Name: targeted rna sequencing assay to characterize gene expression and genomic alterations
Uploaded: 2016-08-04T13:00:00.000+00:00
Duration: 11 min 52 s
Description: Watch this Scientific Journal Video about Targeted RNA Sequencing Assay to Characterize Gene Expression and Genomic Alterations at JoVE.com

We give special thanks to Ezra Lyon, Eliot Zhu, Michele Wing, Esko Kautto and Eric Samorodnitsky for technical support. We would also like to thank Jenny Badillo for her administrative support for our team. We acknowledge the Ohio Supercomputer Center (OSC) for providing disk space, processing capacity, and support to run our analyses. We thank the Comprehensive Cancer Center (CCC) at The Ohio State University Wexner Medical Center for their administrative support of this work. S.R. and Team are supported by the American Cancer Society (MRSG-12-194-01-TBG), a Prostate Cancer Foundation Young Investigator Award, NHGRI (UM1HG006508-01A1), Fore Cancer Research Foundation, American Lung Association, and Pelotonia.

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S.R. receives funding from Novartis and Ariad Pharmaceuticals for conduct of clinical trials. S.R. immediate family members own stock in Johnson and Johnson.

RNAseq 캡처 RNAseq 및 마이크로 어레이 사이의 중간 전략은 사체의 선택된 부분을 평가하기위한 접근이다. 캡처의 이점은 유전자 변형의 데스크탑 시퀀서, 높은 처리량, 및 검출에 선정 신속한 처리 시간 감소를 포함한다. 이 방법은, 비 코딩 RNA를 (23)의 특성을 단일 염기를 검출하도록 구성 될 수있는 RNA의 스 플라이 싱을 조사하고 유전자 융합체 또는 구조적 재 배열 (24)을 식별하기 위해, 4-6 변종. 또한,이 방법은 파라핀 블록 (24, 25)에 포르말린으로 고정 후의 임베디드 한 임상 샘플 또는 처리에 적용될 수있다. 마이크로 어레이에 비해 RNAseq 캡처 몇 가지 중요한 이점이 있으며, 실시간 정량 PCR, 생거 시퀀싱 및 DNA 시퀀싱. 마이크로 어레이 인해 크로스 하이브리드 및 프로브의 결합이 아닌 특정 높은 배경에 의해 제한된다. 리터와 유전자의 정량고도로 발현 유전자 측정 한 채도 신호의 영향을받는 동안 흐름 식은, 배경 노이즈로 인해 제한된다. RNAseq 캡처 비교, 실시간 PCR 재현하기 어려운 증명한다. 또한, RNAseq이 새로운 성적 증명서의 검출을 허용, 이하 시작 입력 자료를 필요로하며, 생거 시퀀싱 대안 스 플라이 싱 (26) .IN 대비를 검색 할 수 있습니다, RNAseq는 높은 처리량과 낮은 표현의 miRNA의 분석을 할 수 있습니다. 생거 시퀀싱 그러나 새로운 융합의 식별이 선험적 후보 중단의 요구에 의해 방해 알려진 엑손 - 엑​​손 접합 및 체세포 DNA 돌연변이와 융합의 검증을위한 유용한 도구로 입증했다. 비용 효율적인되지 DNA 시퀀싱, 데이터의 큰 저장 공간을 필요로하고, 전사 후 변형 검출 불가능하다. RNAseq 캡처에 관련된 몇 가지 중요한 단계가 있습니다. 우선, 도서관에서의 제품 수율을 향상시킬세척시 RNA / cDNA를 특정 상자성 구슬과 상자성 비즈, 이상 수율의 손실로 이어질 것입니다 구슬을, 건조하지 않도록주의해야합니다. 또한,하지의 밑에 건조 된 cDNA 수율을 감소시킬 수 에탄올, 모든 에탄올 샘플 튜브에서 제거되어 있는지 확인 구슬을. 둘째, 상호 보완적인 프로브의 cDNA 라이브러리의 하이브리드 우리가 사전에 적어도 두 시간 동안 65 ° C로 온난화 워시 버퍼 I과 엄격한 버퍼를 추천 일관된 온도에 따라 달라집니다. 또한, 하이브 리다이 제이션 이후의 결합 및 세척 단계 동안 65 ° C를 유지하기 위해 필수적이다. 여기에 사용되는 프로브는 키나아제, 같은 전사 인자, 집 유지 유전자와 같은 일반 재배에 관여하는 유전자를 포함하는 약물 개발에 대한 관심의 유전자의 엑손을 위해 설계되었다. 또한, 유전자 함량은 사용자 정의 및 캡처 패널 크기는 다를 수 있습니다. 게놈 영역에 새로운 정보가 발생로서 또한, 추가적인 프로브 설계와 T에 추가 될 수있다그는 캡처 패널을 기존. 정렬 측정 평가, 특히 온 - 목표 비율은 대상 영역이 강화되었다 얼마나 잘에 대한 정보를 제공합니다. 낮은 온 - 목표 속도는 원하는 대상 영역을 캡처 및 농축되지 않은 이에 실패 하이브리드 캡처, 때문일 수 있습니다. 이 경우, 라이브러리 세트의 재 혼성화 캡처가 수행되어야한다. 낮은 온 - 레이트 목표는 또한 샘플의 rRNA의 비율을 산출함으로써 확인할 수있다 rRNA의 고갈 실패로 될 수있다. 샘플 내의 rRNA의 높은 비율은 rRNA의 고갈로 시작하는 샘플의 재 조제를 필요로한다. 라이브러리 농도 혼성화 캡처위한 요구 이하로 떨어질 경우, 또한, 상기 샘플 종류와 품질 (: 50 ~ 1,000 ng의 범위)에 대해 시작 입력의 양을 최적화하는 것이 바람직 할 것이다. 대상 RNAseq 응용 프로그램에 대한 몇 가지 장점이 있지만, 또한에 제한 사항이 있습니다중히 여기다. 가난한 RNA의 RIN에 따라 품질이나 분열의 정도와 샘플 시퀀싱 품질 라이브러리를 생성 할 수 없습니다. 여러 그룹이 포르말린 고정 파라핀 포함 된 샘플의 성공을 증명하고있다, 그러나 순서 24,25,27에 대한 통과하지 않습니다 샘플이 있습니다. RNAseq 캡처 알려진 성적 증명서에 초점을 맞추고 있기 때문에, 그것은 소설이나 주석이 달려 있지 않은 성적에 대한 편견 RNAseq의 장점을 잃는다. 또한, SNP 검출을위한 방법 RNAseq는 표현 사체의 돌연변이를 검출 할 수있다. RNAseq 캡처의 미래의 기회는 연구와 임상 응용 프로그램을 포함한다. 긴 비 코딩 RNA의 수천 생물학에서의 역할의 최근 발견은 초점을 맞춘 특성화가 필요합니다. 암, 감염성 질환, 및 비 침습적 검사로 인간 질병의 특성을 병원에서 RNAseq 캡처 연구 테스트를 넘어 확장 할 수 있으며, 임상 분석으로 번역합니다. 게놈 서열과 함께 approaCHES, RNAseq 캡처 연구하고 표현 게놈의 특성을 통합 할 수 있습니다.

