Background: In 2018, the Next-Generation Sequencing Committee of SWGDAM queried bioinformatic and statistical interpretation method developers regarding data needs for the development of sequence-based probabilistic genotyping software. Methods: Based on this engagement, a set of 74 mixture samples was conceived and created using 11 single-source samples. The allelic overlap among these samples was evaluated and sample combinations of varying complexity were selected, aiming to represent the variability observed in forensic casework. Results: The samples were distributed into a 96-well plate design containing several features: (1) three-person mixtures of 1% to 5% minor components in triplicate with varying levels of input DNA to provide information on sensitivity and reproducibility, (2) three-person mixtures containing degraded DNA of either only the major contributor or all three contributors, (3) four- and five-person mixtures with varying ratios and donors, (4) a single-source dilution series. Conclusions: Mixture samples were prepared and have been sequenced thus far with three commercially available kits targeting forensic short tandem repeat (STR) and single nucleotide polymorphism (SNP) markers, with FASTQ data files and metadata publicly available at doi.org/10.18434/M32157.

Background/Objectives: Traditionally, Sanger sequencing was used to characterize reference materials and confirm discordant allele calls from different STR typing kits at the National Institute of Standards and Technology (NIST). Sequencing can also identify genomic variations within polymerase chain reaction (PCR) amplicons containing STRs, particularly variants that result in null alleles and alleles that do not migrate within allele sizing bins provided by kit manufacturers. Methods: Sanger methods are low-throughput, time- and labor-intensive, and require additional procedures for analysis of heterozygous alleles. To address these limitations, a quicker, more straightforward protocol that uses next-generation sequencing (NGS) was developed. Results: This research provides the criteria used to individually sequence thirty-five autosomal STR loci, with PCR primer locations chosen to increase amplicon length and maximize the likelihood of detecting variants in the flanking region. The list of targeted sequences, associated primers, and chromosomal coordinates is also included. Conclusions: By applying NGS technology to forensic samples containing variant alleles, additional information can be obtained about their molecular basis, and this information can be published and shared across the forensic community. The development of this protocol can increase awareness and encourage the integration of NGS technology into forensic laboratories to improve forensic DNA typing for human identification.

Obtaining an accepted minimum amount of extracted and purified DNA is critical for generating complete forensic short tandem repeat (STR) profiles. Certain sample types encountered in forensic laboratories may pose challenges in obtaining the desired target amount of extracted DNA, which can be compounded when an extraction yields low recovery, resulting in a loss of the genetic material necessary for downstream forensic DNA typing. While multiple studies have evaluated the impact of extraction chemistries on yield, they often focus on “end‐to‐end” profiling success rather than quantifying the discrete loss of DNA during the extraction step. This study presents a quantitative framework using digital PCR (dPCR) to benchmark extraction efficiency by comparing known amounts of DNA pre‐ and post‐extraction. Extraction efficiency was evaluated using silica spin column and magnetic resin‐based protocols across five input amounts, with three sample types: whole blood, human cells, and pre‐extracted DNA. Results demonstrate significant differences in efficiency between protocols for cellular samples, with both methods exhibiting increased variability at the 1 ng threshold. This data also revealed that previously extracted DNA (SRM 2372a) fails to accurately represent cellular extraction dynamics, serving instead as a control for purification‐related loss (e.g., column retention) rather than lysis efficiency.