SMARTer for NGS. SMARTer Solutions 다카라코리아바이오메디칼

Transcription

1 SMARTer for NGS SMARTer Solutions 다카라코리아바이오메디칼

2 Contents Next Generation Sequencing SMARTer Sequencing Kits Single Cell RNA-Seq & mrna-seq SMARTer Ultra Low Input RNA Kit for Sequencing -v3 SMARTer Ultra Low RNA Kit for the Fluidigm C1System Total RNA-seq solutions SMARTer Stranded RNA-Seq Kit SMARTer Universal Low Input RNA Kit RiboGone: rrna removal for low input samples DNA sequencing solutions (NEW) Library Quantification Kit (NEW) SMARTer 선택가이드

3 Sequencing Cell ( 세포 ) > Nucleus ( 핵 ) > Gene ( 유전자 ) > Base sequence ( 염기서열 ) Base sequence ( 염기서열 ) - A (Adenine), T (Thymine), G (Guanine), C (Cytokine) 의조합배열 - 염기가어떻게조합되어있냐에따라세포의기능및성질이결정 염기서열을결정하기위한연구활동 = Sequencing!!

4 Next Generation Sequencing (NGS)? Sequencing 의역사 1953년 DNA 구조규명 (Watson&Crick) 1975년 1세대염기서열분석 (Fredric Sanger) 1990년 인간지놈프로젝트 1995년 Shotgun sequencing 2001년 인간지놈프로젝트완료 2006년 차세대염기서열분석 (NGS) - 454Life Science (Roche) - Solexa 2007년 Illumina의 Solexa 합병 1 세대염기서열분석 년, Fredric Sanger - dideoxy 염기서열분석법 - 화학적으로변형뉴클레오타이드이용 차세대염기서열분석 (NGS) 2, 3 세대 - 짧은시간내에대용량염기서열분석 - DNA 서열을증폭하고형광표식을캡쳐하여 reading 2014 년 Next-Next generation sequencing - PacBio RSII System Roche/454 Illumina/Solexa ABI/Solid

5 Next Generation Sequencing (NGS)? NGS Platform Illumina : MiSeq, HiSeq, NextSeq Thermo Fisher: Ion torrent-proton, PGM Pacific Biosciences: PACBIO RS II SOLiD sequencing Roche GS Junior Illumina MiSeq Ion torrent PGM PacBio RSII NGS 는 Sanger sequencing 에비하여저렴한비용으로더빠르고더많은 sequencing 데이터를생산할수있다.

6 NGS Application DNA-Seq RNA-Seq RNA-Seq DNA Whole Genome ChiP DNA Targeted DNA RNA mrna (PolyA+RNA) Total RNA Non-coding RNA Non-coding RNA Small RNA, microrna DNA * Cost-effect * Fast * Ultra throughput * Cloning free * Short read

7 NGS 적용분야 1. De novo assembly - 다양한종의 Whole Genome Sequencing (WGS) 2. Target Sequencing (Sequence Capture) - 특정유전체의일부분을선택적으로분석 3. RNA Seq Analysis (Transcriptome) - 유전자와 exon 의발현및발현된유전자의각종변이등을한번에연구 4. Epigenomics - Epigenetics: 유전되는 DNA 서열설명이불가능한부분의해석가능 5. Chip-seq - 단백질에 binding 된 DNA 를분리하여 biding site 동정 6. Bioinformatics - 방대한양의 sequence 를다룰수있는생물정보학분석 도표출처 : AllSeq Inc

8 Clontech 중점제품 For low-input sample, The Transcriptome (RNA-Seq) ChIP-Seq (DNA-Seq)

9 SMARTER SEQUENCING KITS

10 NGS Workflow <NGS workflow> - Clontech 제품은어디에쓰이나요? cdna 합성, Library Preparation 에서사용됩니다! Library preparation for NGS RNA Isolation cdna Synthesis Library construction