Whole transcriptome or RNA sequencing (RNAseq) is an unbiased sequencing method to assess all RNA products. The goal of targeted RNAseq (Capture) is a focused evaluation of selected transcripts with increased sensitivity, dynamic range, reduced cost or scale, and increased throughput compared to standard RNAseq. Similar to standard RNAseq, targeted enrichment approaches can be used to evaluate gene expression, multiple RNA species such as mRNA, microRNA (miRNA), lncRNA1, other noncoding RNAs2, gene fusions3, and mutations4-6.
Capture involves hybridization of complementary oligonucleotides to enrich cDNA libraries for sequencing. The rationale for RNAseq Capture is similar to microarray approaches where complementary oligonucleotides or probes are hybridized to samples and then measured for relative abundance. For microarray technologies, expression is based on relative signal measured for transcripts binding to these probes. Microarrays are thus limited by range, potential background noise from non-specific binding, and cross-hybridization of probes. Furthermore, arrays have limited dynamic range for low and highly expressed transcripts compared to RNAseq1. Microarrays are widely utilized due to their reduced cost and high throughput capacity compared to RNAseq. 
Here, we demonstrate a method for RNAseq Capture that offers a middle ground between RNAseq and microarray approaches for evaluating the transcriptome. RNAseq Capture has intermediate throughput, greater dynamic range and sensitivity, and is scaled for fast turnaround on desktop sequencers. RNAseq Capture also requires reduced computational resources in terms of storage space and data processing.

