Sequence-based diversity of 23 autosomal STR loci in Koreans investigated using an in-house massively parallel sequencing panel

Kim, Eun Hye,Lee, Hwan Young,Kwon, So Yeun,Lee, Eun Young,Yang, Woo Ick,Shin, Kyoung-Jin Elsevier 2017 Forensic science international. Genetics Vol.30 No.-

<P><B>Abstract</B></P> <P>As DNA databases continue to grow and international cooperation increases, forensic STR loci have expanded to increase the discriminatory power and inter-database compatibility. Current capillary electrophoresis (CE) and/or massively parallel sequencing (MPS)-based commercial STR analysis systems reflect such changing trends of expanding STR loci. Due to the general gains of larger multiplexing and the detection of sequence variation, the application of MPS technology to STR analysis has further improved discrimination and is expected to aid in mixture interpretation by increasing the effective number of alleles. However, high-throughput analysis has rarely been reported for forensic DNA databasing. In this study, we present the sequencing results from 250 Korean samples at 23 commonly used STR loci (D1S1656, TPOX, D2S441, D2S1338, D3S1358, FGA, CSF1PO, D5S818, D6S1043, D7S820, D8S1179, D10S1248, TH01, vWA, D12S391, D13S317, Penta E, D16S539, D18S51, D19S433, D21S11, Penta D, and D22S1045) using an in-house assay designed for MPS. All amplicons in the multiplex exhibited a size range of 77 to 217 base pairs, and the barcoded library for the MPS run was easily prepared using a PCR-based library preparation method followed by sequencing on a MiSeq System (Illumina). We compared the STR genotyping results with those obtained using CE and scrutinized the sequence variations in both the targeted STR and flanking regions. MPS results of 23 autosomal STRs were 99.97% concordant with those of CE results. D12S391 and D21S11 exhibited, respectively, the highest number of alleles and genotypes by the MPS analysis. Single nucleotide polymorphisms and insertion and deletions (Indels) were observed in the flanking regions of D1S1656, D2S441, D5S818, D7S820, D13S317, D16S539, D21S11, and Penta D. Consequently, an MPS analysis of an expanded set of STRs, as demonstrated in the population statistics of a Korean population, will be of great practical use in forensic genetics.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Twenty three autosomal STRs were investigated in 250 Koreans using an in-house MPS panel. </LI> <LI> MPS results of 23 autosomal STRs were 99.97% concordant with those of CE results. </LI> <LI> D12S391 and D21S11 exhibited, respectively, the highest number of alleles and genotypes by the MPS analysis. </LI> <LI> SNPs and/or Indels were observed in the flanking region of D1S1656, D2S441, D5S818, D7S820, D13S317, D16S539, D21S11, and Penta D. </LI> <LI> The developed MPS panel can be successfully applied to forensic DNA databasing and casework analysis. </LI> </UL> </P>

Sequence Variations of 31 Y-Chromosomal Short Tandem Repeats Analyzed by Massively Parallel Sequencing in Three U.S. Population Groups and Korean Population

Mi Hyeon Moon,Sae Rom Hong,Kyoung-Jin Shin 대한의학회 2022 Journal of Korean medical science Vol.37 No.6

