Ancient DNA work was performed in the specialized ancient DNA (aDNA) facilities of the Department of Genetics, University of Szeged, Hungary with strict clean-room conditions. In order to authenticate the results, we considered the latest recommendations of (Llamas et al. 2017) throughout of the experiments.DNA extraction:Note: This protocol is based on (Rohland and Hofreiter 2007), supplemented with partial predigestion (Damgaard et al. 2015) and using a GuHCl Binding Buffer modified after (Gamba et al. 2016).- 100 mg bone powder from tooth root, petrous bone or other dense bone was predigested in 1 ml 0,5 M EDTA 100 \u00b5g\/ml Proteinase K for 30 minutes at 48 oC, to increase the proportion of endogenous DNA (Damgaard et al. 2015),- Then DNA solubilisation was done overnight at 48 oC in 1 ml extraction buffer containing 0.45 M EDTA, 250 \u00b5g\/ml Proteinase K, 1% Triton X-100, and 50 mM DTT.- DNA was bound to silica (Rohland and Hofreiter 2007) adding 6 ml Binding Buffer (5,83 M GuHCl, 105 mM NaOAc, 46,8% isopropanol, 0,06% Tween-20) and 150 \u00b5l silica suspension to the 1 ml extract, and the pH was adjusted between 4-6 with HCl.- After 3 hours binding at room temperature silica was pelleted, and washed twice with 80% ethanol, then DNA was eluted in 100 \u00b5l TE buffer.NGS library constructionNote: We used the double stranded library protocol of (Meyer and Kircher 2010) with double indexing (Kircher, Sawyer, and Meyer 2012), except that all purifications were done with MinElute columns . We also applied partial UDG treatment of (Rohland et al. 2015), but decreased the recommended USER and UGI concentrations to half (0.03 U\/\u03bcL) and at the same time increased the incubation time from 30 to 40 minutes. This modification removed uracils with comparable efficiency to the original method.DNA free negative control libraries were also made to detect possible contamination during handling or present in materials.Partial UDG treatment: AB1reagent\nvolume (\u03bcL)\nper sample\n2Buffer Tango (10X)\n 6\n3dNTPs (25 mM each)\n 0,24\n4 ATP (100 mM)\n 0,6\n5USER (1 U\/\u03bcL NEB)\n 1,8\n6DNA extract\n 51,36\n7 Total:\n 60 \u03bcL\nThe reaction was incubated at 37oC for 40 minutes in PCR machine, with 40 oC lid temerature.Then 1,8 \u03bcL UGI (Uracil Glycosylase Inhibitor, 2U\/\u03bcL NEB) was added to the reaction, which was further incubated at 37oC for 40 minutes.Blunt-End Repair:- To each reaction we added 3 \u03bcL\u00a0 T4 polynucleotide kinase (10 U\/\u03bcL) and 1,2 \u03bcL T4 DNA polymerase-t (5 U\/\u03bcL), then incubated in PCR machine at 25\u00b0C for 15 minutes, followed by incubation at 12\u00b0C for 5 minutes and cooling to 4\u00b0C.- 350 ul MinElute PB buffer (QIAGEN) was added to the reaction, then it was purified on MinElute columns. DNA was eluted in 20 ul EB prewarmed to 55 oC.Adapter ligation: AB1reagent\nvolume (\u03bcL)\nper sample\n2 T4 DNA ligase buffer (10X)\n 4\n3 PEG-4000 (50%)\n 4\n4 adapter mix (50 \u03bcM each)\n 0,5 \n5 T4 DNA ligase (5 U \/\u03bcL)\n 1\n6 H2O\n 10,5 \n7 DNA\n 20 ul\n8 Total:\n 40ul\n- We incubated the reaction at 22\u00b0C for 30 minutes in PCR machine.- Then 200 \u03bcL PB Buffer was added followed by MinElute purification. DNA was eluted in 20 ul EB.Adapter Fill-In:Note: We assembled the reaction below without library DNA, and the elution step from above was centrifuged directly on the fill-in reaction mix. AB1reagent\nvolume (\u03bcL)\nper sample\n2 H2O\n 14.1\n3 ThermoPol reaction buffer (10X)\n 4\n4 dNTPs (25 mM each)\n 0.4\n5 Bst polymerase, large fragment (8 U\/\u03bcL)\n 1.5\n6 Library DNA (from adapter ligation)\n 20\n7 Total:\n 40ul\n- The reaction was incubated at 37\u00b0C for 20 minutes, then 200 ul PB was added followed by MinElute purification. Library was eluted in 20 ul EB.Library preamplification:Libraries were preamplified in 2 x 50 \u00b5l reactions containing 800 nM each of IS7 and IS8 primers, 200 \u00b5M dNTP mix, 2 mM MgCl2, 0,02 U\/\u00b5l GoTaq G2 Hot Start Polymerase (Promega) and 1X GoTaq buffer, followed by MinElute purification. PCR conditions were\u00a0 96 oC 6 min, 11 cycles of\u00a0 94 oC 30 sec, 58 oC 30 sec, 72 oC 30 sec, followed by a final extension of 64 oC 10 min. Libraries were eluted from the column in 50 \u00b5l 55 oC EB buffer (Qiagen), and concentration was measured with Qubit (Termo Fisher Scientific). Libraries below 5 ng\/\u00b5l concentration were reamplified in the same reaction for additional 5-12 cycles, depending on concentration, in order to obtain 50 \u00b5l preamplified library with a concentration between 10-50 ng\/\u00b5l.