<table><tbody><tr><td>Thermomixer R</td><td>Eppendorf</td><td>21516-166</td><td></td></tr><tr><td>Centrifuge 5417R</td><td>Eppendorf</td><td>5417R</td><td></td></tr><tr><td>miRNeasy Mini Kit</td><td>Qiagen</td><td>217004</td><td></td></tr><tr><td>Molecular Biology Grade Ethanol</td><td>Sigma Aldrich</td><td>E7023-6X500ML</td><td></td></tr><tr><td>Thermoblock 24 x 1.5 ml</td><td>Eppendorf</td><td>21516-166</td><td></td></tr><tr><td>MiSeq Reagent Kit v2 (300-cycles)</td><td>Illumina</td><td>MS-102-2002</td><td></td></tr><tr><td>MiSeq Desktop Sequencer</td><td>Illumina</td><td></td><td></td></tr><tr><td>PhiX Control v3</td><td>Illumina</td><td>FC-110-3001</td><td></td></tr><tr><td>TruSeq Stranded Total RNA Kit with RiboZero Gold SetA</td><td>Illumina</td><td>RS-122-2301</td><td></td></tr><tr><td>25 rxn xGen&amp;reg; Universal Blocking Oligo - TS-p5</td><td>IDT</td><td>127040822</td><td></td></tr><tr><td>25 rxn xGen&amp;reg; Universal Blocking Oligo - TS-p7(6nt)</td><td>IDT</td><td>127040823</td><td></td></tr><tr><td>25 rxn xGen&amp;reg; Universal Blocking Oligo - TS-p7(8nt)</td><td>IDT</td><td>127040824</td><td></td></tr><tr><td>Agencourt&amp;reg; AMPure&amp;reg; XP - PCR Purification beads&amp;nbsp;</td><td>Beckman-Coulter</td><td>A63880</td><td></td></tr><tr><td>Dynabeads&amp;reg; M-270 Streptavidin</td><td>Life Technologies</td><td>65305</td><td></td></tr><tr><td>COT Human DNA, Fluorometric Grade, 1 mg</td><td>Roche Applied Science</td><td>05480647001</td><td></td></tr><tr><td>Qubit&amp;reg; Assay Tubes&amp;nbsp;</td><td>Life Technologies</td><td>Q32856</td><td></td></tr><tr><td>Qubit&amp;reg; dsDNA HS Assay Kit</td><td>Life Technologies</td><td>Q32851</td><td></td></tr><tr><td>SeqCap&amp;reg; EZ Hybridization and Wash Kits&amp;nbsp; (24 or 96 reactions)</td><td>Roche NimbleGen&amp;nbsp;</td><td>05634261001 or 05634253001&amp;nbsp;</td><td></td></tr><tr><td>Qubit&amp;reg; 2.0 Fluorometer&amp;nbsp;</td><td>Life Technologies</td><td>Q32866</td><td></td></tr><tr><td>10 x 2 ml IDTE pH 8.0 (1x TE Solution)</td><td>IDT</td><td></td><td></td></tr><tr><td>Tween20 BioXtra</td><td>Sigma</td><td>P7949-500ML</td><td></td></tr><tr><td>Nuclease Free Water</td><td>Life Technologies</td><td>AM9937</td><td></td></tr><tr><td>C1000 Touch&amp;trade; Thermal Cycler with 96&amp;ndash;Well Fast Rection Module</td><td>Biorad</td><td>185-1196</td><td></td></tr><tr><td>SeqCap EZ Hybridization and Wash Kits</td><td>Roche Applied Science</td><td>05634253001</td><td></td></tr><tr><td>SuperScript II Reverse Transcription 200 U/&amp;#956;l</td><td>Life Technologies</td><td>18064-014</td><td></td></tr><tr><td>D1000 ScreenTape</td><td>Agilent Technol. Inc.</td><td>5067-5582</td><td></td></tr><tr><td>Agencourt RNAClean XP - 40 ml</td><td>Beckman Coulter Inc</td><td>A63987</td><td></td></tr><tr><td>RNA ScreenTape</td><td>Agilent Technol. Inc.</td><td>5067-5576</td><td></td></tr><tr><td>RNA ScreenTape Ladder</td><td>Agilent Technol. Inc.</td><td>5067-5578</td><td></td></tr><tr><td>RNA ScreenTape Sample Buffer</td><td>Agilent Technol. Inc.</td><td>5067-5577</td><td></td></tr><tr><td>Sodium Hydroxide</td><td>Sigma</td><td>72068-100ML</td><td></td></tr><tr><td>DynaBeads MyOne Streptavidin T1</td><td>Life Technologies</td><td>65602</td><td></td></tr><tr><td>DYNAMAG -96 SIDE EACH</td><td>Life Technologies</td><td>12331D</td><td></td></tr><tr><td>Chloroform</td><td>Sigma</td><td>C2432-1L</td><td></td></tr><tr><td>KAPA HotStart ReadyMix</td><td>KAPA Biosystems</td><td>KK2602</td><td></td></tr><tr><td>NanoDrop 2000 Spectrophotometer</td><td>Thermo Scientific</td><td></td><td></td></tr><tr><td>My Block Mini Dry Bath</td><td>Benchmark</td><td>BSH200</td><td></td></tr><tr><td>D1000 Reagents</td><td>Agilent Technol. Inc.</td><td>5067- 5583</td><td></td></tr><tr><td>Vacufuge Plus</td><td>Eppendorf</td><td>022829861&amp;nbsp;</td><td></td></tr></tbody></table>

targeted rna sequencing assay to characterize gene expression and genomic alterations