Background: Rapidly mutating (RM) Y-chromosomal short tandem repeats (Y-STRs) have been demonstrated to increase the possibility of distinguishing between male relatives due to a higher mutation rate than conventional Y-STRs. Massively parallel sequencing (MPS) can be useful for forensic DNA typing as it allows the detection of sequence variants of many forensic markers. Here, we present sequence variations of 31 Y-STRs including nine RM Y-STRs (DYF387S1, DYF399S1, DYF404S1, DYS449, DYS518, DYS570, DYS576, DYS612, and DYS627), their frequencies, distribution, and the gain in the number of alleles using MPS. Methods: We constructed a multiplex MPS assay capable of simultaneously amplifying 32 Y-chromosomal markers, producing amplicons ranging from 85–274 bp. Barcoded libraries from 220 unrelated males from four populations—African Americans, Caucasians, Hispanics, and Koreans—were generated via two-step polymerase chain reaction and sequenced on a MiSeq system. Genotype concordance between the capillary electrophoresis (CE) and MPS method and sequence variation of Y-STRs were investigated. Results: In total, 195 alleles were increased by MPS compared to CE-based alleles (261 to 456). The DYS518 marker showed the largest increase due to repeat region variation (a 3.69-fold increase). The highest increase in the number of alleles due to single nucleotide polymorphisms in the flanking region was found in DYF399S1. RM Y-STRs had more diverse sequences than conventional Y-STRs. Furthermore, null alleles were observed in DYS576 due to primer-binding site mutation, and allele drop-outs in DYS449 resulted from low marker coverage of less than the threshold. Conclusion: The results suggest that the expanded and discriminative MPS assay could provide more genetic information for Y-STRs, especially for RM Y-STRs, and could advance male individualization. Compiling sequence-based Y-STR data for worldwide populations would facilitate the application of MPS in the field of forensic genetics and could be applicable in solving male-related forensic cases.

Detection of Innate and Artificial Mitochondrial DNA Heteroplasmy by Massively Parallel Sequencing: Considerations for Analysis

김문영,조소희,이지현,서희진,이숭덕 대한의학회 2018 Journal of Korean medical science Vol.33 No.52

Background: Mitochondrial heteroplasmy, the co-existence of different mitochondrial polymorphisms within an individual, has various forensic and clinical implications. But there is still no guideline on the application of massively parallel sequencing (MPS) in heteroplasmy detection. We present here some critical issues that should be considered in heteroplasmy studies using MPS. Methods: Among five samples with known innate heteroplasmies, two pairs of mixture were generated for artificial heteroplasmies with target minor allele frequencies (MAFs) ranging from 50% to 1%. Each sample was amplified by two-amplicon method and sequenced by Ion Torrent system. The outcomes of two different analysis tools, Torrent Suite Variant Caller (TVC) and mtDNA-Server (mDS), were compared. Results: All the innate heteroplasmies were detected correctly by both analysis tools. Average MAFs of artificial heteroplasmies correlated well to the target values. The detection rates were almost 90% for high-level heteroplasmies, but decreased for low-level heteroplasmies. TVC generally showed lower detection rates than mDS, which seems to be due to their own computation algorithms which drop out some reference-dominant heteroplasmies. Meanwhile, mDS reported several unintended low-level heteroplasmies which were suggested as nuclear mitochondrial DNA sequences. The average coverage depth of each sample placed on the same chip showed considerable variation. The increase of coverage depth had no effect on the detection rates. Conclusion: In addition to the general accuracy of the MPS application on detecting heteroplasmy, our study indicates that the understanding of the nature of mitochondrial DNA and analysis algorithm would be crucial for appropriate interpretation of MPS results.

An XPDL-Based Workflow Control-Structure and Data-Sequence Analyzer

( Kwanghoon Pio Kim ) 한국인터넷정보학회 2019 KSII Transactions on Internet and Information Syst Vol.13 No.3

