Ancient DNA Technologies: Complete Reference Guide & Cheat Sheet

Introduction to Ancient DNA (aDNA)

Ancient DNA (aDNA) refers to genetic material recovered from archaeological, paleontological, and historical specimens. This field merges molecular biology, archaeology, and bioinformatics to reveal insights about past populations, evolution, and human history. The analysis of aDNA has revolutionized our understanding of human migrations, extinct species, disease evolution, and adaptation processes. Despite significant challenges such as DNA degradation, contamination, and limited sample availability, technological advances in the past two decades have transformed aDNA from a niche specialty to a mainstream scientific discipline. This cheat sheet provides a comprehensive overview of ancient DNA technologies, methodologies, applications, and best practices for researchers and practitioners.

Core Concepts and Terminology

Key aDNA Characteristics

Preservation Factors

• Temperature: Colder environments better preserve DNA (permafrost, caves)
• Humidity: Dry conditions (desert) or waterlogged anaerobic environments
• pH: Neutral to slightly alkaline conditions are optimal
• Microbial Activity: Less microbial action means better preservation
• Time: Generally, older samples contain less intact DNA
• Specimen Type: Dense materials (petrous bone, teeth) preserve DNA better

Sample Collection and Handling

Optimal Sample Types

1. Petrous Portion of Temporal Bone: Highest DNA yield in humans/animals
2. Teeth: Especially dentine and cementum layers
3. Dense Cortical Bone: Better preservation than cancellous (spongy) bone
4. Hair with Root Sheath: Contains nuclear DNA
5. Plant Seeds/Tissues: In dry preservation contexts
6. Coprolites: For dietary and microbiome studies
7. Sediments: For environmental DNA studies

Contamination Prevention Protocol

• Field Collection:

  • Use sterile tools and gloves
  • Change gloves between samples
  • Minimize handling
  • Store in sterile containers
  • Avoid exposure to direct sunlight
  • Keep cool if possible

• Laboratory Processing:

  • Physical cleaning (UV, bleach treatment of surfaces)
  • Sample surface removal (2-3mm)
  • Dedicated aDNA facilities with:
    • Positive air pressure
    • HEPA filtration
    • UV irradiation
    • Separate pre- and post-PCR areas
    • PPE (coveralls, face masks, hairnets, shoe covers, double gloves)
    • Unidirectional workflow

Extraction Technologies

Classic Extraction Methods

1. Phenol-Chloroform Extraction

   • Involves organic separation of DNA from proteins
   • Limited sensitivity for highly degraded DNA

2. Silica-Based Methods

   • Binds DNA to silica in presence of chaotropic salts
   • Modified versions more effective for aDNA
   • Examples: QIAquick PCR Purification Kit (modified)

Specialized aDNA Extraction Protocols

1. Dabney Protocol (2013)

   • Optimized for ultrashort DNA fragments
   • Uses extended binding buffer and modified silica columns
   • Significantly higher recovery of fragments <50bp

2. Rohland & Hofreiter Method

   • Uses guanidinium thiocyanate buffer
   • Optimized for challenging samples like cave sediment

3. Glocke & Meyer Protocol

   • Pre-digestion step to remove contamination
   • Optimized for highly contaminated samples
   • Multiple digestion steps

4. DNA from Sediments

   • Phosphate buffers for clay-rich samples
   • DNA binding to clay particles requires specialized approaches

Pre-Treatment Innovations

• EDTA Demineralization: Dissolves bone mineral matrix
• Proteinase K Digestion: Breaks down proteins bound to DNA
• N-phenacylthiazolium Bromide (PTB): Breaks DNA-protein crosslinks
• Bleach Surface Decontamination: Removes external contamination
• UV Irradiation of Surface: Damages contaminant DNA

Library Preparation Methods

Single-Stranded vs. Double-Stranded DNA Libraries

Key Library Preparation Protocols

1. Meyer & Kircher Protocol (2010)

   • Double-stranded approach
   • Illumina platform compatibility
   • Widely used standard method

2. Gansauge & Meyer Protocol (2013)

   • Single-stranded approach
   • Significantly higher DNA recovery
   • Critical breakthrough for very old samples

3. Swift Accel-NGS 2S

   • Commercial kit with modifications for aDNA
   • Reduces chimera formation

4. NEBNext Ultra II DNA

   • Commercial kit optimized for low-input DNA
   • Requires modifications for aDNA

Unique Molecular Identifiers (UMIs)