RNA 서열 (RNAseq)는 유전자 발현 돌연변이 유전자 융합체, 및 비 암호화 RNA를 검출하고 특성화하는 데에 이용 될 수있는 다목적 방법이다. 1 억 순서 읽고 등의 mRNA 및 비 암호화 RNA를 여러 RNA 제품을 포함 할 수있다 - 표준 RNAseq (30)가 필요합니다. 우리는 타겟 RNAseq (캡처) 바탕 화면 시퀀서를 사용하여 선택 RNA 제품에 초점을 맞춘 연구를 허용하는 방법을 보여줍니다. RNAseq 캡처 그렇지 않으면 기존의 RNAseq 방법을 사용하여 놓칠 수있다, 주석이 달려 있지 않은 저, 또는 일시적으로 표현 된 성적 증명서를 특성화 할 수 있습니다. 여기에서 우리는 세포 라인, 리보솜 RNA 고갈, cDNA 합성, 바코드 라이브러리, 바탕 화면 시퀀서에 하이브리드 및 목표 성적 증명서의 캡처 및 다중 서열의 준비에서 RNA의 추출을 설명합니다. 또한 품질 관리 평가, 정렬 검출 융합 유전자 발현의 정량화 및 단일 NUC의 식별을 포함하는 연산 파이프 라인을 분석, 개요leotide 변형. 이 분석은 유전자 발현, 유전자 융합, 돌연변이의 특성을 타겟팅 사체 서열을 허용한다.