11 SMART 기술이란? Full length cdna 를합성하기위한 Clontech 의독자적기술 - Switching Mechanism At 5 -end of RNA Transcript - 나노그람 (ng) 수준의 total RNA 로부터고품질의 cdna 를합성 Terminal Transferase ( 말단전이활성 ) Template Switching ( 주형전환활성 ) LD-PCR by Advantage 2 Polymerase Simple 1-tube cdna 합성 linkers/adapters ligation 불필요 Total RNA 로실험진행 Powerful full-length cdna transcripts 소량의 total RNA 로실험가능 Quality Full length cdna 조제

12 왜 SMART 기술이필요한가? Low-Input sample 에최적이다! NGS Library 제작의한계 * 적은량의 mrna 또는 Total RNA * Adaptor 를부착해야한다 (Ligation) 극복! Clontech SMARTer 는,, Simple Powerful Quality * 피코그람수준의 RNA 로부터 cdna 합성 * Full length 의 cdna 획득 * Ligation 과정없이 Adaptor 부착 ( 고효율 )

13 샘플별선택가이드 NGS workflow 샘플시료 Starting sample rrna 제거 SMARTer cdna 합성 Library 제작 Library 정량 NGS 분석 mrna (PolyA+) SMARTer Ultra Low Input-v3 RNA Single Cell Total RNA RiboGone SMARTer Ultra Low Input-FluidigmC1 SMARTer Universal Low Input Library Prep Library Quantific ation kit (for illumina) NGS 분석 -Illumina, -Ion torrent SMARTer stranded RNA-Seq DNA ChIP DNA DNA SMART -ChIP Seq

14 기기별선택가이드 아래의기기를보셨다면! 제품추천은이렇게! Illumina MiSeq, HiSeq. Ion torrent PGM Fluidigm C1 PacBio RSII Single cell & mrna Ultra Low Input-v3 Ultra Low Input-v3 Ultra Low RNA Kit for the Fluidigm C1 Total RNA Universal Low Input Stranded RNA-Seq Universal Low Input cdna Synthesis kit (634925, ) ChIP DNA DNA ChIP-Seq Kit Library quantification Kit

15 샘플별선택가이드 Single Cell RNA-Seq & mrna-seq SMARTer Ultra Low Input RNA Kit for Sequencing -v3 SMARTer Ultra Low RNA Kit for the Fluidigm C1System Total RNA-seq solutions SMARTer Stranded RNA-Seq Kit SMARTer Universal Low Input RNA Kit RiboGone: rrna removal for low input samples DNA sequencing solutions - DNA SMART: a novel method for generating ChIP-Seq libraries from low quantities of cells Library Quantification Kit

16 SINGLE CELL RNA-SEQ SMARTer Ultra Low Input RNA Kit

17 Single Cell 분석의중요성 왜 Single cell 을분석하나? New cell type identification Developmental biology cell lineage study Stem cells and ips-cell lineage studies Understanding regulatory pathways Understanding tumor heterogeneity 각각의 Single cell 은저마다다른발현형 (expression) 을보인다

18 SMARTer Ultra Low Input Oligo dt Primer: SMART CDS Primer Highly sensitive, selective Transcriptome analysis directly from cells or single cell Uniform transcript coverage Minimal 3 5 bias Blocked IS-PCR primer No sequencing reads of SMARTer Oligo mrna, Single Cell Oligo dt Priming Ultra Low! - SMARTer Ultra Low Input RNA Kit for Sequencing v3 - SMARTer Ultra Low Input RNA Kit for the Fluidigm C1 System

19 SMARTer Ultra Low Input RNA-Seq-v3 SMARTer Ultra Low Input RNA Kit for Sequencing v3 최고의민감도 : 1-1,000 cells, 10 pg 10 ng mrna 시간단축 : Purification 과정감소 (Single SPRI-bead purification of ds cdna) cdna 증폭효율향상 : SeqAmp DNA Polymerase NGS 기기적용성 : Illumina and Ion Torrent High-quality RNA-seq data