• Short, random nucleotide sequences added during library preparation
• Allow identification of PCR duplicates
• Critical for accurate quantification and error correction
• Implementation:
  1. Add during adapter ligation
  2. Track during bioinformatic processing
  3. Collapse identical reads with same UMI

Enrichment Technologies

Target Enrichment Methods

1. Hybridization Capture

   • Principle: Uses DNA/RNA baits complementary to targets
   • Approaches:
     • Array-based capture (MYbaits, Agilent SureSelect)
     • In-solution capture (more common for aDNA)
   • Design Types:
     • Whole genome capture (e.g., human DNA from microbial background)
     • Targeted capture (specific genes, chromosomes)
     • Exome capture (all coding regions)

2. PCR-Based Enrichment

   • Limited utility for highly fragmented DNA
   • Used for specific targets with well-preserved samples
   • Multiplex PCR allows multiple targets

3. CRISPR-Cas Systems

   • Emerging technology for targeted enrichment
   • Can be more specific than hybridization capture
   • Examples: CATCH, CRISPR-Cap

Common Enrichment Targets

• Mitochondrial DNA:

  • Higher copy number than nuclear DNA
  • Used for maternal lineage analysis
  • Complete mitogenome more informative than HVR only

• Y-Chromosome:

  • Paternal lineage information
  • Lower success rate than mtDNA due to copy number

• Autosomal DNA:

  • Whole-genome or selected SNPs
  • Population genetics, phenotypic traits

• Pathogen DNA:

  • Disease-causing organisms in archaeological remains
  • Custom capture designs for specific pathogens
  • Examples: Yersinia pestis (plague), M. tuberculosis, M. leprae

Sequencing Technologies for aDNA

Platform Comparison for aDNA Applications

Sequencing Considerations for aDNA

• Read Length: Short reads (75-150bp) optimal for fragmented aDNA
• Sequencing Depth: Higher coverage compensates for damage/contamination
  • Shotgun: 0.1-1X for screening, >1X for analysis
  • Targeted: 20-100X minimum coverage
• Paired-End Sequencing: Improves mapping quality
• Platforms with Lower GC Bias: Important for accurate representation
• Multiplexing Strategy: Index design to avoid cross-contamination

Bioinformatic Analysis Workflows

Raw Data Processing

1. Quality Control

   • FastQC/MultiQC for sequence quality assessment
   • Adapter trimming (Cutadapt, AdapterRemoval)
   • Quality filtering
   • Length filtering (typically keep >30bp)

2. aDNA-Specific Processing

   • Damage pattern assessment (mapDamage, DamageProfiler)
   • UMI processing if applicable (UMI-tools)
   • Read merging for overlapping pairs (FLASH, PEAR)

3. Mapping to Reference

   • BWA-aln with ancient DNA parameters (-l 1024)
   • Bowtie2 with –very-sensitive-local
   • Specialized mappers (e.g., paleomix)
   • Consider closely related species for extinct organisms

4. Post-Mapping Processing

   • Remove duplicates (Picard, samtools)
   • Remove low-quality mappings (MapQ filtering)
   • Rescale quality scores at damaged positions
   • Local realignment around indels

Authentication Methods

1. Damage Pattern Analysis

   • C→T transitions at 5′ ends (G→A at 3′ ends)
   • Damage increases with sample age
   • Tools: mapDamage, DamageProfiler, PMDtools

2. Fragment Length Distribution

   • Authentic aDNA shows shorter fragment length
   • Typically 30-70bp average for ancient samples

3. Contamination Estimation

   • Nuclear DNA: Heterozygosity on X chromosome in males (ANGSD)
   • mtDNA: Mismatches at haplotype-defining sites (Schmutzi, contamMix)
   • Reference-free methods: DICE, AuthentiCT

4. Sex Determination

   • Ratio of X/Y/autosomal coverage
   • Consistent sex assignment across methods

Population Genetics Analysis

1. Variant Calling Approaches

   • Genotype likelihood methods (ANGSD)
   • Pseudo-haploid calls (random allele sampling)
   • Joint calling with modern references (GATK with modifications)

2. Key Analysis Methods

   • Principal Component Analysis: Project onto modern variation
   • ADMIXTURE/Structure: Ancestry proportions
   • f-statistics: Population relationships (f3, f4, qpAdm)
   • Phylogenetic methods: Maximum likelihood, Bayesian
   • Identity-by-descent: Relatedness estimation

