causes a slightly basic environment and is thus perfect conditions for preserving bones and DNA. Previous studies on these remains revealed kinship relations between many individuals [44, 45]. These results are based on genetic fingerprinting, mtDNA, and Y-haplotypes [46, 47]. In the present work, a triad of father, mother, and daughter [44, 45] was chosen for the investigations. Besides kinship calculations, STR fingerprints are used for personal identification due to the unique pattern of STRs. In this study, a genetic STR fingerprint multiplex analysis is used to ensure the authenticity of DNA extracts by monitoring for potential contaminations from the laboratory staff.
Considering the rules of Mendelian inheritance, the known kinship relation between the chosen individuals is helpful for revealing potential false negative results. Especially in aDNA analyses, the phenomenon of allelic dropout is common. Large alleles are more frequently affected by allelic dropout than short alleles are, depending on the degree of DNA fragmentation of the remains .
In the present work, the presence/absence situation of 30 Alu loci was investigated for three members of a prehistoric family (father, left femur DO 1911; mother, left femur DO 3756; daughter, left femur DO 3750) and two modern individuals of Caucasian origin as positive controls. A presence band is defined as the Alu locus where the element is inserted, resulting in a long amplification fragment, an absence band as the locus where the Alu element is not inserted, which appears as a shorter fragment on the gel. We show that it is possible to amplify, Alu loci, including flanking regions with fragment lengths up to 500 bp, for the 3,000-year-old remains in the Lichtenstein cave. We also demonstrate an alternative approach for cases in which, due to DNA degradation, the classic PCR approach failed to amplify the longer presence fragments. Additionally, we give a brief statement about questions to be raised in further investigations.
The homozygous results for the prehistoric individuals are represented only by “+” or “‐”, to include the possibility of allelic dropout events. In contrast, the homozygous results for the modern samples are indicated by “+/+” or “-/-”, due to the reliability of analyses of modern DNA. In this study, a “definite result” is defined as successful product amplification for all three samples (father, mother and daughter) per Alu locus based on both molecular approaches (FAP and if necessary IAP, see Table 1) upon condition that the family situation is congruent. Alu loci that are marked yellow show the incongruence of the family situation in relation to Mendelian inheritance, or the amplification failed completely. This is best explained by the phenomenon of allelic dropout, which is known and common in aDNA analyses. The presence band is periodically not amplified because allelic dropout usually impacts larger alleles. A low number of intact targets is one reason why some alleles may not occur at all or may not reach the detection limits of the devices of electrophoresis .
Proof of kinship in the prehistoric samples and authenticity
Heptaplex based fingerprint results for all investigated individuals
Additional fingerprint results for the prehistorical individuals
DO 1911 (F)
DO 3756 (M)
DO 3750 (D)
The study clearly demonstrates the possibility of presence/absence analyses of TEs in 3,000-year-old human remains from the Lichtenstein cave. These and earlier results indicate and prove the high quality of DNA preservation and the applicability of molecular analyses using the remains from this cave [44, 45, 52], but could not yet show the amplification of 500 bp fragments. Of 30 loci, we initially achieved 22 definite results (FAP in Table 1). With additional amplification using internal Alu primers, we could add four more results (Alu_3, Alu_14, Alu_19 and Alu_20), thus, 26 definite results (FAP and IAP in Table 1). The following Alu loci were incongruent with the family situation: Alu_2, Alu_7, Alu_16, Alu_26 and Alu_27. Amplification with internal Alu primers could place Alu_16, Alu_26 and Alu_27 in congruence with the family situation. The proposed verification technique is to check for possible presence bands by amplification with an internal Alu primer to get short target sequences of ~150 bp. Fragment lengths of more than 200 bp tend to be affected more often by allelic dropout events; therefore, short amplicons should be used. Thus, in further analyses, results that show only absence bands should be subjected to this strategy. Even less well-preserved DNA can be analyzed by this approach. The present study constitutes the basis for further investigations with more Alu loci and larger samples for microevolutionary studies in Central Europe. Such large-scale investigations would provide insight into Alu SINE-based microevolutionary processes in humans during the last few thousand years and help us comprehend the evolutionary dynamics of our genome. Current projects, like the 1,000 Human Genome Project, investigate human genetic variation and the interrelation of genotypes and phenotypes as well as variants in annotated genes and inherited genetic disorders [13, 53]. Through computational biology, the 1,000 Genome Project recently provided a genome-wide catalog of Alu polymorphisms for human populations . A database with these group-specific insertions of polymorphic Alu elements is useful for future analyses with a larger dataset of Bronze Age Lichtenstein individuals – for instance, to investigate the geographic origin of Lichtenstein family members, who belong to the longest known family tree in the world. Through large-scale Alu element analyses of many individuals from the Bronze Age Lichtenstein cave, we may be able to detect human variability and evolution within one geographic region on a timeline. These data would represent a great supplement to recent human population genetic studies based on TEs.
