이 작품은 어린이 디스커버리 연구소 부여 MC-II-2006-1 (RDM과 TED), NIH Epigenetics 로드맵 부여 [1R01DA025744-01 및 3R01DA025744-02S1] (RDM 및 FLMV), U01AG023746 (SC), Saigh에 의해 지원되었다 재단 (FLMV와 TED), 1K08CA140720-01A1와 알렉 스의 레모네이드가 &#39;A&#39;상 지원 (TED)하라. 우리는 게놈 분석과 관련하여 도움이 의학의 워싱턴 대학 대학원에서 유전학의학과 게놈 기술 교통 센터에 감사드립니다. 센터는 부분적으로 연구 자원에 대한 NationalCenter (NCRR), 건강 (NIH)의 국립 연구소의 한 구성 요소에서 # UL1RR024992 NCI 암센터 지원 부여 Siteman 암 센터 # P30 CA91842 의해 ICTS / CTSA 그랜트에 의해 지원되고 의학 연구에 대한 NIH 로드맵. 이 책자는 전적으로 저자의 책임이며 반드시 NCRR 또는 NIH의 공식 견해를 대변하지 않습니다.

<ol>
	<li>Vallania, F. L. M., Druley, T. E., Ramos, E., Wang, J., Borecki, I., Province, M., Mitra, 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=High-throughput+discovery+of+rare+insertions+and+deletions+in+large+cohorts.">High-throughput discovery of rare insertions and deletions in large cohorts.</a> Genome Research. 20, 1391-1397 (2010).</li><li>Shendure, J., Mitra, R., Varma, C., Church, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advanced+Sequencing+Technologies:+Methods+and+Goals.">Advanced Sequencing Technologies: Methods and Goals.</a> Nature Reviews of Genetics. 5, 335-344 (2004).</li><li>The 1000 Genomes Project Consortium. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+map+of+human+genome+variation+from+population-scale+sequencing.">A map of human genome variation from population-scale sequencing.</a> Nature. 467, 1061-1073 (2010).</li><li>Manolio, T. A., Collins, F. S., Cox, N. J., Goldstein, D. B., Hindorff, L. A., Hunter, D. J., McCarthy, M. I., Ramos, E. M., Cardon, L. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Finding+the+missing+heritability+of+complex+diseases.">Finding the missing heritability of complex diseases.</a> Nature. 461, 747-753 (2009).</li><li>Reich, D. E., Lander, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+allelic+spectrum+of+human+disease.">On the allelic spectrum of human disease.</a> Trends Genet. 17, 502-510 (2001).</li><li>Cohen, J. C., Kiss, R. S., Pertsemlidis, A., Marcel, Y. L., McPherson, R., Hobbs, H. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+rare+alleles+contribute+to+low+plasma+levels+of+HDL+cholesterol.">Multiple rare alleles contribute to low plasma levels of HDL cholesterol.</a> Science. 305, 869-872 (2004).</li><li>Ji, W., Foo, J. N., O'Roak, B. J., Zhao, H., Larson, M. G., Simon, D. B., Newton-Cheh, C., State, M. W., Levy, D., Lifton, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rare+independent+mutations+in+renal+salt+handling+genes+contribute+to+blood+pressure+variation.">Rare independent mutations in renal salt handling genes contribute to blood pressure variation.</a> Nat. Genet. 40, 592-599 (2008).</li><li>Ahituv, N., Kavaslar, N., Schackwitz, W., Ustaszewska, A., Martin, J., Hebert, S., Doelle, H., Ersoy, B., Kryukov, G., Schmidt, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Medical+sequencing+at+the+extremes+of+human+body+mass.">Medical sequencing at the extremes of human body mass.</a> Am. J. Hum. Genet. 80, 779-791 (2007).</li><li>Druley, T. E., Vallania, F. L., Wegner, D. J., Varley, K. E., Knowles, O. L., Bonds, J. A., Robison, S. W., Doniger, S. W., Hamvas, A., Cole, F. S., Fay, J. C., Mitra, 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=Quantification+of+rare+allelic+variants+from+pooled+genomic+DNA.">Quantification of rare allelic variants from pooled genomic DNA.</a> Nat. Methods. 6, 263-265 (2009).</li><li>Mitra, R. D., Butty, V., Shendure, J., Housman, D., Church, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Digital+Genotyping+and+Haplotyping+with+Polymerase+Colonies.">Digital Genotyping and Haplotyping with Polymerase Colonies.</a> Proc. Natl. Acad. Sci. 100, 5926-5931 (2003).</li></ol>

관심의 어떠한 충돌 선언 없습니다.

