Forensic DNA analysis is frequently challenged by low-template, degraded, and inhibitor-rich samples, which can compromise the performance of standard short tandem repeat (STR) methods. High-throughput SNP-based technologies, such as Illumina Infinium microarrays, offer advantages in certain applications, particularly complex kinship testing; however, their applicability to forensic casework remains insufficiently characterised. This study evaluates the performance of an Illumina Infinium high-throughput screening (HTS) iSelect Custom microarray panel (Rita version 3) under conditions representative of challenging forensic samples.

DNA samples from the Coriell repository were tested across a range of input quantities (200 ng, 50 ng, and 1 ng) to assess sensitivity. Inhibition studies were performed using three common forensic inhibitors; haematin, humic acid, and collagen across multiple concentrations and DNA inputs. The potential mitigating effect of bovine serum albumin (BSA) was also investigated. Genotyping performance was evaluated using call rate, GenCall metrics, signal intensity, and reproducibility between replicates following analysis with the GenomeStudio 2.0 software.

The custom microarray demonstrated robust performance at reduced DNA inputs, with optimal call rates (>0.99) achieved even at 1 ng. The assay showed high tolerance to haematin, with minimal impact observed up to 2000 µM and partial degradation only at higher concentrations, exceeding the inhibitor resistance commonly reported for STR and sequencing methods. In contrast, humic acid exhibited a DNA input-dependent inhibitory effect, although the addition of BSA significantly improved genotyping success under moderate inhibition. Collagen produced variable and solvent-dependent effects, underscoring the complexity of inhibitor interactions in forensic analyses. Additional investigations are extending this work to degraded DNA samples, where preliminary observations indicate robust performance, and to DNA mixtures, which are notoriously complex for SNP microarray-based methods.

Although generated using the custom Rita array, these findings can be beneficial for other Illumina HTS microarrays, including the Global Screening Array (GSA), extending their relevance to applications such as ancestry inference, kinship analysis, and investigative genetic genealogy. Collectively, these results support the integration of SNP microarray technologies into forensic workflows for the analysis of samples of lower quality than previously expected, leading the way to further studies on real casework samples.

Authors

• Sasitaran Iyavoo (University of Lincoln, United Kingdom)
• Sharlize Pedroza Matute (University of Lincoln, United Kingdom)
• Kiera Turvey (AttoGroup Ltd, United Kingdom)
• Thomas Haizel (AttoGroup Ltd, United Kingdom)