Samples and DNA extraction
The skeletal material used for the present thesis originates from the Bronze Age Lichtenstein cave near Osterode in Lower Saxony, Germany. All bone material from the cave is stored at -20 °C in the Department of Historical Anthropology and Human Ecology of the Göttingen University, Lower Saxony, Germany. The DNA of the ancient individuals was extracted from three different members of a prehistoric family: father (left femur DO 1911), mother (left femur DO 3756) and daughter (left femur DO 3750). In all three cases, the DNA was extracted from the middle of the diaphysis. The modern DNA of the person from the United States of America was extracted from lymphocytes (CAU_1), and was provided with full written consent. This sample was ordered from “The Interstate Companies” (Memphis, Tennessee, USA) blood bank. The DNA of the modern positive control CAU_2 was extracted from cells of the buccal mucosa.
aDNA extraction from skeletal material with the QIAvac-24-plus
Fragments about 1 cm2 in size are sawed out of the middle of the diaphysis of the left femora. All outer surfaces of the fragments are removed to minimize the risk of a contamination with modern human DNA from, e.g., the excavation personal. The fragments are crushed with a steel mortar before they are powdered in a swing mill for 3 min at 24 swings per second. Afterwards, 0.25 g of the powder is transferred into a 15 ml FalconTube and 3900 μl of EDTA UltraPure™ 0.5 M pH 8 (Invitrogen™) and 100 μl of Proteinase K (600mAnson-U/ml) are added. This mixture is incubated for 18 h at 37 °C in a rotator. Now, an additional 50 μl of Proteinase K are added and the mixture is incubated at 56 °C for 2 h in a rotator. 50 μl of SDS (10 mg/ml) are added, followed by an incubation time of 5 min at 65 °C. The lysate is centrifuged at 3300rcf for 3 min to sediment surplus organic material. The lysate is transferred into a 50 ml FalconTube that contains 16 ml PB-Buffer (Qiagen) and 100 μl sodium acetate buffer (pH 5.2, 3 M, Sigma). After manually mixing the lysate, it is centrifuged at 3300rcf for 3 min. The DNA clean-up is conducted with minElute spin columns and funnels for large volumes using the QIAvac-24-plus (Qiagen). Deviating from the protocol, three wash steps with PE-buffer (Qiagen) are performed. The DNA is eluted in 60 μl RNase-free water (also cf. ).
The DNA of the blood sample from CAU_1 is extracted with the Wizard Genomic DNA Purification Kit (Promega) following the producer’s protocol for extraction from whole blood samples (300 μl).
Buccal mucosa swab sample
A buccal mucosa swab from CAU_2 is transferred into a 2 ml reaction tube. 400 μl of G2-buffer (Qiagen) and 10 μl of Proteinase K are added, followed by incubation for 1 h at 56 °C and 350 rpm on an Eppendorf thermomixer comfort. Afterwards, 200 μl of the lysate is transferred into a clean tube and 1 ml of PB-buffer and 100 μl of sodium acetate buffer are added. After manually mixing the lysate, it is centrifuged at 3300rcf for 3 min. Now the DNA is cleaned up with minElute spin columns and large volume funnels as described above.
