Ancient DNA: Methods and Protocols (49 page)

171–175, 225

Quantification ................................... 3 8, 121, 123, 126, 128, PCR negative controls ............................... 1 14, 136–139

147, 149–151, 153, 174, 186, 187, 190–192, 198

post- .......................................................4, 5, 7, 138, 139

pre- .......................................................................... 5, 31

R

quantitative PCR (qPCR) .........................38, 62, 65, 69, Rabbit serum albumin (RSA) ........... 8, 32, 89, 113, 135–137

121–132, 147, 149–151, 153, 158–160, 164, 167,

RAxML .............................................................................3 2

173, 174, 181, 186, 190, 193

Replication ......................................... 3 , 5, 18, 105, 139, 178

real time ................................................8 2, 121–132, 160

Reproducibility .................................................... 5 , 126, 152

second step ......................................................... 1 35–140

RNA ..... ....................................................1 22, 180, 181, 212

singleplex ................................................... 1 34, 137, 139

RNAse. ........................................................................ 7 4, 77

two-step ..................................................... 1 28, 134, 171

RSA.
See
Rabbit serum albumin (RSA)

PEC .
See
Primer extension capture (PEC)

PEG.
See
Polyethylene glycol (PEG)

S

Permafrost

preserved DNA ............................................... 9 , 21, 111

Sample preparation ................... 7 –9, 14, 15, 24–25, 103, 144

Phenol

Sample storage ................................................... 7 –9, 81, 118

-chloroform ................................... 1 3–19, 52, 73, 76, 82, SDS.
See
Sodium dodecyl sulphate (SDS)

83, 87

Sediment ..................................................................... 2 1, 37

Phylogenetic analysis ..........................3 2, 105, 212, 229–239

DNA .........................................................5 7–62, 72, 123

Phylogeny ............................................................ 3 0, 33, 230

Sensitivity ..................................... 3 , 129, 130, 133, 175, 225

Phylogeographic .........................................9 3, 107, 190, 229

Sequencing ....................................... 454 , 157, 168, 175, 181

Phylogeography ....................................................... 1 94–195

coverage ...................................... 1 78, 179, 193, 198, 200

Phytolith ............................................................................7 2

DNA-sequencing ............... 1 44, 150, 153, 175, 197–226

Pipeline ............................................. 1 68, 206, 212, 222, 224

error .................................... 1 94, 201, 206, 208, 211, 220

Pleistocene ........................................................... 8 7–91, 108

genomic ...................... 1 23, 143, 144, 155, 190, 192, 193

Pollen ... ..............................................................................7 2

library ........................................ 1 44, 150, 153, 155, 172, Polyethylene glycol (PEG) ........................18, 152, 159, 161, 173, 186, 204

162, 166, 182, 184

multiplex ............................................................ 2 02, 209

Polymerase

paired-end ..........................................1 98, 201, 206–211

Bst- ......................146, 149, 152, 160, 163, 166, 182, 184

platform ..................................... 1 34, 145, 156, 157, 172, T4- ............................................................ 146, 162, 165

178, 179, 182, 198–200, 212, 224

Polynucleotide kinase ..............................1 45, 148, 159, 162, Sanger- ....................................... 138, 143, 155, 171, 230

165, 182, 184

shotgun ...................................................... 1 56, 177, 197

Preamplified ................................................................... 1 73

Silica

Precipitation .............................................. 4 4, 58, 60, 73, 82, based .................... 3 8, 43, 69, 73, 89, 94, 96, 98, 105, 172

84, 98, 116

Siliconized tubes ...........................................2 6, 99, 163, 169

precipitated ..................................... 4 2, 47, 48, 61, 83, 85

Sloth .... .................................................................. 3 7, 51–55

Preservation conditions ............................................. 3 , 9, 72

Sodium dodecyl sulphate (SDS) ........................... 14, 16–18, Primer

43, 44, 47, 61, 68, 74, 82

dimer .................................... 3 2, 118, 131, 140, 167, 173

Sodium hypochlorite ............................................. 8 , 14, 103

extension .............................................1 50, 156, 179, 180

Soil ............................................................. 2 2, 37, 57–62, 72

Primer3 .................................................................... 103, 172

Solexa .............................................. 1 23, 134, 143, 145, 156, Primer extension capture

