1、测序前文库制备 - Illumina 测序 平台报告人： 刘竹琳Email: supportneb-Tel： 010 82378266-22005Roche 4542006Illumina Solexa2007ABI Solid2010Illumina HiSeq 20002014Illumina HiSeq X Ten二代测序平台测序应用 基因组学 全基因组 De novo 测序 全基因组重测序 外显子 & 目标区域捕获测序 转录组学 转录组测序 数字基因表达谱测序 Small RNA 测序 长链非编码 RNA 测序（ LncRNA-Seq） Circ RNA 测序 表观组学 甲基化测序 C
2、hIP 测序测序流程NEBNext Reagents for IlluminaIlluminaDNAGenomicDNA原理应用ChIP DNARNADirectional原理应用Non-DirectionalSmall RNA原理应用DNA Library Prep for IlluminaFragmentation End Repair dA Tailing Adaptor Ligation PCR Enrichment片段化基因组 DNA末端补平磷酸化（ T4、 Klenow、PNK、 dNTP）加 A 尾（ Klenowexo-、 dNTP）连接接头（ T4 DNA Ligase）PC
3、R（ Q5、 dNTP）DNA Library Prep for Illumina - FragmentationFragmentation End Repair dA Tailing Adaptor Ligation PCR Enrichment片段化基因组 DNA末端补平磷酸化（ T4、 Klenow、PNK、 dNTP）加 A 尾（ Klenowexo-、 dNTP）连接接头（ T4 DNA Ligase）PCR（ Q5、 dNTP）What is DNA fragmentation？DNA fragmentation is the separation or breaking of D
4、NA strands into pieces. Methods of DNA Fragmentation Nebulization: Illumina(100-200bp)/ Roche454(500-800bp)Sonication : ABI SOLiD(100bp)Enzyme-based: NEBNEBNextTM dsDNA Fragmentase 简介 由两种酶组成，一种切刻双链中的一条链，另一种在切刻的对侧切割 能将 dsDNA 切割成 100-800 bp，片段大小取决于反应时间 片段化的 DNA 末端是粘性末端， 5磷酸基， 3羟基 DNA 的量与酶的比例是常数，反应时间长短
5、决定片段大小DNA 片段化的方法 酶学法Vvn endonuclease mutantT7 endonuclease mutant 组成： 两种内切酶 创伤弧菌 核酸酶突变蛋白质（ Vibrio vulnificus nuclease mutant protein） T7 核酸内切酶突变蛋白质（ T7 endonuclease mutant protein）NEBNextTM dsDNA FragmentaseNEBNextTM dsDNA Fragmentase 切割原理dRibo5 3dRibo dRibo dRibo dRibo dRibo dRibo dRibodRibodRibodR
6、ibodRibodRibodRibodRibodRibo53 dsDNA Fragmentase generates dsDNA breaks in a time -dependent manner The resulting DNA fragments contain short overhangs, 5-phosphates, and 3-hydroxyl groups. Vvn endonuclease mutantT7 endonuclease mutantFragmentase Reaction Protocol 1. 建立反应体系，不要加入Fragmentase 2. 放置冰上 5
7、分钟 3. 加入Fragmentase 4. 37 温育，温育时间根据情况而定 5. 终止反应：加入5 ul 0.5 M EDTA 6. 用于 DNA 末端修复、分离不同大小片段、分析基因组 DNAWGA DNAPCR 产物 （ 1-6 kb）RT-PCR 产物（ 1-6 kb）GC-rich 基因组 DNAAT-rich 基因组 DNAM0348S 50 次反应 100 ul ￥ 1149.00M0348L 250次反应 500 ul ￥ 4569.00加入顺序： H2O、 DNA、 buffer、 BSAFragmentase 优势Figure 3: Relative size distr
8、ibution of E. coli DNA fragments with NEBNext dsDNA Fragmentase vs. Nebulization as seen using the Bioanalyzer 2100. The dsDNA Fragmentase sample was incubated for 30 minutes at 37C with 0.5 g of DNA per l of NEBNext dsDNA Fragmentase in 1X Fragmentase Reaction Buffer with 100 g/ml BSA. The Nebulize
9、r sample was prepared by nebulization of DNA in 50% glycerol for 6 minutes at 35 psi.优势易缩小或放大体系，高通量易控制片段大小价格便宜使用方便DNA末端均一DNA Library Prep for Illumina End RepairWhat is end repair？End repair is used for blunting and phosphorylation of DNA ends.T4 DNA Polymerase3 5exonuclease activity 5 3 polymerase
10、activity T4 PNKAddition of 5-phosphates 35 53 3355Remove 3overhangs 533553 53Fill-in 5overhangs Fragmentation End Repair dA Tailing PCR EnrichmentAdaptor Ligation片段化基因组 DNA末端补平磷酸化（ T4、 Klenow、PNK、 dNTP）加 A 尾（ Klenowexo-、 dNTP）连接接头（ T4 DNA Ligase）PCR（ Q5、 dNTP）DNA Library Prep for Illumina dA Tailing
11、Fragmentation End Repair dA Tailing Adaptor Ligation PCR Enrichment片段化基因组 DNA末端补平磷酸化（ T4、 Klenow、PNK、 dNTP）加 A 尾（ Klenowexo-、 dNTP）连接接头（ T4 DNA Ligase）PCR（ Q5、 dNTP）What is dA Tailing?Adds a single A to the 3end of the fragmented, blunt, phosphorylated DNA Klenow Fragment (35 exo)AADNA Library Prep
12、for Illumina Adaptor Ligation Fragmentation End Repair dA Tailing Adaptor Ligation PCR Enrichment片段化基因组 DNA末端补平磷酸化（ T4、 Klenow、PNK、 dNTP）加 A 尾（ Klenowexo-、 dNTP）连接接头（ T4 DNA Ligase）PCR（ Q5、 dNTP）What is adaptr ligation?Ligation of adaptors onto the A-tailed Sample DNA T4 DNA LigaseTAATDNA Library Pr
13、ep for Illumina PCR EnrichmentFragmentation End Repair dA Tailing Adaptor Ligation PCR Enrichment片段化基因组 DNA末端补平磷酸化（ T4、 Klenow、PNK、 dNTP）加 A 尾（ Klenowexo-、 dNTP）连接接头（ T4 DNA Ligase）PCR（ Q5、 dNTP）What is PCR Enrichment ?Amplification of Adaptor-ligated sample DNA5 ng: 13-15 Cycle50 ng: 10 Cycle1 ug:
14、0-6 CycleNEBNext Reagents for IlluminaIlluminaDNAGenomicDNA原理应用ChIP DNARNADirectional原理应用Non-DirectionalSmall RNA原理应用Original NEBNext Ultra for IlluminaOptimized for 5 ng 1 g input of genomic DNA (the first NEBNext kits required 1 5 g).Clean-up steps were minimized.Each step was optimized: Combined
15、steps: End Repair and dA Tailing Novel ligation reagents: New Ligase Master Mix (Blunt/TA), optimized NEBNext Adaptor Novel polymerase reagents: NEBNext Q5 High-Fidelity PCR Master MixFast (2.5 - 3 hrs) and minimal (15 min) hands on timeThe original Ultra DNA kit was released in Oct. 2012, and it wa
16、s the first streamlined kit on the market for Illumina library preparation.Original Ultra (NEB #E7370)End Repair/dA-TailingAdaptorLigationClean-up/Size Selection Clean-upAmplificationEvolving Library Prep NeedsWe developed the Ultra II DNA Library Prep Kit to achieve: High quality, reliable library
17、preparation with o Lower input amountso Challenging sample types like FFPE DNA Higher library yields Uniform GC content coverage Fewer PCR cycles in library amplification PCR-Free workflows at lower input amountsThe new kit also had to: Be fast and easy to use Be automatable Work well with high qual
18、ity DNA Work well with a broad range of input amounts, up to 1gDevelopment of NEBNext Ultra IIEnd Repair/dA-TailingAdaptorLigationClean-up/Size Selection Clean-upAmplificationNEBNext Ultra II Ligase MMNEBNext Ultra II End Prep Master Mix and BufferNEBNext Ultra II Q5 HiFi Master MixThe popular, fast
19、, streamlined workflow has been retained.The result: very substantial improvements in performanceThe reagents for each step have been reformulated Ultra II (NEB #E7645).Ultra II Performance Summary Higher yields Lower input amounts and a broad input range (500 pg to 1 g) Fewer PCR cycles required Ev
20、en better GC coverageFFPE DNAFFPE DNA can be of very low and variable quality and it can be challenging to make high quality libraries, especially when input amounts are low.NEBNext Ultra II enables construction of high quality libraries from FFPE DNA samples, even with low input amountsLibraries we
21、re prepared using Ultra II from 17-30 ng of human DNA extracted from the FFPE tissue samples listed, amplified using 10 cycles of PCR and sequenced on the Illumina MiSeq. Reads were mapped to the hg19 reference genome using Bowtie 2.2.4. % Mapped: The percentage of reads mapped to Human GRCh37 refer
22、ence. % Mapped in Pairs: The percentage of reads whose mate pair was also aligned to the reference. % Duplication: The percentage of mapped sequence that is marked as duplicate. % Chimeras: The percentage of reads that map outside of a maximum insert size or that have the two ends mapping to differe
23、nt chromosomes. Bioanalyzer traces of each library show high quality libraries with minimal adaptor-dimer.Illumina 测序样本准备 -片段选择AMPure XP beads低起始量，高产量超快速优化流程，减少手动操作时间适用于所有种类的样本更适用于目的基因捕获建库有试剂盒和模块包装功能验证，严格质控NEBNext DNA Prep for IlluminaOrder informationNEBNext Adaptor The structure of the NEBNext ada
24、ptor is different than the Illumina adaptors. The NEBNext adaptor is a hairpin loop structure while the Illumina adaptor has a Y shape The NEBNext Adaptor does not include barcodes.The NEBNext Adaptor is ligated with higher efficiency than the TruSeq adaptor.This increased ligation efficiency leads
25、to higher library yields compared to TruSeq libraries.NEBNext Primer Each of the NEBNext Index Primers contains a different 6 base index (also termed a barcode). These indexes are the same as the indexes used by Illumina. Each of the barcodes was equally represented after sequencing, and less than 0
26、.1% cross-contamination was observed.BC = 6-base Barcode/indexP5 and P7 are sequences required for subsequent steps in the Illumina workflow, and these are the names used by Illumina for these sequences.Increased Library Yields with NEBNextAdaptors vs TruSeq Adaptors NEBNext Oligos for Illumina Adaptor USER Enzyme NEBNext Universal PCR Primer for Illumina NEBNext Index Primer for IlluminaFragmentase UltraII Kit Oligos+ +NEBNext Reagents for IlluminaIlluminaDNAGenomicDNA原理应用ChIP DNARNADirectional原理应用Non-DirectionalSmall RNA原理应用