발병률과 일반적인 복잡한 phenotypes과 같은 비만 8, 고콜레스테롤혈증 4, 고혈압 7, 다른 사람과 같은 질병의 치료 반응은 드문 변이의 개인 프로필에 의해 검토됨 수있다는 것을 증가하는 증거가있다. 영향을받는 인구의 집계 이러한 변형은 깊은 진단 및 치료에 영향을 미칠 것이다 유전자 및 경로를 식별하지만, 별도로 영향을받는 개인을 분석하는 것은 매번 금지 비용하실 수 있습니다. 인구 기반의 분석은 여러 loci에서 유전자 변화를 관측을위한보다 효율적인 방법을 제공합니다. 우리는 인구에 걸쳐 유전적 변이의 유형을 식별하기위한 가시가 소프트웨어 패키지와 결합하여 소설 풀링된-DNA 시퀀싱 프로토콜을 소개합니다. 우리는 있었다 희귀 변종을 포함, 947 개인 대형 풀링된 인구 내에서 사소한 대립 유전자를 식별하고 quantifying에이 방법의 정확성을 입증풀링된 시퀀싱에서 드 노보 전화 개별 pyrosequencing에 의해 확인되었습니다. 우리의 전략은 주로 긍정의 설립 모든 실험 내에서 부정적인 통제에 의해 다른 프로토콜과 다릅니다. 이것은 파편이 다른 접근 방식 1에 비해 훨씬 더 높은 정확성과 파워를 얻을 수 있습니다. allele 당 25 배 이상의 최적 범위는 풀 크기에만 비늘이 선형적으로 이러한 요구 사항 등 대형 수영장의 분석이 가능하게, 독립적으로 수영장의 크기의 고정됩니다. 우리의 접근 방식은 매우 유연하며 관심의 표현형을뿐 아니라 그러한 혼합 세포 인구 및 종양 biopsies 같은 자연적으로 이기종있는 샘플에 적용할 수 있습니다. 같은 exome 또는 게놈과 같은 대형 대상 지역의 풀링된 시퀀싱에서 계속 늘어나는 관심을 감안할 때, 우리 도서관 준비와 가시가 분석 맞춤 캡처 및 전체 - exome 시퀀싱과 호환지만 가시가 패키지에 정렬 유틸리티는 위해 설계되지 않았습니다 큰레퍼런스 시퀀스. 따라서, 우리는 성공적으로 풀링된 샘플 (라모스 외., 제출)에서 전화 변종이어서 게놈 차원의 정렬을 위해, Novoalign, 동적 프로그래밍 aligner를 이용했습니다. 따라서, 우리의 풀링된 시퀀싱 전략은 대상 시퀀스의 증가 금액과 큰 수영장을 성공적으로 확장할 수 있습니다.

<table border="1"><tbody><tr><td> 시약 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td><td> 섹션 </td></tr><tr><td> PfuUltra 하이 피델리티 </td><td> 애질런트 </td><td> 600,384 </td><td> 1.4 </td></tr><tr><td> 베타인 </td><td> 시그마 </td><td> B2629 </td><td> 1.4 </td></tr><tr><td> M13mp18 ssDNA 벡터 </td><td> 코 </td><td> N4040S </td><td> 1.5 </td></tr><tr><td> pGEM-T 쉬운 </td><td> Promega </td><td> A1360 </td><td> 1.5 </td></tr><tr><td> T4 폴리 뉴클레오 타이드의 키나제 </td><td> 코 </td><td> M0201S </td><td> 2.2 </td></tr><tr><td> T4 Ligase </td><td> 코 </td><td> M0202S </td><td> 2.2 </td></tr><tr><td> 폴리에틸렌 글리콜 8,000 MW </td><td> 시그마 </td><td> P5413 </td><td> 2.2 </td></tr><tr><td> Bioruptor의 sonicator </td><td> Diagenode </td><td> UCD-200-TS</td><td> 2.3 </td></tr></tbody></table>