157, 159, 163, 165

(PEC) ...........................................150, 156, 179–181

SOLiD ..................................... 1 23, 143, 181, 187, 223, 224

Protein 2 ......................................... 14, 17, 38, 46, 60, 61, 73, Sonicator ................................................................. 1 82, 184

76, 81– 83, 223, 233

Spin-columns ............................................ 4 6, 47, 58, 61, 66, Proteinase K ........................................ 1 4, 16–18, 23, 25, 26, 145, 148–150, 161, 162, 165

31, 34, 43–45, 47, 58, 59, 61, 66–68, 73, 74

SPRI beads .............................................................. 1 83, 187

ANCIENT DNA: METHODS AND PROTOCOLS 247

Index

Streptavidin .............................................1 52, 161, 163, 168, Transversion .............................................................. 9 0, 238

181, 182, 186, 187

Trimming .................................................1 98, 204, 206–211

Subfossil .................................................................... 3 8, 190

trimmed ..................................................... 2 05, 209, 224

Subsampling .......................................................... 2 9, 88, 89

Tris

Substitution ....................................... 9 0, 108, 201, 213, 214, -HCl ....................... 1 4, 23, 39, 44, 58, 59, 61, 73, 82, 95, 225, 232, 233, 238

147, 160, 161

SYBR

Tris-acetate-EDTA (TAE) .......................113, 115, 135, 138

Green ..........................................1 22, 124, 126, 128–130

Tris-borate-EDTA (TBE) .............................. 115, 135, 138

Tris-EDTA (TE) ......................39, 40, 44, 47, 52, 72, 73, 75, T

76, 82, 84, 89, 95, 97, 98, 103, 104, 113, 135, 147,

TAE.
See
Tris-acetate-EDTA (TAE)

150, 151, 153, 160–163, 165–167, 182, 186, 187

Tag(s) ... .................................................................... 155, 199

Triton-X100 ......................................................................95

Tagging .................................................................... 1 72, 200

Tween-20 .................................... 39, 40, 147, 153, 160–163, Taq

165–168, 182, 183

AmpliTaq .................................. 1 12, 114, 118, 124, 126, U

130, 135–137, 139, 146, 149, 152, 160, 164, 168, 173

AmpliTaq Gold ......................... 1 12, 114, 118, 124, 126, Uracil

130, 135–137, 139, 146, 149, 152, 160, 164, 168, 173

DNA-glycosylase (UDG) .......................2, 112, 145, 152

Hifi Taq ..................................................................... 1 14

N-glycosylase (UNG) ................................ 112, 125, 130

High Fidelity Taq ...................................3 2, 89, 112, 114

UV irradiation ........................................................... 8 , 9, 14

Target

enrichment ................................................ 1 50, 177–188

V

specific ....................................................................... 1 90

Vivaflow ............................................................................9 7

TBE.
See
Tris-borate-EDTA (TBE)

Vivaspi . .................................................................. 6 6, 67, 69

TE.
See
Tris-EDTA (TE)

TempNet ......................................................................... 1 05

W

TOPO-TA cloning ...............................5 3, 89, 114, 116, 118

Transition ..................................... 2 , 8, 53, 55, 112, 145, 238

Waterlogged ............................................................... 7 1, 72

• Cover_978-1-61779-515-2
  
  • FrontMatter
    
    • Ancient DNA
      
      • Preface
        
      • Contents
        
      • Contributors
        
• Chapter 1: Setting Up an Ancient DNA Laboratory
  
  • Chapter 1: Setting Up an Ancient DNA Laboratory
    
    • 1. Introduction
      
      • 1.1. Difficulties of aDNA Work
        
        • 1.1.1. Postmortem Degradation
          
        • 1.1.2. DNA Survival and Antediluvian DNA
          
        • 1.1.3. Contamination
          
    • 2. Guidelines for aDNA Research
      
    • 3. Setting Up an Ancient DNA Laboratory
      
      • 3.1. Setting Up the Ancient DNA Workspace
        
      • 3.2. Reagents and Materials
        
      • 3.3. Maintaining a Sterile Space
        
      • 3.4. Sample Preparation and Storage
        
    • 4. Notes
      
    • References
      
• Chapter 2: A Phenol–Chloroform Protocol for Extracting DNA from Ancient Samples
  
  • Chapter 2: A Phenol–Chloroform Protocol for Extracting DNA from Ancient Samples
    
