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Cited by 79Forensic and Genetic ResearchPaleopathology and ancient diseasesForensic Anthropology and Bioarchaeology Studies

Successful enrichment and recovery of whole mitochondrial genomes from ancient human dental calculus

Open access
Ozga, Andrew T., Nieves‐Colón, Maria A., Honap, Tanvi P., Sankaranarayanan, Krithivasan, Hofman, Courtney A., Milner, George R., Lewis, Cecil M., Stone, Anne C. +1 more·2016·American Journal of Physical Anthropology
DOI 10.1002/ajpa.22960PMC PMC4866892
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Abstract

Objectives: Archaeological dental calculus is a rich source of host‐associated biomolecules. Importantly, however, dental calculus is more accurately described as a calcified microbial biofilm than a host tissue. As such, concerns regarding destructive analysis of human remains may not apply as strongly to dental calculus, opening the possibility of obtaining human health and ancestry information from dental calculus in cases where destructive analysis of conventional skeletal remains is not permitted. Here we investigate the preservation of human mitochondrial DNA (mtDNA) in archaeological dental calculus and its potential for full mitochondrial genome (mitogenome) reconstruction in maternal lineage ancestry analysis. Materials and Methods: Extracted DNA from six individuals at the 700‐year‐old Norris Farms #36 cemetery in Illinois was enriched for mtDNA using in‐solution capture techniques, followed by Illumina high‐throughput sequencing. Results: Full mitogenomes (7–34×) were successfully reconstructed from dental calculus for all six individuals, including three individuals who had previously tested negative for DNA preservation in bone using conventional PCR techniques. Mitochondrial haplogroup assignments were consistent with previously published findings, and additional comparative analysis of paired dental calculus and dentine from two individuals yielded equivalent haplotype results. All dental calculus samples exhibited damage patterns consistent with ancient DNA, and mitochondrial sequences were estimated to be 92–100% endogenous. DNA polymerase choice was found to impact error rates in downstream sequence analysis, but these effects can be mitigated by greater sequencing depth. Discussion: Dental calculus is a viable alternative source of human DNA that can be used to reconstruct full mitogenomes from archaeological remains. Am J Phys Anthropol 160:220–228, 2016. © 2016 The Authors American Journal of Physical Anthropology Published by Wiley Periodicals, Inc.

AI snapshot

Written from the paper’s full text.

TL;DR

Archaeological dental calculus from six individuals at the 700-year-old Norris Farms #36 cemetery yielded full mitochondrial genomes after in-solution mtDNA capture and Illumina sequencing. The study concludes dental calculus is a viable alternative source of ancient human mtDNA, including for individuals whose bone had previously failed PCR-based mtDNA analysis.

Key findings

  • Full mitogenomes (7–34× coverage) were reconstructed from dental calculus for all six individuals, including three (NF-15, NF-217, NF-262) that had previously tested negative for DNA preservation in bone by conventional PCR.
  • Mitochondrial haplogroup assignments were consistent with previously published findings and with paired dentine from two individuals; all assignments were consistent with Native American maternal ancestry.
  • Dental calculus total DNA yield exceeded dentine by an average of >25-fold; paired calculus/dentine comparisons showed 84-fold (NF-47) and 15-fold (NF-217) higher yields in calculus.
  • DNA polymerase choice affected error rates: in dental calculus, Phusion HS II libraries had a nearly 2-fold lower substitution error rate (1.0%) than Platinum Taq HiFi (1.7%) or KAPA HiFi Uracil+ (1.8%); higher sequencing depth mitigated these effects.

Methods

  • DNA was extracted from six archaeological dental calculus samples from Norris Farms #36 (~700 BP, Illinois), two paired dentine samples, and two modern dental calculus samples; archaeological and dentine libraries were enriched for mtDNA using in-solution capture and sequenced on Illumina MiSeq and HiSeq platforms.
  • Shotgun libraries were constructed using three DNA polymerases—Platinum Taq High Fidelity, Phusion Hot Start II, and KAPA HiFi Uracil+—and pre-capture shotgun sequencing was performed for four archaeological and two modern libraries to assess starting mtDNA content.
  • Reads were processed with SeqPrep, mapped to the revised Cambridge Reference Sequence using BWA, filtered with Picard, assessed for damage with MapDamage 2.0, estimated for contamination with contamMix, and assigned haplogroups with HaploGrep 2.0.

