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Comparison between magnetic bead and qPCR library normalisation methods for forensic MPS genotyping.
Bhavik Mehta1,2, Samantha Venables3,4, Paul Roffey3,4
1National Centre for Forensic Studies, Faculty of Education, Science, Technology and Mathematics (ESTeM), University of Canberra, Canberra, Bruce, ACT 2617, Australia. bhavik.mehta@hotmail.com.
International Journal of Legal Medicine
|April 19, 2017
Summary
Massively parallel sequencing (MPS) in forensic science offers comprehensive DNA profiling. Quantitative polymerase chain reaction (qPCR) normalization proved superior to bead normalization for MPS library preparation, with NEBNext® showing higher genotype concordance.
Area of Science:
- Forensic Science
- Genetics
- Molecular Biology
Background:
- Massively parallel sequencing (MPS) is emerging as a powerful tool in forensic science, enabling simultaneous analysis of numerous DNA markers.
- The ForenSeq™ DNA Signature Kit on MiSeq FGx™ platform allows for the profiling of autosomal STRs, X and Y chromosome STRs, identity SNPs, ancestry SNPs, and phenotype SNPs.
Purpose of the Study:
- To compare the manufacturer-recommended magnetic bead normalization method with quantitative polymerase chain reaction (qPCR) for MPS library preparation.
- To evaluate two different qPCR chemistries, KAPA® and NEBNext®, for their performance in MPS library preparation.
- To establish a robust MPS workflow suitable for operational forensic laboratories.
Main Methods:
- Comparison of magnetic bead normalization versus qPCR-based normalization for MPS library preparation.
- Evaluation of KAPA® and NEBNext® qPCR chemistries.
- Assessment of genotype concordance and overall workflow efficiency.
Main Results:
- Quantitative polymerase chain reaction (qPCR) normalization significantly outperformed the magnetic bead normalization method.
- The NEBNext® qPCR chemistry demonstrated higher genotype concordance compared to the KAPA® chemistry.
- An optimized MPS workflow was established for forensic applications.
Conclusions:
- qPCR-based normalization is a more effective method for MPS library preparation in forensic science than bead-based normalization.
- NEBNext® offers superior performance for genotype concordance in this MPS workflow.
- The developed MPS workflow is adaptable for routine use in forensic laboratories.