RNAseq 캡처 개략적 강조 주요 단계가도 1에 도시되어있다. 공지 된 돌연변이와 함께 4 개의 암 세포주는 RNAseq 캡처 기술의 유효성을 증명 하였다 (K562 ABL1 융합, RET 융합과 LC2와, EOL1 PDGFRalpha 융합 및 RT-으로 4 FGFR3 융합과). 네 개의 샘플을 함께 모으고 100 혈압 FASTQ 파일을 생성하는 데스크탑 시퀀서에 판독 2 배속으로 서열화 하였다. 인간 사체 1) 품질 관리 평가 2) 정렬 3) 유전자 발현의 정량 분석​​, 4) 융합 호출 및 5) 변형 호출 : FASTQ 파일 다섯 가지 주요 구성 요소를 포함하는 RNAseq 분석 파이프 라인을 통해 실행 하였다. 정렬 파일 (BAM)은 단일 염기 변이를 호출 유전자 발현을 계산하는데 사용된다. 융합은 (자신의 정렬 수행) 등 TopHat 융합 등 융합 발신자를 사용하여 호출되고, 출력은 날기를 주석이g 융합 탐지 소프트웨어. 유전자 RNAseq에서 표현과 캡처의 비교 (10)에 의해 표적 성적 증명서의 농축을 보여줍니다 캡처 방법 (그림 2A)를 사용하여 1,000 배. RNAseq 비교 포착하여 타겟 사체 영역에 매핑 된 읽기 또한,도 2b는 퍼센트의 증가를 나타낸다. 품질 관리 대책의 평가는 그림 3에 표시됩니다. 캡처 RNAseq은 사체 (3A, 94 % 대 93 %)에 정렬의 관점에서 동일하게 수행하고 삽입 크기 (3B, 174 bp의 대 (162) BP)를 의미한다. 캡쳐 방법을 사용하여, 엑손 영역의 높은 비율은 서열화되어있다 (3C 77 % 대 60 %), 반대로 인트론 영역의 하부 비율 서열화된다 (3D, 4 % 대 20 %). 샘플 당 총 읽기 수는 3E에 도시된다 </str예상대로 50 배 더 캡처보다 읽기를 통해 사연&gt;, 그리고 RNAseq 생성. 마지막으로, 각 샘플에 존재 rRNA의 서열의 비율 RNAseq (3 층, 4 % 대 15 %)에 비해 포획 방법을 이용하여 낮았다. 표 1에 나타낸 용융 검출 출력은 표준화 된 융합 판독지지 생성된다. RNAseq 4 개의 모든 세포주 융합체 검출에 성공 캡처. 캡처 및 RNAseq의 영역을 중첩라는 단일 염기 변이의 비교는 그림 4에 표시됩니다.이 대상 영역 내에서 캡처 및 RNAseq 사이 변종의 높은 일치도를 보여줍니다. <img alt="그림 1" src="/files/ftp_upload/54090/54090fig1.jpg" /> 그림 1. 도식 RNAseq 캡처 단계의.이 실험 데모에서는, RNA가 먼저 deple입니다역전사 효소를 사용하여 화학적 분열 보완 DNA (cDNA의)의 합성 다음 리보솜 RNA의 테드. 다음으로, cDNA를 폴리아 데 닐화 및 라이브러리를 생성하기 위해 플랫폼 별 어댑터 양단에 결찰된다. 적절한 어댑터 만의 cDNA 라이브러리는 다음 PCR에 의해 증폭된다. 라이브러리는 사용자 정의 올리고 뉴클레오티드 프로브에 하이브리드 및 자기 구슬을 사용하여 캡처됩니다. 캡처 라이브러리이 소량 차세대 시퀀싱 충분를 한 번 증폭되어야한다. 다중 라이브러리는 병렬로 순서화 될 수있다. 시퀀싱 데이터는 유전자 융합, 발현 또는 돌연변이 같은 관심의 RNA 이벤트를 분석한다. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54090/54090fig1large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> <img alt="그림 2" src="/files/ftp_upload/54090/54090fig2.jpg" /> 그림 2. CRNAseq. A, 측정 네 개의 암 세포 라인 K562, LC2, EOL1 및 RT-4 캡처 및 RNAseq 사이의 유전자 발현 비교 대 캡처에서 표적 유전자의 omparison 매핑 만 읽고 (FPKM) (로그 스케일) 당 킬로 당 읽습니다. 관심의 표적 유전자가 아닌 타겟 유전자 (회색). B, 대상 영역에 매핑이 네 가지 암 세포주에서 RNAseq 라이브러리 대 캡처에 증가 읽기의 비율 비교 (파란색) 강화됩니다. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54090/54090fig2large.jpg" target="_blank">여기를 클릭하십시오이의 더 큰 버전을 볼 수 있습니다 이 그림.</a> <img alt="그림 3" src="/files/ftp_upload/54090/54090fig3.jpg" /> 네 개의 대표 암 세포주에서 RNAseq 대 캡처. A는, 비율로는 사체에 매핑을 읽고 시퀀싱 메트릭 그림 3, <stroNG&gt; B가, 라이브러리 삽입 크기를 의미한다. C를,의 비율은 엑손 지역에서 읽습니다. D의 비율이 인트론 지역에서 읽습니다. E를, 총 순서가. F, 리보솜 RNA에 매핑을 읽는 비율이 읽습니다. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54090/54090fig3large.jpg" target="_blank">을 보려면 여기를 클릭하십시오 이 그림의 더 큰 버전.</a> <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td rowspan="2"> 세포주 </td><td rowspan="2"> 퓨전 </td><td rowspan="2"> 도서관 유형 </td><td rowspan="2"> 총 읽어 </td><td rowspan="2"> 대상 읽고에 </td><td colspan="3"> 지원 정규화 퓨전 읽고 (NFSR) </td></tr><tr><td> TophatFusion </td><td> ChimeraScan </td><td> TR쪽으로 </td></tr><tr><td> K562 </td><td> BCR-ABL </td><td> RNAseq </td><td> 150300482 </td><td> 279438 </td><td> 0 </td><td> (438) </td><td> 0 </td></tr><tr><td></td><td></td><td> 포착 </td><td> 9341148 </td><td> 7566087 </td><td> 598 </td><td> (343) </td><td> 0 </td></tr><tr><td> LC2 </td><td> CCDC6-RET </td><td> RNAseq </td><td> 128861790 </td><td> 307566 </td><td> 0 </td><td> 97 </td><td> 0 </td></tr><tr><td></td><td></td><td> 포착 </td><td> 12320692 </td><td> 10314284 </td><td> (71) </td><td> (44) </td><td> 6 </td></tr><tr><td> EOL1 </td><td> FIP1L1-PDGFRA </td><td> RNAseq </td><td> 135321406 </td><td> 225222 </td><td> 0 </td><td> 0 </td><td> (170) </td></tr><tr><td></td><td></td><td> 포착 </td><td> 9317418 </td><td> 7680818 </td><td> (143) </td><td> 0 </td><td> (7) </td></tr><tr><td> RT4 </td><td> FGFR3<html> -TACC3 </td><td> RNAseq </td><td> 161350024 </td><td> 208741 </td><td> 0 </td><td> (131) </td><td> 469 </td></tr><tr><td></td><td></td><td> 포착 </td><td> 8305950 </td><td> 6563574 </td><td> (358) </td><td> (88) </td><td> (34) </td></tr></tbody></table> K562, LC2, EOL1 및 RT-4의 RNAseq 대 캡처 표 1. 퓨전 감지.이 표는이 데모에 사용되는 네 개의 암 세포 라인과 세 가지 융합 탐지 알고리즘, TopHat2, ChimeraScan 및 TRUP를 표시합니다. 이 표는보다 큰 60000000가 RNAseq 활용 읽기에 비해 읽기 미만 1,000 만 총을 사용하여 캡처와 융합을 감지 할 수있는 능력을 보여줍니다. 키나아제 판독하여 융착지지 융합 나누어 계산 하였다 판독지지 1 백만 곱 읽는다. <img alt="&lt;/ HTML">&quot;그림 4&quot;SRC = &quot;/ 파일 / ftp_upload / 54090 / 54090fig4.jpg&quot;/&gt; RNAseq 대 캡처를 요구 그림 4. SNV.이 벤 다이어그램은 네 개의 세포주의 각각에 대해 캡처 및 RNAseq에 의해 검출 된 단일 염기 변형 (SNVs)의 수를 표시 (K562, LC2, EOL1 및 RT-4). 이 높은 대상 영역 내에서 캡처 및 RNAseq 사이 SNVs의 일치 보여줍니다. K562 (81.3 %), LC2 (78.3 %), EOL1 (89.5 %)과 RT-4 (73.9 %)을 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54090/54090fig4large.jpg" target="_blank">의 더 큰 버전을 보려면 여기를 클릭하십시오 이 그림.</a>

Watch this Scientific Journal Video about Targeted RNA Sequencing Assay to Characterize Gene Expression and Genomic Alterations at JoVE.com

Targeted RNA Sequencing Assay to Characterize Gene Expression and Genomic Alterations

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 ...

targeted-rna-sequencing-assay-to-characterize-gene-expression-genomic

Universidad San Sebastian

Research

JoVE Journal

Biology

10.3K 조회수.  The Ohio State University. 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.