20 SMARTer Ultra Low Input RNA-Seq-v3 Ion Torrent Illumina

21 샘플별선택가이드 Single Cell RNA-Seq & mrna-seq SMARTer Ultra Low Input RNA Kit for Sequencing -v3 SMARTer Ultra Low RNA Kit for the Fluidigm C1System Total RNA-seq solutions SMARTer Stranded RNA-Seq Kit SMARTer Universal Low Input RNA Kit RiboGone: rrna removal for low input samples DNA sequencing solutions - DNA SMART: a novel method for generating ChIP-Seq libraries from low quantities of cells Library Quantification Kit

22 TOTAL RNA-SEQ SOLUTION SMARTer Stranded RNA-Seq Kit SMARTer Universal Low Input RiboGone: rrnaremoval for low input samples

23 Total RNA-Seq challenges Total RNA NGS 의한계점? Ribosomal RNA Long Protocols Input challenges Random primer 로 cdna 합성 원하지않는 RNA 까지모두합성, Sequencing Total RNA에는,,, mrna (messenger RNA, Transcript) : 의미있는 sequence read rrna (Ribosomal RNA) : 원하지않는 sequence read : Total RNA중의 90% 가 rrna

24 RiboGone-Mammalian: rrna 제거 RNA Isolation cdna Synthesis Library construction Ribo + Gone! : Goodbye ribosomal RNA ~ Human, mouse, rat total RNA sample로부터 rrna, mtrna 제거 소량의 (low input) RNA 에도적용가능 : 10 ng 100 ng FFPE Tissue와같은 degraded sample도적용가능 Oligo dt priming 제품의경우, mrna만선택적으로 cdna 합성하므로사용하지않아도됨

25 RiboGone-Mammalian: rrna 제거 RNA Isolation cdna Synthesis Library construction Ribo + Gone! : Goodbye ribosomal RNA ~ RiboGone 을사용하여, rrna 를제거한효과!

26 FFPE Challenges FFPE sample 로부터 RNA-Seq 을하고싶을경우? Ribosomal RNA (rrna) Very poor quality RNA (RIN<2) Small amount of sample (few ngs) 이럴때어떤제품을추천할까요? SMARTer Universal Low Input 제품을추천! FFPE

27 SMARTer Universal Low Input RNA Kit for Sequencing 샘플 : 절단, 분해된 RNA 에특화 - sheared rrna-depleted total RNA (200pg-10ng) Random primer 이용 NGS Platform Low Input Library Prep Kit Illumina NGS Ion Express -Ion Torrent NGS FFPE, LCM sample Random priming Universal Low Input - SMARTer Universal Low Input RNA Kit for sequencing

28 SMARTer Stranded RNA-Seq Kit 샘플 : rrna 제거 total RNA Poly(A) enriched total RNA 100pg-100ng Random primer 이용 Illumina 용 Library 까지제작 (4 시간 ) cdna 합성과정에서 Illumina adaptor 부착 Total RNA Random priming mrna 정보유지 SMARTer stranded - SMARTer Stranded RNA-Seq Kit - mrna strand-specific - Illumina 용 Library 제작 (~4 시간 )

29 샘플별선택가이드 Single Cell RNA-Seq & mrna-seq SMARTer Ultra Low Input RNA Kit for Sequencing -v3 SMARTer Ultra Low RNA Kit for the Fluidigm C1System Total RNA-seq solutions SMARTer Stranded RNA-Seq Kit SMARTer Universal Low Input RNA Kit RiboGone: rrna removal for low input samples DNA sequencing solutions - DNA SMART: a novel method for generating ChIP-Seq libraries from low quantities of cells Library Quantification Kit

30 NEW! DNA SEQUENCING SOLUTIONS DNA SMART ChIP-Seq Kit

31 DNA SMART ChIP-Seq Kit ChIP (Chromatin Immuno Precipitation)? - Protein-DNA 상호작용과 Chromatin 구조를분석하기위한실험 - Target protein에대한항체를이용하여 Protein-DNA Complex를면역침강 - 이후, 단백질과결합한 DNA를분리하여 PCR, microarray, NGS 분석 DNA SMART ChIP-Seq Kit DNA Protein ChIP 실험으로부터추출한 DNA 로부터 Illumina NGS 용 Library 를제작 - is able to generate libraries from DNA isolated by ChIP - has high sensitivity (works with lower amounts of DNA) making it very helpful in studies that are using fewer cells or have low abundant transcription factors.