A workflow process (or business process) management system helps to define, execute, monitor and manage workflow models deployed on a workflow-supported enterprise, and the system is compartmentalized into a modeling subsystem and an enacting subsystem, in general. The modeling subsystem’s functionality is to discover and analyze workflow models via a theoretical modeling methodology like ICN, to graphically define them via a graphical representation notation like BPMN, and to systematically deploy those graphically defined models onto the enacting subsystem by transforming into their textual models represented by a standardized workflow process definition language like XPDL. Before deploying those defined workflow models, it is very important to inspect its syntactical correctness as well as its structural properness to minimize the loss of effectiveness and the depreciation of efficiency in managing the corresponding workflow models. In this paper, we are particularly interested in verifying very large-scale and massively parallel workflow models, and so we need a sophisticated analyzer to automatically analyze those specialized and complex styles of workflow models. One of the sophisticated analyzers devised in this paper is able to analyze not only the structural complexity but also the data-sequence complexity, especially. The structural complexity is based upon combinational usages of those control-structure constructs such as subprocesses, exclusive-OR, parallel-AND and iterative-LOOP primitives with preserving matched pairing and proper nesting properties, whereas the data-sequence complexity is based upon combinational usages of those relevant data repositories such as data definition sequences and data use sequences. Through the devised and implemented analyzer in this paper, we are able eventually to achieve the systematic verifications of the syntactical correctness as well as the effective validation of the structural properness on those complicate and large-scale styles of workflow models. As an experimental study, we apply the implemented analyzer to an exemplary large-scale and massively parallel workflow process model, the Large Bank Transaction Workflow Process Model, and show the structural complexity analysis results via a series of operational screens captured from the implemented analyzer.

고대 유전자에 대한 두 종류의 DNA 분리 방법의 비교 연구: 실리카 현탁액 방법 및 초원심분리 농축 방법

Lee, Eun-jung,Maixner, Frank,Zink, Albert 한국분석과학회 2018 분석과학 Vol.31 No.2

This study compared two methods for preparing ancient DNA (aDNA) for the construction of successful shotgun libraries that may be applied to massive parallel sequencing. For the comparative analysis, the DNA of prehistoric rib samples from Hungary was extracted using either a manually prepared silica suspension or the Amicon Ultracel-15 10K ultracentrifugal device (Millipore). After the extraction of the same amount of bone powder (about 150 mg) from three samples by each method, the amount of extracted double-stranded DNA and the subsequent degree of construction of the shotgun library were analyzed. The Amicon device method was rapid and easier to perform and resulted in an approximately 11-fold higher DNA recovery than that obtained using the silica suspension. The shotgun library constructed using DNA templates prepared by the Amicon device was more successful than that constructed from templates isolated using the silica suspension. The comparative study of these two aDNA extraction methods showed that the Amicon device has the advantages of saving time, process simplicity, and high efficiency. 이 연구에서는 대규모 병렬형 염기서열 분석 (massively parallel sequencing)에 적용할 샷건 라이브러리 (shotgun library)를 성공적으로 제작하기 위해 두 가지 유형의 고대 DNA (ancient DNA, aDNA) 분리 방법을 비교하였다. 헝가리 선사 시대 늑골 뼈 시료로 실리카 현탁액을 이용한 추출법과 Amicon Ultracel-15 10K 초원심 분리 장치(Millipore)를 이용한 추출법을 비교하였다. 약 150 mg의 뼛가루에서 각각의 방법으로 3 회 반복 추출한 후 이중 가닥 DNA (double stranded DNA, ds DNA)의 양을 측정하였다. 초원심분리 농축 방법은 실리카 현탁액을 사용하는 것보다 더 빠르고, 더 쉬운 공정이며 약 11 배 높은 DNA 회수율을 나타냈다. 또한 초원심 분리 장치로 획득한 DNA 주형은 실리카 현탁액으로 획득한 것보다 샷건 라이브러리가 훨씬 성공적으로 만들어졌다. 두 종류의 aDNA 추출 방법을 비교한 본 연구는 Amicon 장치를 사용하는 분리법이 시간의 절약, 단순한 프로세스 및 높은 효율 등의 장점을 지니고 있음을 보여주었다.