\u00a0Double indexing:50 ng preamplified libraries were double indexed according to (Kircher, Sawyer, and Meyer 2012) in a 50 \u00b5l PCR reaction containing 1 x KAPA HiFi HotStart ReadyMix (Kapa Biosystems) and 1000 nM each of P5 and P7 indexing primers. PCR conditions were 98 oC 3 min, 6 cycles of 98 oC 20 sec, 66 oC 10 sec, 72 oC 15 sec followed by a final extension of 72 oC 30sec. Indexed libraries were MinElute purified and their concentration was measured with Qubit, and size distribution was checked on Agilent 2200 TapeStation Genomic DNA ScreenTape.Note: Lately we use 30 ng preamplified libraries and just 5 PCR cycles to avoid overamplification.Mitochondrial DNA capture and sequencing:Biotinilated mtDNA baits were prepared from three overlapping long-range PCR products as described in (Maricic, Whitten, and P\u00e4\u00e4bo 2010), but using the following primer pairs, L14759-H06378, L10870-H14799, L06363-H10888, described in (Haak et al. 2010).Capture was done according to (Maricic, Whitten, and P\u00e4\u00e4bo 2010) with the following modifications: Just four blocking oligos, given below were used in 3 \u00b5M (each) final concentration:\u00a0BO1.P5.part1F: AATGATACGGCGACCACCGAGATCTACAC-Phosphate,BO2.P5.part2F ACACTCTTTCCCTACACGACGCTCTTCCGATCT-Phosphate,BO4.P7.part1 R GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-Phosphate,BO6.P7.part2 R CAAGCAGAAGACGGCATACGAGAT-Phosphate.- For one capture 300 ng biotinilated bait was used with 30 \u00b5l Dynabeads MyOne Streptavidin C1 magnetic beads (Thermo Fisher Scientific).- Double indexed libraries of 20 samples\u00a0 (300 ng each) were mixed and concentrated on MinElute columns, then captured together in a 64 \u00b5l hybridization reaction. When fewer samples were enriched, we used proportionally smaller amounts of baits.- After washing, bead-bound enriched libraries were resuspended in 20 \u00b5l water and released\u00a0 from the beads in a 60 \u00b5l PCR reaction containing 1 X KAPA HiFi HotStart ReadyMix and 2000 nM each of IS5- IS6 library primers. PCR conditions were: 98 oC 1 min, 10 cycles of 98 oC 20 sec, 60 oC 30 sec, 72 oC 30 sec, followed by a final extension of 72 oC 30 sec. The captured and amplified library mix was purified on MinElute column and eluted in 15 \u00b5l EB.- Before sequencing, libraries were quantified with Qubit, and quality checked and Agilent 2200 TapeStation Genomic DNA ScreenTape. Sequencing was done at the SeqOmics Biotechnology Ltd., using MiSeq sequencer with MiSeq Reagent Kit v3 (Illumina, MS-102-3003) generating 2x150bp paired-end sequences.Note: Lately we perform low coverage shotgun sequencing prior to enrichment to estimate endogenous DNA content. Then 5-7 libraries with similar endogenous content are enriched together.Data analysisThe adapters of paired-end reads were trimmed with the cutadapt software (Martin 2011) in paired end mode. Read quality was assessed with FastQC (S. Andrews 2016). Sequences shorter than 25 nucleotide were removed from this dataset. The resulting analysis-ready reads were mapped to the GRCh37.75 human genome reference sequence using the Burrows Wheeler Aligner (BWA) v0.7.9 software (Li and Durbin 2009) with the BWA mem algorithm in paired mode and default parameters. Aligning to the GRCh37.75 human reference genome that also contains the mtDNA revised Cambridge Reference Sequence (rCRS, NC_012920.1) (R. M. Andrews et al. 1999) helped to avoid the forced false alignment of homologous nuclear mitochondrial sequences (NumtS) to rCRS, though the proportion of NumtS, derived from low copy nuclear genome, is expexted to