Alu loci and primer design
Alu loci were chosen based on earlier publications with a population-genetics focus [23–26]. The site-specific Alu sequence was determined by using RepeatMasker . An additional 500 bp flanking sequence on each site was extracted from the human reference genome (hg38) in NCBI . The locus-specific primers were designed with PrimerSelect, version 10.1.2 (DNASTAR). The primer characteristics are a strong 5′ and a weak 3′ end by not exceeding a length of 30 bp; further, primer dimerization and hairpin formation were avoided to enhance the specificity and sensitivity of the reaction . The total length of the target sequence (including the Alu insert) should be as short as possible, which usually resulted in amplicons of 450 bp to 500 bp (cf. also Additional file 4 for detailed information).
The internal Alu primers were designed based on a highly conserved region of the Alu sequence that is specific to the respective subfamily. Therefore, randomly selected Alu inserts of the respective AluY subfamily were aligned. The amplification always includes the Alu head.
A full list of the primer sequences is shown in the Additional file 5.
All PCRs are conducted under the same conditions apart from the annealing temperatures. Depending on the energy profiles and melting temperatures of the primer sets and based on preliminary primer tests, different annealing temperatures, varying from 52 to 60 °C, are chosen. The amplification is conducted using the following cycling program: Initial hot start at 95 °C for 5 min; 40 cycles with denaturation at 94 °C for 1 min, annealing at 52 – 60 °C for 1 min, elongation at 72 °C for 1 min; a final soak at 10 °C for 10 min. The PCR is composed of 12.5 μl of Multiplex PCR Mastermix (Qiagen), 1 μl each forward and reverse primer, (both 20 μM working solution), 5 μl DNA for aDNA samples and 0.5 μl DNA (plus 4.5 μl RNase free water) for modern DNA samples and 5.5 μl of RNase-free water to get a final volume of 25 μl per reaction.
The amplification with an internal Alu primer was performed with an elongation time of 20 s. All other parameters are identical to the classical PCR approach.
For proof of authenticity, every DNA extract used in the study here presented was subjected to STR-typing by a multiplex amplification as described previously . Deviating from this work, the sex discriminating amelogenin gene is arranged in the blue dye panel. The reaction mix is composed of 12.5 μl Multiplex PCR Mastermix (Qiagen), 2.85 μl multiplex primer mix, 4.65 μl RNase-free water and 5 μl DNA extract.
Gel electrophoresis and fragment length estimation
Each amplification result is checked by ethidium bromide stained agarose gel electrophoresis (2.5 %). The fragment length determination is performed with a 50 bp molecular ladder (Invitrogen). For the electrophoresis, usually a voltage of 120 V and a run time of 30 min are applied.
The STR products are separated in a 50 cm capillary on an ABI 3500 Genetic Analyzer (Applied Biosystems) using POP-7™ Polymer for 3500/3500xL Genetic Analyzers and the 3500 Data collection Software (all Applied Biosystems). Allele determination is performed with GeneMapper Software 5 (Applied Biosystems).