비디오: Targeted RNA Sequencing Assay to Characterize Gene Expression and Genomic Alterations

Using RNA-sequencing to Detect Novel Splice Variants Related to Drug Resistance in In Vitro Cancer Models

Drug resistance remains a major problem in the treatment of cancer for both hematological malignancies and solid tumors. Intrinsic or acquired resistance can be caused by a range of mechanisms, including increased drug elimination, decreased drug uptake, drug inactivation and alterations of drug targets. Recent data showed that other than by well-known genetic (mutation, amplification) and epigenetic (DNA hypermethylation, histone post-translational modification) modifications, drug resistance mechanisms might also be regulated by splicing aberrations. This is a rapidly growing field of investigation that deserves future attention in order to plan more effective therapeutic approaches. The protocol described in this paper is aimed at investigating the impact of aberrant splicing on drug resistance in solid tumors and hematological malignancies. To this goal, we analyzed the transcriptomic profiles of several in vitro models through RNA-seq and established a qRT-PCR based method to validate candidate genes. In particular, we evaluated the differential splicing of DDX5 and PKM transcripts. The aberrant splicing detected by the computational tool MATS was validated in leukemic cells, showing that different DDX5 splice variants are expressed in the parental vs. resistant cells. In these cells, we also observed a higher PKM2/PKM1 ratio, which was not detected in the Panc-1 gemcitabine-resistant counterpart compared to parental Panc-1 cells, suggesting a different mechanism of drug-resistance induced by gemcitabine exposure.

Using RNA-sequencing to Detect Novel Splice Variants Related to Drug Resistance in In Vitro Cancer Models

Here we describe a protocol aimed at investigating the impact of aberrant splicing on drug resistance in solid tumors and hematological malignancies. To this goal, we analyzed the transcriptomic profiles of parental and resistant in vitro models through RNA-seq and established a qRT-PCR based method to validate candidate genes.

에 약물 저항 관련 소설 스플 라이스 변종을 감지하는 RNA 시퀀싱을 사용하여<em&gt; 체외</em&gt; 암 모델

Drug resistance remains a major problem in the treatment of cancer for both hematological malignancies and solid tumors. Intrinsic or acquired resistance ...

연구

암 연구학

Rare Event Detection Using Error-corrected DNA and RNA Sequencing

Conventional next-generation sequencing techniques (NGS) have allowed for immense genomic characterization for over a decade. Specifically, NGS has been used to analyze the spectrum of clonal mutations in malignancy. Though far more efficient than traditional Sanger methods, NGS struggles with identifying rare clonal and subclonal mutations due to its high error rate of ~0.5&#8211;2.0%. Thus, standard NGS has a limit of detection for mutations that are &#62;0.02 variant allele fraction (VAF). While the clinical significance for mutations this rare in patients without known disease remains unclear, patients treated for leukemia have significantly improved outcomes when residual disease is &#60;0.0001 by flow cytometry. In order to mitigate this artefactual background of NGS, numerous methods have been developed. Here we describe a method for Error-corrected DNA and RNA Sequencing (ECS), which involves tagging individual molecules with both a 16 bp random index for error-correction and an 8 bp patient-specific index for multiplexing. Our method can detect and track clonal mutations at variant allele fractions (VAFs) two orders of magnitude lower than the detection limit of NGS and as rare as 0.0001 VAF.

Next-generation sequencing (NGS) is a powerful tool for genomic characterization that is limited by the high error rate of the platform (~0.5&#8211;2.0%). We describe our methods of error-corrected sequencing that allow us to obviate the NGS error rate and detect mutations at variant allele fractions as rare as 0.0001.

드문 이벤트 감지 오류 수정 DNA와 RNA 시퀀싱을 사용 하 여

Conventional next-generation sequencing techniques (NGS) have allowed for immense genomic characterization for over a decade. Specifically, NGS has been ...

유전학

Analysis of Combinatorial miRNA Treatments to Regulate Cell Cycle and Angiogenesis

Lung cancer (LC) is the leading cause of cancer-related deaths worldwide. Similar to other cancer cells, a fundamental characteristic of LC cells is unregulated proliferation and cell division. Inhibition of proliferation by halting cell cycle progression has been shown to be a promising approach for cancer treatment, including LC. 
miRNA therapeutics have emerged as important post-transcriptional gene regulators and are increasingly being studied for use in cancer treatment. In recent work, we utilized two miRNAs, miR-143 and miR-506, to regulate cell cycle progression. A549 non-small cell lung cancer (NSCLC) cells were transfected, gene expression alterations were analyzed, and apoptotic activity due to the treatment was finally analyzed. Downregulation of cyclin-dependent kinases (CDKs) were detected (i.e., CDK1, CDK4 and CDK6), and cell cycle halted at the G1/S and G2/M phase transitions. Pathway analysis indicated potential antiangiogenic activity of the treatment, which endows the approach with multifaceted activity. Here, described are the methodologies used to identify miRNA activity regarding cell cycle inhibition, induction of apoptosis, and effects of treatment on endothelial cells by inhibition of angiogenesis. It is hoped that the methods presented here will support future research on miRNA therapeutics and corresponding activity and that the representative data will guide other researchers during experimental analyses.

