저자 아무 승인 있다.

<ol>
	<li>Aronson, S. J., Rehm, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Building+the+foundation+for+genomics+in+precision+medicine.">Building the foundation for genomics in precision medicine.</a> Nature. 526 (7573), 336-342 (2015).</li><li>Ball, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Designing+a+GWAS:+power,+sample+size,+and+data+structure.">Designing a GWAS: power, sample size, and data structure.</a> Genome-Wide Association Studies and Genomic Prediction. , 37-98 (2013).</li><li>Kwon, J. M., Goate, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+candidate+gene+approach.">The candidate gene approach.</a> Alcohol research and health. 24 (3), 164-168 (2000).</li><li>Oeth, P., Mistro, G. 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Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pretreatment+epidermal+growth+factor+receptor+(EGFR)+T790M+mutation+predicts+shorter+EGFR+tyrosine+kinase+inhibitor+response+duration+in+patients+with+non-small-cell+lung+cancer.">Pretreatment epidermal growth factor receptor (EGFR) T790M mutation predicts shorter EGFR tyrosine kinase inhibitor response duration in patients with non-small-cell lung cancer.</a> Journal of clinical oncology. 30 (4), 433-440 (2012).</li><li>Singhal, N., Kumar, M., Kanaujia, P. K., Virdi, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MALDI-TOF+mass+spectrometry:+an+emerging+technology+for+microbial+identification+and+diagnosis.">MALDI-TOF mass spectrometry: an emerging technology for microbial identification and diagnosis.</a> Frontiers in microbiology. 6, 791 (2015).</li><li>Cricca, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+genotyping+of+high-risk+Human+Papillomavirus+by+MALDI-TOF+Mass+Spectrometry-based+method.">High-throughput genotyping of high-risk Human Papillomavirus by MALDI-TOF Mass Spectrometry-based method.</a> New Microbiologica. 38 (2), 211-223 (2015).</li><li>Ashley, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+Variation+at+the+Th2+Immune+Gene+IL13+is+Associated+with+IgE-mediated+Paediatric+Food+Allergy.">Genetic Variation at the Th2 Immune Gene IL13 is Associated with IgE-mediated Paediatric Food Allergy.</a> Clinical &amp; Experimental Allergy. 47 (8), 1032-1037 (2017).</li><li>Bousman, C. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+NRG1+and+DAOA+genetic+variation+on+transition+to+psychosis+in+individuals+at+ultra-high+risk+for+psychosis.">Effects of NRG1 and DAOA genetic variation on transition to psychosis in individuals at ultra-high risk for psychosis.</a> Translational psychiatry. 3 (4), e251 (2013).</li><li>Moffatt, M. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+large-scale,+consortium-based+genomewide+association+study+of+asthma.">A large-scale, consortium-based genomewide association study of asthma.</a> New England Journal of Medicine. 363 (13), 1211-1221 (2010).</li><li>Granada, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+genome-wide+association+study+of+plasma+total+IgE+concentrations+in+the+Framingham+Heart+Study.">A genome-wide association study of plasma total IgE concentrations in the Framingham Heart Study.</a> Journal of Allergy and Clinical Immunology. 129 (3), 840-845 (2012).</li><li>Oeth, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=iPLEX+assay:+Increased+plexing+efficiency+and+flexibility+for+MassARRAY+system+through+single+base+primer+extension+with+mass-modified+terminators.">iPLEX assay: Increased plexing efficiency and flexibility for MassARRAY system through single base primer extension with mass-modified terminators.</a> Sequenom application note. 27, (2005).</li><li>Ellis, J. A., Ong, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+MassARRAY+System+for+Targeted+SNP+Genotyping.">The MassARRAY System for Targeted SNP Genotyping.</a> Genotyping: Methods and Protocols. , 77-94 (2017).</li><li>Johnson, A. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SNAP:+A+web-based+tool+for+identification+and+annotation+of+proxy+SNPs+using+HapMap.">SNAP: A web-based tool for identification and annotation of proxy SNPs using HapMap.</a> Bioinformatics. 24 (24), 2938-2939 (2008).</li></ol>

저자는 공개 없다.

