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Updated: Nov 9, 2025

Optimized Bone Sampling Protocols for the Retrieval of Ancient DNA from Archaeological Remains
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Systematic benchmark of ancient DNA read mapping.

Adrien Oliva1, Raymond Tobler1, Alan Cooper2

  • 1Australian Centre for Ancient DNA, School of Biological Sciences, The University of Adelaide, South Australia, 5005, Australia.

Briefings in Bioinformatics
|April 9, 2021
PubMed
Summary

Benchmarking DNA mapping strategies for ancient DNA (aDNA) studies reveals that specific parameterizations in NovoAlign, BWA-aln, and BWA-mem reduce reference bias. Using an IUPAC reference genome further improved unbiased results in aDNA mapping.

Keywords:
alignmentancient DNAbenchmarkingreference bias

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Area of Science:

  • Genomics
  • Bioinformatics
  • Population Genetics

Background:

  • Current genome assembly relies on mapping short DNA sequences to a reference genome.
  • This process faces computational challenges and introduces reference bias, particularly affecting ancient DNA (aDNA) studies with degraded DNA fragments.
  • Existing aDNA mapping strategies have not significantly evolved despite advancements in software.

Purpose of the Study:

  • To benchmark the performance of 30 distinct DNA read mapping strategies across four software packages (BWA-aln, BWA-mem, NovoAlign, Bowtie2).
  • To quantify the impact of reference bias in ancient DNA studies.
  • To evaluate new software and parameterizations for improved aDNA mapping.

Main Methods:

  • Simulated ancient DNA reads from three human populations were used.
  • Thirty mapping strategies were tested using BWA-aln, BWA-mem, NovoAlign, and Bowtie2.
  • Mapping precision and reference bias were quantified, with a focus on downstream population genetic analyses.

Main Results:

  • Specific parameterizations of NovoAlign, BWA-aln, and BWA-mem demonstrated high mapping precision and low reference bias.
  • Filtering reads by mapping quality further reduced bias.
  • Unbiased results with NovoAlign were achieved using an IUPAC reference genome, highlighting the value of population genetic data in mapping.

Conclusions:

  • Optimized mapping strategies can significantly mitigate reference bias in ancient DNA studies.
  • The use of an IUPAC reference genome, incorporating population genetic information, offers a promising approach for unbiased aDNA mapping.
  • These findings suggest a need to update current gold-standard mapping practices for ancient DNA research.