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NSPlex: an efficient method to analyze non-specific peaks amplified using commercial STR kits

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Abstract

Commercial short tandem repeat (STR) kits exclusively contain human-specific primers; however, various non-human organisms with high homology to the STR kit's primer sequences can cause cross-reactivity. Owing to the proprietary nature of the primers in STR kits, the origins and sequences of most non-specific peaks (NSPs) remain unclear. Such NSPs can complicate data interpretation between the casework and reference samples; thus, we developed “NSPlex”, an efficient method to discover the biological origins of NSPs. We used leftover STR kit amplicons after capillary electrophoresis and performed advanced bioinformatics analyses using next-generation sequencing followed by BLAST nucleotide searches. Using our method, we could successfully identify NSP generated from PCR amplicons of a sample mixture of human DNA and DNA extracted from matcha powder (finely ground powder of green tea leaves and previously known as a potential source of NSP). Our results showed our method is efficient for NSP analysis without the need for the primer information as in commercial STR kits.

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Acknowledgements

We would like to thank Editage (www.editage.com) for the English language editing.

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No funding was received to assist with the preparation of this manuscript.

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Authors and Affiliations

  1. DNA Center Kashiwa Branch, Criminal Identification Division, National Police Agency, 6-3-1 Kashiwanoha, Kashiwa, Chiba, 277-0882, Japan

    Yukinobu Kutsuwada

  2. Division of Human Genetics, Center for Molecular Medicine, Jichi Medical University, Tochigi, Japan

    Yukinobu Kutsuwada, Hidetoshi Tsuda, Kazuhisa Watanabe, Ayumi Matsumoto & Sadahiko Iwamoto

  3. Forensic Science Laboratory, Okayama Prefectural Police Headquarter, Okayama, Japan

    Sho Tomotake

  4. Fourth Biology Section, First Department of Forensic Science, National Research Institute of Police Science, Chiba, Japan

    Natsuko Mizuno

Authors
  1. Yukinobu Kutsuwada
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  2. Sho Tomotake
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  3. Hidetoshi Tsuda
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  4. Kazuhisa Watanabe
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  5. Ayumi Matsumoto
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  6. Sadahiko Iwamoto
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  7. Natsuko Mizuno
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Correspondence to Yukinobu Kutsuwada.

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The original online version of this article was revised: The original article contains a spelling error in author name. The author “Sho Tomoteke” should be corrected to “Sho Tomotake”.

Supplementary Information

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414_2024_3234_MOESM1_ESM.pdf

Suppl. Fig. 1 Graphical scheme of next-generation sequencing fragment preparation amplified by the fluorescent STR kit. A labeled fragment was adenylated at the 3’ end by Taq polymerase followed by end-repair to convert the adenylated fragment into a blunt-end fragment. Double-stranded fragments with unique dual indexes (UDI) were generated by two-step ligation of adapter sequences using separate enzymes. Ligated amplicons were enriched by additional PCR amplification. Finally, the enriched DNA library was created for next-generation sequencing into paired-end reads. P5 and P7 represent platform-specific sequence binding to the flow cell, and i5 and i7 represent sample-specific sequences to identify the sample origin (PDF 28 KB)

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Kutsuwada, Y., Tomotake, S., Tsuda, H. et al. NSPlex: an efficient method to analyze non-specific peaks amplified using commercial STR kits. Int J Legal Med (2024). https://doi.org/10.1007/s00414-024-03234-y

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