여기, 다중화 된 유전형 질량 분석을 사용 하 여 방법을 설명 합니다. 대표 결과 MALDI TOF 질량 분석4 분석 결과 화학 재료의 표13에 나열와 결합 하는 PCR를 사용 하 여 생성 되었다. 이 플랫폼으로 실험실에서 총 1,255 개인 40 h 이내 9 Snp에 대 한 11,295 genotypes 생성 했습니다.
우리는 생성 SNP 후보 유전자 IL13 발견 임상 코 호트 ?...

인간의 복잡 한 질병에 유전 이체 질병 민감성에 기여 하 고 이러한 변형 측정 이해 병 인, 고 위험 환자 그룹, 및 치료 조치를 식별 하는 데 유용 있을 수 있습니다. 실제로, 정밀 의학의 약속은 환자 서브 그룹1을 식별 하는 게놈 정보를 이용 하 여 의존 합니다. 불행히도, 공간 내에 복잡 한 질병 생물학, 어디 질병 고기 상당한 유전이 성분, 낮은 penetrance, 가변 표현력에 의해 underpinned 아 르, 일대 소설 식별을 게놈 넓은 접근에 대 한 요구 사항 크기 후보 들은 엄청나게 큰2입니다. 또한, 대상된 후보 유전자 접근 질병 병 인3특정 유전자/경로 대 한 선험적 가설으로 시작합니다. 경로 분석 도구 일반적으로 탐구 될 수많은 후보 경로 생성 한 확인된 대상 loci의 이상 조사 하는 데 사용 됩니다. 여기 다중화 유전형 접근 인간의 코 호트 연구4에 적합 한 분석 결과 함께 Snp의 수백 수만의 조사에 대 한 수 있도록 설명 합니다. 이 방식은 상대적으로 높은 통해-넣어, 수백 수 소설 발견 연구에 대 한 genotyped DNA 샘플의 수천 및 특정 경로 조사를 허용 합니다. 여기에 설명 된 메서드는 유용 식별 위험 대립 유전자 및 임상 특성의 그들의 협회에 대 한 상대적으로 신속 하 고 저렴 한 방식으로 합니다. 이 플랫폼은 미생물 감염7 및 인간 유 두 종 바이러스8등에 대 한 검사 및 진단 목적5,6, 최근, 매우 유리한 되었습니다.
이 프로토콜은 조사, 즉유전자의 세트의 선택과 시작., 일반적으로 문학 검색, 또는 질병 과정;에 참여에 대 한 선험적 가설을 통해 결정 대상 지역 또는 아마도 검색 게놈 넓은 협회 (GWA) 연구의 주요한 연결으로 복제에 대 한 선택. 유전자 집합에서 연구원 태그 Snp의 세련 된 목록을 선택 합니다. 즉, 결합 불균형 (LD), 또는 지역에서 변종 사이 상호 관계는 haplotype 이라고 높은 LD에 Snp의 그룹에 대 한 대표 '태그 SNP' 식별 하 데 사용 됩니다. 지역의 높은 LD는 유전형 한 SNP는 haplotype에서 모든 Snp에 변화를 표현 하기 위해 충분 한 Snp는 함께 상속 자주는 것을 의미 합니다. 또는, 많은 지역, 예를 들어에서 Snp의 최종 목록에 다음과 같은 경우., GWA 연구에 대 한 복제,이 프로세스 해야 하지. 멀티플렉스 유전형 분석 결과 다음 설계 되어야 합니다 이러한 목표 주위는 증폭 뇌관 연장 뇌관의 질량 다른의 고 해석할 생산 제품 질량 스펙트럼. 이 매개 변수는 멀티플렉스 유전형 분석 결과 디자인 도구에 의해 쉽게 구현 됩니다. 이 디자인에서 앞으로 역 뇌관 관심의 마커를 대상 하는 SNP를 포함 하는 시퀀스를 증폭에 사용 됩니다. 확장 뇌관은 Snp에 직접 인접 연결 하 고 단일, 대량 수정, '터미네이터' 기반은 SNP에 보완을 추가 됩니다. 터미네이터 자료 추가 DNA의 확장을 방지합니다. 기지의 대량 수정 파편을 질량 분석에 의해 감지 되는 단일 자료에 의해 다른 수 있습니다. 유전형 화학을 포함 하는 접시 측정 질량 분석 플랫폼에 대 한 칩에 적용 됩니다. 시스템에 의해 감지 원시 유전형 호출에 적절 한 품질 제어를 적용 한 후 데이터는 수출 고 연관 질병 고기에 대 한 테스트에 통계 분석을 위해 사용 될 수 있습니다.

