This PDF file includes:

• Note S1. Ancient DNA recovery and treatment.
• Note S2. Decontamination methods and initial screening.
• Note S3. Data generation and data processing.
• Note S4. Sex determination.
• Note S5. Mitochondrial contamination estimates.
• Note S6. Reconstruction of the mitochondrial genomes.
• Note S7. Phylogenetic analysis of the mitochondrial genomes.
• Note S8. Characterization of present-day human DNA contamination in the nuclear genome.
• Note S9. Genetic relationships and effect of present-day human DNA contamination, sequencing errors, and reference bias.
• Note S10. Split time estimates.
• Note S11. Discordance between the nuclear and mitochondrial divergence of HST to other Neandertals.
• Note S12. Likelihood of a recent mitochondrial replacement in Neandertals.
• Table S1. Overview of DNA extracts and libraries prepared from the HST femur.
• Table S2. Overview of DNA extracts and libraries prepared for Scladina I-4A.
• Table S3. DNA content in the libraries prepared from HST extracts prepared following different decontamination methods (set 1 in table S1).
• Table S4. DNA content in the libraries prepared from the bone powder treated with sodium hypochlorite.
• Table S5. DNA content in the initial libraries prepared from the untreated extracts from Scladina I-4A.
• Table S6. Present-day human DNA contamination estimates after three decontamination methods applied to bone powder from the HST femur.
• Table S7. Present-day human DNA contamination estimates from Scladina I-4A mtDNA based on differences between Neandertals and modern humans.
• Table S8. Sequencing summary statistics for HST with the following filters: length (≥35 bp) and mapping quality (≥25).
• Table S9. Sequencing summary statistics for HST with the following filters: length (≥30 bp) and mapping quality (≥25).
• Table S10. Sequencing summary statistics for Scladina I-4A with the following filters: length (≥35 bp) and mapping quality (≥25).
• Table S11. Sequencing summary statistics for Scladina I-4A with the following filters: length (≥30 bp) and mapping quality (≥25).
• Table S12. Sequencing statistics of the negative controls for HST (see table S1).
• Table S13. Sequencing statistics of the negative controls for Scladina I-4A (see table S2).
• Table S14. Summary of HST mtDNA sequencing.
• Table S15. Summary of Scladina I-4A mtDNA sequencing.
• Table S16. Coverage statistics for all sequences from HST within the alignability track, map35_L100.
• Table S17. Coverage statistics for HST sequences with a C-to-T substitution within the three first or last positions of either ends.
• Table S18. Coverage statistics for all sequences from Scladina I-4A within the alignability track, map35_L100.
• Table S19. Coverage statistics for Scladina I-4A sequences with a C-to-T substitution within the three first or last positions of either ends.
• Table S20. Present-day human DNA contamination estimates from HST mtDNA.
• Table S21. Present-day human DNA contamination estimates from Scladina I-4A mtDNA based on differences between Neandertals and modern humans.
• Table S22. Present-day human DNA contamination estimates from Scladina I-4A mtDNA based on differences between Scladina I-4A and modern humans.
• Table S23. Present-day human DNA contamination estimates on mtDNA in the blank libraries of HST based on differences between HST and modern humans.
• Table S24. Present-day human DNA contamination estimates on mtDNA in the blank libraries of Scladina I-4A based on differences between Neandertals and modern humans.
• Table S25. Best substitution models according to the three model selection measures computed by jModelTest 2.1.10.
• Table S26. Marginal likelihoods of the different tested clock and tree models obtained from a path sampling approach using only the coding region of the mitochondrial sequences.
• Table S27. Marginal likelihoods of the different tested clock and tree models obtained from a path sampling approach using the full mitochondrial genome sequences.
• Table S28. Estimates of molecular age and divergence times.
• Table S29. Present-day human DNA contamination estimates for HST nuclear DNA based on deamination rates on the last positions of the molecules.
• Table S30. Present-day human DNA contamination estimates for Scladina I-4A nuclear DNA based on deamination rates on the last positions of the molecules.
• Table S31. Relationship between sequence length and present-day human DNA contamination estimate based on deamination rates in HST nuclear DNA sequences.
• Table S32. Present-day human DNA contamination estimates based on the sharing of derived alleles with a modern human.
• Table S33. Genome-wide counts of the three possible allelic configurations informative about the underlying topologies relating Vindija 33.19 and the Altai Neandertal to HST and Scladina I-4A before correcting for reference bias or contamination (see tables S40 and S41 for corrected results and fig. S17 for a description of these allelic configurations).
• Table S34. Comparison of alignments to hg19 and panTro4.
• Table S35. Excess of ancestral alleles in Late Neandertals compared to Vindija 33.19 at sites that are derived in the Altai Neandertal genome but ancestral in the genomes of an Mbuti and a Denisovan.
• Table S36. Effect of the modified alignment procedure on the allele sharing with the Altai Neandertal.
• Table S37. Alleles seen in Vindija 87 at positions that are heterozygous in Vindija 33.19.