Limitations

  • Stated: greater sequencing depth and a larger sample size are necessary to determine whether the lower cytosine deamination observed in dental calculus compared to dentine is a broader trend; improvements in capture enrichment efficiency are also needed.
  • Evident from the text: the archaeological sample is small (six individuals, only two paired dentine samples, two modern calculus samples), and four of 18 dental calculus libraries were excluded due to chimera formation.
  • Evident from the text: mtDNA proportions in dental calculus were low and variable, and the two modern calculus samples differed by nearly two orders of magnitude in total human DNA (9.1% vs. 0.1%), limiting comparative inference.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.1002/ajpa.22960

Cites

51 of 51 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

22 of 54 cited sources checked · 8 supported · 1 flagged · accuracy not yet measured

  • Premisecitation

    Three of the six individuals from whom mitogenomes were reconstructed had previously tested negative for DNA preservation in bone using conventional PCR techniques.

    “including three individuals who had previously tested negative for DNA preservation in bone using conventional PCR techniques”
    • Also stated in: § Study population · plain
      “Of these, ribs from three individuals (NF‐15, NF‐217, and NF‐262) were previously unsuccessful for mtDNA haplogroup analysis and HVRI sequencing using targeted PCR”
    Quote in the paperSubstantial claim1 of 2 sources checked
    • cites Stone et al., 1996 source
    • cites Stone and Stoneking, 1998 source

      The Introduction describes an earlier PCR-based screening of 100 adults and says some were reexamined, but it does not identify three individuals who previously tested negative. The Results report mtDNA findings for a much larger sample, not six reconstructed mitogenomes. Searches for prior negative PCR tests and the three-of-six figure found no passage establishing the claim. (LLM, conf=0.74)

      Experimental — accuracy not yet measured
  • Findingcitation

    Whole mitogenome reconstruction from dental calculus was successful for the three individuals (NF-15, NF-217, and NF-262) that had previously failed to yield amplifiable mtDNA from bone.

    “Importantly, whole mitogenome reconstruction from dental calculus was successful for the three individuals (NF‐15, NF‐217, and NF‐262) that had previously failed to yield amplifiable mtDNA from bone (Stone and Stoneking, 1998).”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites Stone and Stoneking, 1998 source

      The Results report that 44 of 152 Norris Farms samples did not contain sufficient DNA for analysis, and Subjects and Methods says DNA was extracted primarily from bone. The paper does not identify NF-15, NF-217, or NF-262 as failed samples, so it cannot verify the failure attributed to those three individuals. The cited paper does not report the dental-calculus reconstruction, which appears to be the citing authors’ own finding. (LLM, conf=0.84)

      Experimental — accuracy not yet measured
  • Premisecitation

    Platinum Taq HiFi has been shown to efficiently amplify over >90% of uracil sites.

    “Platinum Taq HiFi has also been shown to efficiently amplify over >90% of uracil sites (Heyn et al., 2010).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites Heyn et al., 2010 source
  • Study design and conductown data

    The authenticity of the human reads obtained from archaeological dental calculus and dentine was assessed by examining damage patterns typical of ancient DNA.

    “The authenticity of the human reads obtained from archaeological dental calculus and dentine was assessed by examining damage patterns typical of ancient DNA (Briggs et al., 2007; Ginolhac et al., 2011; Jonsson et al., 2013).”
    Quote in the paperSubstantial claim3 of 3 sources checked
    • cites Briggs et al., 2007 source

      The Abstract and Results and Discussion describe characteristic ancient-DNA damage patterns, supporting their use as patterns to examine in the citing study. But Results and Discussion cautions that contaminating modern human DNA can show typical misincorporations, so damage patterns alone cannot reliably establish the authenticity of human reads. (LLM, conf=0.86)

      Experimental — accuracy not yet measured
    • cites Ginolhac et al., 2011 source
    • cites Jonsson et al., 2013 source
  • Premisecitation

    Dental calculus is primarily a calcified microbial biofilm.

    “The biological content of dental calculus is primarily microbial in origin (Warinner et al., 2014b)”
    • Also stated in: § Discussion · plain
      “Dental calculus is a microbial biofilm that acquires human DNA and proteins passively, primarily through host secretions and immunity‐associated processes, such as NETosis (Ryder, 2010; Brinkmann and Zychlinsky, 2012).”
    Quote in the paperSubstantial claim1 of 3 sources checked
    • cites Warinner et al., 2014b

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

    • cites Ryder, 2010 source
    • cites Brinkmann and Zychlinsky, 2012 source

      The Periodontitis section discusses bacteria in the gingival crevice and neutrophil extracellular traps, but not dental calculus. Searches for dental calculus, calcified biofilm, and microbial composition found no passage in our copy addressing the claim. The printed sentence cites Warinner et al. (2014b), not this source. (LLM, conf=0.75)

      Experimental — accuracy not yet measured
  • May combine two claims. 32
  • Classified as probably not a substantial claim. 24

  • May combine two claims.