Bacterial Communities from the Water Column and the Surface Sediments along a Transect in the East Sea

이정규,최근형 (사)한국해양생명과학회 2021 한국해양생명과학회지 Vol.6 No.1

We determined the composition of water and sediment bacterial assemblages from the East Sea using 16S rRNA gene sequencing. Total bacterial reads were greater in surface waters (<100 m) than in deep seawaters (>500 m) and sediments. However, total OTUs, bacterial diversity, and evenness were greater in deep seawaters than in surface waters with those in the sediment comparable to the deep sea waters. Proteobacteria was the most dominant bacterial phylum comprising 67.3% of the total sequence reads followed by Bacteriodetes (15.8%). Planctomycetes, Verrucomicrobia, and Actinobacteria followed all together consisting of only 8.1% of the total sequence. Candidatus Pelagibacter ubique considered oligotrophic bacteria, and Planctomycetes copiotrophic bacteria showed an opposite distribution in the surface waters, suggesting a potentially direct competition for available resources by these bacteria with different traits. The bacterial community in the warm surface waters were well separated from the other deep cold seawater and sediment samples. The bacteria exclusively associated with deep sea waters was Actinobacteriacea, known to be prevalent in the deep photic zone. The bacterial group Chromatiales and Lutibacter were those exclusively associated with the sediment samples. The overall bacterial community showed similarities in the horizontal rather than vertical direction in the East Sea.

Bacterial Communities from the Water Column and the Surface Sediments along a Transect in the East Sea

Lee, Jeong-Kyu,Choi, Keun-Hyung The Korean Society of Marine Life Science 2021 한국해양생명과학회지 Vol.6 No.1

We determined the composition of water and sediment bacterial assemblages from the East Sea using 16S rRNA gene sequencing. Total bacterial reads were greater in surface waters (<100 m) than in deep seawaters (>500 m) and sediments. However, total OTUs, bacterial diversity, and evenness were greater in deep seawaters than in surface waters with those in the sediment comparable to the deep sea waters. Proteobacteria was the most dominant bacterial phylum comprising 67.3% of the total sequence reads followed by Bacteriodetes (15.8%). Planctomycetes, Verrucomicrobia, and Actinobacteria followed all together consisting of only 8.1% of the total sequence. Candidatus Pelagibacter ubique considered oligotrophic bacteria, and Planctomycetes copiotrophic bacteria showed an opposite distribution in the surface waters, suggesting a potentially direct competition for available resources by these bacteria with different traits. The bacterial community in the warm surface waters were well separated from the other deep cold seawater and sediment samples. The bacteria exclusively associated with deep sea waters was Actinobacteriacea, known to be prevalent in the deep photic zone. The bacterial group Chromatiales and Lutibacter were those exclusively associated with the sediment samples. The overall bacterial community showed similarities in the horizontal rather than vertical direction in the East Sea.

Clinical Impact of Somatic Variants in Homologous Recombination Repair-Related Genes in Ovarian High-Grade Serous Carcinoma

최민철,황소현,김세화,정상근,박현,주원덕,송승훈,이찬,김태헌,강혜윤,안희정 대한암학회 2020 Cancer Research and Treatment Vol.52 No.2

Purpose In this study, we investigated the frequencies of mutations in DNA damage repair genes including BRCA1, BRCA2, homologous recombination genes and TP53 gene in ovarian highgrade serous carcinoma, alongside those of germline and somatic BRCA mutations, with the aim of improving the identification of patients suitable for treatment with poly(ADPribose) polymerase inhibitors. Materials and Methods Tissue samples from 77 Korean patients with ovarian high-grade serous carcinoma were subjected to next-generation sequencing. Pathogenic alterations of 38 DNA damage repair genes and TP53 gene and their relationships with patient survival were examined. Additionally, we analyzed BRCA germline variants in blood samples from 47 of the patients for comparison. Results BRCA1, BRCA2, and TP53 mutations were detected in 28.6%, 5.2%, and 80.5% of the 77 patients, respectively. Alterations in RAD50, ATR, MSH6, MSH2, and FANCA were also identified. At least one mutation in a DNA damage repair gene was detected in 40.3% of patients (31/77). Germline and somatic BRCA mutations were found in 20 of 47 patients (42.6%), and four patients had only somatic mutations without germline mutations (8.5%, 4/47). Patients with DNA damage repair gene alterations with or without TP53mutation, exhibited better disease-free survival than those with TP53 mutation alone. Conclusion DNA damage repair genes were mutated in 40.3% of patients with high-grade serous carcinoma, with somatic BRCAmutations in the absence of germline mutation in 8.5%. Somatic variant examination, along with germline testing of DNA damage repair genes, has potential to detect additional candidates for PARP inhibitor treatment.