<table><tbody><tr><td>Genomic DNA&amp;nbsp;</td><td>-</td><td>-</td><td>1 &amp;mu;L at a concentration of 5-10 ng/&amp;mu;L</td></tr><tr><td>Primers: forward and reverse amplification and extension</td><td>IDT</td><td>-</td><td>see manuscript section 1.2.1 on design of primers</td></tr><tr><td>Deionized water&amp;nbsp;</td><td>E.g. Milli-Q water&amp;nbsp;</td><td>-</td><td>deionized with 18.2 M&amp;Omega;.cm resistivity</td></tr><tr><td>Genotyping reagent kit. iPLEX Gold Chemistry reagent set&amp;nbsp;</td><td>Agena Bioscience</td><td>#10148-2</td><td>includes all reagents for reactions in 2.2.1, 2.3.1 and 2.4.2 , chip and resin</td></tr><tr><td>PCR plates (384-well)</td><td>Abgene</td><td>#ABGAB-1384</td><td>For the MassARRAY system plates by Abgene are compatible</td></tr><tr><td>Micropipettes</td><td></td><td></td><td>single and 8-channel</td></tr><tr><td>Centrifuge&amp;nbsp;</td><td></td><td></td><td>compatible with 384-well plates</td></tr><tr><td>Thermocycler</td><td></td><td></td><td>compatible with PCR programs as detailed in 2.2.4, 2.3.2 and 2.4.3</td></tr><tr><td>Dimple resin plate&amp;nbsp;</td><td>Agena Bioscience</td><td></td><td>6mg, 384-well</td></tr><tr><td>Plate rotator&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>MassARRAY Analyzer 4 System</td><td>Agena Biosciences</td><td></td><td>MALDI-TOF (matrix-assisted laser desorption/ionization &amp;ndash; time of flight) Mass Spectrometer.</td></tr><tr><td>RS1000 Nanodispenser</td><td>Agena Biosciences</td><td></td><td></td></tr><tr><td>Assay Design Suite</td><td>Agena Biosciences</td><td></td><td>Tool used to design the multiplex genotyping assays</td></tr><tr><td>Hot Start Taq</td><td></td><td></td><td>DNA polymerase enzyme&amp;nbsp;</td></tr><tr><td>Resin&amp;nbsp;</td><td>Agena Biosciences</td><td></td><td>Supplied with iPLEX kit</td></tr></tbody></table>

candidate gene testing in clinical cohort studies with multiplexed genotyping and mass spectrometry