    • 1. Introduction
      
    • 2. Materials
      
      • 2.1. Sample Preparation
        
      • 2.2. Chelation
        
      • 2.3. Digestion
        
      • 2.4. Phase Separation
        
      • 2.5. Concentration
        
    • 3. Methods
      
      • 3.1. Sample Preparation
        
      • 3.2. Chelation
        
      • 3.3. Digestion
        
      • 3.4. Phase Separation
        
      • 3.5. Concentration
        
    • 4. Notes
      
    • References
      
• Chapter 3: DNA Extraction of Ancient Animal Hard Tissue Samples via Adsorption to Silica Particles
  
  • Chapter 3: DNA Extraction of Ancient Animal Hard Tissue Samples via Adsorption to Silica Particles
    
    • 1. Introduction
      
    • 2. Materials
      
      • 2.1. DNA Release from Bony Specimen
        
      • 2.2. DNA Purification and Concentration
        
    • 3. Methods
      
      • 3.1. Preparing the Silica Suspension
        
      • 3.2. Preparing the Columns
        
      • 3.3. Sample Preparation and DNA Release
        
      • 3.4. DNA Adsorption to Silica, Washing Steps, and Elution
        
    • 4. Notes
      
    • References
      
• Chapter 4: Case Study: Recovery of Ancient Nuclear DNA from Toe Pads of the Extinct Passenger Pigeon*
  
  • Chapter 4: Case Study: Recovery of Ancient Nuclear DNA from Toe Pads of the Extinct Passenger Pigeon*
    
    • 1. Introduction
      
    • 2. Materials and Methods
      
    • 3. Results and Discussion
      
    • References
      
• Chapter 5: Extraction of DNA from Paleofeces
  
  • Chapter 5: Extraction of DNA from Paleofeces
    
    • 1. Introduction
      
    • 2. Materials
      
      • 2.1. Laboratory Supplies (per Sample)
        
      • 2.2. Laboratory Equipment
        
      • 2.3. Solutions and Buffers (per Sample)
        
    • 3. Methods
      
      • 3.1. Preparing the Silica Suspension
        
      • 3.2. Paleofeces DNA Extraction
        
    • 4. Notes
      
    • References
      
• Chapter 6: DNA Extraction from Keratin and Chitin
  
  • Chapter 6: DNA Extraction from Keratin and Chitin
    
    • 1. Introduction
      
    • 2. Materials
      
      • 2.1. General Requirements
        
      • 2.2. For Silica-Column Purification ( 3.3)
        
      • 2.3. For Organic Purification ( 3.4)
        
    • 3. Methods
      
      • 3.1. Tissue Pre-Preparation
        
      • 3.2. Tissue Digestion
        
      • 3.3. DNA Purification: Silica Method ( see Note 5)
        
      • 3.4. DNA Purification: Organic Extraction ( see Note 8)
        
    • 4. Notes
      
    • References
      
• Chapter 7: Case Study: Ancient Sloth DNA Recovered from Hairs Preserved in Paleofeces*
  
  • Chapter 7: Case Study: Ancient Sloth DNA Recovered from Hairs Preserved in Paleofeces*
    
    • 1. Introduction
      
    • 2. Materials and Methods
      
    • 3. Results and Discussion
      
    • References
      
• Chapter 8: Ancient DNA Extraction from Soils and Sediments
  
  • Chapter 8: Ancient DNA Extraction from Soils and Sediments
    
    • 1. Introduction
      
    • 2. Materials
      
      • 2.1. Large Extraction
        
      • 2.2. Small Extraction (Materials)
        
    • 3. Methods
      
      • 3.1. Large Extraction
        
      • 3.2. Small Extraction
        
    • 4. Notes
      
    • References
      
• Chapter 9: DNA Extraction from Fossil Eggshell
  
  • Chapter 9: DNA Extraction from Fossil Eggshell
    
    • 1. Introduction
      
    • 2. Materials
      
      • 2.1. Eggshell Sampling
        
      • 2.2. Eggshell Digestion
        
      • 2.3. Eggshell Extraction
        
    • 3. Methods
      
      • 3.1. Eggshell Sampling
        
      • 3.2. Eggshell Digestion
        
      • 3.3. DNA Purification: Silica Method
        
    • 4. Notes
      
    • References
      
• Chapter 10: Ancient DNA Extraction from Plants
  
  • Chapter 10: Ancient DNA Extraction from Plants
    
    • 1. Introduction
      
    • 2. Materials
      
      • 2.1. CTAB Protocol
        
      • 2.2. PTB Protocol
        
    • 3. Methods
      
      • 3.1. CTAB Protocol
        
      • 3.2. PTB Protocol
        
    • 4. Notes
      
    • References
      
• Chapter 11: DNA Extraction from Formalin-Fixed Material
  
  • Chapter 11: DNA Extraction from Formalin-Fixed Material
    
    • 1. Introduction
      
    • 2. Materials
      
    • 3. Methods
      
      • 3.1. Tissue Pre-Preparation
        
      • 3.2. Tissue Digestion
        
    • 4. Notes
      
    • References
      
• Chapter 12: Case Study: Ancient DNA Recovered from Pleistocene-Age Remains of a Florida Armadillo*
  