miRNA therapeutics have significant potential in regulating cancer progression. Demonstrated here are analytical approaches used for identification of the activity of a combinatorial miRNA treatment in halting cell cycle and angiogenesis.

조합 미르 세포 주기 조절 및 신생 치료의 분석

Lung cancer (LC) is the leading cause of cancer-related deaths worldwide. Similar to other cancer cells, a fundamental characteristic of LC cells is ...

Defining Gene Functions in Tumorigenesis by Ex vivo Ablation of Floxed Alleles in Malignant Peripheral Nerve Sheath Tumor Cells

The development of new drugs that precisely target key proteins in human cancers is fundamentally altering cancer therapeutics. However, before these drugs can be used, their target proteins must be validated as therapeutic targets in specific cancer types. This validation is often performed by knocking out the gene encoding the candidate therapeutic target in a genetically engineered mouse (GEM) model of cancer and determining what effect this has on tumor growth. Unfortunately, technical issues such as embryonic lethality in conventional knockouts and mosaicism in conditional knockouts often limit this approach. To overcome these limitations, an approach to ablating a floxed embryonic lethal gene of interest in short-term cultures of malignant peripheral nerve sheath tumors (MPNSTs) generated in a GEM model was developed.
This paper describes how to establish a mouse model with the appropriate genotype, derive short-term tumor cultures from these animals, and then ablate the floxed embryonic lethal gene using an adenoviral vector that expresses Cre recombinase and enhanced green fluorescent protein (eGFP). Purification of cells transduced with adenovirus using fluorescence-activated cell sorting (FACS) and the quantification of the effects that gene ablation exerts on cellular proliferation, viability, the transcriptome, and orthotopic allograft growth is then detailed. These methodologies provide an effective and generalizable approach to identifying and validating therapeutic targets in vitro and in vivo. These approaches also provide a renewable source of low-passage tumor-derived cells with reduced in vitro growth artifacts. This allows the biological role of the targeted gene to be studied in diverse biologic processes such as migration, invasion, metastasis, and intercellular communication mediated by the secretome.

Defining Gene Functions in Tumorigenesis by Ex vivo Ablation of Floxed Alleles in Malignant Peripheral Nerve Sheath Tumor Cells

Here, we present a protocol for performing gene knockouts that are embryonic lethal in vivo in genetically engineered mouse model-derived tumors and then assessing the effect that the knockout has on tumor growth, proliferation, survival, migration, invasion, and the transcriptome in vitro and in vivo.

악성 말초 신경 시스 종양 세포에서 Floxed 대립 유전자의 Ex vivo 절제에 의한 종양 발생에서 유전자 기능 정의

The development of new drugs that precisely target key proteins in human cancers is fundamentally altering cancer therapeutics. However, before these ...

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

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.

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

Efforts  to detect and investigate key oncogenic mutations have proven valuable to  facilitate the appropriate treatment for cancer patients. The ...

생물학

Multiplexed Analysis of Retinal Gene Expression and Chromatin Accessibility Using scRNA-Seq and scATAC-Seq

Powerful next generation sequencing techniques offer robust and comprehensive analysis to investigate how retinal gene regulatory networks function during development and in disease states. Single-cell RNA sequencing allows us to comprehensively profile gene expression changes observed in retinal development and disease at a cellular level, while single-cell ATAC-Seq allows analysis of chromatin accessibility and transcription factor binding to be profiled at similar resolution. Here the use of these techniques in the developing retina is described, and MULTI-Seq is demonstrated, where individual samples are labeled with a modified oligonucleotide-lipid complex, enabling researchers to both increase the scope of individual experiments and substantially reduce costs.

Here, the authors showcase the utility of MULTI-seq for phenotyping and subsequent paired scRNA-seq and scATAC-seq in characterizing the transcriptomic and chromatin accessibility profiles in retina.

scRNA-Seq 및 scATAC-Seq 를 이용한 망막 유전자 발현 및 크로마틴 접근성의 다중화 분석

Powerful next generation sequencing techniques offer robust and comprehensive analysis to investigate how retinal gene regulatory networks function during ...