detection of rare genomic variants from pooled sequencing using splinter

DNA 시퀀싱 기술이 현저하게 최근 2에 진출했습니다로서 임의의 두 개인 사이의 유전적 변이의 양을 이전에 3 생각보다 큰 것이 점점 더 분명되었다. 대조적으로, 배열 기반 genotyping은 4,5 흔한 질환의 phenotypic 다양성에 일반적인 순서 변종 큰 공헌을 식별하는 데 실패했습니다. 함께 촬영이 관찰 공통 질병 / 일반 및 복잡한 phenotypes의 &quot;누락 heritability&quot;대부분의 대신 희귀 또는 개인의 DNA 변종 6-8 중 개인의 개인 프로필에 의한 것을 제안 희귀 변종 가설의 발전을 이끈 것은 . 그러나 드문 변형 복잡한 phenotypes 영향을 얼마나 특성화하는 것은 많은 게놈 loci에 많은 영향을받는 개인의 분석을 필요로하고, 이상적으로 영향을받지 일대에서 비슷한 설문 조사에 비교됩니다. 오늘날의 플랫폼이 제공하는 시퀀싱 전력에도 불구하고많은 게놈의 loci 및 필요한 후속 전산 분석 인구 기반 조사는 많은 수사관을위한 금지 남아있다. 이 요구를 충족하기 위해, 우리는 풀링된 시퀀싱 기법 1,9와 결과 데이터에서 고도로 정확하고 희귀한 변종 검출을위한 새로운 소프트웨어 패키지 1을 개발했습니다. 영향을받는 개인 및 설문 조사 단일 시퀀싱 라이브러리에서 여러 대상 지역에서 유전 변이의 정도의 전체 인구에서 풀 genomes에 대한 능력은 기존의 단일 샘플 시퀀싱 방법론에 뛰어난 비용 및 시간 절감 효과를 제공합니다. 25 배 이상의 allele 당 평균 시퀀싱 보험 혜택과 함께, 우리의 사용자 정의 알고리즘, 가시가 1로 최대의 수영장에서 높은 민감도와 특이성과 길이가 무려 기본 쌍에 삽입, 삭제 및 대체를 호출하기위한 내부 변형 호출 제어 전략을 사용 500 개인의 돌연변이 allele. 여기 풀링을 준비하는 방법을 설명도서관을 equencing 것은 풀링 시퀀싱 분석 (위해 가시 패키지를 사용하는 방법에 대한 단계별 지침은 다음 <a href="http://www.ibridgenetwork.org/wustl/splinter" target="_blank">http://www.ibridgenetwork.org/wustl/splinter</a> ). 우리는 947 개인의 풀링된 시퀀싱 간의 비교를 보여, 누구의도 인당 시퀀싱의 20킬로바이트 이상에서 게놈 차원의 배열을 받았습니다. 태그가의 genotyping과 풀링된 샘플로 불리는 새로운 변종 사이의 일치가 우수했다. 이 방법은 쉽게 게놈 loci과 개인의 숫자의 개수까지 확장할 수 있습니다. 연구하에 인구를 모방 비율에서 내부 긍정과 부정 amplicon 컨트롤을 통합함으로써, 알고리즘은 최적의 성능을 보정 할 수 있습니다. 이 전략은 또한 하이브리드화 캡처 또는 개별 고유의 바코드와 함께 사용하도록 수정할 수와 같은 종양의 DNA와 같은 천연 이기종 샘플의 시퀀싱에 적용할 수 있습니다.

Watch this Scientific Journal Video about Detection of Rare Genomic Variants from Pooled Sequencing Using SPLINTER at JoVE.com

Detection of Rare Genomic Variants from Pooled Sequencing Using SPLINTER

풀링된 유전자 시퀀싱은 큰 무리에서 복잡한 phenotypes와 관련된 희귀한 변종을 탐지하기위한 빠르고 비용 효율적인 전략이다. 여기 가시가 소프트웨어 패키지를 사용하여 32 암 관련 유전자의 풀링, 차세대 시퀀싱의 전산 분석을 설명합니다. 이 방식은 확장성, 그리고 관심의 표현형에 적용됩니다.

파편을 사용하여 풀링된 장면에서 드문 게놈 변종의 탐지

As DNA sequencing technology has markedly advanced in recent years2, it has become increasingly evident that the amount of genetic variation between any ...

detection-rare-genomic-variants-from-pooled-sequencing-using

Research

JoVE Journal

Biology

15.0K 조회수.  Washington University School of Medicine. 풀링된 유전자 시퀀싱은 큰 무리에서 복잡한 phenotypes와 관련된 희귀한 변종을 탐지하기위한 빠르고 비용 효율적인 전략이다. 여기 가시가 소프트웨어 패키지를 사용하여 32 암 관련 유전자의 풀링, 차세대 시퀀싱의 전산 분석을 설명합니다. 이 방식은 확장성, 그리고 관심의 표현형에 적용됩니다.