Cloning and sequencing
Cloning of PCR products is conducted with the Blue/White-Selection based pGEM®-T Easy Vector System (Promega). Deviating from the manufacturer’s protocol, 300 μl SOC medium (Invitrogen) is used to suspend the cells. Additionally, 50 μl – 100 μl of the cell suspension are plated. The Colony-PCR Mastermix is identical to the other PCRs except for the PCR primers. The primers pUC/M13 forward and reverse (Promega) are used in working concentrations of 20 μM. One colony replaces the DNA inset. The Colony PCR is conducted with the following program: Initial denaturation at 94 °C for 3 min; 30 cycles with denaturation at 94 °C for 30 s, annealing at 55 °C for 1 min, elongation at 72 °C for 50 s; final elongation at 72 °C for 2 min and final soak at 10 °C for 10 min. PCR products are purified with an isopropanol purification protocol: the PCR product is incubated with 83 μl HPLC water, 100 μl isopropanol (100 %) and 10 μl sodium acetate (3 M) for 10 min, then centrifuged at 13,200 rpm for 10 min in a conventional tabletop microcentrifuge. The supernatant is discarded and 150 μl of ethanol (70 %) is added. After another 10 min of centrifugation at 13,200 rpm, the supernatant is discarded, the pellet is dried and the desired amount of RNase-free water is added for resuspension. The sequencing reaction is composed of 4 μl Sequencing Buffer (5x), 2 μl BigDyeTerminator v1.1, 0.3 μl primer (20 μM), 6.7 μl HPLC water and 7 μl purified PCR product. Sequencing is performed in forward and reverse direction with the following program: Initial heating step at 94 °C for 3 min; 33 cycles with denaturation at 94 °C for 30 s, annealing at 55 °C for 1 min and elongation at 72 °C for 2.5 min; soak at 10 °C. Sequencing products are purified with NucleoSeq® columns (Macherey-Nagel). The products are separated in a 50 cm capillary on an ABI 3500 Genetic Analyzer (Applied Biosystems) using POP-7™ Polymer for 3500/3500xL Genetic Analyzers and the 3500 Data collection Software (all Applied Biosystems). The sequences are edited in BioEdit version 7.2.5  and submitted to a BLAST analysis. Finally, sequence data with the following accession numbers were deposited in GenBank: KU323383-KU323387.
All single RPIs are multiplied to get a combined RPI.
long interspersed elements
short interspersed elements
The Alu SINE-based work was supported by the University of Göttingen with funding provided by the Lower Saxony Ministry for Science and Culture (Ministerium für Wissenschaft und Kultur, MWK) to OP. The research on the skeletal remains from the Lichtenstein cave was funded by the MWK to SH. We want to thank Stefan Flindt from the Landkreis Osterode am Harz (Kreisarchäologie) Lower Saxony for enabling this research by allowing the use of the invaluable sample material. His long-term cooperation in research on human remains from the Lichtenstein cave is highly appreciated.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
- McClintock B. The origin and behavior of mutable loci in maize. Proc Natl Acad Sci U S A. 1950;36:344–55.View ArticlePubMedPubMed CentralGoogle Scholar
- McClintock B. Induction of Instability at Selected Loci in Maize. Genetics. 1953;38:579–99.PubMedPubMed CentralGoogle Scholar
- Doolittle WF, Sapienza C. Selfish genes, the phenotype paradigm and genome evolution. Nature. 1980;284:601–3.View ArticlePubMedGoogle Scholar
- Callinan PA, Batzer MA. Retrotransposable Elements and Human Disease. In: Volff JN, editor. Genome and Disease. Genome Dyn, vol. 1. Basel: Karger; 2006. p. 104–15.View ArticleGoogle Scholar
- Volff JN. Turning junk into gold: domestication of transposable elements and the creation of new genes in eukaryotes. Bioessays. 2006;28:913–22.View ArticlePubMedGoogle Scholar
