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