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


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.


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



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)


  1. 1.
    Kayser M, de Knijff P (2011) Improving human forensics through advances in genetics, genomics and molecular biology. Nat Rev Genet 12(3):179–192CrossRefPubMedGoogle Scholar
  2. 2.
    Butler K, Peck M, Hart J, Schanfield M, Podini D (2011) Molecular “eyewitness”: forensic prediction of phenotype and ancestry. Forensic Sci Int Genet Suppl Ser 3:e498–e499CrossRefGoogle Scholar
  3. 3.
    Budowle B, van Daal A (2008) Forensically relevant SNP classes. BioTechniques 44(5):603–610CrossRefPubMedGoogle Scholar
  4. 4.
    Kayser M (2015) Forensic DNA phenotyping: predicting human appearance from crime scene material for investigative purposes. Forensic Sci Int Genet 18:33–48CrossRefPubMedGoogle Scholar
  5. 5.
    Daniel R, Santos C, Phillips C, Fondevila M, van Oorschot RAH, Carracedo Á, Lareu MV, McNevin D (2015) A SNaPshot of next generation sequencing for forensic SNP analysis. Forensic Sci Int Genet 14(0):50–60CrossRefPubMedGoogle Scholar
  6. 6.
    Churchill JD, Schmedes SE, King JL, Budowle B (2016) Evaluation of the Illumina® Beta version ForenSeq™ DNA Signature prep kit for use in genetic profiling. Forensic Sc Int Genet 20:20–29CrossRefGoogle Scholar
  7. 7.
    Børsting C, Morling N (2015) Next generation sequencing and its applications in forensic genetics. Forensic Sci Int Genet 18:78–89CrossRefPubMedGoogle Scholar
  8. 8.
    Silvia AL, Shugarts N, Smith J (2016) A preliminary assessment of the ForenSeq™ FGx System: next generation sequencing of an STR and SNP multiplex. Int J Leg Med 131(1):1–14Google Scholar
  9. 9.
    Mehta B, Daniel R, Phillips C, Doyle S, Elvidge G, McNevin D (2016) Massively parallel sequencing of customised forensically informative SNP panels on the MiSeq. Electrophoresis 37(21):2832–2840CrossRefPubMedGoogle Scholar
  10. 10.
    Børsting C, Fordyce SL, Olofsson J, Mogensen HS, Morling N (2014) Evaluation of the ion Torrent™ HID SNP 169-plex: a SNP typing assay developed for human identification by second generation sequencing. Forensic Sci Int Genet 12(0):144–154CrossRefPubMedGoogle Scholar
  11. 11.
    Jäger AC, Alvarez ML, Davis CP, Guzmán E, Han Y, Way L, Walichiewicz P, Silva D, Pham N, Caves G (2017) Developmental validation of the MiSeq FGx forensic genomics system for targeted next generation sequencing in forensic DNA casework and database laboratories. Forensic Sci Int Genet 28:52–70CrossRefPubMedGoogle Scholar
  12. 12.
    Harris JK, Sahl JW, Castoe TA, Wagner BD, Pollock DD, Spear JR (2010) Comparison of normalization methods for construction of large, multiplex amplicon pools for next-generation sequencing. Appl Environ Microbiol 76(12):3863–3868CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Trujillano D, Weiss ME, Köster J, Papachristos EB, Werber M, Kandaswamy KK, Marais A, Eichler S, Creed J, Baysal E (2015) Validation of a semiconductor next-generation sequencing assay for the clinical genetic screening of CFTR. Molecular genetics & genomic medicine 3(5):396–403CrossRefGoogle Scholar
  14. 14.
    Quail MA, Smith M, Coupland P, Otto TD, Harris SR, Connor TR, Bertoni A, Swerdlow HP, Gu Y (2012) A tale of three next generation sequencing platforms: comparison of Ion Torrent, Pacific Biosciences and Illumina MiSeq sequencers. BMC Genomics 13(1):341CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Illumina (2016) ForenSeqTM Universal Analysis Software guide (v15053876 01)Google Scholar
  16. 16.
    Fordyce SL, Mogensen HS, Børsting C, Lagacé RE, Chang C-W, Rajagopalan N, Morling N (2015) Second-generation sequencing of forensic STRs using the Ion Torrent™ HID STR 10-plex and the Ion PGM™. Forensic Sci Int Genet 14(0):132–140CrossRefPubMedGoogle Scholar
  17. 17.
    Hayden MJ, Wa SLKK (2013) Method of Producing a Normalised Nucleic Acid Library Using Solid State Capture Material. Google Patents, U.S. Patent Application No. 14/430, 786Google Scholar

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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