- Piskurek O, Jackson DJ. Transposable Elements: From DNA Parasites to Architects of Metazoan Evolution. Genes. 2012;3:409–22.View ArticlePubMedPubMed CentralGoogle Scholar
- Heras SR, López MC, Olivares M, Thomas MC. The L1Tc non-LTR retrotransposon of Trypanosoma cruzi contains an internal RNA-pol II-dependent promoter that strongly activates gene transcription and generates unspliced transcripts. Nucleic Acids Res. 2007;35(7):2199–214.View ArticlePubMedPubMed CentralGoogle Scholar
- Estecio MRH, Gallegos J, Dekmezian M, Lu Y, Liang S, Issa JPJ. SINE Retrotransposons Cause Epigenetic Reprogramming of Adjacent Gene Promoters. Mol Cancer Res. 2012;10(10):1332–42.View ArticlePubMedPubMed CentralGoogle Scholar
- Sasaki T, Nishihara H, Hirakawa M, Fujimura K, Tanaka M, Kokubo N, Kimura-Yoshida C, Matsuo I, Sumiyama K, Saitou N, Shimogori T, Okada N. Possible involvement of SINEs in mammalian-specific brain formation. Proc Natl Acad Sci U S A. 2008;105(11):4220–25.View ArticlePubMedPubMed CentralGoogle Scholar
- Piskurek O, Okada N. Poxviruses as possible vectors for horizontal transfer of retroposons from reptiles to mammals. Proc Natl Acad Sci U S A. 2007;104:12046–51.View ArticlePubMedPubMed CentralGoogle Scholar
- Lander ES, Linton LM, Birren B, Nusbaum C, Zody MC, Baldwin J, et al. Initial sequencing and analysis of the human genome. Nature. 2001;409:860–921.View ArticlePubMedGoogle Scholar
- De Koning AP, Gu W, Castoe TA, Batzer MA, Pollock DD. Repetitive Elements May Comprise Over Two-Thirds of the Human Genome. PLoS Genet. 2011;67:183–93.Google Scholar
- Abecasis GR, Altshuler D, Auton LD, Durbin RM, Gibbs RA, Hurles ME, et al. A map of human genome variation from population-scale sequencing. Nature. 2010;467(7319):1061–73.View ArticlePubMedGoogle Scholar
- Rishishwar L, Tellez Villa CE, Jordan IK. Transposable element polymorphisms recapitulate human evolution. Mobile DNA. 2015;6:21.View ArticlePubMedPubMed CentralGoogle Scholar
- Vassetzky NA, Kramerov DA. SINEBase: a database and tool for SINE analysis. Nucleic Acids Res. 2013;41:D83–9.View ArticlePubMedPubMed CentralGoogle Scholar
- Jurka J, Kapitonov VV, Pavlicek A, Klonowski P, Kohany O, Walichiewicz J. Repbase Update, a database of eukaryotic repetitive elements. Cytogenet Genome Res. 2005;110:462–7.View ArticlePubMedGoogle Scholar
- Okada N, Hamada M, Ogiwara I, Ohshima K. SINEs and LINEs share common 3′ sequences: A review. Gene. 1997;205:229–43.View ArticlePubMedGoogle Scholar
- Okada N, Shedlock AM, Nikaido M. Retroposon mapping in molecular systematics. Methods Mol Biol. 2004;260:189–226.PubMedGoogle Scholar
- Shedlock AM, Kazuhiko T, Okada N. SINEs of speciation: tracking lineages with retroposons. TRENDS Ecol Evol. 2004;19(10):545–53.View ArticlePubMedGoogle Scholar
- Shedlock AM, Okada N. SINE insertions: powerful tools for molecular systematics. Bioessays. 2000;22(2):148–60.View ArticlePubMedGoogle Scholar
- Schmitz J, Ohme M, Zischler H. SINE insertions in cladistic analyses and the phylogenetic affiliations of Tarsius bancanus to other primates. Genetics. 2001;157(2):777–84.
- Deininger PL. Alu elements: know the SINEs. Genome Biol. 2011;12:236.View ArticlePubMedPubMed CentralGoogle Scholar
- Furano AV. The biological properties and evolutionary dynamics of mammalian LINE-1 retrotransposons. Prog Nucleic Acids Res Mol Biol. 2000;64:255–94.View ArticleGoogle Scholar
- Okada N, Shedlock AM, Nikaido M. Retroposon Mapping in Molecular Systematics. In: Mobile Genetic Elements: Protocols and Genomic Applications, Methods in Molecular Biology. Totowa, New Jersey, U.S.A.: Humana Press; 2004. vol. 260. p. 189–226.