3. Specialized aDNA Software

   • EAGER/nf-core/eager: Processing pipeline for aDNA
   • ATLAS: Genotype likelihood estimation for aDNA
   • admixtools: Suite for f-statistics
   • Schmutzi: Contamination estimation and consensus calling

Common Applications and Case Studies

Human Evolution and Migration

• Out of Africa Expansions

  • Early human dispersal patterns
  • Interbreeding with archaic hominins

• Agricultural Transitions

  • Neolithic expansion in Europe
  • Steppe migrations and Indo-European spread

• Recent Population History

  • Colonial era admixture
  • Historical demographic changes

Ancient Pathogen Genomics

• Epidemic Diseases

  • Plague (Yersinia pestis) evolution
  • Historical tuberculosis and leprosy strains
  • Ancient viral DNA (HBV, smallpox)

• Host-Pathogen Coevolution

  • Immune gene adaptation
  • Virulence changes over time

Extinct Species Analysis

• Megafauna Extinction

  • Woolly mammoth genomics
  • Causes of population decline
  • Genetic diversity before extinction

• Archaic Humans

  • Neanderthal and Denisovan genomes
  • Genetic contributions to modern humans

Environmental and Climate Studies

• Ancient Sedimentary DNA

  • Paleoenvironmental reconstruction
  • Ecosystem changes
  • Species presence/absence

• Ancient Ice Core DNA

  • Long-term biodiversity records
  • Climate change impacts

Common Challenges and Solutions

Best Practices and Guidelines

Sample Selection Strategy

1. Preliminary Screening

   • Sample multiple individuals when possible
   • Prioritize best preservation contexts
   • Consider pilot studies with few samples before large studies

2. Non-Destructive Assessment

   • ZooMS (Zooarchaeology by Mass Spectrometry) for species ID
   • Bone density/collagen preservation as DNA proxy
   • pXRF for elemental composition screening

3. Ethical Considerations

   • Indigenous community consultation and approval
   • Minimal destruction sampling approaches
   • Long-term sample preservation plans
   • Data sharing agreements

Analytical Best Practices

1. Authentication Standards

   • Multiple authentication methods required
   • Replicate extractions for critical samples
   • Independent laboratory confirmation for extraordinary claims
   • Publishing all authentication metrics

2. Reporting Standards

   • Detailed methods descriptions
   • Repository submission of raw data
   • Contamination estimates included
   • Coverage statistics and mapping parameters

3. Interpretation Guidelines

   • Consider temporal and geographic sampling biases
   • Integrate with archaeological/historical context
   • Acknowledge limitations in conclusions
   • Clearly separate data from interpretation

Resources for Further Learning

Key Publications

• Methodological Papers

  • Dabney et al. (2013). “Complete mitochondrial genome sequence of a Middle Pleistocene cave bear reconstructed from ultrashort DNA fragments.”
  • Gansauge & Meyer (2013). “Single-stranded DNA library preparation for the sequencing of ancient or damaged DNA.”
  • Orlando et al. (2021). “Ancient DNA analysis.”
  • Pinhasi et al. (2015). “Optimal Ancient DNA Yields from the Inner Ear Part of the Human Petrous Bone.”

• Review Articles

  • Skoglund & Mathieson (2018). “Ancient genomics of modern humans: The first decade.”
  • Brunson & Reich (2019). “The Promise of Paleogenomics Beyond Our Own Species.”
  • Fellows Yates et al. (2021). “Reproducible, portable, and efficient ancient genome reconstruction with nf-core/eager.”

Online Resources and Tools

• Databases

  • Allen Ancient DNA Resource (AADR)
  • Ancient Genomes
  • AncientMetagenomeDir

• Software Collections

  • nf-core/eager – Reproducible aDNA pipeline
  • EAGER – Original aDNA pipeline
  • Paleomix – Pipeline for aDNA processing

• Educational Resources

  • Ancient DNA virtual labs and protocols
  • Workshop materials from leading aDNA centers
  • Specialized summer schools and courses

Professional Organizations

• Society for Archaeological Sciences
• International Society for Evolution, Medicine, and Public Health
• Society for Molecular Biology and Evolution
• SPAAM Community (Standards, Precautions and Advances in Ancient Metagenomics)

This cheat sheet provides a comprehensive overview of the current state of ancient DNA technologies, methodologies, and best practices. The field continues to evolve rapidly, with new methods and applications emerging regularly. Researchers should stay current with the latest literature and community standards as they develop their own ancient DNA research programs.