Mitochondrial genome sequencing with short overlapping amplicons on MiSeq FGx system
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...
| Main Authors: | , , , |
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| Format: | Article |
| Language: | English |
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Oxford University Press
2021-08-01
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| Series: | Forensic Sciences Research |
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| Online Access: | http://dx.doi.org/10.1080/20961790.2021.1963514 |
| _version_ | 1797766784379518976 |
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| author | Yang Xin Rulin Jia Suhua Zhang Fei Guo |
| author_facet | Yang Xin Rulin Jia Suhua Zhang Fei Guo |
| author_sort | Yang Xin |
| collection | DOAJ |
| description | 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 FGxTM 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. |
| first_indexed | 2024-03-12T20:30:13Z |
| format | Article |
| id | doaj.art-5b073640731f438fb922e3d65ed5c3ac |
| institution | Directory Open Access Journal |
| issn | 2096-1790 2471-1411 |
| language | English |
| last_indexed | 2024-03-12T20:30:13Z |
| publishDate | 2021-08-01 |
| publisher | Oxford University Press |
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| series | Forensic Sciences Research |
| spelling | doaj.art-5b073640731f438fb922e3d65ed5c3ac2023-08-02T00:08:58ZengOxford University PressForensic Sciences Research2096-17902471-14112021-08-010011210.1080/20961790.2021.19635141963514Mitochondrial genome sequencing with short overlapping amplicons on MiSeq FGx systemYang Xin0Rulin Jia1Suhua Zhang2Fei Guo3Forensic Science College, Criminal Investigation Police University of ChinaForensic Science College, Criminal Investigation Police University of ChinaShanghai Key Laboratory of Forensic Medicine, Shanghai Forensic Service Platform, Academy of Forensic Science, Ministry of JusticeShanghai Key Laboratory of Forensic Medicine, Shanghai Forensic Service Platform, Academy of Forensic Science, Ministry of JusticeWith 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 FGxTM 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.http://dx.doi.org/10.1080/20961790.2021.1963514forensic sciencesmassively parallel sequencingmiseq fgxsystemmitochondrial genomexibedegraded samples |
| spellingShingle | Yang Xin Rulin Jia Suhua Zhang Fei Guo Mitochondrial genome sequencing with short overlapping amplicons on MiSeq FGx system Forensic Sciences Research forensic sciences massively parallel sequencing miseq fgx system mitochondrial genome xibe degraded samples |
| title | Mitochondrial genome sequencing with short overlapping amplicons on MiSeq FGx system |
| title_full | Mitochondrial genome sequencing with short overlapping amplicons on MiSeq FGx system |
| title_fullStr | Mitochondrial genome sequencing with short overlapping amplicons on MiSeq FGx system |
| title_full_unstemmed | Mitochondrial genome sequencing with short overlapping amplicons on MiSeq FGx system |
| title_short | Mitochondrial genome sequencing with short overlapping amplicons on MiSeq FGx system |
| title_sort | mitochondrial genome sequencing with short overlapping amplicons on miseq fgx system |
| topic | forensic sciences massively parallel sequencing miseq fgx system mitochondrial genome xibe degraded samples |
| url | http://dx.doi.org/10.1080/20961790.2021.1963514 |
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