- Carrol ML, Roy-Engel AM, Nguyen SV, Salem AH, Vogel E, Vincent B, Myers J et al. Large-scale Analysis of the Alu Ya5 and Yb8 Subfamilies and their Contribution to Human Genomic Diversity. J Mol Biol. 2001;311:17–40.View ArticleGoogle Scholar
- Carter AB, Salem AH, Hedges DJ, Nguyen Keegan C, Kimball B, Walker JA, et al. Genome wide analysis of the human Alu Yb8 lineage. Hum Genomics. 2004;1:167–78.PubMedPubMed CentralGoogle Scholar
- Otieno AC, Carter AB, Hedges DJ, Walker JA, Ray DA, Garber RK, Anders BA et al. Analysis of the human Alu Ya-lineage. J Mol Biol. 2004;342:109–18.View ArticlePubMedGoogle Scholar
- Garber RK, Hedges DJ, Herke SW, Hazard NW, Batzer MA. The Alu Yc1 subfamily: sorting the wheat from the chaff. Cytogenet Genome Res. 2005;110:537–42.View ArticlePubMedGoogle Scholar
- Nikaido M, Piskurek O, Okada N. Toothed whale monophyly reassessed by SINE insertion analysis: The absence of lineage sorting effects suggest a small population of a common ancestral species. Mol Phylogenet Evol. 2007;43:216–24.View ArticlePubMedGoogle Scholar
- Nishihara H, Maruyama S, Okada N. Retroposon analysis and recent geological data suggest near-simultaneous divergence of the three superorders of mammals. Proc Natl Acad Sci U S A. 2009;106(13):5235–40.View ArticlePubMedPubMed CentralGoogle Scholar
- Churakov G, Kriegs JO, Baertsch R, Zemann A, Brosius J, Schmitz J. Mosaic retroposon insertion patterns in placental mammals. Genome Res. 2009;19(5):868–75.View ArticlePubMedPubMed CentralGoogle Scholar
- Matzke A, Churakov G, Berkes P, Arms EM, Kelsey D, Brosius J, et al. Retroposon Insertion Patterns of neoavian Birds: Strong Evidence for an Extensive Incomplete Lineage Sorting Era. Mol Biol Evol. 2012;29(6):1497–501.View ArticlePubMedGoogle Scholar
- Watkins WS, Rogers AR, Ostler CT, Wooding S, Bamshad MJ, Brassington AM, et al. Genetic variation among world populations: inferences from 100 Alu insertion polymorphisms. Genome Res. 2003;13(7):1607–18.View ArticlePubMedPubMed CentralGoogle Scholar
- Kajikawa M, Okada N. LINEs Mobilize SINEs in the Eel through a Shared 3′ Sequence. Cell. 2002;111(3):433–44.View ArticlePubMedGoogle Scholar
- Dewannieux M, Esnault C, Heidmann T. LINE-mediated retrotransposition of marked Alu sequences. Nat Genet. 2003;35:41–8.View ArticlePubMedGoogle Scholar
- Simonti CN, Capra JA. The evolution of the human genome. Curr Opin Genet Dev. 2015;35:9–15.View ArticlePubMedGoogle Scholar
- Nekrutenko A, Li WH. Transposable elements are found in a large number of human protein-coding genes. Trends Genet. 2001;17:619–21.View ArticlePubMedGoogle Scholar
- Cordaux R, Batzer MA. The impact of retrotransposons on human genome evolution. Nat Rev Genet. 2009;10(10):691–703.View ArticlePubMedPubMed CentralGoogle Scholar
- Hummel S. Ancient DNA Typing. Methods, Strategies and Applications. Berlin, Germany: Springer; 2003.
