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NGS for the analysis of microsatellite allele variants in Nguni and Sotho-Tswana populations

NGS for the analysis of microsatellite allele variants in Nguni and Sotho-Tswana populations. Jo-Anne Laurence 1 Brendan Wilhelmi 1 , Adrienne L. Edkins 1, 2 1 Department of Biochemistry and Microbiology, Rhodes University

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NGS for the analysis of microsatellite allele variants in Nguni and Sotho-Tswana populations

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  1. NGS for the analysis of microsatellite allele variants in Nguni and Sotho-Tswana populations Jo-Anne Laurence1 Brendan Wilhelmi1, Adrienne L. Edkins1, 2 1Department of Biochemistry and Microbiology, Rhodes University 2 Biomedical Biotechnology Research Unit (BioBRU), Rhodes University Clearance by the Ethical Standards Committee of Rhodes University (2013Q1-5)

  2. Introduction

  3. The usefulness of DNA “It has long been an axiom of mine that the little things are infinitely the most important” SHERLOCK HOLMES • 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 • 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 • Method of Typing: • Analysis of Short Tandem Repeats (STRs) (Metzker, M. , 2010; Jobling & Gill, 2004 ; Kayser & de Knijff, 2011)

  4. 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 Example of DNA Profiling using STRs • 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? (Kayser and de Knijff, 2011)

  5. Project Aims • To develop a 2-step PCR reaction for the analysis of STRs using Next Generation Sequencing (NGS) • To use this protocol to identify and characterise STR variants, • and to investigate the relationship between specific STR sub-allele frequencies and biogeographic ancestry

  6. OBJECTIVES Methods and Results

  7. OBJECTIVE 1: Identify suitable STR loci • Likely sub-allelic structure • 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

  8. OBJECTIVE 2: Optimise PCR Reactions M13 • Multiplex amplification of STRs using universal (M13)-tailed primers • 1° PCR MULTIPLEX SINGLEPLEX 1° MULTIPLEX 2° MULTIPLEX M 1° Bp 250 200 150 Bp 300 250 200 M 2° Expected size increase of 70 bp • 2° PCR • Addition of fusion primer with population specific MID Fusion primer (Performed on Subject 0) Bp 250 200 150 M1 2 345 6M 1º D21S11 (4) D7S820 (5) vWA (1) D3S1358 (3) D13S317 (6) D2S441 (2)

  9. OBJECTIVE 3: DNA sample Collection • 144 individuals from Nguni and Sotho-Tswana populations (72 from each) • Buccal sample collection • DNA stabilisation and extraction - Isohelix DDK-50 • Quantification of DNA by Nanodrop Isohelix SK1 Swabs

  10. OBJECTIVE 5: Sample Processing • For all the DNA samples collected 00 • 2° PCR • 1° PCR • 150 ng template DNA • M13-tailed STR specific primers Subjects 1-18 (Nguni) 300 250 200 M 0 N 1 2 3 4 5 6 7 8 9 10 11 12 1314 15 16 1718 • 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 • NGS & • Analysis • GS Junior sequencing NGS • Amplicon Variant Analyzer and Population Statistics 0 - positive control (Subject 0) N - negative control

  11. OBJECTIVE 6: Sample Analysis • Organising and analysing the data using the GS Amplicon Variant Analyzer • Determination of Forensic Parameters • Match Probability (MP) • Power of Exclusion (PE) • Power of Discrimination (PD) • Repeat-Number-Based TypingvsSequence-Based Typing. • Determination of Genotype:Sub-Population Relationship

  12. Preliminary Results • Table 2: Number of reads (sequences) following NGS • Example Analysis: D21S11 • 13 unique alleles observed • Variant? • Deletion?

  13. Conclusions Summary of Work • 2-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 variants • Population-specific SNP identification  Predictive power • Method can be modified to analyse other regions of DNA

  14. Acknowledgements 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) • SandisaImbewu Project (Rhodes University ) • National Research Foundation

  15. References • Metzker, M. (2010) Sequencing Technologies – The Next Generation. Nature Reviews: Genetics,11, pp. 31-46 • Jobling, M. a and Gill, P. (2004) Encoded Evidence: Dna in Forensic Analysis. Nature Reviews: Genetics. 5. 739-751 • Kayser, M. and De Knijff, P., (2011). Improving human forensics through advances in genetics, genomics and molecular biology. Nature reviews. Genetics, 12(3), pp.179–92 • 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 • 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 • 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.

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