Jo-Anne Laurence 1

B rendan Wi lhe lmi 1 , Adr ienne L . Edk ins 1 , 2

1 D e p a r t m e n t o f B i o c h e m i s t r y a n d M i c r o b i o l o g y, R h o d e s U n i v e r s i t y2 B i o m e d i c a l B i o t e c h n o l o g y R e s e a r c h U n i t ( B i o B R U ) , R h o d e s U n i v e r s i t y

C l e a r a n c e b y t h e E t h i c a l S t a n d a r d s C o m m i t t e e o f R h o d e s U n i v e r s i t y ( 2 0 1 3 Q 1 - 5 )

NGS for the analysis of microsatellite allele variants

in Nguni and Sotho-Tswana populations

Introduction

“It has long been an axiom of mine that the little things are infinitely the most important”

SHERLOCK HOLMES

The usefulness of DNA

(Metzker, M. , 2010; Jobling & Gill, 2004 ; Kayser & de Knijff, 2011)

• Clinical diagnostics• Disease-causal variants• Human population history• Revealing familial relationships• Forensic Applications

⁻ Identification of crime suspects ⁻ Naming of missing persons⁻ Biogeographic ancestry prediction⁻ Prediction of externally visible characteristics

• Criminal Law (Forensic Procedures) Amendment Act 37 of 2013 “DNA Act” passed into law this year

• DNA Database: DNA profiles for convicted individuals, crime scenes and suspects

Method of Typing: Analysis of Short Tandem Repeats (STRs)

Autosomal Short Tandem Repeats (STRs)

Non-coding regions Analysis: - Multiplex PCR of a panel of STRs- Separation of products based on size using capillary

electrophoresis

(Kayser and de Knijff, 2011)

Example of DNA Profiling using STRsSTR John Mary

1 vWA 18,18 15,132 D21S11 8,7 9,53 D7D820 5,5 5,84 D13D317 13,7 5,7

RECENT STUDIES• By using a different detection platform

- Compatible and comparable results- Higher power of discriminations

• Sequence variations within STRs- Indels- Single Nucleotide Polyporphisms (SNPs)

Suitable for SA?

Project Aims

1. To develop a 2-step PCR reaction for the analysis of STRs using Next Generation Sequencing (NGS)

2. To use this protocol to identify and characterise STR variants,

3. and to investigate the relationship between specific STR sub-allele frequencies and biogeographic ancestry

Methods and Results

OBJECTIVES

OBJECTIVE 1: Identify suitable STR loci

1. Likely sub-allelic structure

2. Projected multiplexing and NGS pooling success• Similar primer annealing temperatures • Limited Primer Interactions (FastPCR 6.3)• Amplicon sizes

- 200-400 bp- Range of <150 bp

STR MutationRate

HapMap Project STRbase Literature

SNPs African Data? Seq Variation Seq. Variation

D21S11 0.19% none n/a yes yes3,4,5

VWA 0.17% none n/a yes yes3,4,5

D13S317 0.14% 1 reported absent yes yes3,5

D3S1358 0.12% not found n/a yes yes3,4,5

D7S820 0.10% 3 reported 2 of 3 no yes3,5

D2S441 unknown none reported n/a n/a yes3

OBJECTIVE 2: Optimise PCR Reactions

Fusion primer

M13

1° PCR

Multiplex amplification of STRs using universal (M13)-tailed primers

2° PCRAddition of fusion primer with population specific MID

Bp

250

200

150

M 1 2 3 4 5 6 M 1º

D21S11 (4)

D7S820 (5)vWA (1)

D3S1358 (3)

D13S317 (6)D2S441 (2)

SINGLEPLEX MULTIPLEX

Bp

300

250

200

M 2°Bp

250

200

150

Expected size

increase of 70 bp

M 1°1° MULTIPLEX 2° MULTIPLEX

(Performed on Subject 0)

