Sequencing and Standards for 

Characterization of the 

Mitochondrial Genome

Kevin Kiesler, M.S.

Research Biologist - NIST Applied Genetics Group

Forensics @NIST

November 7, 2018
Outline

• Sanger sequencing and the “next generation”

• Reference materials and mtDNA sequencing

• Population sequencing project
NIST Disclaimer

• Disclaimer - Points of view in this document are those of the authors and 

do not necessarily represent the official position or policies of the U.S. 

Department of Commerce or the Department of Justice. Certain 

commercial equipment, instruments, and materials are identified in order 

to specify experimental procedures as completely as possible. In no case 

does such identification imply a recommendation or endorsement by NIST, 

nor does it imply that any of the materials, instruments, or equipment 

identified are necessarily the best available for the purpose. 

• All work presented has been reviewed and approved by the NIST Human 

Subjects Protections Office.
Mitochondrial DNA Sequencing - Introduction

• Mitochondrial DNA advantages

• High copy number

• Very small quantity of evidence required

• Single nucleotide variants (SNPs or SNVs) = profile

• Small region of DNA analyzed

• Short PCR amplicons

• Applicable to challenging/degraded/limited samples

• Mass disaster, missing persons

• Example: World Trade Center victim identification – 44,000 mtDNA profiles

• Disadvantage

• Low power of discrimination using “Control Region”

• 26,127 CR genotypes in EMPOP database for matching

• Can be improved with whole mtGenome analysis

Mitochondrial Genome

Diagram

Image credit: Emmanuel Douzery

Control Region
Sanger Sequencing Workflow - mtGenome

Extract 

DNA

Quantitate 

DNA

PCR 

Template 

for 

Sequencing

Purify Sequencing 

Reactions Purify

Size 

Separation 

(CE)

Data 

Analysis Variant List

1 2 3 4 5 6 7 8 9

8

Reactions

Per Sample

> 100

Reactions

Per Sample

Lyons et al. BMC Genomics 2013, 14:881
Next Generation Sequencing Workflow

Extract DNA Quantitate 

DNA

PCR 

Template for 

Sequencing

Library 

Preparation

QC & 

Quantify Sequence Data Analysis Variant List

1 2 3 4 5 6 7 8

Manual Procedure

Two

reactions

per Sample

Step 3: Amplify mtGenome

with long PCR (2 reactions) Step 4: Library preparation Step 6: Sequence on MiSeq

Several steps,

one well per

sample

Image credit:

www.Illumina.com Fendt et al. BMC Genomics 2009, 10:139 Image credit - www.thermofisher.com
Next Generation Sequencing Workflow

Extract DNA Quantitate 

DNA

Automated 

Library 

Preparation

Library

Quantitation

(manual)

Automated 

Template 

Preparation

Sequencing Data Analysis Variant List

Automated Procedure

(Applied Biosystems Precision ID mtDNA Whole Genome Panel)

Automated

Reaction

Setup

Image credit:

www.wright.edu/~oleg.paliy/NGS.html

Step 4: Prepare

templated beads

Sample 1

Sample 2

Sample 3

Sample 4

Sample 5

Sample 6

Sample 7

Sample 8

Step 3: Amplify mtGenome with 162 primer pairs in 2 reactions

eight samples at a time on Ion Chef 

Step 5: Sequence

on Ion Torrent

S5 or PGM

Step 6: Align

NGS reads

to reference

genome (rCRS)

Image credit:

www.thermofisher.com

1 2 3 4 5 6 7 8
Sequencing Instruments at NIST

• Multiple platforms

• Orthogonal measurements

• Characterize Standard Reference Materials

Ion Torrent

S5

Ion Chef

Illumina 

MiSeq FGx

Ion Torrent 

PGM

Applied 

Biosystems

SOLiD
NIST Mitochondrial Sequencing SRMs

• SRM 2392

• Three components 

• Component A: DNA from cell line CHR

• Component B: DNA from cell line 9947A

• Component C: Cloned fragment from HV1 region of CHR containing C-stretch

• SRM 2392-I

• One component

• DNA from cell line HL60

• Characterized with Sanger methods

• Released in 2001
Sanger-Based Sequence Agrees With NGS Values

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Previously no software available that

correctly handled these C-stretch

insertions with forensic nomenclature.

Reference material

Technology
NGS Can Detect Low Level Heteroplasmy

• Level is below what we can reliably see with Sanger methods

SRM 2392 Component A (CHR)

Position 1,393 R (17 % A)

SRM 2392-I (HL-60)

Position 2,445 Y (7 % C)
Multiple Orthogonal Measurements

• Great approach for certifying reference materials!

SRM 2392

Component 

A Component B SRM 2392-I

Nucleotide 64 T 1393 A 7861 C 2445 5149

PGM Edge 26.8 21.2 72.7 10.6 5.1

PGM NIST 1 24.3 15.6 45.0 7.5 9.1

PGM NIST 2 25.0 17.5 65.7 7.7 7.7

PGM NIST 3 29.7 16.5 59.4 7.7 7.7

PGM NIST HiQ 33.2 15.2 77.7 7.7 8.4

MiSeq Edge 33.0 19.0 87.4 10.7 6.8

MiSeq NIST 31.6 17.9 88.4 9.1 6.4

HiSeq BC 30.6 16.9 88.3 7.4 7.1

SOLiD NIST 29.0 16.7 87.3 7.3 7.0

Average 29.2 17.4 74.6 8.4 7.3

St. Dev. 3.1 1.7 14.5 1.3 1.1
Multiple Orthogonal Measurements

• Great approach for characterizing reference materials!