    Premisecitation

    Other sources of ancient human DNA (coprolites, quids, aprons) are rare, preserved only under exceptional circumstances, and many have culturally, geographically, or temporally limited distributions.

    “However, in most cases such sources are rare, being preserved only under exceptional circumstances, and many have culturally, geographically, or temporally limited distributions.”
    Quote in the paperSubstantial claim1 of 3 sources checked
    • cites Poinar et al., 2001 source

      The Conclusions (page unavailable) describe paleofeces from dry caves and rock shelters as a relatively abundant source of ancient DNA, contrary to the claim that coprolites are rare. The paper does not establish the claimed cultural, geographic, or temporal limits for quids and aprons. (LLM, conf=0.86)

      Checked by a reasoning model — may be wrong.

      Experimental — accuracy not yet measured
    • cites Gilbert et al., 2008b

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

    • cites LeBlanc et al., 2007 source

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Nondestructive extraction methods can be problematic if DNA preservation is poor or if surface contamination is high.

    “However, these methods can be problematic if DNA preservation is poor or if surface contamination is high.”
    Quote in the paperNot a substantial claim1 of 3 sources checked
    • cites Rohland et al., 2004 source

      The Results and discussion report comparatively low DNA yields and contaminating sequences, including storage-related sequences that fell from 18% of clones in the first extraction to none in later extractions. The section also says specimen preparation treatments may affect DNA quality. It does not establish that poor DNA preservation or high surface contamination specifically makes nondestructive extraction problematic. (LLM, conf=0.82)

      Experimental — accuracy not yet measured
    • cites Bolnick et al., 2012

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

    • cites Hofreiter, 2012

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • Classified as probably not a substantial claim. May combine two claims.

    Premisecitation

    Authentic ancient DNA exhibits characteristic damage including short DNA fragment lengths, a high rate of cytosine deamination at the termini of DNA fragments, and an enrichment of purine bases at DNA breakage sites.

    “Briefly, authentic ancient DNA is known to exhibit characteristic damage that includes short DNA fragment lengths, a high rate of cytosine deamination at the termini of DNA fragments, and an enrichment of purine bases at DNA breakage sites.”
    Quote in the paperSubstantial claim2 of 3 sources checked
    • cites Briggs et al., 2007 source

      Not checked in the free run.

    • cites Ginolhac et al., 2011 source
    • cites Jonsson et al., 2013 source

      The Introduction reports elevated cytosine deamination at 5′ overhangs and damage-related substitutions near read ends, supporting the terminal-deamination part of the claim. It also mentions strand breaks, but our copy does not state that ancient DNA fragments are characteristically short or that purines are enriched at breakage sites. (LLM, conf=0.87)

      Experimental — accuracy not yet measured

  • Classified as probably not a substantial claim. May combine two claims.

    Study design and conductcitation

    Ancient DNA damage patterns were assessed on unmerged reads using MapDamage 2.0, and read quality scores were modified with the rescale option to account for post-mortem damage.

    “Ancient DNA damage patterns were assessed on unmerged reads using MapDamage 2.0 (Jonsson et al., 2013), and read quality scores were modified with the rescale option to account for post‐mortem damage.”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites Jonsson et al., 2013 source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductcitation

    Reads originating from four of the 18 dental calculus libraries were impacted by chimera formation during pooled post-capture amplification and were excluded from further analysis.

    “Reads originating from four of the 18 dental calculus libraries were impacted by chimera formation during pooled post‐capture amplification (Kircher et al., 2012; Hawkins et al., 2015) and were excluded from further analysis (NF‐15c KAPA HiFi Uracil+, NF‐108c Phusion HS II, and NF‐47c Phusion HS II and KAPA HiFi Uracil+).”
    Quote in the paperNot a substantial claim1 of 2 sources checked
    • cites Kircher et al., 2012 source

      Not checked in the free run.

    • cites Hawkins et al., 2015 source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    The parent material dental plaque is estimated to contain >200,000,000 microbial cells per milligram.

    “its parent material, dental plaque, is estimated to contain >200,000,000 microbial cells per milligram (Socransky and Haffajee, 2005)”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites Socransky and Haffajee, 2005 source

      The section “The first component: microbial composition of subgingival biofilms” gives an average of 2.1 × 10⁸ organisms per milligram, which exceeds 200,000,000. It specifies subgingival plaque and milligrams of wet weight; the citing sentence presents the estimate for dental plaque without those qualifiers. (LLM, conf=0.82)

      Experimental — accuracy not yet measured

  • Classified as probably not a substantial claim. May combine two claims.

    Study design and conductcitation

    MtDNA haplogroup assignment was performed on positions with a read depth > 1 (coverage filtered) using HaploGrep 2.0, followed by manual confirmation of haplogroup-defining mutations.