- Höss M, Jaruga P, Zastawny TH, Dizdaroglu M, Pääbo S. DNA damage and DNA sequence retrieval from ancient tissues. Nucleic Acid Res. 1996;24(7):1304–7.View ArticlePubMedPubMed CentralGoogle Scholar
- Haack K, Hummel S, Hummel B. Ancient DNA fragments longer than 300 bp. Anthrop Anz. 2000;58:51–6.Google Scholar
- Burger J, Hummel S, Herrmann B, Henke W. DNA preservation: A microsatellite-DNA study on ancient skeletal remains. Electrophoresis. 1999;20:1722–8.View ArticlePubMedGoogle Scholar
- Lindahl T. Instability and decay of the primary structure of DNA. Nature. 1993;32(422):709–11.View ArticleGoogle Scholar
- Schultes T. Typisierung alter DNA zur Rekonstruktion von Verwandtschaft in einem bronzezeitlichen Skelettkollektiv. 2000. Dissertation, University of Göttingen.Google Scholar
- Schilz F. Molekulargenetische Verwandtschaftsanalysen am prähistorischen Skelettkollektiv der Lichtensteinhöhe. 2006. Dissertation, University of Göttingen.Google Scholar
- Schultes T, Hummel S, Herrmann B. Ancient DNA-typing approaches for the determination of kinship in a disturbed collective burial site. Anthrop Anz. 2000;58(1):37–44.Google Scholar
- Schilz F, Hummel S, Herrmann B. Design of a multiplex PCR for genotyping 16 short tandem repeats in degraded DNA samples. Anthrop Anz. 2004;62(4):369–78.Google Scholar
- ensembl.org. http://www.ensembl.org/index.html.
- Nelson DL, Ledbetter SA, Corbo L, Victoria MF, Ramirez-Solis R, Webster TD, Ledbetter DH, Caskey CT. Alu polymerase chain reaction: A method for rapid isolation of human-specific sequences from complex DNA sources. Proc Natl Acad Sci U S A. 1989;86:6686–90.View ArticlePubMedPubMed CentralGoogle Scholar
- Kass DH, Batzer MA. Inter-Alu Polymerase Chain Reaction: Advancements and Applications. Anal Biochem. 1995;228:185–93.View ArticlePubMedGoogle Scholar
- allstr Autosomal Database for short tandem repeats. http://allstr.de/allstr/searchMarker.seam. Accessed 3rd September 2015.
- Hummel S, Schmidt D, Kremeyer B, Herrmann B, Oppermann M. Detection of the CCR5- Δ32 HIV resistance gene in Bronze Age skeletons. Genes Immun. 2005;6:371–4.View ArticlePubMedGoogle Scholar
- Konkel MK, Walker JA, Hotard AB, Ranck MC, Fontenot CC, Storer J et al. Sequence Analysis and Characterization of Active Human Alu subfamilies Based on the 1000 Genomes Pilot Project. Genome Biology and Evolution. 2015; doi:10.1093/gbe/evv167.
- Frischalowski M, Seidenberg V, Großkopf B, Wulf FW, Hummel S. Molekulargenetische Untersuchung des Verwandtschaftsverhältnisses von möglichen Mutter-Kind-Bestattungen aus dem frühzeitlichen Eldagsen. Nachrichten aus Niedersachsens Urgeschichte. 2015;84:(in press).
- Smit AFA, Hubley R, Green P. RepeatMasker Open-4.0. 2013-2015. http://www.repeatmasker.org.
- National Center for Biotechnology Information. http://www.ncbi.nlm.nih.gov/assembly/GCF_000001405.26.
- Seidenberg V, Schilz F, Pfister D, Georges L, Fehren-Schmitz L, Hummel S. A new miniSTR heptaplex system for genetic fingerprinting of ancient DNA from archaeological human bone. J Archaeol Sci. 2012;39(10):3224–9.View ArticleGoogle Scholar
- Hall TA. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/97/NT. Nucl Acids Symp. 1999;41:95–8.Google Scholar
- Brenner CH. Symbolic Kinship Program. Genetics. 1997;145:535–42.PubMedPubMed CentralGoogle Scholar
- Gjertson DW, Brenner CH, Baur MP, Carracedo A, Guidet F, Luque JA, et al. ISFG: Recommendations on biostatistics in paternity testing. Forensic Sci Int Genet. 2007;1:223–31.View ArticlePubMedGoogle Scholar