SRM 2392

Component 

A Component B SRM 2392-I

Nucleotide 64 T 1393 A 7861 C 2445 5149

PGM Edge 26.8 21.2 72.7 10.6 5.1

PGM NIST 1 24.3 15.6 45.0 7.5 9.1

PGM NIST 2 25.0 17.5 65.7 7.7 7.7

PGM NIST 3 29.7 16.5 59.4 7.7 7.7

PGM NIST HiQ 33.2 15.2 77.7 7.7 8.4

MiSeq Edge 33.0 19.0 87.4 10.7 6.8

MiSeq NIST 31.6 17.9 88.4 9.1 6.4

HiSeq BC 30.6 16.9 88.3 7.4 7.1

SOLiD NIST 29.0 16.7 87.3 7.3 7.0

Average 29.2 17.4 74.6 8.4 7.3

St. Dev. 3.1 1.7 14.5 1.3 1.1

87 %

☺

It can also educate you about your technology.
Multiple Orthogonal Measurements

• Great approach for characterizing reference materials! SOFTWARE
Conclusions

• Reference materials 

• Can identify technical limitations/bias

• Often need multiple measurements

• Orthogonal techniques

• Help to select best procedures
Population Scale Sequencing
Project Goals

• Submit forensic-quality whole mtGenome data to EMPOP

• Database used for match statistics

• Current version (V4, Release 11)

• n = 26,127 control region sequences

• n = 256 whole genome sequences

• NIST population samples (n > 1,000)

• African American, Asian, Caucasian, Hispanic 

• Sequencing plan

• Start with Caucasian population

• ≈ 440 mtGenomes
Project Plans

• What instrument do we use?

• What protocol/chemistry do we use?

• What analysis procedure do we use?

• Software, data review, etc.

Ion Torrent S5

Ion Chef

Illumina MiSeq FGx
Project Planning: Instrument Selection

• Considerations

• Cost

• Time/labor

• Automation

 $-

 $5,000

 $10,000

 $15,000

 $20,000

 $25,000

 $30,000

S5XL Automated

Library Prep

S5XL Manual

Library Prep

MiSeq ILMN

Method

MiSeq AFDIL

Method

Cost Per 96 Samples (Approximate)
Project Planning: Protocol Selection

• Options

• Illumina mtGenome procedure

• Long PCR primers developed by Dr. Mark Wilson’s lab

• TaKaRa LA Taq

• Illumina Nextera XT library preparation

• Illumina MiSeq v2 2x150 cartridge (per protocol)

• mtGenome procedure used by Armed Forces DNA Identification Lab (AFDIL)

• Long PCR primers from Fendt et al., BMC Genomics 2009, 10:139

• TaKaRa LA Taq (GC Buffer & BSA)

• Kapa HyperPlus Library Kit

• Illumina V3 2x300 cartridge
More Consistent Coverage Depth with Kapa HyperPlus

AFDIL method allows higher multiplexing with less likelihood of dropout sites

Illumina Whole mtGenome Method (Nextera Library Kit)

AFDIL Whole mtGenome Method (Kapa Hyper Plus Library Kit)
Haplogroup Estimation from EMPOP

• No surprise haplogroups

Sample Missing Mutations Private Mutations Haplogroup Continent

GT38086 none T16189C H1c21 Europe (H)

GT38087 none none T2b6b Europe

GT38089 none T16189C J1c8a Europe

GT38091 none C10933T A15467G V Europe

GT38092 none C198T A9327G A13801G T15670C H5e1a1 Europe (H)

GT38093 none none H1o Europe

GT38094 none A11252G H65 Europe (H)

GT38095 -573.1C G709A C9727T I1a1b Europe (I1a1)

GT38097 none G8027A G15301A K1a1b1 Europe

GT38098 A16183C -309.1C -309.2C C16111T T152C H1b1 Europe

GT38100 none -309.1C G4655A H1 Europe

GT38106 none T2416C A8817G H2a2a Europe

GT38107 none C3388A C8788T U5a1a1 Europe

GT38108 none T4373C T15313C H1+16189 Europe

JA44327 -573.1C G16474T I 2 Europe (I)

JM28315 -309.1C none K2a6 Europe

JT52345 none A16138G A73G H5a1c1a Europe

JT52346 none none H5g Europe

MT97121 none T16093C G7762A J1c2 Europe

MT97122 none none T2b2b Europe

MT97123 none A16158G T1a1 Europe (T1a)

MT97124 T16093C -309.1C -524.3A -524.4C K1a Europe

MT97125 none T152C U5a2d1a Europe (U5a)
Informatics

• Forensic mtDNA nomenclature is challenging!

• Commercial software now available

• Softgenetics GeneMarker HTS

• Compatible with forensic nomenclature

• EMPOP formatted report

• CLC Genomics Workbench

• AFDIL / Qiagen – developed AQME Tool

• ThermoFisher Scientific

• Converge mtDNA Analysis (released October 2018)
Conclusions

• Mitochondrial Genome Protocol

• Cost and data quality directed decision process

• Selected AFDIL-developed procedure for reference-quality samples

• Even coverage

• Higher multiplexing

• Degraded samples will need a different procedure

• Analysis method must be high-throughput

• High accuracy required for EMPOP submission
Thank You!

Questions?

• Funding

• NIST Special Programs Office: Forensic DNA

• FBI Biometrics Center of Excellence: Forensic DNA Typing as a Biometric tool. 

Contact info:

Kevin.Kiesler@NIST.gov

 Acknowledgements 

Armed Forces DNA Idenification Laboratory (AFDIL)

Kim Andreaggi

Charla Marshall

NIST Applied Genetics Group NGS Team

Dr. Peter Vallone, Group leader

Lisa Borsuk

Sarah Riman

Becky Steffen

Katherine Gettings
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