    “MtDNA haplogroup assignment was performed on positions with a read depth > 1 (coverage filtered) using HaploGrep 2.0 (Kloss‐Brandstatter et al., 2011), followed by manual confirmation of haplogroup‐defining mutations provided in Phylotree mtDNA tree Build 16 (van Oven and Kayser, 2009).”
    Quote in the paperSubstantial claim1 of 2 sources checked
    • cites Kloss‐Brandstatter et al., 2011

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

    • cites van Oven and Kayser, 2009 source

  • May combine two claims.

    Findingown data

    Haplogroup assignments from dental calculus matched those for paired dentine samples in this study and matched bone samples previously analyzed by targeted PCR of the HVRI region.

    “haplogroup assignments matched those for paired dentine samples in this study and matched bone samples previously analyzed by targeted PCR of the HVRI region (Stone and Stoneking, 1998) (Table 4).”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites Stone and Stoneking, 1998 source

  • May combine two claims.

    Premisecitation

    The Norris Farms #36 cemetery was selected for analysis because bone samples from this site had been previously investigated using ancient DNA techniques, and mtDNA haplogroup assignments and HVRI sequences are available for 108 and 52 individuals, respectively, making it the most intensively studied pre-Contact North American site to date.

    “The Norris Farms #36 cemetery was selected for analysis because bone samples from this site had been previously investigated using ancient DNA techniques (Stone and Stoneking, 1993, 1998, 1999), and mtDNA haplogroup assignments and HVRI sequences are available for 108 and 52 individuals, respectively, making it the most intensively studied pre‐Contact North American site to date.”
    Quote in the paperSubstantial claim2 of 3 sources checked
    • cites Stone and Stoneking, 1993 source
    • cites Stone and Stoneking, 1998 source

      Split into 2 atoms — 1 confirmed, 1 partially supported, 0 disputed, 0 misattributed, 0 unverifiable. Expand the claim to see per-atom verdicts.

      Experimental — accuracy not yet measured
    • cites Stone and Stoneking, 1999 source

      Split into 2 atoms — 0 confirmed, 2 partially supported, 0 disputed, 0 misattributed, 0 unverifiable. Expand the claim to see per-atom verdicts.

      Experimental — accuracy not yet measured

  • Classified as probably not a substantial claim. May combine two claims.

    Premisecitation

    Previous studies have reported lower error rates and higher extension termination rates for Pfu-based polymerases (such as Phusion) compared to Taq-based and other uracil-tolerant polymerases.

    “Previous studies have reported lower error rates and higher extension termination rates for Pfu‐based polymerases (such as Phusion) compared to Taq‐based and other uracil‐tolerant polymerases (Heyn et al., 2010; Ginolhac et al., 2011; Wales et al., 2014).”
    Quote in the paperSubstantial claim3 of 3 sources checked
    • cites Heyn et al., 2010 source

      In Results, ‘PEP on artificial templates’ (page not provided), Pfu showed much higher extension termination on uracil-containing templates than the Taq-based polymerases tested. The paper calls Pfu a proofreading enzyme, but does not report comparative error rates or test Phusion; its termination finding is also specific to the lesions and templates studied. (LLM, conf=0.88)

      Experimental — accuracy not yet measured
    • cites Ginolhac et al., 2011 source

      In the 'mapDamage options' section (page not supplied), the paper reports fewer cytosine-to-thymine misincorporations in a Phusion-amplified ancient-DNA library than in one amplified with high-fidelity Taq. That supports a narrower claim about damage-related misincorporations, not general polymerase error rates. Our copy does not report extension-termination rates or comparisons with other uracil-tolerant polymerases. (LLM, conf=0.87)

      Experimental — accuracy not yet measured
    • cites Wales et al., 2014 source

      The Results (DNA Polymerases, Fidelity) report significantly lower substitution error rates for Phusion than for the other tested polymerases. The Results (Compatibility with damaged DNA) and Discussion say Phusion stalls at uracil in damaged templates while the others read through it, but the paper does not report comparative extension-termination rates. It also identifies PfuTurbo Cx as a uracil-tolerant comparator, so the finding should not be generalized to all Pfu-based polymerases. (LLM, conf=0.85)

      Experimental — accuracy not yet measured

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Of the six individuals, ribs from three (NF-15, NF-217, and NF-262) were previously unsuccessful for mtDNA haplogroup analysis and HVRI sequencing using targeted PCR, while three other rib samples (NF-47, NF-95, and NF-108) successfully produced results for mtDNA haplogroup markers, HVRI sequences, and amelogenin gene sex identification markers.