복잡 한 질병 종종 질병 민감성에 기여 하는 여러 일반적인 유전자 변종에 의해 underpinned 아 르. 여기, 우리는 질량 분석, 있는 멀티플렉스 유전형 분석 결과 사용 하 여 임상 동료에 있는 유전자 통로 협회 조사 비용 효율적인 태그 단일 염기 다형성 (SNP) 접근을 설명 합니다. 우리는 예를 들어 음식 알레르기 후보 소재 시 Interleukin13 (IL13)를 조사합니다. 이 메서드는 효율적으로 지역 내의 공유 결합 불균형 (LD)을 이용 하 여 범위를 최대화 합니다. 선택 된 LD Snp는 다음에 설계 다중화 분석 결과 40 다른 Snp 동시에 분석할 수 있도록 비용 효율성을 향상. 연쇄 반응 (PCR) 단일 뉴클레오티드 확장명 대상 loci를 증폭 하는 사용 되 고 있는 amplicons 다음 매트릭스 보조 레이저 탈 착/이온화 시간의 flight(MALDI-TOF) 질량 분석을 사용 하 여 측정 됩니다. 원시 출력 소프트웨어, 엄격한 품질 관리 정 및 컷-오프를 사용 하 여 호출 하는 유전자 형을 분석 하 고 높은 확률 genotypes 되 고 데이터 분석에 대 한 출력.

위에서 설명한 프로토콜, genotyped 우리는 Th2 면역 유전자 IL13 의 음식 알레르기의 경우 및 컨트롤9일대에 걸쳐 Snp를 태그. 우리 음식 알레르기 위험을 증가 하는 관심의 영역 내에 유전자 변형 여부를 테스트 하는 조상과 다른 잠재적인 covariates에 대 한 조정 하는 로지스틱 회귀 분석을 적용. 표 10 9 한 변형 rs1295686 도전...

Watch this Scientific Journal Video about Candidate Gene Testing in Clinical Cohort Studies with Multiplexed Genotyping and Mass Spectrometry at JoVE.com

Candidate Gene Testing in Clinical Cohort Studies with Multiplexed Genotyping and Mass Spectrometry

후보 유전자 다중화 유전형과 질량 분석 임상 코 호트 연구에서 테스트

복잡 한 인간의 질병에 기여 하는 유전 이체의 소설 메커니즘을 식별 수 있습니다. 여기, 후보 유전자 또는 유전자 통로 분석 낮은 비용 범위를 극대화 하 고 코 호트 기반 연구 의무가 멀티플렉스 유전형 접근 방식을 설명 합니다.

Complex diseases are often underpinned by multiple common genetic variants that contribute to disease susceptibility. Here, we describe a cost-effective ...

candidate-gene-testing-clinical-cohort-studies-with-multiplexed

Universidad de Cartagena UDEC

Research

JoVE Journal

Genetics

10.1K 조회수.  Max-Delbrück Center for Molecular Medicine. 복잡 한 인간의 질병에 기여 하는 유전 이체의 소설 메커니즘을 식별 수 있습니다. 여기, 후보 유전자 또는 유전자 통로 분석 낮은 비용 범위를 극대화 하 고 코 호트 기반 연구 의무가 멀티플렉스 유전형 접근 방식을 설명 합니다.

비디오: Candidate Gene Testing in Clinical Cohort Studies with Multiplexed Genotyping and Mass Spectrometry

Determining the Likelihood of Variant Pathogenicity Using Amino Acid-level Signal-to-Noise Analysis of Genetic Variation

Advancements in the cost and speed of next generation genetic sequencing have generated an explosion of clinical whole exome and whole genome testing. While this has led to increased identification of likely pathogenic mutations associated with genetic syndromes, it has also dramatically increased the number of incidentally found genetic variants of unknown significance (VUS). Determining the clinical significance of these variants is a major challenge for both scientists and clinicians. An approach to assist in determining the likelihood of pathogenicity is signal-to-noise analysis at the protein sequence level. This protocol describes a method for amino acid-level signal-to-noise analysis that leverages variant frequency at each amino acid position of the protein with known protein topology to identify areas of the primary sequence with elevated likelihood of pathologic variation (relative to population &#34;background&#34; variation). This method can identify amino acid residue location &#34;hotspots&#34; of high pathologic signal, which can be used to refine the diagnostic weight of VUSs such as those identified by next generation genetic testing.