비디오: Detection of Rare Genomic Variants from Pooled Sequencing Using SPLINTER

Isolation of Genomic DNA from Mouse Tails

마우스 뒷면에서 게놈 DNA의 분리

연구

생물학

Extraction of High Molecular Weight Genomic DNA from Soils and Sediments

The soil microbiome is a vast and relatively unexplored reservoir of genomic diversity and metabolic innovation that is intimately associated with nutrient and energy flow within terrestrial ecosystems. Cultivation-independent environmental genomic, also known as metagenomic, approaches promise unprecedented access to this genetic information with respect to pathway reconstruction and functional screening for high value therapeutic and biomass conversion processes. However, the soil microbiome still remains a challenge largely due to the difficulty in obtaining high molecular weight DNA of sufficient quality for large insert library production. Here we introduce a protocol for extracting high molecular weight, microbial community genomic DNA from soils and sediments. The quality of isolated genomic DNA is ideal for constructing large insert environmental genomic libraries for downstream sequencing and screening applications. 
The procedure starts with cell lysis. Cell walls and membranes of microbes are lysed by both mechanical (grinding) and chemical forces (&beta;-mercaptoethanol). Genomic DNA is then isolated using extraction buffer, chloroform-isoamyl alcohol and isopropyl alcohol. The buffers employed for the lysis and extraction steps include guanidine isothiocyanate and hexadecyltrimethylammonium bromide (CTAB) to preserve the integrity of the high molecular weight genomic DNA. Depending on your downstream application, the isolated genomic DNA can be further purified using cesium chloride (CsCl) gradient ultracentrifugation, which reduces impurities including humic acids. The first procedure, extraction, takes approximately 8 hours, excluding DNA quantification step. The CsCl gradient ultracentrifugation, is a two days process. During the entire procedure, genomic DNA should be treated gently to prevent shearing, avoid severe vortexing, and repetitive harsh pipetting.

A methodology to isolate high molecular weight and high quality genomic DNA from soil microbial community is described.

소일스 및 세디먼츠에서 높은 분자량의 게놈의 DNA의 추출

The soil microbiome is a vast and relatively unexplored reservoir of genomic diversity and metabolic innovation that is intimately associated with ...

Competitive Genomic Screens of Barcoded Yeast Libraries

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily. The tempo of these advances is accelerating, promising greater depth and breadth. In light of these extraordinary advances, the need for fast, parallel methods to define gene function becomes ever more important. Collections of genome-wide deletion mutants in yeasts and E. coli have served as workhorses for functional characterization of gene function, but this approach is not scalable, current gene-deletion approaches require each of the thousands of genes that comprise a genome to be deleted and verified. Only after this work is complete can we pursue high-throughput phenotyping. Over the past decade, our laboratory has refined a portfolio of competitive, miniaturized, high-throughput genome-wide assays that can be performed in parallel. This parallelization is possible because of the inclusion of DNA 'tags', or 'barcodes,' into each mutant, with the barcode serving as a proxy for the mutation and one can measure the barcode abundance to assess mutant fitness. In this study, we seek to fill the gap between DNA sequence and barcoded mutant collections. To accomplish this we introduce a combined transposon disruption-barcoding approach that opens up parallel barcode assays to newly sequenced, but poorly characterized microbes. To illustrate this approach we present a new Candida albicans barcoded disruption collection and describe how both microarray-based and next generation sequencing-based platforms can be used to collect 10,000 - 1,000,000 gene-gene and drug-gene interactions in a single experiment.

We have developed comprehensive, unbiased genome-wide screens to understand gene-drug and gene-environment interactions. Methods for screening these mutant collections are presented.

Barcoded 효모 게놈 라이브러리의 경쟁력 화면

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily.  The tempo of these advances is ...

