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International Journal of Legal Medicine

, Volume 132, Issue 1, pp 125–132 | Cite as

Comparison between magnetic bead and qPCR library normalisation methods for forensic MPS genotyping

  • Bhavik MehtaEmail author
  • Samantha Venables
  • Paul Roffey
Methods Paper

Abstract

Massively parallel sequencing (MPS) is fast approaching operational use in forensic science, with the capability to analyse hundreds of DNA identity and DNA intelligence markers in multiple samples simultaneously. The ForenSeq™ DNA Signature Kit on MiSeq FGx™ (Illumina) workflow can provide profiles for autosomal short tandem repeats (STRs), X chromosome and Y chromosome STRs, identity single nucleotide polymorphisms (SNPs), biogeographical ancestry SNPs and phenotype (eye and hair colour) SNPs from a sample. The library preparation procedure involves a series of steps including target amplification, library purification and library normalisation. This study highlights the comparison between the manufacturer recommended magnetic bead normalisation and quantitative polymerase chain reaction (qPCR) methods. Furthermore, two qPCR chemistries, KAPA® (KAPA Biosystems) and NEBNext® (New England Bio Inc.), have also been compared. The qPCR outperformed the bead normalisation method, while the NEBNext® kit obtained higher genotype concordance than KAPA®. The study also established an MPS workflow that can be utilised in any operational forensic laboratory.

Keywords

Forensic DNA profiling Next generation sequencing (NGS) Massively parallel sequencing (MPS) Illumina MiSeq FGx Library normalisation Quantitative polymerase chain reaction (qPCR) 

Notes

Acknowledgements

The authors gratefully acknowledge funding support from the Specialist Operations- Forensics, Australian Federal Police. We would also like to acknowledge Dr. Eric Wenger and Slazana Ristveska from Specialist Operations—Forensics, Australian Federal Police for their consultation support.

Compliance with ethical standards

Conflict of interest

The authors declared that they have no conflict of interest.

Supplementary material

414_2017_1591_MOESM1_ESM.xlsx (14 kb)
ESM 1 (XLSX 14 kb)
414_2017_1591_MOESM2_ESM.docx (18 kb)
Supplementary Table 1 The total reads in each run. (DOCX 18 kb)
414_2017_1591_MOESM3_ESM.docx (20 kb)
Supplementary Table 2 Genetic markers with low genotype concordance; concordance of >50% is indicated in bold. (DOCX 19 kb)

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Copyright information

© Springer-Verlag Berlin Heidelberg 2017

Authors and Affiliations

  • Bhavik Mehta
    • 1
    • 2
    Email author
  • Samantha Venables
    • 1
    • 2
  • Paul Roffey
    • 1
    • 2
  1. 1.National Centre for Forensic Studies, Faculty of Education, Science, Technology and Mathematics (ESTeM)University of CanberraBruceAustralia
  2. 2.Specialist Operations – Forensics, Australian Federal PoliceMajuraAustralia

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