Amino acid-level signal-to-noise analysis determines the prevalence of genetic variation at a given amino acid position normalized to background genetic variation of a given population. This allows for identification of variant &#34;hotspots&#34; within a protein sequence (signal) that rises above the frequency of rare variants found in a population (noise).

유전 변이의 아미노산 수준 신호-잡음 분석을 사용 하 여 Variant Pathogenicity의 가능성 확인

Advancements in the cost and speed of next generation genetic sequencing have generated an explosion of clinical whole exome and whole genome testing. ...

연구

유전학

Detection and Monitoring of Tumor Associated Circulating DNA in Patient Biofluids

Complications associated with upfront and repeat surgical tissue sampling present the need for minimally invasive platforms capable of molecular sub-classification and temporal monitoring of tumor response to therapy. Here, we describe our dPCR-based method for the detection of tumor somatic mutations in cell free DNA (cfDNA), readily available in&#160;patient biofluids. Although limited in the number of mutations that can be tested for in each assay, this method provides a high level of sensitivity and specificity. Monitoring of mutation abundance, as calculated by MAF, allows for the evaluation of tumor response to therapy, thereby providing a much-needed supplement to radiographic imaging.

Here, we present a protocol to detect tumor somatic mutations in circulating DNA present in patient biological fluids (biofluids). Our droplet digital polymerase chain reaction (dPCR)-based method enables quantification of the tumor mutation allelic frequency (MAF), facilitating a minimally invasive complement to diagnosis and temporal monitoring of tumor response.

환자 생체 유체에서 종양 관련 순환 DNA의 검출 및 모니터링

Complications associated with upfront and repeat surgical tissue sampling present the need for minimally invasive platforms capable of molecular ...

암 연구학

Pre-Implantation Genetic Testing for Aneuploidy on a Semiconductor Based Next-Generation Sequencing Platform

Next-generation sequencing has gained increasing importance in the clinical application in the determination of genetic variants. In the pre-implantation genetic test, this technique has its unique advantages in scalability, throughput, and cost. For the pre-implantation genetic test for aneuploidy analysis, the semiconductor-based next-generation sequencing (NGS) system presented here provides a comprehensive approach to determine structural genetic variants at a minimum resolution of 8 Mb. From sample acquisition to the final report, the working process requires multiple steps with close adherence to protocols. Since various critical steps could determine the outcome of amplification, quality of the library, coverage of reads, and output of data, descriptive information with visual demonstration other than words could offer more detail to the operation and manipulation, which may have a great impact on the results of all critical steps. The methods presented herein will display the procedures involved in whole genome amplification (WGA) of biopsied Trophectoderm (TE) cells, genomic library construction, sequencer management, and finally, generating copy number variants' reports.

The protocol presents the overall in-lab procedures required in pre-implantation genetic testing for aneuploidy on a semiconductor-based next-generation sequencing platform. Here we present the detailed steps of whole genome amplification, DNA fragment selection, library construction, template preparation, and sequencing working flow with representative results.

반도체 기반 차세대 시퀀싱 플랫폼에서 Aneuploidy에 대한 이식 전 유전자 검사

Next-generation sequencing has gained increasing importance in the clinical application in the determination of genetic variants. In the pre-implantation ...

종양의 치료 표적을 식별하고 비교하기 위한 게놈 규모 스크리닝

Genetic Profiling and Genome-Scale Dropout Screening to Identify Therapeutic Targets in Mouse Models of Malignant Peripheral Nerve Sheath Tumor