Selective Capture of 5-hydroxymethylcytosine from Genomic DNA

5-methylcytosine (5-mC) constitutes ~2-8% of the total cytosines in human genomic DNA and impacts a broad range of biological functions, including gene expression, maintenance of genome integrity, parental imprinting, X-chromosome inactivation, regulation of development, aging, and cancer1. Recently, the presence of an oxidized 5-mC, 5-hydroxymethylcytosine (5-hmC), was discovered in mammalian cells, in particular in embryonic stem (ES) cells and neuronal cells2-4. 5-hmC is generated by oxidation of 5-mC catalyzed by TET family iron (II)/&alpha;-ketoglutarate-dependent dioxygenases2, 3. 5-hmC is proposed to be involved in the maintenance of embryonic stem (mES) cell, normal hematopoiesis and malignancies, and zygote development2, 5-10. To better understand the function of 5-hmC, a reliable and straightforward sequencing system is essential. Traditional bisulfite sequencing cannot distinguish 5-hmC from 5-mC11. To unravel the biology of 5-hmC, we have developed a highly efficient and selective chemical approach to label and capture 5-hmC, taking advantage of a bacteriophage enzyme that adds a glucose moiety to 5-hmC specifically12.
Here we describe a straightforward two-step procedure for selective chemical labeling of 5-hmC. In the first labeling step, 5-hmC in genomic DNA is labeled with a 6-azide-glucose catalyzed by &beta;-GT, a glucosyltransferase from T4 bacteriophage, in a way that transfers the 6-azide-glucose to 5-hmC from the modified cofactor, UDP-6-N3-Glc (6-N3UDPG). In the second step, biotinylation, a disulfide biotin linker is attached to the azide group by click chemistry. Both steps are highly specific and efficient, leading to complete labeling regardless of the abundance of 5-hmC in genomic regions and giving extremely low background. Following biotinylation of 5-hmC, the 5-hmC-containing DNA fragments are then selectively captured using streptavidin beads in a density-independent manner. The resulting 5-hmC-enriched DNA fragments could be used for downstream analyses, including next-generation sequencing.
Our selective labeling and capture protocol confers high sensitivity, applicable to any source of genomic DNA with variable/diverse 5-hmC abundances. Although the main purpose of this protocol is its downstream application (i.e., next-generation sequencing to map out the 5-hmC distribution in genome), it is compatible with single-molecule, real-time SMRT (DNA) sequencing, which is capable of delivering single-base resolution sequencing of 5-hmC.

Described is a two-step labeling process using &beta;-glucosyltransferase (&beta;-GT) to transfer an azide-glucose to 5-hmC, followed by click chemistry to transfer a biotin linker for easy and density-independent enrichment. This efficient and specific labeling method enables enrichment of 5-hmC with extremely low background and high-throughput epigenomic mapping via next-generation sequencing.

게놈 DNA에서 도보로 5 hydroxymethylcytosine의 선택적 캡처

5-methylcytosine (5-mC) constitutes ~2-8% of the total cytosines in human genomic DNA and impacts a broad range of biological functions, including gene ...

Array Comparative Genomic Hybridization (Array CGH) for Detection of Genomic Copy Number Variants

Array CGH for the detection of genomic copy number variants has replaced G-banded karyotype analysis. This paper describes the technology and its application in a clinical diagnostic service laboratory. DNA extracted from a patient&#8217;s sample (blood, saliva or other tissue types) is labeled with a fluorochrome (either cyanine 5 or cyanine 3). A reference DNA sample is labeled with the opposite fluorochrome. There follows a cleanup step to remove unincorporated nucleotides before the labeled DNAs are mixed and resuspended in a hybridization buffer and applied to an array comprising ~60,000 oligonucleotide probes from loci across the genome, with high probe density in clinically important areas. Following hybridization, the arrays are washed, then scanned and the resulting images are analyzed to measure the red and green fluorescence for each probe. Software is used to assess the quality of each probe measurement, calculate the ratio of red to green fluorescence and detect potential copy number variants.

Array Comparative Genomic Hybridization Array CGH for Detection of Genomic Copy Number Variants

Array CGH for the detection of genomic copy number variants has replaced G-banded karyotype analysis. This paper describes the technology and its application in a diagnostic service laboratory.

게놈 복사 번호 변종의 탐지를위한 배열 비교 게놈 교잡 (배열 CGH)

Array CGH for the detection of genomic copy number variants has replaced G-banded karyotype analysis. This paper describes the technology and its ...

