Forensic Application of SNP-based Resequencing Array for Individual Identification

Introduction

  • Single Nucleotide Polymorphism (SNP) methodologies gaining attention in forensics.

  • Compared to Short Tandem Repeats (STR), SNPs have lower mutation rates and provide information on race and phenotypes.

  • Biallelic characteristics of SNPs allow for automated allele calling systems.

  • Suitable for analyzing small or degraded forensic samples.

  • Limitations include a small number of SNPs that can be analyzed simultaneously and a lack of statistical analysis methods.

Background and Significance of SNPs

  • SNPs offer a promising alternative to STR for forensic identification.

  • Key advantages include:

    • Lower mutation rates provide more stable genetic markers.

    • Capable of retrieving data from highly degraded DNA.

    • Automation potential enhances speed and accuracy of results.

  • Need for ongoing research to address limitations in SNP typing and statistical methodologies.

Analysis Methods and Techniques

Types of SNP Identification Methods

  1. Direct discrimination strategies:

    • Hybridization, primer extension, enzymatic cleavage, and ligation.

  2. Instrumental identification approaches:

    • SNaPshot, microarray technologies, and mass spectrometry.

Factors Affecting SNP Detection

  • Accuracy and sensitivity of different platforms.

  • Number of SNPs detectable.

  • Potential for automation.

SNP Selection and Testing

  • Study focused on a SNP-based microarray system (AccuID).

  • Developed to analyze 169 SNP markers selected from the 1000 genomes database.

  • Criteria for marker selection:

    • Fixation index (Fst) < 0.04 to maximize discrimination power.

    • Linkage disequilibrium (LD) R2 value < 0.3.

    • Minor allele frequency (MAF) between 0.4 – 0.5.

  • Results indicated over 120 SNPs successfully typed from degraded samples.

Methods

Sample Preparation

  • Human genomic DNA samples were obtained for testing.

  • Samples included various DNA types like femur bones, hair, and buccal swabs.

  • Specific DNA extraction methods employed:

    • Phenol/chloroform for bone samples.

    • QIAamp DNA Investigator Kit for other samples.

Multiplex PCR Protocol

  • Multiplex amplification carried out using 169 pooled primer sets.

  • Thermal cycling parameters outlined to ensure successful amplification.

Chip Array Protocol

  • Utilized GeneChip CustomSeq Resequencing array for analysis.

  • Hybridization, staining, and scanning steps followed standard manufacturer protocols.

Results

Accuracy and Sensitivity Tests

  • Concordance Test Results:

    • 144 out of 169 SNPs matched between sequencing and AccuID with 100% concordance.

  • Sensitivity Test:

    • Success in typing SNP markers with as low as 0.01 ng of DNA at a 76.5% typing rate.

Degradation Studies

  • Evaluated typing success with artificially degraded DNA.

    • Success rates remained high even with significant degradation, showcasing resilience of SNP methodology.

Applications and Further Development

  • Demonstrated capability for forensic applications, especially in harsh sample conditions.

  • Potential for expanding array to include more informative markers, such as phenotype predicting markers and lineage markers.

  • Future directions include increasing the marker count for broader forensic applications and improving automation.

Conclusions

  • SNP analysis demonstrates significant promise for forensic identification, particularly in degraded samples.

  • Ongoing research and technology improvements expected to enhance analysis accuracy and usability in forensic science.

  • The results support SNP methods as competitive alternatives to traditional STR systems for forensic applications.