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高通量技术在基因组与微生物学研究中 的 应用 策略与解决 方案 刘贵 明 中科院北京基因组研究所

高通量技术在基因组与微生物学研究中 的 应用 策略与解决 方案 刘贵 明 中科院北京基因组研究所. 内容： 微生物基因组拼接算法和策略 微生物基因组的 pangenome 微生物转录组 微生物基因组的甲基化检测 宏 基因组 16S 测序 WGS 测序 单细胞测序. Diversity of the microbial universe. Acquire genes from environment Conjugation Transformation Phage infection (transduction).

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高通量技术在基因组与微生物学研究中 的 应用 策略与解决 方案 刘贵 明 中科院北京基因组研究所

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  1. 高通量技术在基因组与微生物学研究中的 应用策略与解决方案 刘贵明 中科院北京基因组研究所

  2. 内容： 微生物基因组拼接算法和策略 微生物基因组的pangenome 微生物转录组 微生物基因组的甲基化检测 宏基因组 16S测序 WGS 测序 单细胞测序

  3. Diversity of the microbial universe Acquire genes from environment Conjugation Transformation Phage infection (transduction)

  4. Molecular evolutionary mechanisms that shape bacterial species diversity: one genome, pan-genome and metagenome. a. Intra-species b .inter-species c. population dynamic mechanisms manipulate the genomic diversity of bacterial species d. Metagenomics embraces The community as the unit of study Microbiology in the post-genomic era. Nature Reviews Microbiology 6, 419-430 (June 2008)

  5. 高通量测序平台

  6. 基因组拼接(genome assembly) 1. DNA Shear & Sequence DNA 2. Construct assembly graph from overlapping reads 3. Simplify assembly graph 4. Detangle graph with long reads, mates, and other links

  7. 拼接算法 De Bruijn Graph Overlap-Layout-Consensus A Overlap B Layout C Consensus soapdenovo

  8. Long read assembly (Pacbio) At k = 50, the graph is tangled with hundreds of contigs. (B) k = 1,000 significantly simplifies the graph. (C) At k = 5,000, the graph is fully resolved into a single contig. The advantages of SMRT sequencing. Roberts et al. Genome Biology 2013, 14:405

  9. Illumina平台文库构建 500 bp pair end 2-20K mate pair 40K fosmid library

  10. De novo assembly Soapdenovo input data : Illumina 2. ALLPATHS-LG input data: 180bp +Mate pair or Illumina + PacBio(hybrid assembly) A fill fragments->unipaths-> Error correct

  11. Reference-guided assembly Read-mapped assembly-mapped

  12. Multi-reference assisted chromosome assembly Reference-assisted chromosome assembly. Korbinianet al. PNAS.2011: 10249–10254

  13. GapClose based pair end/mate pair reads Toward almost closed genomes with GapFiller. Boetzeret al. Genome Biology 2012, 13:R56

  14. 微生物基因组的拼接方案 1. Illumina (SOAPdenovo) Insert size: 180bp, 500bp, 2K, 5K 和40K Read length: 2X100bp 2. Pacbio+Illumina(Hybrid assembly, WGS, http://wgs-assembler.sourceforge.net/) Insert size(Illumina): pair end (500bp) Read length: 2X100bp; >5Kb 3. Pacbio Only Read length: >5Kb

  15. Hybrid assembly Hybrid Error Correction & Assembly Trim/correct SR sequence Compute an SR layout for each LR 1. map SRs to LRs 2. Trim LRs at coverage gaps 3. compute consensus for each LR Co-assembly corrected LRs and SRs -WGS assembler can suport 16Kb reads Hybrid error correction and de novo assembly of single-molecule sequencing reads. Nat Biotechnol. ; 30(7): 693–700.

  16. Assemblies from different strategy Contigsizes for various combinations of sequencing technologies Assemblies are for E. coli C227-11 (assemblies including Illumina and PacBio CCS) and E. coli JM221 (assemblies including 454). Both genomes have similar repeat content, PacBio read length, and coverage. Assemblies of only second-generation data are comparable and average N50 ≈ 100 Kbp. By comparison, adding 25X or 50X of PBcR to these data sets increases N50 as much as 5 fold and pushes the maximum contig size greater than 1 Mbp (for the PBcR/CCS combination).