Malignant Peripheral Nerve Sheath Tumors (MPNSTs) are derived from Schwann cells or their precursors. In patients with the tumor susceptibility syndrome neurofibromatosis type 1 (NF1), MPNSTs are the most common malignancy and the leading cause of death. These rare and aggressive soft-tissue sarcomas offer a stark future, with 5-year disease-free survival rates of 34-60%. Treatment options for individuals with MPNSTs are disappointingly limited, with disfiguring surgery being the foremost treatment option. Many once-promising therapies such as tipifarnib, an inhibitor of Ras signaling, have failed clinically. Likewise, phase II clinical trials with erlotinib, which targets the epidermal growth factor (EFGR), and sorafenib, which targets the vascular endothelial growth factor receptor (VEGF), platelet-derived growth factor receptor (PDGF), and Raf, in combination with standard chemotherapy, have also failed to produce a response in patients. 
In recent years, functional genomic screening methods combined with genetic profiling of cancer cell lines have proven useful for identifying essential cytoplasmic signaling pathways and the development of target-specific therapies. In the case of rare tumor types, a variation of this approach known as cross-species comparative oncogenomics is increasingly being used to identify novel therapeutic targets. In cross-species comparative oncogenomics, genetic profiling and functional genomics are performed in genetically engineered mouse (GEM) models and the results are then validated in the rare human specimens and cell lines that are available. 
This paper describes how to identify candidate driver gene mutations in human and mouse MPNST cells using whole exome sequencing (WES). We then describe how to perform genome-scale shRNA screens to identify and compare critical signaling pathways in mouse and human MPNST cells and identify druggable targets in these pathways. These methodologies provide an effective approach to identifying new therapeutic targets in a variety of human cancer types.

저자 스포트라이트: 게놈 스케일 shRNA 스크리닝을 통한 악성 말초 신경초 종양에 대한 새로운 치료 표적 찾기 

We have developed a cross-species comparative oncogenomics approach utilizing genomic analyses and functional genomic screens to identify and compare therapeutic targets in tumors arising in genetically engineered mouse models and the corresponding human tumor type.

악성 말초 신경초 종양의 마우스 모델에서 치료 표적을 식별하기 위한 유전자 프로파일링 및 게놈 규모 드롭아웃 스크리닝

Malignant Peripheral Nerve Sheath Tumors (MPNSTs) are derived from Schwann cells or their precursors. In patients with the tumor susceptibility syndrome ...

An Allele-specific Gene Expression Assay to Test the Functional Basis of Genetic Associations

The number of significant genetic associations with common complex traits is constantly increasing. However, most of these associations have not been understood at molecular level. One of the mechanisms mediating the effect of DNA variants on phenotypes is gene expression, which has been shown to be particularly relevant for complex traits1.
This method tests in a cellular context the effect of specific DNA sequences on gene expression. The principle is to measure the relative abundance of transcripts arising from the two alleles of a gene, analysing cells which carry one copy of the DNA sequences associated with disease (the risk variants)2,3. Therefore, the cells used for this method should meet two fundamental genotypic requirements: they have to be heterozygous both for DNA risk variants and for DNA markers, typically coding polymorphisms, which can distinguish transcripts based on their chromosomal origin (Figure 1). DNA risk variants and DNA markers do not need to have the same allele frequency but the phase (haplotypic) relationship of the genetic markers needs to be understood. It is also important to choose cell types which express the gene of interest. This protocol refers specifically to the procedure adopted to extract nucleic acids from fibroblasts but the method is equally applicable to other cells types including primary cells.
DNA and RNA are extracted from the selected cell lines and cDNA is generated. DNA and cDNA are analysed with a primer extension assay, designed to target the coding DNA markers4. The primer extension assay is carried out using the MassARRAY (Sequenom)5 platform according to the manufacturer's specifications. Primer extension products are then analysed by matrix-assisted laser desorption/ionization time of-flight mass spectrometry (MALDI-TOF/MS). Because the selected markers are heterozygous they will generate two peaks on the MS profiles. The area of each peak is proportional to the transcript abundance and can be measured with a function of the MassARRAY Typer software to generate an allelic ratio (allele 1: allele 2) calculation. The allelic ratio obtained for cDNA is normalized using that measured from genomic DNA, where the allelic ratio is expected to be 1:1 to correct for technical artifacts. Markers with a normalised allelic ratio significantly different to 1 indicate that the amount of transcript generated from the two chromosomes in the same cell is different, suggesting that the DNA variants associated with the phenotype have an effect on gene expression. Experimental controls should be used to confirm the results.