be orders of magnitudes lower than mtDNA in aDNA libraries. Samtools v1.1 (Li et al. 2009) was used for sorting and indexing BAM files. PCR duplicates were removed with Picard Tools v 1.113 (Broad Institute 2016). Ancient DNA damage patterns were assessed using MapDamage 2.0 (J\u00f3nsson et al. 2013), and read quality scores were modified with the rescale option to account for post-mortem damage. Freebayes v1.02 (Garrison and Marth 2012) was used to identify variants and generate variant call format (VCF) files with the parameters -q 10 (exclude nucleotids with Andrews, S. 2016. \u201cFastQC: A Quality Control Tool for High Throughput Sequence Data.\u201d babraham bioinformatics. http:\/\/www.bioinformatics.babraham.ac.uk\/projects\/fastqc.Broad Institute. 2016. \u201cPicard Tools.\u201d https:\/\/broadinstitute.github.io\/picard\/. http:\/\/broadinstitute.github.io\/picard\/.Damgaard, Peter B et al. 2015. \u201cImproving Access to Endogenous DNA in Ancient Bones and Teeth.\u201d Scientific Reports 5: 11184. http:\/\/dx.doi.org\/10.1038\/srep11184%5Cn10.1038\/srep11184%5Cnhttp:\/\/www.nature.com\/articles\/srep11184#supplementary-information.Gamba, Cristina et al. 2016. \u201cComparing the Performance of Three Ancient DNA Extraction Methods for High-Throughput Sequencing.\u201d Molecular Ecology Resources 16(2): 459\u201369.Garrison, Erik, and Gabor Marth. 2012. \u201cHaplotype-Based Variant Detection from Short-Read Sequencing.\u201d arXiv preprint arXiv:1207.3907: 9. http:\/\/arxiv.org\/abs\/1207.3907.Haak, Wolfgang et al. 2010. \u201cAncient DNA from European Early Neolithic Farmers Reveals Their near Eastern Affinities.\u201d PLoS Biology 8(11).J\u00f3nsson, H\u00e1kon et al. 2013. \u201cMapDamage2.0: Fast Approximate Bayesian Estimates of Ancient DNA Damage Parameters.\u201d In Bioinformatics, , 1682\u201384.Kircher, Martin, Susanna Sawyer, and Matthias Meyer. 2012. \u201cDouble Indexing Overcomes Inaccuracies in Multiplex Sequencing on the Illumina Platform.\u201d Nucleic Acids Research 40(1).Li, Heng et al. 2009. \u201cThe Sequence Alignment\/Map Format and SAMtools.\u201d Bioinformatics (Oxford, England) 25(16): 2078\u201379. http:\/\/bioinformatics.oxfordjournals.org\/cgi\/content\/long\/25\/16\/2078 (August 20, 2016).Li, Heng, and Richard Durbin. 2009. \u201cFast and Accurate Short Read Alignment with Burrows-Wheeler Transform.\u201d Bioinformatics (Oxford, England) 25(14): 1754\u201360. http:\/\/bioinformatics.oxfordjournals.org\/cgi\/content\/long\/25\/14\/1754 (August 22, 2016).Llamas, Bastien et al. 2017. \u201cFrom the Field to the Laboratory: Controlling DNA Contamination in Human Ancient DNA Research in the High-Throughput Sequencing Era.\u201d STAR: Science & Technology of Archaeological Research 3(1): 1\u201314. http:\/\/dx.doi.org\/10.1080\/20548923.2016.1258824.Maricic, T, M Whitten, and S P\u00e4\u00e4bo. 2010. \u201cMultiplexed DNA Sequence Capture of Mitochondrial Genomes Using PCR Products.\u201d PLoS ONE 5: e14004. http:\/\/dx.doi.org\/10.1371\/journal.pone.0014004.Martin, Marcel. 2011. \u201cCutadapt Removes Adapter Sequences from High-Throughput Sequencing Reads.\u201d EMBnet.journal 17(1): 10\u201312.McKenna, Aaron et al. 2010. \u201cThe Genome Analysis Toolkit: A MapReduce Framework for Analyzing next-Generation DNA Sequencing Data.\u201d Genome Research 20(9): 1297\u20131303.Meyer, Matthias, and Martin Kircher. 2010. \u201cIllumina Sequencing Library Preparation for Highly Multiplexed Target Capture and Sequencing.\u201d Cold Spring Harbor Protocols 5(6).Rohland, Nadin et al. 2015. \u201cPartial Uracil-DNA-Glycosylase Treatment for Screening of Ancient DNA.\u201d Philosophical transactions of the Royal Society of London. Series B, Biological sciences 370(1660): 20130624. http:\/\/www.ncbi.nlm.nih.gov\/pubmed\/25487342.Rohland, Nadin, and Michael Hofreiter. 2007. \u201cAncient DNA Extraction from Bones and Teeth.\u201d Nature Protocols 2(7): 1756\u201362. http:\/\/www.nature.com\/nprot\/journal\/v2\/n7\/full\/nprot.2007.247.html%5Cnhttp:\/\/www.nature.com\/nprot\/journal\/v2\/n7\/pdf\/nprot.2007.247.pdf.