Validation of novel forensic DNA markers using multiplex microhaplotype sequencing. (July 2020)

• Record Type: Journal Article
• Authors: Gandotra, Neeru; Speed, William C.; Qin, Wenyi; Tang, Yishuo; Pakstis, Andrew J.; Kidd, Kenneth K.; Scharfe, Curt
• Is Part Of: Forensic science international. Volume 47(2020)
• Publication Date: 2020-07
• Subjects: Microhaplotype -- Forensics -- Diverse populations -- SNPs -- Massively parallel sequencing -- MPS; Forensic genetics -- Periodicals; Génétique légale -- Périodiques; Forensic genetics; Electronic journals; Periodicals; 614.1
• DOI: 10.1016/j.fsigen.2020.102275
• Languages: English
• ISSNs: 1872-4973

Abstract

Highlights: A novel 90 MH panel was established and 155 unrelated individuals were sequenced. The 90 MH have a high Effective Number of Alleles and yield good ancestry inference. A balance of population-specific MH markers is appropriate for informative panels. Microhaplotype sequencing can uncover many novel SNPs in diverse populations. Web-based tools were developed to support customized and expandable MH analysis. Abstract: Microhaplotypes (MH) are comprised of multiple single nucleotide polymorphisms (SNPs) that are located within 300 bases of genomic sequence. Improved tools are needed to facilitate broader application of microhaplotypes in a diverse range of populations and forensic settings. We designed an assay for multiplex sequencing of 90 microhaplotypes (mMHseq) that include 46 MH loci with high Effective Number of Alleles (Ae ) from previous studies [1 ], and 44 high Ae MH loci containing between four to fourteen SNPs that were identified from the 1000 Genomes (1KG) Project. The unique design of mMHseq integrates a novel method for multiplex amplification from small DNA amounts, and multiplex sequencing of 48 samples in a single MiSeq run to detect all relevant MH variation. Assay performance was evaluated in a cohort of 156 individuals from seven different world populations from Africa, Asia, and Europe. Three of those populations from East Africa (Chagga, Sandawe, and Zaramo) and one from Eastern Europe (Adygei) had sufficient individuals sequenced by the assay to be included in statistical analyses with the 26 1KG populations. For those 30 populations the mean global average Ae was 5.08 (range: 2.7–11.54) and mean informativeness for biogeographic variation (In ) was 0.30 (range: 0.08-0.70). Eighty-five novel SNPs were detected in 58 of the 90 microhaplotypes. Open-source, web-based software was developed to visualize haplotype phase data for each microhaplotype and individual. Our approach for multiplex microhaplotype sequencing can be customized and expanded as novel loci are being discovered.