Genetic associations often remain unexplained at a functional level. This method aims to assess the effect of phenotype-associated genetic markers on gene expression by analyzing cells heterozygous for transcribed SNPs. The technology allows accurate measurement by MALDI-TOF mass spectrometry to quantify allele-specific primer extension products.

유전자 협회의 기능 기초를 테스트하는 Allele 특정 유전자 발현 분석

The number of significant genetic associations with common complex traits is constantly increasing. However, most of these associations have not been ...

생물학

Integration of Wet and Dry Bench Processes Optimizes Targeted Next-generation Sequencing of Low-quality and Low-quantity Tumor Biopsies

All next-generation sequencing (NGS) procedures include assays performed at the laboratory bench (&#34;wet bench&#34;) and data analyses conducted using bioinformatics pipelines (&#34;dry bench&#34;). Both elements are essential to produce accurate and reliable results, which are particularly critical for clinical laboratories. Targeted NGS technologies have increasingly found favor in oncology applications to help advance precision medicine objectives, yet the methods often involve disconnected and variable wet and dry bench workflows and uncoordinated reagent sets. In this report, we describe a method for sequencing challenging cancer specimens with a 21-gene panel as an example of a comprehensive targeted NGS system. The system integrates functional DNA quantification and qualification, single-tube multiplexed PCR enrichment, and library purification and normalization using analytically-verified, single-source reagents with a standalone bioinformatics suite. As a result, accurate variant calls from low-quality and low-quantity formalin-fixed, paraffin-embedded (FFPE) and fine-needle aspiration (FNA) tumor biopsies can be achieved. The method can routinely assess cancer-associated variants from an input of 400 amplifiable DNA copies, and is modular in design to accommodate new gene content. Two different types of analytically-defined controls provide quality assurance and help safeguard call accuracy with clinically-relevant samples. A flexible &#34;tag&#34; PCR step embeds platform-specific adaptors and index codes to allow sample barcoding and compatibility with common benchtop NGS instruments. Importantly, the protocol is streamlined and can produce 24 sequence-ready libraries in a single day. Finally, the approach links wet and dry bench processes by incorporating pre-analytical sample quality control results directly into the variant calling algorithms to improve mutation detection accuracy and differentiate false-negative and indeterminate calls. This targeted NGS method uses advances in both wetware and software to achieve high-depth, multiplexed sequencing and sensitive analysis of heterogeneous cancer samples for diagnostic applications.

An integrated system for targeted next-generation sequencing of oncology specimens is described. This cross-platform system is optimized for low-quality and low-quantity tumor biopsies, accommodates low DNA inputs, includes well-characterized multi-variant controls, and features a novel variant caller that is informed by quantitative pre-analytical quality control measures.

낮은 품질과 낮은 수량 종양 생검의 습식 및 건식 벤치 프로세스의 통합을 대상으로 최적화하여 차세대 시퀀싱

All next-generation sequencing (NGS) procedures include assays performed at the laboratory bench (&#34;wet bench&#34;) and data analyses conducted using ...

