13. 最新分子生物学技术

Why Another Generation of Sequencers?
● Next (2nd) Generation Sequencing weaknesses: • PCR steps introduce bias (duplicates, PCR errors) • alternating phases of nucleotide incorporation and signal detection • dephasing, quality decreasing towards the end • short reads • big expensive machines, expensive chemicals • long run times ● Make things bigger, better, faster, cheaper: • less input DNA, simpler library preparation, less reagents (no “washing”) • smaller, less expensive machines • reduced run time • longer reads • better quality • higher throughput
(A–C) Scatter plots compare the normalized average number of sequence reads for S. cerevisiae (x-axis) and S. paradoxus (y-axis). Genes with statistically significant differences in read counts (FDR < 5%, minimum 1.5-fold difference) are plotted as open circles with black edges. Translation efficiency is defined here as the number of ribosome protected fragment reads (RPF) divided by the number of mRNA-seq reads covering an ORF.
3’
5’ 5’ 5’ 5’ 5’ 5’ 5’ 5’ 5’ 四 套 5’ 反 5’ 应 体 5’ 系
5’ 测序模板 ATGCGGTACCAT 1--dATP, dCTP, dGTP, dTTP. + ddATP TA TACGCCA TACGCCATGGTA 2--dATP, dCTP, dGTP, dTTP. + ddCTP TAC TACGC TACGCC 3--dATP, dCTP, dGTP, dTTP. + ddGTP TACG TACGCCATG TACGCCATGG
Third Generation Sequencing
• Single molecule sequencing (no amplification needed) • Oxford Nanopore: Read fewer but longer sequences • In 1-2 years, the cost of sequencing a human genome will drop below $1000, storage will cost more than sequencing • Personal genome sequencing might become a key component of public health in every developed country • Bioinformatics will be key to convert data into knowledge
Sanger双脱氧链终止法
原理：双脱氧（2'，3'）-核苷酸可以象2'-脱氧核苷酸那样直接 掺入新合成的DNA链中，但因3’端不具OH基，DNA链合成至 此中断。由于双脱氧核苷酸在每个DNA分子中掺入的位置不 同，故可根据不同长度的DNA片段测定出核苷酸序列。
不能和下一个核苷 酸通过磷酸二酯键 18 连接起来
Advanced molecular biology digs into single-cell level!
Genomics
Transcriptome Proteomics
Metabolomics
Micro-pipetting: meiocytes, pollen………
Fluorescence activated cell sorting (FACS)
22
Next generation sequencing/Second generation seqeucing/Pyrosequencing
Illumina sequencing
The Next Generation
• When does "current generation" become "last generation", and "next generation” become "current generation", and can "third generation" become "next generation"? • Should we stop saying "current" and "next" and start saying "first" and "second"? • I like the idea of always having the "next gen" name.
Single molecule sequencing
–Advantages •Few or no enzymes involved in preparation of the DNA –Reduces cost, time and potential biases/errors •In some systems, no enzymes involved in reading the DNA. •Can often read RNA directly with the same system/method. •Some single molecule systems allow the direct identification of nucleotide modifications. •Helicos – “True Single Molecule Sequencing” (tSMSTM ) system –Single base, reversible dye terminator extension reactions •Pacific BioSciences – Single Molecule Real Time (SMRTTM) sequencing –Dyes that are phospholinked to the nucleotide, very sensitive fluorescent detection in zero mode waveguides •Oxford Nanopores – GridION and MinION systems –Direct reading of unlabeled DNA by threading it through a nanopore
• Traditional sequencing: 384 reads ~1kb / 3 hours • 454 (Roche): 1M reads 450-1000bp / 10-24 hours • HiSeq (Illumina): 100-200M reads of 50-100bp / 3-8 days * 16 samples • SOLiD (Applied Biosystems) >100M reads of 50-60bp / 2-8 days * 12 samples • Ion Torrent (Roche): 5-10M reads of 200-400bp / < 2 hours
T TACGCCAT TACGCCATGGT
Байду номын сангаас
4--dATP, dCTP, dGTP, dTTP. + ddTTP
2. 自动测序仪
Conney等人于1987年设计了不同荧光染料标记引物，然后 做链终止测序，用激光扫描阅读序列。
红色引物+T反应系统（引物+DNA+dNTP+ddTTP）
黑色引物+G反应系统（引物+DNA+dNTP+ddGTP） 绿色引物+A反应系统（引物+DNA+dNTP+ddATP） 兰色引物+C反应系统（引物+DNA+dNTP+ddCTP）
最新分子生物学技术
1. Single-Cell genomic/Transcriptome/Proteomics/Metabolomics 2. Next Generation Sequencing (NGS)
3. Ribosome profiling
4. Protein microarray 5. Chromosome conformation capture
Ribosome footprint profiling
Interpreting ribosome occupancy profiles.
Protein synthesis in vivo
Comparing regulatory divergence of ribosome occupancy, mRNA abundance, and translation efficiency.
Microfluidics
Whole-genome amplification (WGA)
GenomePlex Whole Genome Amplification
Next generation sequencing (NGS)
Next generation sequencing/Second generation seqeucing/Pyrosequencing
过程：制备ss-DNA→与引物
退火→分为4个反应系统→每个 系统中加入dNTP（其中dATP 常带同位素标记）和一种双脱 氧核苷酸→DNA聚合酶定序反 应→反应产物变性后电泳→凝 胶干燥→放射自显影。
该法亦适合mRNA的序列分析。 GC富集区常出现“隐影”或
“停止”现象，如在反应中加 入dITP（脱氧三磷酸次黄嘌呤） 或脱氧三磷酸-7-脱氧鸟苷可解 决GC富集区的测序。
Protein Microarray (Protein chip)
Concentrations of mRNAs within a cell are poorly correlated with the actual abundances of the corresponding proteins
Ribosome profiling
A method based on deep sequencing of ribosome-protected mRNA fragments. Purification and sequencing of these fragments provides a “snapshot” of all the ribosomes active in a cell at a specific time point. This information can determine what proteins are being actively translated in a cell, useful for: • Investigating translational control • Measuring gene expression • Determining the rate of protein synthesis • Predicting protein abundance