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Massively parallel sequencing of the entire control region and targeted coding region SNPs of degraded mtDNA using a simplified library preparation method.

Authors
 Eun Young Lee  ;  Hwan Young Lee  ;  Se Yoon Oh  ;  Sang-Eun Jung  ;  In Seok Yang  ;  Yang-Han Lee  ;  Woo Ick Yang  ;  Kyoung-Jin Shin 
Citation
 FORENSIC SCIENCE INTERNATIONAL-GENETICS, Vol.22 : 37-43, 2016 
Journal Title
 FORENSIC SCIENCE INTERNATIONAL-GENETICS 
ISSN
 1872-4973 
Issue Date
2016
MeSH
Asian Continental Ancestry Group/genetics ; Base Sequence ; DNA Fingerprinting/methods ; DNA Primers ; DNA, Mitochondrial/analysis ; DNA, Mitochondrial/genetics* ; Forensic Genetics/methods ; Genomic Library* ; Haplotypes ; High-Throughput Nucleotide Sequencing/methods* ; Humans ; Multiplex Polymerase Chain Reaction/methods* ; Polymorphism, Single Nucleotide ; Sequence Analysis, DNA
Keywords
Haplogroup ; Library preparation ; Mitochondrial DNA ; Next-generation sequencing
Abstract
The application of next-generation sequencing (NGS) to forensic genetics is being explored by an increasing number of laboratories because of the potential of high-throughput sequencing for recovering genetic information from multiple markers and multiple individuals in a single run. A cumbersome and technically challenging library construction process is required for NGS. In this study, we propose a simplified library preparation method for mitochondrial DNA (mtDNA) analysis that involves two rounds of PCR amplification. In the first-round of multiplex PCR, six fragments covering the entire mtDNA control region and 22 fragments covering interspersed single nucleotide polymorphisms (SNPs) in the coding region that can be used to determine global haplogroups and East Asian haplogroups were amplified using template-specific primers with read sequences. In the following step, indices and platform-specific sequences for the MiSeq(®) system (Illumina) were added by PCR. The barcoded library produced using this simplified workflow was successfully sequenced on the MiSeq system using the MiSeq Reagent Nano Kit v2. A total of 0.4 GB of sequences, 80.6% with base quality of >Q30, were obtained from 12 degraded DNA samples and mapped to the revised Cambridge Reference Sequence (rCRS). A relatively even read count was obtained for all amplicons, with an average coverage of 5200 × and a less than three-fold read count difference between amplicons per sample. Control region sequences were successfully determined, and all samples were assigned to the relevant haplogroups. In addition, enhanced discrimination was observed by adding coding region SNPs to the control region in in silico analysis. Because the developed multiplex PCR system amplifies small-sized amplicons (<250 bp), NGS analysis using the library preparation method described here allows mtDNA analysis using highly degraded DNA samples.
Full Text
http://www.sciencedirect.com/science/article/pii/S187249731630014X
DOI
10.1016/j.fsigen.2016.01.014
Appears in Collections:
1. College of Medicine (의과대학) > Dept. of Forensic Medicine (법의학과) > 1. Journal Papers
1. College of Medicine (의과대학) > Dept. of Pathology (병리학교실) > 1. Journal Papers
Yonsei Authors
Shin, Kyoung Jin(신경진) ORCID logo https://orcid.org/0000-0002-1059-9665
Yang, Woo Ick(양우익) ORCID logo https://orcid.org/0000-0002-6084-5019
Yang, In Seok(양인석) ORCID logo https://orcid.org/0000-0001-5224-2587
Lee, Eun Young(이은영)
Lee, Hwan Young(이환영)
Jung, Sang Eun(정상은)
URI
https://ir.ymlib.yonsei.ac.kr/handle/22282913/146988
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