  • Chapter 12: Case Study: Ancient DNA Recovered from Pleistocene-Age Remains of a Florida Armadillo*
    
    • 1. Introduction
      
    • 2. Materials and Methods
      
    • 3. Results and Discussion
      
      • 3.1. Sample Preservation
        
      • 3.2. Troubleshooting the Experiment
        
      • 3.3. Challenges of a Warm Environment
        
    • References
      
• Chapter 13: Nondestructive DNA Extraction from Museum Specimens
  
  • Chapter 13: Nondestructive DNA Extraction from Museum Specimens
    
    • 1. Introduction
      
    • 2. Materials
      
    • 3. Methods
      
      • 3.1. Incubation
        
      • 3.2. DNA Purification
        
      • 3.3. Sample Curation
        
    • 4. Notes
      
    • References
      
• Chapter 14: Case Study: Using a Nondestructive DNA Extraction Method to Generate mtDNA Sequences
  
  • Chapter 14: Case Study: Using a Nondestructive DNA Extraction Method to Generate mtDNA Sequences from Historical Chimpanzee Specimens*
    
    • 1. Introduction
      
    • 2. Materials and Methods
      
      • 2.1. Sample Preparation
        
      • 2.2. DNA Extraction and Amplification
        
      • 2.3. Phylogenetic Analysis
        
    • 3. Results and Discussion
      
    • References
      
• Chapter 15: PCR Amplification, Cloning, and Sequencing of Ancient DNA
  
  • Chapter 15: PCR Amplification, Cloning, and Sequencing of Ancient DNA
    
    • 1. Introduction
      
    • 2. Methods
      
      • 2.1. Required Reagents
        
      • 2.2. Agarose Gel Visualization
        
      • 2.3. PCR Purification
        
      • 2.4. BigDye (Applied Biosystems) Sequencing
        
      • 2.5. Cloning (Using the TOPO-TA Kit, Invitrogen)
        
    • 3. Methods
      
      • 3.1. Master Mix Setup
        
      • 3.2. Agarose Gel Visualization and PCR Purification
        
      • 3.3. Direct Sequencing
        
      • 3.4. Cloning
        
    • 4. Notes
      
    • References
      
• Chapter 16: Quantitative Real-Time PCR in aDNA Research
  
  • Chapter 16: Quantitative Real-Time PCR in aDNA Research
    
    • 1. Introduction
      
    • 2. Materials
      
    • 3. Methods
      
      • 3.1. Preparation of qPCR Standards
        
      • 3.2. Setting Up the qPCR Reaction
        
      • 3.3. Thermocycling on a qPCR Platform
        
    • 4. Notes
      
    • References
      
• Chapter 17: Multiplex PCR Amplification of Ancient DNA
  
  • Chapter 17: Multiplex PCR Amplification of Ancient DNA
    
    • 1. Introduction
      
    • 2. Materials
      
      • 2.1. PCR Reagents
        
      • 2.2. Agarose Gel Visualization and PCR Purification
        
    • 3. Methods
      
      • 3.1. Primer Design
        
      • 3.2. First-Step PCR Setup
        
      • 3.3. Second-Step PCR Setup
        
      • 3.4. Agarose Gel Visualization and PCR Purification
        
    • 4. Notes
      
    • References
      
• Chapter 18: Preparation of Next-Generation Sequencing Libraries from Damaged DNA
  
  • Chapter 18: Preparation of Next-Generation Sequencing Libraries from Damaged DNA
    
