An alternate workflow for preparing Precision ID Ancestry and Identity Panel libraries for Illumina sequencing

Area of Science:

• Forensic genetics
• Molecular biology
• Next-generation sequencing (NGS)

Background:

• Single nucleotide polymorphisms (SNPs) offer advantages over short tandem repeats (STRs) in forensic science, including smaller amplicon sizes and predictive capabilities.
• Commercial workflows exist for SNP genotyping, but an alternative using Illumina chemistry was explored.

Purpose of the Study:

• To develop and validate an alternative workflow for genotyping forensically relevant SNPs using Illumina sequencing chemistry.
• To optimize the workflow for compatibility with diverse sample types, including casework samples.

Main Methods:

• Commercial genomic DNA was amplified using different uracil-tolerant polymerase master mixes.
• Amplicons were prepared into libraries and sequenced on an Illumina MiniSeq system.
• Reads were analyzed using a published pipeline to determine genotypes and read depth.

Main Results:

• Phusion U Multiplex PCR Master Mix demonstrated superior performance in genotyping the targeted SNPs (P <0.0001).
• The optimized workflow successfully genotyped 93.9% of SNPs across commercial DNA, buccal swabs, and environmental samples.
• The workflow showed high compatibility with diverse sample matrices.

Conclusions:

• A robust and straightforward workflow for SNP genotyping using Illumina chemistry was developed.
• This alternative method is suitable for forensic laboratories processing both reference and casework samples.
• The workflow enhances the utility of SNPs in forensic investigations.