    “Of these, ribs from three individuals (NF‐15, NF‐217, and NF‐262) were previously unsuccessful for mtDNA haplogroup analysis and HVRI sequencing using targeted PCR, while three other rib samples (NF‐47, NF‐95, and NF‐108) successfully produced results for mtDNA haplogroup markers, HVRI sequences, and amelogenin gene sex identification markers (Stone et al., 1996; Stone and Stoneking, 1998).”
    Quote in the paperNot a substantial claim1 of 2 sources checked
    • cites Stone et al., 1996 source
    • cites Stone and Stoneking, 1998 source

      Split into 2 atoms — 0 confirmed, 2 partially supported, 0 disputed, 0 misattributed, 0 unverifiable. Expand the claim to see per-atom verdicts.

      Experimental — accuracy not yet measured

  • May combine two claims.

    Findingown data

    For paired samples NF-47 and NF-217, calculus DNA yields exceeded dentine by 84-fold and 15-fold, respectively.

    “Comparison of calculus and dentine pairs for NF‐47 and NF‐217 likewise produced similar results, with calculus DNA yields exceeding that of dentine by 84‐fold and 15‐fold, respectively (Table 1).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Prior to enrichment, the proportion of human mtDNA in dentine was higher (NF-47, 0.0385%; NF-217, 0.0003%), but still low.

    “The proportion of human mtDNA in dentine prior to enrichment was higher (NF‐47, 0.0385%; NF‐217, 0.0003%), but still low (Table 3).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    On average, the relative proportion of human mtDNA in dereplicated post-capture DNA libraries was 0.41% for dental calculus and 1.37% for dentine.

    “On average, the relative proportion of human mtDNA in dereplicated post‐capture DNA libraries for dental calculus and dentine was 0.41 and 1.37%, respectively (Table 3).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Only 0.2% of sites (n = 29) were found to be variable across the NF-217 mitogenome, and all but one were transitions.

    “Only 0.2% of sites (n = 29) were found to be variable across the NF‐217 mitogenome, and all but one were transitions.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Each of the damage patterns (short fragment lengths, cytosine deamination, and purine enrichment) was evident in both the pre- and post-capture data sets.

    “Each of these patterns was evident in both our pre‐ and post‐capture data sets.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In paired samples, post-capture libraries contained 14-15% more damage in dental calculus samples and 59-72% more damage in dentine samples.

    “In paired samples, post‐capture libraries contained 14–15% more damage in dental calculus samples and 59–72% more damage in dentine samples.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Post-capture median fragment lengths in dentine samples were 27-102 bp longer than prior to capture, a substantial increase.

    “post‐capture median fragment lengths in dentine samples were 27–102 bp longer than prior to capture, a substantial increase.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Although rich in total DNA, the proportion of human DNA in dental calculus is low and variable.

    “Although rich in total DNA, the proportion of human DNA in dental calculus is low and variable.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    KAPA HiFi Uracil+, which was sequenced at much higher depth, had fewer variable sites in the consensus sequences, despite exhibiting the highest error rate of the three enzymes.

    “By contrast, KAPA HiFi Uracil+, which was sequenced at much higher depth, had fewer variable sites in the consensus sequences, despite exhibiting the highest error rate of the three enzymes.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Post-capture median DNA fragment lengths for the same samples were longer, and differences were observed between dental calculus and dentine.

    “Post‐capture median DNA fragment lengths for the same samples were longer, and differences were observed between dental calculus and dentine (Table 3).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data

    Dental calculus is a viable alternative source of human DNA that can be used to reconstruct full mitogenomes from archaeological remains.

    “Dental calculus is a viable alternative source of human DNA that can be used to reconstruct full mitogenomes from archaeological remains.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationreasoning

    The ratio of mtDNA to total human DNA is not constant in dental calculus and differs by more than an order of magnitude.

    “Consequently, the ratio of mtDNA to total human is not constant in dental calculus and differs by more than an order magnitude.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    Taken together, the results are consistent with ancient DNA and support the authenticity of the human reads in the data sets.

    “Taken together, these results are consistent with ancient DNA and support the authenticity of the human reads in our data sets.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim. May combine two claims.

    Study design and conductnone given

    Prior to extraction, dental calculus samples were UV-irradiated for 1 min on each side, and dentine samples were cleaned by mechanical abrasion followed by UV-irradiation.

    “Prior to extraction, dental calculus samples (16–25 mg) were UV‐irradiated for 1 min on each side, and dentine samples (87–98 mg) were cleaned by mechanical abrasion of the tooth root surface using a Dremel rotary tool, followed by UV‐irradiation for 1 min on each side.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Interpretationreasoning

    The reasons for the variable ratio of mtDNA to total human DNA in dental calculus are unclear, but may be related to differences in the cell types contributing to the human DNA component of dental calculus.

