Assessing a High Throughput SNP Assay for Closed Scenario Kinship Investigation

Published

May 14, 2025

Author(s)

Kevin Kiesler, Carolyn Steffen, Lauren Mullen, Peter Vallone

Abstract

Researchers at the U.S. National Institute of Standards and Technology (NIST) have conducted experiments using the ForenSeq Kintelligence-HT kit, a highly multiplexed single nucleotide polymorphism (SNP) panel intended for use in disaster victim identification scenarios. The kit works with the MiSeq FGx DNA Sequencing System to assay more than 10,000 SNP loci, with 9,867 SNPs for the primary task of kinship estimation, and 106 X-chromosome SNPs, 85 Y-chromosome SNPs, 56 ancestry informative SNPs (aiSNPs), 24 phenotype informative SNPs (piSNPs), and 94 individual identification SNPs (iiSNPs) for additional functionality. The system includes a dedicated Universal Analysis Software, which offers a local server and an offline database for sample comparison and kinship calculations. The Kintelligence-HT kit is designed to analyze up to 36 high-quality antemortem reference samples or 12 postmortem-type samples per sequencing flowcell, providing throughput needed for disaster scenarios. NIST experimental samples included parent-parent-child trios (n = 2) and pairs of alleged brothers (n = 4) as well as degraded DNA with degradation index ranging from 2.7 to 43.5, and low-input DNA of 100 pg and 500 pg. UAS (v2.6) kinship calculations reflected the relationships between first-degree relatives. Analysis of challenging degraded DNA yielded a slightly lower average SNP locus call rate of 95.5 % (± 1.8 %), relative to non-degraded samples (97.9 % ± 0.3 %) in the same sequencing run. Low DNA-input samples had lower average SNP locus call rates at 100 pg input (84.8 % ± 3.1 %) than samples run with 500 pg input (93.6 % ± 6.5 %) or 1000 pg input (99.3 % ± 0.2 %). Challenging degraded or low input samples were successfully searched in the UAS 2.6 database for kinship analysis. Multiplexing 36 samples on a single flowcell run resulted in an average SNP locus call rate of 88.7 % (± 4.5 %) for kinship markers. Lower SNP call rates may create uncertainty in kinship assessment by suppressing the estimate of shared DNA segments.

Proceedings Title

Proceedings of the 30th Congress of the International Society for Forensic Genetics

Conference Dates

September 9-13, 2024

Conference Location

Santiago De Compostela, ES

Conference Title

30th Congress of the International Society for Forensic Genetics

Pub Type

Conferences

Download Paper

https://doi.org/10.15304/cc.2025.1869

Local Download

Keywords

Single Nucleotide Polymorphism, Disaster Victim Identification, Kinship.

Citation

Kiesler, K. , Steffen, C. , Mullen, L. and Vallone, P. (2025), Assessing a High Throughput SNP Assay for Closed Scenario Kinship Investigation, Proceedings of the 30th Congress of the International Society for Forensic Genetics, Santiago De Compostela, ES, [online], https://doi.org/10.15304/cc.2025.1869, https://tsapps.nist.gov/publication/get_pdf.cfm?pub_id=958805 (Accessed September 15, 2026)

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