32 SMART RTase 숨겨진기능 Reverse Transcriptase (RTase)? - RNA-dependent DNA polymerase - RNA로부터 complementary DNA (cdna) 를합성하는효소 - Retrovirus (HIV, MMLV) 의자가유전체복제에서유래 RTase 의기본특성 (a) RNA-dependent DNA polymerase (b) ribonuclease H (c) DNA-dependent DNA polymerase Virus 유래 RTase 의특성 SMARTer Reverse Transcriptase 유래 용도 Moloney murine leukemia virus (M-MLV) first strand cdna synthesis 반응온도 37 RNaseH RNaseH 활성이낮다 출처 : Wikipedia

33 DNA SMART ChIP-Seq Kit

34 DNA SMART ChIP-Seq Kit Target User - Illumina 기기보유고객 - Epigenetics 연구자 Category>Epigenetics 제품사용고객

35 샘플별선택가이드 Single Cell RNA-Seq & mrna-seq SMARTer Ultra Low Input RNA Kit for Sequencing -v3 SMARTer Ultra Low RNA Kit for the Fluidigm C1System Total RNA-seq solutions SMARTer Stranded RNA-Seq Kit SMARTer Universal Low Input RNA Kit RiboGone: rrna removal for low input samples DNA sequencing solutions - DNA SMART: a novel method for generating ChIP-Seq libraries from low quantities of cells Library Quantification Kit

36 NEW! LIBRARY QUANTIFICATION Library Quantification Kit (for Illumina)

37 Library Quantification Kit (for Illumina) Library Quantification Kit qpcr 방법을이용한 Illumina Library 정량키트 Too much or too less = poor quality data Sensitive: qpcr 방법을사용함으로써기존의방식보다높은민감도 Robust: GC-contents 의함량에관계없이높은정확성과효율로정량 Accurate

38 Library Quantification Kit (for Illumina) POINT! TARGET! - Illumina NGS 사용자라면누구나!! - SMARTer 제품을사용하지않는고객에게도추천가능!

39 SMARTER 선택가이드

40 Clontech SMARTer 제품군 Clontech SMARTer 는,, * 피코그람수준의 RNA 로부터 cdna 합성 * Full length 의 cdna 획득 * Ligation 과정없이 Adaptor 부착 ( 고효율 ) Simple Powerful Quality 극소량 (Low Input) Sample 로부터 NGS 용 Library 제작에최적! 다수의 NGS Platform 이인정한최고의제품 일반 SMARTer cdna 합성제품도 NGS 고객에게추천가능! SMARTer PCR cdna Synthesis Kit (code , ) SMARTer RACE 5'/3' Kit (code , )

41 샘플별선택가이드 NGS workflow 샘플시료 Starting sample rrna 제거 SMARTer cdna 합성 Library 제작 Library 정량 NGS 분석 mrna (PolyA+) SMARTer Ultra Low Input-v3 RNA Single Cell Total RNA RiboGone SMARTer Ultra Low Input-FluidigmC1 SMARTer Universal Low Input Library Prep Library Quantific ation kit (for illumina) NGS 분석 -Illumina, -Ion torrent SMARTer stranded RNA-Seq DNA ChIP DNA DNA SMART -ChIP Seq

42 기기별선택가이드 아래의기기를보셨다면! 제품추천은이렇게! Illumina MiSeq, HiSeq. Ion torrent PGM Fluidigm C1 PacBio RSII Single cell & mrna Ultra Low Input-v3 Ultra Low Input-v3 Ultra Low RNA Kit for the Fluidigm C1 Total RNA Universal Low Input stranded RNA-Seq Universal Low Input cdna Synthesis kit (634925, ) ChIP DNA DNA ChIP-Seq Kit Library quantification Kit

43 기기별선택가이드 * SMARTer Ultra Low RNA Kit for the Fluidigm C1 System (code , )

44