    “The reasons for this are unclear, but may be related to differences in the cell types contributing to the human DNA component of dental calculus.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisecitation

    Dental calculus contains human DNA and proteins.

    “In addition to microbes (bacteria and archaea), dental calculus also contains trace amounts of dietary (plant and animal) biomolecules and microfossils, viral DNA (primarily bacteriophages, or bacteria‐infecting viruses), and human DNA and proteins (Warinner et al., 2014a, 2014b).”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites Warinner et al., 2014a

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

    • cites Warinner et al., 2014b

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • Classified as probably not a substantial claim.

    Premisecitation

    In-solution capture enrichment is known to bias towards the recovery of longer DNA fragments.

    “In‐solution capture enrichment is known to bias towards the recovery of longer DNA fragments (Dabney et al., 2013a)”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Dabney et al., 2013a

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • Classified as probably not a substantial claim. May combine two claims.

    Premisecitation

    Dental calculus is an abundant, nearly ubiquitous, and long-term reservoir of ancient host-associated biomolecules, including human DNA.

    “Recently, dental calculus has been identified as an abundant, nearly ubiquitous, and long‐term reservoir of ancient host‐associated biomolecules, including human DNA (Warinner et al., 2014b, 2015a).”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites Warinner et al., 2014b

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

    • cites Warinner et al., 2015a

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • May combine two claims.

    Premisecitation

    Human DNA was initially detected within archaeological dental calculus by targeted PCR amplification of mtDNA, followed by haplogroup inference using restriction enzyme digestion or conventional cloning and Sanger sequencing of HVRI.

    “Human DNA was initially detected within archaeological dental calculus by targeted PCR amplification of mitochondrial DNA (mtDNA), followed by haplogroup inference using restriction enzyme digestion (Black et al., 2011) or conventional cloning and Sanger sequencing of the hypervariable region I (HVRI) (De La Fuente et al., 2013).”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites Black et al., 2011

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

    • cites De La Fuente et al., 2013 source

  • May combine two claims.

    Premisecitation

    Dental calculus is the richest known source of aDNA in the archaeological record, with reported DNA yields routinely exceeding 100 ng mg−1 in well-preserved samples.

    “Given that dental calculus is the richest known source of aDNA in the archaeological record, with reported DNA yields routinely exceeding 100 ng mg−1 in well‐preserved samples (Warinner et al., 2015a)”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Warinner et al., 2015a

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • May combine two claims.

    Interpretationown data

    At present, dentine presents a preferred tissue for mitogenome reconstruction, but in cases where it is not possible to analyze dentine, dental calculus is a suitable substitute that requires only slightly more sequencing effort to achieve equivalent results.

    “Thus, at present, dentine presents a preferred tissue for mitogenome reconstruction, but in cases where it is not possible to analyze dentine, dental calculus is a suitable substitute that requires only slightly more sequencing effort to achieve equivalent results.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based). May combine two claims.

    Study design and conductnone given

    The Norris Farms #36 skeletal remains are unaffiliated with existing Native American tribes, and permission for destructive analysis was granted by the Illinois State Museum.

    “The Norris Farms #36 skeletal remains are unaffiliated with existing Native American tribes, and permission for destructive analysis was granted by the Illinois State Museum.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Premisecitation

    Destructive analysis of skeletal remains is a highly sensitive topic for many repatriation committees and institutions holding skeletal remains.

    “Destructive analysis of skeletal remains is a highly sensitive topic for many repatriation committees and institutions holding skeletal remains (Dongoske, 1996; Killion and Molloy, 1999; Kosslak, 1999; O'Rourke et al., 2005; Cast et al., 2010)”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites Dongoske, 1996 source
    • cites Killion and Molloy, 1999

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

    • cites Kosslak, 1999 source

  • Classified as probably not a substantial claim.

    Study design and conductnone given

    A non-template extraction control (negative control) was processed alongside the experimental samples during all analytical steps to monitor for possible contamination.

    “A non‐template extraction control (negative control) was processed alongside the experimental samples during all analytical steps to monitor for possible contamination.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Human DNA in dental calculus originates primarily from GCF, saliva, and blood.

    “suggesting that GCF, saliva, and blood, respectively, are the primary sources of human biomolecules in dental calculus (Warinner et al., 2014b)”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Warinner et al., 2014b

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • Classified as probably not a substantial claim.

    Study design and conductcitation

    To estimate the proportion of contaminant mitochondrial sequences, the authors implemented the Bayesian-based tool ContamMix.

    “To estimate the proportion of contaminant mitochondrial sequences in our data set, we implemented the Bayesian‐based tool ContamMix (Fu et al., 2013).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Fu et al., 2013

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • May combine two claims.

