A SNaPshot of next generation sequencing for forensic SNP analysis

Runa Daniel, Carla Santos, Christopher Phillips, Manuel Fondevila, Roland Van Oorschot, Angel Carracedo, Maria Lareu, Dennis MCNEVIN

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    Abstract

    Forensic phenotyping can provide useful intelligence regarding the biogeographical ancestry (BGA) and externally visible characteristics (EVCs) of the donor of an evidentiary sample. Currently, single nucleotide polymorphism (SNP) based inference of BGA and EVCs is performed most commonly using SNaPshot®, a single base extension (SBE) assay. However, a single SNaPshot multiplex PCR is limited to 30-40 SNPs. Next generation sequencing (NGS) offers the potential to genotype hundreds to thousands of SNPs from multiple samples in a single experimental run. The PCR multiplexes from five SNaPshot assays (SNPforID 52plex, SNPforID 34plex, Eurasiaplex, IrisPlex and an unpublished BGA assay) were applied to three different DNA template amounts (0.1, 0.2 and 0.3 ng) in three samples (9947A and 007 control DNAs and a male donor). The pooled PCR amplicons containing 136 unique SNPs were sequenced using Life Technologies' Ion Torrent™ PGM system. Approximately 72 Mb of sequence was generated from two 10 Mb Ion 314™ v1 chips. Accurate genotypes were readily obtained from all three template amounts. Of a total of 408 genotypes, 395 (97%) were fully concordant with SNaPshot across all three template amounts. Of those genotypes discordant with SNaPshot, six Ion Torrent sequences (1.5%) were fully concordant with Sanger sequencing across the three template amounts. Seven SNPs (1.7%) were either discordant between template amounts or discordant with Sanger sequencing. Sequence coverage observed in the negative control, and, allele coverage variation for heterozygous genotypes highlights the need to establish a threshold for background levels of sequence output and heterozygous balance. This preliminary study of the Ion Torrent PGM system has demonstrated considerable potential for use in forensic DNA analyses as a low to medium throughput NGS platform using established SNaPshot assays.
    Original languageEnglish
    Pages (from-to)50-60
    Number of pages11
    JournalForensic Science International: Genetics
    Volume14
    Issue number4
    DOIs
    Publication statusPublished - 2015

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    Single Nucleotide Polymorphism
    Genotype
    Ions
    Multiplex Polymerase Chain Reaction
    DNA
    Intelligence
    Alleles
    Technology
    Polymerase Chain Reaction

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    Daniel, R., Santos, C., Phillips, C., Fondevila, M., Van Oorschot, R., Carracedo, A., ... MCNEVIN, D. (2015). A SNaPshot of next generation sequencing for forensic SNP analysis. Forensic Science International: Genetics, 14(4), 50-60. https://doi.org/10.1016/j.fsigen.2014.08.013
    Daniel, Runa ; Santos, Carla ; Phillips, Christopher ; Fondevila, Manuel ; Van Oorschot, Roland ; Carracedo, Angel ; Lareu, Maria ; MCNEVIN, Dennis. / A SNaPshot of next generation sequencing for forensic SNP analysis. In: Forensic Science International: Genetics. 2015 ; Vol. 14, No. 4. pp. 50-60.
    @article{611451d0f3434e748bb4af47719c66eb,
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    abstract = "Forensic phenotyping can provide useful intelligence regarding the biogeographical ancestry (BGA) and externally visible characteristics (EVCs) of the donor of an evidentiary sample. Currently, single nucleotide polymorphism (SNP) based inference of BGA and EVCs is performed most commonly using SNaPshot{\circledR}, a single base extension (SBE) assay. However, a single SNaPshot multiplex PCR is limited to 30-40 SNPs. Next generation sequencing (NGS) offers the potential to genotype hundreds to thousands of SNPs from multiple samples in a single experimental run. The PCR multiplexes from five SNaPshot assays (SNPforID 52plex, SNPforID 34plex, Eurasiaplex, IrisPlex and an unpublished BGA assay) were applied to three different DNA template amounts (0.1, 0.2 and 0.3 ng) in three samples (9947A and 007 control DNAs and a male donor). The pooled PCR amplicons containing 136 unique SNPs were sequenced using Life Technologies' Ion Torrent™ PGM system. Approximately 72 Mb of sequence was generated from two 10 Mb Ion 314™ v1 chips. Accurate genotypes were readily obtained from all three template amounts. Of a total of 408 genotypes, 395 (97{\%}) were fully concordant with SNaPshot across all three template amounts. Of those genotypes discordant with SNaPshot, six Ion Torrent sequences (1.5{\%}) were fully concordant with Sanger sequencing across the three template amounts. Seven SNPs (1.7{\%}) were either discordant between template amounts or discordant with Sanger sequencing. Sequence coverage observed in the negative control, and, allele coverage variation for heterozygous genotypes highlights the need to establish a threshold for background levels of sequence output and heterozygous balance. This preliminary study of the Ion Torrent PGM system has demonstrated considerable potential for use in forensic DNA analyses as a low to medium throughput NGS platform using established SNaPshot assays.",
    keywords = "Biogeographical ancestry (BGA), Externally visible characteristics (EVCs), Molecular photofitting, Next generation sequencing (NGS), Single nucleotide polymorphisms (SNPs), SNaPshot",
    author = "Runa Daniel and Carla Santos and Christopher Phillips and Manuel Fondevila and {Van Oorschot}, Roland and Angel Carracedo and Maria Lareu and Dennis MCNEVIN",
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    Daniel, R, Santos, C, Phillips, C, Fondevila, M, Van Oorschot, R, Carracedo, A, Lareu, M & MCNEVIN, D 2015, 'A SNaPshot of next generation sequencing for forensic SNP analysis', Forensic Science International: Genetics, vol. 14, no. 4, pp. 50-60. https://doi.org/10.1016/j.fsigen.2014.08.013

    A SNaPshot of next generation sequencing for forensic SNP analysis. / Daniel, Runa; Santos, Carla; Phillips, Christopher; Fondevila, Manuel; Van Oorschot, Roland; Carracedo, Angel; Lareu, Maria; MCNEVIN, Dennis.