Massively Parallel Sequencing of Chikso (Korean Brindle Cattle) to Discover Genome-Wide SNPs and InDels

최정우,이성진,Xiaoping Liao,박새롬,전현정,정원홍,Paul Stothard,Yeon Soo Park,이정구,이경태,김상환,오재돈,김남신,Tae-Hun Kim,Hak Kyo Lee 한국분자세포생물학회 2013 Molecules and cells Vol.36 No.3

Since the completion of the bovine sequencing projects, a substantial number of genetic variations such as single nucleotide polymorphisms have become available across the cattle genome. Recently, cataloguing such genetic variations has been accelerated using massively parallel sequencing technology. However, most of the recent studies have been concentrated on European Bos taurus cattle breeds, resulting in a severe lack of knowledge for valuable native cattle genetic resources worldwide. Here, we present the first whole-genome sequencing results for an endangered Korean native cattle breed, Chikso, using the Illumina HiSeq 2,000 sequencing platform. The genome of a Chikso bull was sequenced to approximately 25.3-fold coverage with 98.8% of the bovine reference genome sequence (UMD 3.1) covered. In total, 5,874,026 single nucleotide polymorphisms and 551,363 insertion/deletions were identified across all 29 autosomes and the X-chromosome, of which 45% and 75% were previously unknown, respectively. Most of the variations (92.7% of single nucleotide polymorphisms and 92.9% of insertion/deletions) were located in intergenic and intron regions. A total of 16,273 single nucleotide polymorphisms causing missense mutations were detected in 7,111 genes throughout the genome, which could potentially contribute to variation in economically important traits in Chikso. This study provides a valuable resource for further investigations of the genetic mechanisms underlying traits of interest in cattle, and for the development of improved genomics-based breeding tools.

Genome Analysis of the Domestic Dog (Korean Jindo) by Massively Parallel Sequencing

Kim, Ryong Nam,Kim, Dae-Soo,Choi, Sang-Haeng,Yoon, Byoung-Ha,Kang, Aram,Nam, Seong-Hyeuk,Kim, Dong-Wook,Kim, Jong-Joo,Ha, Ji-Hong,Toyoda, Atsushi,Fujiyama, Asao,Kim, Aeri,Kim, Min-Young,Park, Kun-Hyan Oxford University Press 2012 DNA research Vol.19 No.3

<P>Although pioneering sequencing projects have shed light on the boxer and poodle genomes, a number of challenges need to be met before the sequencing and annotation of the dog genome can be considered complete. Here, we present the DNA sequence of the Jindo dog genome, sequenced to 45-fold average coverage using Illumina massively parallel sequencing technology. A comparison of the sequence to the reference boxer genome led to the identification of 4 675 437 single nucleotide polymorphisms (SNPs, including 3 346 058 novel SNPs), 71 642 indels and 8131 structural variations. Of these, 339 non-synonymous SNPs and 3 indels are located within coding sequences (CDS). In particular, 3 non-synonymous SNPs and a 26-bp deletion occur in the <I>TCOF1</I> locus, implying that the difference observed in cranial facial morphology between Jindo and boxer dogs might be influenced by those variations. Through the annotation of the Jindo olfactory receptor gene family, we found 2 unique olfactory receptor genes and 236 olfactory receptor genes harbouring non-synonymous homozygous SNPs that are likely to affect smelling capability. In addition, we determined the DNA sequence of the Jindo dog mitochondrial genome and identified Jindo dog-specific mtDNA genotypes. This Jindo genome data upgrade our understanding of dog genomic architecture and will be a very valuable resource for investigating not only dog genetics and genomics but also human and dog disease genetics and comparative genomics.</P>