    Premisecitation

    Shotgun metagenomics confirmed the presence of both mitochondrial and nuclear DNA within archaeological dental calculus and provided an estimate of its overall relative abundance: approximately 0.5% of all identifiable DNA sequences in one deeply sequenced sample.

    “The subsequent application of shotgun metagenomics using next‐generation sequencing (NGS) technology further confirmed the presence of both mitochondrial and nuclear DNA within archaeological dental calculus, and moreover provided an estimate of its overall relative abundance: approximately 0.5% of all identifiable DNA sequences in one deeply sequenced sample (Warinner et al., 2014b).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Warinner et al., 2014b

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • May combine two claims.

    Interpretationreasoning

    The finding that the proportion of mitochondrial DNA is lower in dental calculus compared to dentine is consistent with the fact that dental calculus is primarily a calcified microbial biofilm.

    “This is consistent with the fact that dental calculus is primarily a calcified microbial biofilm.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Study design and conductnone given

    DNA was extracted from archaeological dental calculus in a dedicated ancient DNA facility at the University of Oklahoma LMAMR in accordance with established contamination control precautions and workflows.

    “DNA was extracted from archaeological dental calculus in a dedicated ancient DNA facility at the University of Oklahoma Laboratories of Molecular Anthropology and Microbiome Research (LMAMR) in accordance with established contamination control precautions and workflows.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Study design and conductcitation

    Contamination levels were estimated using contamMix.

    “Contamination levels were estimated using contamMix (Fu et al., 2013).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Fu et al., 2013

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Interpretationcitation

    Host immune response and the NET activity of neutrophils at the site of plaque formation may influence the ratio of mitochondrial to nuclear DNA present within individual dental calculus samples.

    “Host immune response and the NET activity of neutrophils at the site of plaque formation may thus influence the ratio of mitochondrial to nuclear DNA present within individual dental calculus samples.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Study design and conductnone given

    Nuclear mitochondrial sequences (NumtS) were identified as any reads exhibiting superior alignment to the human nuclear genome, and removed.

    “Nuclear mitochondrial sequences (NumtS) were identified as any reads exhibiting superior alignment to the human nuclear genome, and removed.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Interpretationown data

    Hydrolytic damage may be lower in dental calculus than in dentine.

    “This pattern suggests that hydrolytic damage may be lower in dental calculus than in dentine”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    Modern dental calculus was extracted in a modern DNA laboratory using the same protocols except that the decontamination steps were not performed.

    “Modern dental calculus was extracted in a modern DNA laboratory using the same protocols except that the decontamination steps were not performed.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Neutrophils contain 10-15 times fewer mtDNA genome copies compared to peripheral blood mononuclear cells (PBMC).

    “neutrophils, an abundant blood leukocyte involved in host innate immune response to dental plaque and calculus formation (Warinner et al., 2014b), contain 10–15 times fewer mtDNA genome copies compared to peripheral blood mononuclear cells (PBMC) (Maianski et al., 2004)”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites Maianski et al., 2004

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

    • cites Warinner et al., 2014b

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

46 of 46

  • The paper relies on this without support.

    During life, dentine contains exclusively human DNA.

    “which during life should exclusively contain human DNA”

  • The paper relies on this without support.

    Dental calculus is not an integral or vital part of the human body.

    “it is not an integral or vital part of the human body”

  • The paper relies on this without support.

    The dentine samples started with a higher endogenous content in the pre-capture libraries.

    “the dentine samples started with a higher endogenous content in the pre‐capture libraries.”

  • The paper relies on this without support.

    Pre-capture dentine samples started with a higher endogenous content that included a subset of longer, but more heavily damaged, DNA fragments.

    “the pre‐‐capture dentine samples started with a higher endogenous content that included a subset of longer, but more heavily damaged, DNA fragments.”

  • The paper relies on this without support.

    Platinum Taq HiFi is a mixture of predominantly Platinum Taq, a family-A polymerase largely tolerant of uracils, and a smaller quantity of Pyrococcus GB-D polymerase, a family-B polymerase intolerant of uracils.

    “Platinum Taq HiFi is a mixture of predominantly Platinum Taq, a family‐A polymerase that is largely tolerant of uracils, and a smaller quantity of Pyrococcus GB‐D polymerase, a family‐B polymerase that is intolerant of uracils.”

  • The paper relies on this without support.

    Dental calculus is less porous and thus less susceptible to degradation by environmental microbes than dentine or bone.

    “because dental calculus is less porous and thus less susceptible to degradation by environmental microbes than dentine or bone”

  • The paper relies on this without support.

    Dental calculus is abundant, nearly ubiquitous, preserves over long-time periods, and contains both host genomic and acquired health/disease-related information.