• Table S38. Sequencing and alignment errors of Vindija 87 sequences at positions where Vindija 33.19 is homozygous different from the Altai Neandertal, comparing the original alignments to hg19 with our modified alignment procedure.
• Table S39. Summary of the alignments to the two references.
• Table S40. Applying different sequence lengths cutoffs does not affect the allele sharing with the Altai Neandertal after realignments.
• Table S41. Genome-wide counts of the three possible allelic configurations informative about the underlying topologies relating Vindija 33.19 and the Altai Neandertal to HST and Scladina I-4A after correcting for reference bias (see table S33 to compare with uncorrected results and table S42 for results corrected for contamination).
• Table S42. Counts of the three possible allelic configurations informative about the underlying topologies relating Vindija 33.19 and the Altai Neandertal to HST and Scladina I-4A after correcting for both reference bias and contamination.
• Table S43. Summary statistics about the physical distance between the positions used to infer the genetic relationship of HST to Vindija 33.19 and the Altai Neandertal.
• Table S44. Summary statistics about the physical distance between the positions used to infer the genetic relationship of Scladina I-4A to Vindija 33.19 and the Altai Neandertal.
• Table S45. Effective number of independent positions.
• Table S46. Comparison between split time estimates from the Vindija population based on a coalescent divergence model and the F(A|B) statistic for five low-coverage Neandertal genomes.
• Table S47. Split time estimates from the Vindija population based on a coalescent divergence model.
• Table S48. Age estimate for individual B (branch shortening) used to convert the F(A|B) values shown in table S47 into time before present.
• Table S49. Summary of the number of sites and blocks used to compute the F(A|B) statistic and CIs.
• Table S50. Split time estimates between HST or Scladina I-4A and different populations (population B) based on the calibration of the F(A|B) statistic.
• Table S51. Predictions of the mitochondrial TMRCA given different split times between the populations of HST and Vindija 33.19.
• Table S52. Predictions of the mitochondrial TMRCA given different split times between the Vindija 33.19 population and a hypothetical isolated Neandertal population.
• Table S53. Predictions of the mitochondrial TMRCA as done for table S51 but using either the upper or the lower estimates of the Neandertal population size.
• Fig. S1. Length distribution of unique DNA fragments aligned to the human reference genome hg19 with a mapping quality of 25 or above (average length = 33 bp for HST and 25 bp for Scladina I-4A) and mapping uniquely (alignability track, map35_L100).
• Fig. S2. Proportion of spurious alignment for different sequence lengths in the three libraries of HST that represent ~80% of the generated sequences for this specimen.
• Fig. S3. Proportion of spurious alignment in the libraries of Scladina I-4A (same as for HST in fig. S2).
• Fig. S4. Bivariate plot of root length against labio-lingual crown diameter (in millimeter) for the permanent mandibular canine.
• Fig. S5. Bivariate plot of root length against labio-lingual crown diameter (in millimeter) for the permanent maxillary central incisor.
• Fig. S6. Bivariate plot of root pulp volume against total root volume (in cubic millimeter) for the permanent maxillary central incisor.
• Fig. S7. Ratio of sequences aligning to the X chromosome and autosomes.
• Fig. S8. Number of sequences mapping to each chromosome normalized by chromosome length.
• Fig. S9. Deamination patterns from the mtDNA.
• Fig. S10. Maximum parsimony tree built with MEGA6 (Molecular Evolutionary Genetics Analysis, program version 6).
• Fig. S11. Phylogenetic relationship of currently available archaic human mitochondrial genomes reconstructed from a Bayesian analysis with BEAST 2 (Bayesian Evolutionary Analysis Sampling Trees, program version 2).
• Fig. S12. C-to-T substitution frequencies at the end of nuclear DNA sequences (dashed lines), including frequencies conditioned on a C-to-T substitution at the other end (solid lines).
• Fig. S13. Proportion of alleles that are derived in the Altai Neandertal but ancestral in the Vindija 33.19 Neandertal and Denisovan genomes stratified by the allele frequency in the Luhya and Yoruba populations (AFR) of the 1000 genomes dataset.
• Fig. S14. Deamination frequencies on sequences from HST that carry a modern human allele absent from the currently available Neandertal genomes.
• Fig. S15. Deamination frequencies on sequences from Scladina I-4A that carry a modern human allele absent from the currently available Neandertal genomes.
• Fig. S16. Lineage assignment before correcting for the reference bias.
• Fig. S17. Expectations for the genetic relationship of HST and Scladina I-4A to Vindija 33.19 and the Altai Neandertal.
• Fig. S18. Lineage assignment after correcting for the reference bias.
• Fig. S19. Comparison of the expected and observed mitochondrial TMRCA of HST with other European Neandertals.
• Fig. S20. Probability that all sampled Neandertal mtDNAs come from an early modern human population as a function of the admixture rate.
• Fig. S21. Probability that all sampled Neandertal mtDNAs come from an early modern human population as a function of the admixture rate.
• References (38–114)

Download PDF