의학

Infinium Assay for Large-scale SNP Genotyping Applications

Genotyping variants in the human genome has proven to be an efficient method to identify genetic associations with phenotypes. The distribution of variants within families or populations can facilitate identification of the genetic factors of disease. Illumina&#39;s panel of genotyping BeadChips allows investigators to genotype thousands or millions of single nucleotide polymorphisms (SNPs) or to analyze other genomic variants, such as copy number, across a large number of DNA samples. These SNPs can be spread throughout the genome or targeted in specific regions in order to maximize potential discovery. The Infinium assay has been optimized to yield high-quality, accurate results quickly. With proper setup, a single technician can process from a few hundred to over a thousand DNA samples per week, depending on the type of array. This assay guides users through every step, starting with genomic DNA and ending with the scanning of the array. Using propriety reagents, samples are amplified, fragmented, precipitated, resuspended, hybridized to the chip, extended by a single base, stained, and scanned on either an iScan or Hi Scan high-resolution optical imaging system. One overnight step is required to amplify the DNA. The DNA is denatured and isothermally amplified by whole-genome amplification; therefore, no PCR is required. Samples are hybridized to the arrays during a second overnight step. By the third day, the samples are ready to be scanned and analyzed. Amplified DNA may be stockpiled in large quantities, allowing bead arrays to be processed every day of the week, thereby maximizing throughput.

A protocol is described that uses Illumina&#39;s Infinium assays to perform large-scale genotyping. These assays can reliably genotype millions of SNPs across hundreds of individual DNA samples in three days. Once generated, these genotypes can be used to check for associations with a variety of different diseases or phenotypes.

대규모 SNP 유전자형 응용 프로그램에 대한 Infinium 분석

Genotyping variants in the human genome has proven to be an efficient method to identify genetic associations with phenotypes. The distribution of ...

Detecting Somatic Genetic Alterations in Tumor Specimens by Exon Capture and Massively Parallel Sequencing

Efforts to detect and investigate key oncogenic mutations have proven valuable to facilitate the appropriate treatment for cancer patients. The establishment of high-throughput, massively parallel "next-generation" sequencing has aided the discovery of many such mutations. To enhance the clinical and translational utility of this technology, platforms must be high-throughput, cost-effective, and compatible with formalin-fixed paraffin embedded (FFPE) tissue samples that may yield small amounts of degraded or damaged DNA. Here, we describe the preparation of barcoded and multiplexed DNA libraries followed by hybridization-based capture of targeted exons for the detection of cancer-associated mutations in fresh frozen and FFPE tumors by massively parallel sequencing. This method enables the identification of sequence mutations, copy number alterations, and select structural rearrangements involving all targeted genes. Targeted exon sequencing offers the benefits of high throughput, low cost, and deep sequence coverage, thus conferring high sensitivity for detecting low frequency mutations.

We describe the preparation of barcoded DNA libraries and subsequent hybridization-based exon capture for detection of key cancer-associated mutations in clinical tumor specimens by massively parallel "next generation" sequencing. Targeted exon sequencing offers the benefits of high throughput, low cost, and deep sequence coverage, thus yielding high sensitivity for detecting low frequency mutations.

후보 유전자 다중화 유전형과 질량 분석 임상 코 호트 연구에서 테스트

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이 비디오의 챕터

유전 변이의 아미노산 수준 신호-잡음 분석을 사용 하 여 Variant Pathogenicity의 가능성 확인

환자 생체 유체에서 종양 관련 순환 DNA의 검출 및 모니터링

반도체 기반 차세대 시퀀싱 플랫폼에서 Aneuploidy에 대한 이식 전 유전자 검사

악성 말초 신경초 종양의 마우스 모델에서 치료 표적을 식별하기 위한 유전자 프로파일링 및 게놈 규모 드롭아웃 스크리닝

악성 말초 신경초 종양의 마우스 모델에서 치료 표적을 식별하기 위한 유전자 프로파일링 및 게놈 규모 드롭아웃 스크리닝

악성 말초 신경초 종양의 마우스 모델에서 치료 표적을 식별하기 위한 유전자 프로파일링 및 게놈 규모 드롭아웃 스크리닝

유전자 협회의 기능 기초를 테스트하는 Allele 특정 유전자 발현 분석

낮은 품질과 낮은 수량 종양 생검의 습식 및 건식 벤치 프로세스의 통합을 대상으로 최적화하여 차세대 시퀀싱

대규모 SNP 유전자형 응용 프로그램에 대한 Infinium 분석

엑손 캡처 및 대규모 병렬 염기 서열 종양 검체에서 체세포 유전 변경을 검출