    “It is unique among archaeological substrates in that it is abundant, nearly ubiquitous, preserves over long‐time periods, and contains both host genomic and acquired health/disease‐related information.”

  • The paper relies on this without support.

    Dental calculus has substantially higher total DNA content than dentine.

    “its substantially higher total DNA content”
8 of 8

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

  • NF‐47c DNA yield does not match sample size and normalized yield

    Minor

    Multiplying the reported 16.0 mg sample size by the reported 37.5 ng mg−1 yield gives 600.0 ng, not the listed total of 571.2 ng.

    Reported 571.2 · recomputed 600

  • Ancient calculus samples' pre-capture mtDNA proportions are reversed

    Minor

    The text assigns <0.0001% to NF‐47 and 0.0002% to NF‐217, whereas Table 2 reports 0.0002% for NF‐47c and <0.0001% for NF‐217c.

  • Modern samples' human DNA proportions are reversed

    Minor

    The text assigns approximately 9.1% human DNA to MOD‐1 and 0.1% to MOD‐2, while Table 2 assigns those proportions to MOD‐2 and MOD‐1, respectively.

3 of 3

Cites

Flagged first, then by citations

2 of the 53 references couldn’t be matched to a known paper.

Research works printed but not linked: 6

  1. Black J, 2011, Dental calculus as an alternative source of mitochondrial DNA for analysis of skeletal remains, Proc Soc Calif Archaeol, 25, 1
  2. KatzenbergMA.2001. Destructive analyses of human remains in the age of NAGPRA and related legislation. In: Sawchuk L Pfeiffer S editors. Out of the past: the history of human osteology at the University of Toronto. Scarborough: CITD Press. Available at:https://tspace.library.utoronto.ca/citd/Osteology/index.html
  3. Killion TW, 1999, Repatriation's silver lining, SAA Bull, 17, 111
  4. 10.5250/amerindiquar.34.2.131 · DOI
  5. McManamon FP., 2000, Archaeological method and theory: an encyclopedia
  6. Santure SK, 1990, Archaeological investigations at the Morton Village and Norris Farms 36 cemetery

Cited by

Sorted by citations

Related

Authors
Ozga, A. T., Nieves‐Colón, M. A., Honap, T. P., Sankaranarayanan, K., Hofman, C. A., Milner, G. R., Lewis, C. M., Stone, A. C., & Warinner, C.
Affiliations
  • University of Oklahoma (US)
  • School of Human Evolution and Social Change Arizona State University Tempe AZ 85287
  • Arizona State University (US)
  • Pennsylvania State University (US)
  • University of Zurich (CH)
Corresponding author
Warinner, Christina
ORCID
Venue
American Journal of Physical Anthropology
Publisher
Wiley
Published
2016-03-16
Volume
160
Issue
2
Pages
220-228
ISSN
  • 0002-9483 (ISSN-L)
  • 1096-8644
DOI
10.1002/ajpa.22960
PMC
PMC4866892
OpenAlex
W2301949952
Semantic Scholar
6711ad8409b27fbc8f4d53d404e6df8a81286d5e
Cited by
79
References
53
Citations by year
2026: 6 · 2025: 4 · 2024: 4 · 2023: 9 · 2022: 6 · 2021: 11 · 2020: 13 · 2019: 7 · 2018: 10 · 2017: 5 · 2016: 4
FWCI
6.35
Type
journal article
Language
en
Open access
Licence
Locations
  • American Journal of Physical Anthropology (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • Zurich Open Repository and Archive (University of Zurich) (acceptedVersion, cc-by)
  • Europe PMC (PubMed Central) (submittedVersion, cc-by)
  • PubMed Central (submittedVersion, cc-by)
  • NSUWorks (Nova Southeastern University) (submittedVersion, cc-by)
  • MPG.PuRe (Max Planck Society) (submittedVersion)
  • Zurich Open Repository and Archive (University of Zurich) (submittedVersion, cc-by)
  • Zurich Open Repository and Archive (University of Zurich)
Funders
  • National Science Foundation (BCS‐1063939; BCS‐1515163, BCS‐1516633)
  • National Institutes of Health (R01 GM‐089866)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Forensic and Genetic Research
  • Social Sciences › Arts and Humanities › Archeology › Paleopathology and ancient diseases
  • Social Sciences › Arts and Humanities › Archeology › Forensic Anthropology and Bioarchaeology Studies
Keywords
dental calculus, ancient DNA, mitochondrial genome, mtDNA haplogroups, dentin, ancient human remains
MeSH terms
Anthropology, Physical, Archaeology, Dental Calculus, DNA, Mitochondrial, Humans, Sequence Analysis, DNA, History, 15th Century, Genome, Mitochondrial

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-10-26.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-26.

Cited sources are checked by gpt-6-sol.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

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