This manuscript reports Short Tandem Repeat (STR) sequence-based allele frequencies for 1036 samples across 27 autosomal STR loci: D1S1656, TPOX, D2S441, D2S1338, D3S1358, D4S2408, FGA, D5S818, CSF1PO, D6S1043, D7S820, D8S1179, D9S1122, D10S1248, TH01, vWA, D12S391, D13S317, Penta E, D16S539, D17S1301, D18S51, D19S433, D20S482, D21S11, Penta D, and D22S1045. Sequence data were analyzed by two bioinformatic pipelines and all samples have been evaluated for concordance with alleles derived from CE-based analysis at all loci. Each reported sequence includes high-quality flanking sequence and is properly formatted according to the most recent guidance of the International Society for Forensic Genetics. In addition, GenBank accession numbers are reported for each sequence, and associated records are available in the STRSeq BioProject (https://www.ncbi.nlm.nih.gov/bioproject/380127). The D3S1358 locus demonstrates the greatest average increase in heterozygosity across populations (approximately 10 percentage points). Loci demonstrating average increase in heterozygosity from 10 to 5 percentage points include (in descending order) D9S1122, D13S317, D8S1179, D21S11, D5S818, D12S391, and D2S441. The remaining 19 loci each demonstrate less than 5 percentage point increase in average heterozygosity. Discussion includes the utility of this data in understanding traditional CE results, such as informing stutter models and understanding migration challenges, and considerations for population sampling strategies in light of the marked increase in rare alleles for several of the sequence-based STR loci. This NIST 1036 data set is expected to support the implementation of STR sequencing forensic casework by providing high-confidence sequence-based allele frequencies for the same sample set which are already the basis for population statistics in many U.S. forensic laboratories.

This manuscript reports Y-chromosomal short tandem repeat (Y-STR) haplotypes for 1032 male U.S. population samples across 30 Y-STR loci characterized by three capillary electrophoresis (CE) length-based kits (PowerPlex Y23 System, Yfiler Plus PCR Amplification Kit, and Investigator Argus Y-28 QS Kit) and one sequence-based kit (ForenSeq DNA Signature Prep Kit): DYF387S1, DYS19, DYS385 a/b, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS437, DYS438, DYS439, DYS448, DYS449, DYS456, DYS458, DYS460, DYS481, DYS505, DYS518, DYS522, DYS533, DYS549, DYS570, DYS576, DYS612, DYS627, DYS635, DYS643, and Y-GATA-H4. The length-based Y-STR haplotypes include six loci that are not reported in the sequence-based kit (DYS393, DYS449, DYS456, DYS458, DYS518, and DYS627), whereas three loci included in the sequence-based kit are not present in length-based kits (DYS505, DYS522, and DYS612). For the latter, a custom multiplex was used to generate CE length-based data, allowing 1032 samples to be evaluated for concordance across the 30 Y-STR loci included in these four commercial Y-STR typing kits. Discordances between typing methods were analyzed further to assess underlying causes such as primer binding site mutations and flanking region insertions/deletions. Allele-level frequency and statistical information is provided for sequenced loci, excluding the multi-copy loci DYF387S1 and DYS385 a/b, for which locus-specific haplotype-level frequencies are provided instead. The resulting data reveals the degree of information gained through sequencing: 88% of sequenced Y-STR loci contain additional sequence-based alleles compared to length-based data, with the DYS389II locus containing the most additional alleles (51) observed by sequencing. Despite these allelic increases, only minimal improvement was observed in haplotype resolution by sequence, with all four commercial kits providing a similar ability to differentiate length-based haplotypes in this sample set. Finally, a subset of 369 male samples were compared to their corresponding additionally sequenced father samples, revealing the sequence basis for the 50 length-based changes observed, and no additional sequence-based mutations. GenBank accession numbers are reported for each unique sequence, and associated records are available in the STRSeq Y-Chromosomal STR Loci National Center for Biotechnology Information (NCBI) BioProject, accession PRJNA380347. Haplotype data is updated in the Y-STR Haplotype Reference Database (YHRD) for the ‘NIST 1032’ data set to now achieve the level of maximal haplotype of YHRD. All supplementary files including revisions to previously published Y-STR data are available in the NIST Public Data Repository: U.S. population data for human identification markers, DOI 10.18434/t4/1500024.

The US National Institute of Standards and Technology (NIST) analyzed a set of 1036 samples representing four major US population groups (African American, Asian American, Caucasian, and Hispanic) with 94 single nucleotide polymorphisms (SNPs) used for individual identification (iiSNPs). The compact size of iiSNP amplicons compared to short tandem repeat (STR) markers increases the likelihood of successful amplification with degraded DNA samples. Allele frequencies and relevant forensic statistics were calculated for each population group as well as the aggregate population sample. Examination of sequence data in the regions flanking the targeted SNPs identified additional variants, which can be combined with the target SNPs to form microhaplotypes (multiple phased SNPs within a short-read sequence). Comparison of iiSNP performance with and without flanking SNP variation identified four amplicons containing microhaplotypes with observed heterozygosity increases of greater than 15% over the targeted SNP alone. For this set of 1036 samples, comparison of average match probabilities from iiSNPs with the 20 CODIS core STR markers yielded an estimate of 1.7 × 10⁻³⁸ for iiSNPs (assuming independence between all 94 SNPs), which was four orders of magnitude lower (more discriminating) than STRs where internal sequence variation was considered, and 10 orders of magnitude lower than STRs using established capillary electrophoresis length-based genotypes.

In response to the COVID-19 pandemic, the National Institute of Standards and Technology released a synthetic RNA material for SARS-CoV-2 in June 2020. The goal was to rapidly produce a material to support molecular diagnostic testing applications. This material, referred to as Research Grade Test Material 10169, was shipped free of charge to laboratories across the globe to provide a non-hazardous material for assay development and assay calibration. The material consisted of two unique regions of the SARS-CoV-2 genome approximately 4 kb nucleotides in length. The concentration of each synthetic fragment was measured using RT-dPCR methods and confirmed to be compatible with RT-qPCR methods. In this report, the preparation, stability, and limitations of this material are described.

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.