    In: Forensic Science International: Genetics, Vol. 14, No. 4, 2015, p. 50-60.

    Research output: Contribution to journalArticle

    TY - JOUR

    T1 - A SNaPshot of next generation sequencing for forensic SNP analysis

    AU - Daniel, Runa

    AU - Santos, Carla

    AU - Phillips, Christopher

    AU - Fondevila, Manuel

    AU - Van Oorschot, Roland

    AU - Carracedo, Angel

    AU - Lareu, Maria

    AU - MCNEVIN, Dennis

    PY - 2015

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    N2 - Forensic phenotyping can provide useful intelligence regarding the biogeographical ancestry (BGA) and externally visible characteristics (EVCs) of the donor of an evidentiary sample. Currently, single nucleotide polymorphism (SNP) based inference of BGA and EVCs is performed most commonly using SNaPshot®, a single base extension (SBE) assay. However, a single SNaPshot multiplex PCR is limited to 30-40 SNPs. Next generation sequencing (NGS) offers the potential to genotype hundreds to thousands of SNPs from multiple samples in a single experimental run. The PCR multiplexes from five SNaPshot assays (SNPforID 52plex, SNPforID 34plex, Eurasiaplex, IrisPlex and an unpublished BGA assay) were applied to three different DNA template amounts (0.1, 0.2 and 0.3 ng) in three samples (9947A and 007 control DNAs and a male donor). The pooled PCR amplicons containing 136 unique SNPs were sequenced using Life Technologies' Ion Torrent™ PGM system. Approximately 72 Mb of sequence was generated from two 10 Mb Ion 314™ v1 chips. Accurate genotypes were readily obtained from all three template amounts. Of a total of 408 genotypes, 395 (97%) were fully concordant with SNaPshot across all three template amounts. Of those genotypes discordant with SNaPshot, six Ion Torrent sequences (1.5%) were fully concordant with Sanger sequencing across the three template amounts. Seven SNPs (1.7%) were either discordant between template amounts or discordant with Sanger sequencing. Sequence coverage observed in the negative control, and, allele coverage variation for heterozygous genotypes highlights the need to establish a threshold for background levels of sequence output and heterozygous balance. This preliminary study of the Ion Torrent PGM system has demonstrated considerable potential for use in forensic DNA analyses as a low to medium throughput NGS platform using established SNaPshot assays.

    AB - Forensic phenotyping can provide useful intelligence regarding the biogeographical ancestry (BGA) and externally visible characteristics (EVCs) of the donor of an evidentiary sample. Currently, single nucleotide polymorphism (SNP) based inference of BGA and EVCs is performed most commonly using SNaPshot®, a single base extension (SBE) assay. However, a single SNaPshot multiplex PCR is limited to 30-40 SNPs. Next generation sequencing (NGS) offers the potential to genotype hundreds to thousands of SNPs from multiple samples in a single experimental run. The PCR multiplexes from five SNaPshot assays (SNPforID 52plex, SNPforID 34plex, Eurasiaplex, IrisPlex and an unpublished BGA assay) were applied to three different DNA template amounts (0.1, 0.2 and 0.3 ng) in three samples (9947A and 007 control DNAs and a male donor). The pooled PCR amplicons containing 136 unique SNPs were sequenced using Life Technologies' Ion Torrent™ PGM system. Approximately 72 Mb of sequence was generated from two 10 Mb Ion 314™ v1 chips. Accurate genotypes were readily obtained from all three template amounts. Of a total of 408 genotypes, 395 (97%) were fully concordant with SNaPshot across all three template amounts. Of those genotypes discordant with SNaPshot, six Ion Torrent sequences (1.5%) were fully concordant with Sanger sequencing across the three template amounts. Seven SNPs (1.7%) were either discordant between template amounts or discordant with Sanger sequencing. Sequence coverage observed in the negative control, and, allele coverage variation for heterozygous genotypes highlights the need to establish a threshold for background levels of sequence output and heterozygous balance. This preliminary study of the Ion Torrent PGM system has demonstrated considerable potential for use in forensic DNA analyses as a low to medium throughput NGS platform using established SNaPshot assays.

    KW - Biogeographical ancestry (BGA)

    KW - Externally visible characteristics (EVCs)

    KW - Molecular photofitting

    KW - Next generation sequencing (NGS)

    KW - Single nucleotide polymorphisms (SNPs)

    KW - SNaPshot

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    DO - 10.1016/j.fsigen.2014.08.013

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    Daniel R, Santos C, Phillips C, Fondevila M, Van Oorschot R, Carracedo A et al. A SNaPshot of next generation sequencing for forensic SNP analysis. Forensic Science International: Genetics. 2015;14(4):50-60. https://doi.org/10.1016/j.fsigen.2014.08.013