Isolating Potentiated Hsp104 Variants Using Yeast Proteinopathy Models

Many protein-misfolding disorders can be modeled in the budding yeast Saccharomyces cerevisiae. Proteins such as TDP-43 and FUS, implicated in amyotrophic lateral sclerosis, and &#945;-synuclein, implicated in Parkinson&#8217;s disease, are toxic and form cytoplasmic aggregates in yeast. These features recapitulate protein pathologies observed in patients with these disorders. Thus, yeast are an ideal platform for isolating toxicity suppressors from libraries of protein variants. We are interested in applying protein disaggregases to eliminate misfolded toxic protein conformers. Specifically, we are engineering Hsp104, a hexameric AAA+ protein from yeast that is uniquely capable of solubilizing both disordered aggregates and amyloid and returning the proteins to their native conformations. While Hsp104 is highly conserved in eukaryotes and eubacteria, it has no known metazoan homologue. Hsp104 has only limited ability to eliminate disordered aggregates and amyloid fibers implicated in human disease. Thus, we aim to engineer Hsp104 variants to reverse the protein misfolding implicated in neurodegenerative disorders. We have developed methods to screen large libraries of Hsp104 variants for suppression of proteotoxicity in yeast. As yeast are prone to spontaneous nonspecific suppression of toxicity, a two-step screening process has been developed to eliminate false positives. Using these methods, we have identified a series of potentiated Hsp104 variants that potently suppress the toxicity and aggregation of TDP-43, FUS, and &#945;-synuclein. Here, we describe this optimized protocol, which could be adapted to screen libraries constructed using any protein backbone for suppression of toxicity of any protein that is toxic in yeast.

Yeast proteinopathy models are valuable tools to assess the toxicity and aggregation of proteins implicated in disease. Here, we present methods for screening Hsp104 variant libraries for toxicity suppressors. This protocol could be adapted to screen any protein library for toxicity suppressors of any protein that is toxic in yeast.

효력을 더 Hsp104 변종 사용 효모 Proteinopathy 모델을 분리

Many protein-misfolding disorders can be modeled in the budding yeast Saccharomyces cerevisiae. Proteins such as TDP-43 and FUS, implicated in amyotrophic ...

TRAP-rc, Translating Ribosome Affinity Purification from Rare Cell Populations of Drosophila Embryos

Measuring levels of mRNAs in the process of translation in individual cells provides information on the proteins involved in cellular functions at a given point in time. The protocol dubbed Translating Ribosome Affinity Purification (TRAP) is able to capture this mRNA translation process in a cell-type-specific manner. Based on the affinity purification of polysomes carrying a tagged ribosomal subunit, TRAP can be applied to translatome analyses in individual cells, making it possible to compare cell types during the course of developmental processes or to track disease development progress and the impact of potential therapies at molecular level. Here we report an optimized version of the TRAP protocol, called TRAP-rc (rare cells), dedicated to identifying engaged-in-translation RNAs from rare cell populations. TRAP-rc was validated using the Gal4/UAS targeting system in a restricted population of muscle cells in Drosophila embryos. This novel protocol allows the recovery of cell-type-specific RNA in sufficient quantities for global gene expression analytics such as microarrays or RNA-seq. The robustness of the protocol and the large collections of Gal4 drivers make TRAP-rc a highly versatile approach with potential applications in cell-specific genome-wide studies.

TRAP-rc, Translating Ribosome Affinity Purification from Rare Cell Populations of Drosophila Embryos

Translating Ribosome Affinity Purification (TRAP) is able to capture cell-type-specific translation of mRNA. Here we report the first TRAP protocol dedicated to isolation of mRNA in rare cell populations of Drosophila embryos.

희귀 세포 집단에서 TRAP-RC, 번역 리보솜 선호도 정화<em&gt; 초파리</em&gt; 배아

Measuring levels of mRNAs in the process of translation in individual cells provides information on the proteins involved in cellular functions at a given ...