1. Buccal sample collection

2. DNA stabilisation and extraction - Isohelix DDK-50

3. Quantification of DNA by Nanodrop

OBJECTIVE 3: DNA sample Collection

Isohelix SK1 Swabs

144 individuals from Nguni and Sotho-Tswana populations (72 from each) KEY

Marker Population Group

x SothoSotho-Tswanax Tswana

x Pedi

o Xhosa

Ngunio Zulu

o Ndebele

o Swati

For all the DNA samples collected

OBJECTIVE 5: Sample Processing

1° PCR

• 150 ng template DNA• M13-tailed STR specific primers

2° PCR

• 1 pg template DNA• Fusion primers with population specific MIDs

Pool• 10.6 pg/uL DNA per person in pool Used for

emPCR

0 - positive control (Subject 0)N - negative control

NGS &Analysis

• GS Junior sequencing NGS• Amplicon Variant Analyzer and Population Statistics

00

300

250

200

M 0 N 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

Subjects 1-18 (Nguni)2° PCR

1. Organising and analysing the data using the GS Amplicon Variant Analyzer

2. Determination of Forensic Parameters• Match Probability (MP)• Power of Exclusion (PE)• Power of Discrimination (PD)

Repeat-Number-Based Typing vs Sequence-Based Typing.

3. Determination of Genotype:Sub-Population Relationship

OBJECTIVE 6: Sample Analysis

Preliminary Results

Total # Read Nguni Sotho-TswanaRaw Data 145 519 97 420 48 099

Trim 124 538 84 268 40 270

Table 2: Number of reads (sequences) following NGS

Example Analysis: D21S1113 unique alleles observed

Deletion? Variant?

Conclusions

Summary of Work2-step PCR system developed which enables the labelling of

a panel of microsatellites with population-specific primers for use in 454 sequencing.

System used to sequence the STRs from 144 donors belonging to either Nguni or Sotho-Tswana population groups, using the Roche GS Junior System.

Analysis of these results is underway.

Potential applications:A novel DNA profiling method that will provide highly

discriminative results which are both comparable and compatible with existing databases.

- individual-specific MIDs as opposed to population-specific MIDs for simultaneous genetic profiling of hundreds of individuals.

Results obtained from the NGS may reveal novel STR variantsPopulation-specific SNP identification Predictive powerMethod can be modified to analyse other regions of DNA

Special thanks to: My Supervisors: Dr Adrienne Edkins and Dr Brendan Wilhelmi Members of Labs 412, 325 and BioBru 454-sequncing guru: Dr Gwynneth Matcher Garry Jevons and David Penkler for assistance with the subject

database Rhodes University Ethics Committee Those who so kindly donated their DNA Henderson Foundation(Rhodes University) Sandisa Imbewu Project (Rhodes University ) National Research Foundation

Acknowledgements

References1. Metzker, M. (2010) Sequencing Technologies – The Next Generation. Nature Reviews: Genetics,

11, pp. 31-462. Jobling, M. a and Gill, P. (2004) Encoded Evidence: Dna in Forensic Analysis. Nature Reviews:

Genetics. 5. 739-7513. Kayser, M. and De Knijff, P., (2011). Improving human forensics through advances in genetics,

genomics and molecular biology. Nature reviews. Genetics, 12(3), pp.179–924. Oberacher, H., Huber, G., Parson, W. Pitterl, F. Niederstatter, H. and Oberacher, H. (2008)

Increased Forensic Efficiency of DNA Fingerprinting Through Simultaneous Resolution of Length and Nucleotide Variability by High Performance Mass Spectrometry. Human Mutation 29(3), pp. 427-432

5. Pitterl, F. Amory, S., Delport, R., Huber, G., Ludes, B., Oberacher, H., Parson, W., Schmidt, K., and Zimmermann, B. (2009) Increasing the discrimination power of forensic STR tests employing high-performance mass spectrometry, as illustrated in indigenous South African and Central Asian populations. International Journal of Legal Medicine

6. Planz, J. V., Budowle, B., Hall, T., Eisenberg, A.J., Sannes-Lowery, K. A., & Hofstadler, S. A. (2009). Enhancing resolution and statistical power by utilizing mass spectrometry for detection of SNPs within the short tandem repeats. Forensic Science International: Genetics Supplement Series, 2(1), pp. 529–531.