RETRACTED Mitochondrial Genome Sequencing with Short Overlapping Amplicons on Miseq FGx System

Abstract

With the development and maturation of massively parallel sequencing (MPS) technology, the mitochondrial genome (mitogenome) sequencing is increasingly applied in the forensic field. In this study, we employed the strategy of short overlapping amplicons for the whole mitogenome, library preparation with tagmentation using the Nextera® XT DNA Library Preparation Kit, sequencing on the MiSeq FGx TM Forensic Genomics System and analyzing data using the mitochondrial(mtDNA) MSR Plug-in and the mtDNA Variant Analyzer. A total of 27 libraries and 56 libraries were sequenced in a run using MiSeq Reagent Kit v2 and v3, respectively. Results showed more than 1800 × of averaged depth of coverage (DoC) at each position. Concordant haplotypes of 9947 A and 2800 M were obtained at 32 variants. Cross-reactivity was observed with 1 ng primate DNA and 10 ng non-primate DNA but could be easily distinguished. Full and accurate variants were obtained from at least 50 pg input DNA and from minor contributors between 19:1 and 1:19 mixed ratios with known reference profiles. More than 86% variants were detected from ≥200-bp degraded samples but its haplotype was assigned to more ancestral haplogroup. Further, a total of 3 962 variants were observed at 613 nucleotide positions from 103 Xibe mitogenomes with 25:1 ratio of transitions to transversions. Two new transversions (C13735A and A14755C) and two tri-alleles at nps 9824 and 16092 were identified. There were 103 unique mitogenome haplotypes from 103 Chinese Xibe that were assigned to 79 haplogroups. Haplogroup D was the preponderant top-level haplogroup in Xibe followed by F, B, M, A, N, G, C, Z, Y, HV and J. Random match probability (RMP) and haplotype diversity (HD) of the whole mitogenome was calculated as 0.0097 and 1.0000, respectively. Compared with HVS-I only, RMP decreased 33.56%, while the number of haplotypes and HD increased 15.73% and 0.49%, respectively. Principal component analysis (PCA) showed that Xibe was clustered to East and Southeast Asian. As a whole, this MPS strategy is suitable for the whole mitogenome sequencing especially for degraded samples and can facilitate generating mitogenome data to support the routine application in forensic sciences. EMP00726 is the first whole mitogenome dataset from Xibe contributed to the EMPOP. Supplemental data for this article are available online at.

Whole Mitochondrial Genome Detection and Analysis of Two- to Four-Generation Maternal Pedigrees Using a New Massively Parallel Sequencing Panel

“…The distribution of variants in the mtGenome in this study was similar to that in the North Han Chinese population reported by Zhou et al [ 9 ] and in the Shanghai Han Chinese population reported by Ma et al [ 52 ]. Similar distributions of variants in the control region and coding region were observed in previous studies (72.16% ± 1.10% on average) [ 9 , 10 , 23 , 52 ]. In this study, 100% unique haplotypes were observed in the 10 pedigrees when considering the control region, which suggested the high polymorphism of the control region, even if only 1122 bps of the mtDNA sequence were included ( Figure 6 ).…”

“…In this study, 100% unique haplotypes were observed in the 10 pedigrees when considering the control region, which suggested the high polymorphism of the control region, even if only 1122 bps of the mtDNA sequence were included ( Figure 6 ). Meanwhile, some studies also reported an increase in the power of evidence when comparing the mtGenome with the control region, wherein the haplotype diversity increased by 0.02% to 0.21% and the random match probability decreased by 2.02% to 35% [ 9 , 10 , 11 ]. In this study, relatively high polymorphisms were observed in the coding regions of ATP6, ND5, CYB, ND2, and CO1, where there were nine, nine, eight, seven, and seven types of haplotypes, respectively ( Figure 6 ).…”

“…Additionally, the comparison guidelines were constructed for the control region only [ 8 ]. However, several studies have demonstrated that the variants in the coding region of the mtGenome can improve the discrimination power and haplogroup estimation [ 2 , 9 , 10 , 11 ]. Moreover, limited by the sensitivity of electrophoretograms, a minor allele frequency (MAF) of 15~20% has typically been used for point heteroplasmy (PHP) calls [ 11 , 12 ].…”

“…At present, massively parallel sequencing (MPS), whereby a large number of markers and samples can be detected in one sequencing run [ 13 , 14 ], provides a new platform for mtGenome typing. As one of the first forensic genetic markers evaluated using MPS, mtDNA has been studied in populations worldwide [ 9 , 10 , 11 , 15 , 16 ] and in various tissues [ 17 , 18 , 19 , 20 , 21 ] through a variety of MPS multiplexes of the control region and mtGenome. In addition, the MAF of PHP calls ranges from 1% to 10% [ 22 , 23 , 24 , 25 , 26 ], even dropping to as low as 0.15% [ 27 ].…”

“…Both long-amplicon multiplexes (usually divided into two ~8 kb amplicons or one ~9 kb amplicon plus one ~11 kb amplicon) [ 34 , 35 , 36 ] and short-amplicon multiplexes (e.g., the Precision ID mtDNA Whole Genome Panel) [ 10 , 37 ] have been established for the mtDNA mtGenome. Long amplicons can decrease the risk of contamination of nuclear mitochondrial DNA reads (NUMTs) and amplification failure caused by SNPs in the primer-binding region, while the requirement for the input genome DNA (gDNA) is relatively high (usually 1 ng) [ 34 ].…”