Amplification, Next-generation Sequencing, and Genomic DNA Mapping of Retroviral Integration Sites

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of diverse libraries of retroviral integration sites using ligation-mediated PCR (LM-PCR) amplification and next-generation sequencing (NGS), (2) mapping the genomic location of each virus-host junction using BEDTools, and (3) analyzing the data for statistical relevance. Genomic DNA extracted from infected cells is fragmented by digestion with restriction enzymes or by sonication. After suitable DNA end-repair, double-stranded linkers are ligated onto the DNA ends, and semi-nested PCR is conducted using primers complementary to both the long terminal repeat (LTR) end of the virus and the ligated linker DNA. The PCR primers carry sequences required for DNA clustering during NGS, negating the requirement for separate adapter ligation. Quality control (QC) is conducted to assess DNA fragment size distribution and adapter DNA incorporation prior to NGS. Sequence output files are filtered for LTR-containing reads, and the sequences defining the LTR and the linker are cropped away. Trimmed host cell sequences are mapped to a reference genome using BLAT and are filtered for minimally 97% identity to a unique point in the reference genome. Unique integration sites are scrutinized for adjacent nucleotide (nt) sequence and distribution relative to various genomic features. Using this protocol, integration site libraries of high complexity can be constructed from genomic DNA in three days. The entire protocol that encompasses exogenous viral infection of susceptible tissue culture cells to integration site analysis can therefore be conducted in approximately one to two weeks. Recent applications of this technology pertain to longitudinal analysis of integration sites from HIV-infected patients.

We describe a protocol for amplifying retroviral integration sites from the genomic DNA of infected cells, sequencing the amplified virus-host junctions, and then mapping these sequences to a reference genome. We also describe techniques to quantify the distribution of integration sites relative to various genomic annotations using BEDTools.

레트로 바이러스 통합 사이트의 증폭, 차세대 시퀀싱 및 게놈 DNA 매핑

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of ...

Targeted RNA Sequencing Assay to Characterize Gene Expression and Genomic Alterations

RNA sequencing (RNAseq) is a versatile method that can be utilized to detect and characterize gene expression, mutations, gene fusions, and noncoding RNAs. Standard RNAseq requires 30 - 100 million sequencing reads and can include multiple RNA products such as mRNA and noncoding RNAs. We demonstrate how targeted RNAseq (capture) permits a focused study on selected RNA products using a desktop sequencer. RNAseq capture can characterize unannotated, low, or transiently expressed transcripts that may otherwise be missed using traditional RNAseq methods. Here we describe the extraction of RNA from cell lines, ribosomal RNA depletion, cDNA synthesis, preparation of barcoded libraries, hybridization and capture of targeted transcripts and multiplex sequencing on a desktop sequencer. We also outline the computational analysis pipeline, which includes quality control assessment, alignment, fusion detection, gene expression quantification and identification of single nucleotide variants. This assay allows for targeted transcript sequencing to characterize gene expression, gene fusions, and mutations.

We describe a targeted RNA sequencing-based method that includes preparation of indexed cDNA libraries, hybridization and capture with custom probes and data analysis to interrogate selected transcripts for gene expression, mutations, and gene fusions. Targeted RNAseq permits cost-effective, rapid evaluation of selected transcripts on a desktop sequencer.

RNA 시퀀싱 분석은 유전자 발현 및 게놈 변경을 특성화를 대상으로

RNA sequencing (RNAseq) is a versatile method that can be utilized to detect and characterize gene expression, mutations, gene fusions, and noncoding ...

Genome Engineering of Primary Human B Cells Using CRISPR/Cas9

B cells are lymphocytes derived from hematopoietic stem cells and are a key component of the humoral arm of the adaptive immune system. They make attractive candidates for cell-based therapies because of their ease of isolation from peripheral blood, their ability to expand in vitro, and their longevity in vivo. Additionally, their normal biological function&#8212;to produce large amounts of antibodies&#8212;can be utilized to express very large amounts of a therapeutic protein, such as a recombinant antibody to fight infection, or an enzyme for the treatment of enzymopathies. Here, we provide detailed methods for isolating primary human B cells from peripheral blood mononuclear cells (PBMCs) and activating/expanding isolated B cells in vitro. We then demonstrate the steps involved in using the CRISPR/Cas9 system for site-specific KO of endogenous genes in B cells. This method allows for efficient KO of various genes, which can be used to study the biological functions of genes of interest. We then demonstrate the steps for using the CRISPR/Cas9 system together with a recombinant, adeno-associated, viral (rAAV) vector for efficient site-specific integration of a transgene expression cassette in B cells. Together, this protocol provides a step-by-step engineering platform that can be used in primary human B cells to study biological functions of genes as well as for the development of B-cell therapeutics.

Here we provide a detailed, step-by-step protocol for CRISPR/Cas9-based genome engineering of primary human B cells for gene knockout (KO) and knock-in (KI) to study biological functions of genes in B cells and the development of B-cell therapeutics.