    • 1. Introduction
      
    • 2. Materials
      
      • 2.1. End Repair
        
      • 2.2. Adaptor Attachment
        
      • 2.3. Primary Library Amplification
        
      • 2.4. Secondary Library Amplification
        
      • 2.5. Library Quantification
        
    • 3. Methods
      
      • 3.1. aDNA Repair
        
      • 3.2. Adaptor Attachment
        
      • 3.3. Primary Library Amplification
        
      • 3.4. Secondary Library Amplification
        
      • 3.5. Quantification of Library by qPCR
        
    • 4. Notes
      
    • References
      
• Chapter 19: Generating Barcoded Libraries for Multiplex High-Throughput Sequencing
  
  • Chapter 19: Generating Barcoded Libraries for Multiplex High-Throughput Sequencing
    
    • 1. Introduction
      
      • 1.1. Background
        
      • 1.2. Adapter Design
        
    • 2. Materials
      
      • 2.1. Materials Needed for Protocol 1 and Protocol 2
        
      • 2.2. Materials Needed Only for Protocol 1
        
      • 2.3. Materials Needed Only for Protocol 2
        
    • 3. Methods
      
      • 3.1. Prepare Indexing Adapters
        
      • 3.2. Protocol 1
        
        • 3.2.1. Blunt-End Repair
          
        • 3.2.2. Adapter Ligation
          
        • 3.2.3. Prepare Beads
          
        • 3.2.4. Library Immobilization
          
        • 3.2.5. Adapter Fill-In
          
        • 3.2.6. Library Elution
          
        • 3.2.7. Library Quantification
          
        • 3.2.8. Extension and Amplification of Libraries
          
      • 3.3. Protocol 2
        
        • 3.3.1. Blunt-End Repair
          
        • 3.3.2. Adapter Ligation
          
        • 3.3.3. Adapter Fill-In
          
        • 3.3.4. Library Quantification (Optional Step)
          
        • 3.3.5. Extension and Amplification of Libraries
          
        • 3.3.6. Final Quantification of Libraries
          
    • 4. Notes
      
    • References
      
• Chapter 20: Case Study: Targeted high-Throughput Sequencing of Mitochondrial Genomes from Extinct Cave Bears
  
  • Chapter 20: Case Study: Targeted high-Throughput Sequencing of Mitochondrial Genomes from Extinct Cave Bears via Direct Multiplex PCR Sequencing (DMPS)*
    
    • 1. Introduction
      
    • 2. Materials and Methods
      
    • 3. Results and Discussion
      
    • References
      
• Chapter 21: Target Enrichment via DNA Hybridization Capture
  
  • Chapter 21: Target Enrichment via DNA Hybridization Capture
    
    • 1. Introduction
      
    • 2. Materials
      
    • 3. Methods
      
      • 3.1. Hybridization
        
      • 3.2. Immobilization of Target-Enriched Library
        
      • 3.3. Serial Hybridization Captures
        
      • 3.4. Amplification, Quantification, and Pooling Before Sequencing
        
    • 4. Notes
      
    • References
      
• Chapter 22: Case Study: Enrichment of Ancient Mitochondrial DNA by Hybridization Capture*
  
  • Chapter 22: Case Study: Enrichment of Ancient Mitochondrial DNA by Hybridization Capture*
    
    • 1. Introduction
      
    • 2. Methods
      
      • 2.1. DNA Extraction and PCR Screening
        
      • 2.2. Library Preparation and Quantification
        
      • 2.3. Generation of Bait
        
      • 2.4. Serial Hybridization Capture
        
      • 2.5. Sequence Analyses
        
      • 2.6. Phylogenetic Analyses
        
    • 3. Results
      
      • 3.1. Sequence Analyses
        
    • 4. Discussion
      
      • 4.1. Phylogeography
        
    • References
      
• Chapter 23: Analysis of High-Throughput Ancient DNA Sequencing Data
  
  • Chapter 23: Analysis of High-Throughput Ancient DNA Sequencing Data
    
    • 1. Introduction
      
    • 2. Materials
      
      • 2.1. Output Files from the Sequencing Platform
        
      • 2.2. Hardware
        
      • 2.3. Software
        
      • 2.4. Programs and Scripts
        
    • 3. Methods
      
      • 3.1. Separation of Individual Probes in Multiplex Experiments
        
      • 3.2. Removal of Protocol-Specific Library Artifacts
        
      • 3.3. Adapter Trimming and Merging of Paired-End Sequencing Data
        
      • 3.4. Quality Control and Filtering
        
      • 3.5. Identification of Endogenous Molecules
        
      • 3.6. Quantifying Contamination
        
      • 3.7. Handling PCR Duplicates
        
    • 4. Notes
      
    • References