신경과학

Identification of Key Factors Regulating Self-renewal and Differentiation in EML Hematopoietic Precursor Cells by RNA-sequencing Analysis

Hematopoietic stem cells (HSCs) are used clinically for transplantation treatment to rebuild a patient&#39;s hematopoietic system in many diseases such as leukemia and lymphoma. Elucidating the mechanisms controlling HSCs self-renewal and differentiation is important for application of HSCs for research and clinical uses. However, it is not possible to obtain large quantity of HSCs due to their inability to proliferate in vitro. To overcome this hurdle, we used a mouse bone marrow derived cell line, the EML (Erythroid, Myeloid, and Lymphocytic) cell line, as a model system for this study.
RNA-sequencing (RNA-Seq) has been increasingly used to replace microarray for gene expression studies. We report here a detailed method of using RNA-Seq technology to investigate the potential key factors in regulation of EML cell self-renewal and differentiation. The protocol provided in this paper is divided into three parts. The first part explains how to culture EML cells and separate Lin-CD34+ and Lin-CD34- cells. The second part of the protocol offers detailed procedures for total RNA preparation and the subsequent library construction for high-throughput sequencing. The last part describes the method for RNA-Seq data analysis and explains how to use the data to identify differentially expressed transcription factors between Lin-CD34+ and Lin-CD34- cells. The most significantly differentially expressed transcription factors were identified to be the potential key regulators controlling EML cell self-renewal and differentiation. In the discussion section of this paper, we highlight the key steps for successful performance of this experiment.
In summary, this paper offers a method of using RNA-Seq technology to identify potential regulators of self-renewal and differentiation in EML cells. The key factors identified are subjected to downstream functional analysis in vitro and in vivo.

RNA-sequencing and bioinformatics analyses were used to identify significantly and differentially expressed transcription factors in Lin-CD34+ and Lin-CD34- subpopulations of mouse EMLcells. These transcription factors might play important roles in determining the switch between self-renewing Lin-CD34+ and partially differentiated Lin-CD34- cells.

RNA 시퀀싱 분석하여 EML 조혈 전구체 세포의자가 재생 (self-renewal)과 분화를 조절하는 중요한 요소의 식별

Hematopoietic stem cells (HSCs) are used clinically for transplantation treatment to rebuild a patient&#39;s hematopoietic system in many diseases such as ...

Visualizing Genetic Variants, Short Targets, and Point Mutations in the Morphological Tissue Context with an RNA In Situ Hybridization Assay

Because precision medicine is highly dependent on the accurate detection of biomarkers, there is an increasing need for standardized and robust technologies that measure RNA biomarkers in situ in clinical specimens. While grind-and-bind assays like RNAseq and quantitative RT-PCR enable highly sensitive gene expression measurements, they also require RNA extraction and thus prevent valuable expression analysis within the morphological tissue context. The in situ hybridization (ISH) assay described here can detect RNA target sequences as short as 50 nucleotides at single-nucleotide resolution and at the single-cell level. This assay is complementary to the previously developed commercial assay and enables sensitive and specific in situ detection of splice variants, short targets, and point mutations within the tissue. In this protocol, probes were designed to target unique exon junctions for two clinically important splice variants, EGFRvIII and MET&#916;14. The detection of short target sequences was demonstrated by the specific detection of CDR3 sequences of T-cell receptors &#945; and &#946; in the Jurkat T-cell line. Also shown is the utility of this ISH assay for the distinction of RNA target sequences at single-nucleotide resolution (point mutations) through the visualization of EGFR L858R and KRAS G12A single-nucleotide variations in cell lines using automated staining platforms. In summary, the protocol shows a specialized RNA ISH assay that enables the detection of splice variants, short sequences, and mutations in situ for manual performance and on automated stainers.

Visualizing Genetic Variants, Short Targets, and Point Mutations in the Morphological Tissue Context with an RNA In Situ Hybridization Assay

Here, we describe an in situ hybridization assay which enables sensitive and specific detection of sequences as short as 50 nucleotides with single-nucleotide resolution at the single-cell level. The assay, which can be performed manually or automatically, can enable visualization of splice variants, short sequences, and mutations within the tissue context.

유전자 변형, 짧은 목표, 및 교 잡 시험 현장에서 RNA와 형태학 상 조직 맥락에서 점 돌연변이 시각화

Because precision medicine is highly dependent on the accurate detection of biomarkers, there is an increasing need for standardized and robust ...

RNA-Seq

Among different methods to evaluate gene expression, the high-throughput sequencing of RNA, or RNA-seq. is particularly attractive, as it can be performed and analyzed without relying on prior available genomic information. During RNA-seq, RNA isolated from samples of interest is used to generate a DNA library, which is then amplified and sequenced. Ultimately, RNA-seq can determine which genes are expressed, the levels of their expression, and the presence of any previously unknown transcripts.Here, JoVE presents the basic principles behind RNA-seq. We then discuss the experimental and analytical steps of a general RNA-seq protocol. Finally, we examine how researchers are currently using RNA-seq, for example, to compare gene expression between different biological samples, or to characterize protein-RNA interactions.

Next-Gen Transcriptomics Using RNA-Seq

Among different methods to evaluate gene expression, the high-throughput sequencing of RNA, or RNA-seq. is particularly attractive, as it can be performed ...