Introduction

The implementation of MPS technology in forensic genetics has enabled significant improvements in DNA analysis; however, workflows still generally include a quantification step. Eliminating this step could simplify the process, reduce turnaround time, and lower costs. This study evaluated the feasibility of using the ForenSeq MainstAY kit under direct analysis conditions without prior DNA quantification, with the aim of validating its robustness and reliability.

Methodology

A validation study was performed using the ForenSeq MainstAY kit (Qiagen) on reference samples (XX and XY) obtained from buccal swabs, analyzed directly after DNA extraction without prior quantification. Following a series of serial dilution experiments, an input volume of 1 µL of DNA for PCR was established. Libraries were prepared and sequenced on the MiSeq FGx™ platform using a Micro Flow Cell, and data analysis was performed with Universal Analysis Software (UAS).

Quality parameters, percentage of typed loci, and genotype concordance were evaluated across multiple runs performed on different days and by different analysts to assess method repeatability and reproducibility. Positive controls and blanks were included in all runs.

Results

The results demonstrated high method robustness, with complete profiles obtained (27 autosomal STRs and 25 Y-STRs in males), showing 100% locus recovery and full concordance with expected genotypes, except for two replicates that showed deviations.

The method enabled successful amplification and sequencing of 27 autosomal STR loci and 25 Y-chromosome STR loci regardless of the initial DNA concentration. No significant differences in data quality were observed between workflows with and without prior DNA quantification.

Conclusions

The use of the ForenSeq MainstAY kit without prior DNA quantification represents a reliable and robust alternative for routine forensic analysis of standard samples. Eliminating this step simplifies the workflow, reduces processing time, and lowers costs without compromising result quality.

The high reproducibility and concordance observed support its implementation in routine laboratory workflows, particularly in settings where operational efficiency is critical. This approach is applicable to forensic laboratories using MPS technology and contributes to the optimization of analytical processes.

Authors

• Marcos Merchán Valdivia (Citogen S.L.U., Spain)
• Alicia Moré Agudo (Citogen S.L.U., Spain)
• Sandra Pérez Pérez (Citogen S.L.U., Spain)
• Amaya Gorostiza (Citogen S.L.U., Spain)
• Isabel Navarro Vera (Citogen S.L.U., Spain)
• Alba Hernández Maza (Citogen S.L.U., Spain)