  17. Assemblies from different strategy

  18. De Novo SMRT Sequencing Genome size: 124.6 MbGC content: 33.92%Raw data: 11 GbAssembly coverage: 15.37xPolished Contigs: 540Max Contig Length: 12.98 MbN50 Contig Length: 6.19 MbSum of Contig Lengths: 124.57 Mb

  19. Genome annotation genome COG ORF sRNA t/rRNA repeat Prohpage KEGG Tree NR recombination Ka/Ks Syteny InterPor Ortholog Pangenome

  20. Pan-genome Strain-specific genes Pan-genome Pan-genome: the global gene repertoire Pertaining to a species Core genes: genes shared by all the strains Strain-specific genes: genes present in only one strain and absent from all the others Dispensable genes Core genes Dispensable genes: genes shared by some but not all the strains

  21. Mathematical definition of the Pan-genome Open pan-genome: Continuously increasing In size. Examples: E.Coli Streptococcus Close pan-genome: No continuously increasing In size example Bacillus anthracis

  22. The Core and Pan-Genomes of E. coli >80% similarity All pan-genome 20 completely sequenced genomes Vary in size more than 1Mb Core genome: 1976 genes Pan-genome: 17838 genes Removing IS

  23. The Phylogenetic History of the Strains concatenated gene of core genome (1878 genes) and maximum likelihood approach First split group B2 and group D; Group A,B1,S1,S3 and SS emerged more recently.

  24. Recent acquired are enriched in phage and transposable elements

  25. 微生物转录组(RNA-seq) 转录组学( transcriptomics) ，是在RNA 水平上研究 基因转录的整体情况及转录调控. 细菌RNA-seq 1 rRNA和tRNA(MICROBExpress) 2 mRNA没有poly-A mRNA富集 1.16S 和23S rRNA的保守区域 2.核酸外切酶 3.抗体捕获

  26. Strand specific library DSSS protocol workflow. (A) Fragmentation(60-200bp) (B) Dephosphorylation. 5’phosphates are removed from RNA . (C) 3’adapter ligation. (D) Rephosphorylation. (E) 5’ adapter ligation (F ) Reverse transcription (RT) and amplification of library. (G) Sequencing. 3’UTR 5’UTR Antisense Operons FPKM sRNA Transcriptional Start Sites(TSS)

  27. RNA-seq and ChIP-chip–based strategy to identify promoters, transcribed regions, and ssRNAs (A) The different cDNA libraries that were generated and sequenced in this study. (B) Reads to different genome locations (C) RNA-seq, and ChIP-chip data to identify small RNAs, TSSs, promoters, and transcribed regions throughout the chromosome of

  28. PacBio RS系统实现对碱基修饰进行直接测序 N6-methyladenine、N4-methylcytosine 、 5-mC和5-hmC Methylome of G. metallireducensGS-15. (a) three instances of methylated sequence regions. (b) coverage and kinetic score for all genomic positions. (c) MTasespecificities determined from the genomic positions detected as methylated. (d) Summary of detected methylated positions across the genome.

  29. Ecolimethylation MTases targeting motifs in genome(a) and plasmid(b) Nat Biotechnol. 2012 Dec;30(12):1232-9

  30. The RM system associated with M.EcoGIIIregulates the expression of many genes and pathways. Nat Biotechnol. 2012 Dec;30(12):1232-9

  31. Nat Biotechnol. 2012 Dec;30(12):1232-9

  32. 宏基因组（Metagenome）,Handelsman等在一篇研究土壤微生物的文章中宏基因组（Metagenome）,Handelsman等在一篇研究土壤微生物的文章中 首次提出,指“微生物群落中的所有基因组的集合”。 研究内容 (1) 针对 16S rRNA为主要研究对象的核糖体 RNA 研究: 种群分布和种群丰度 (2) 以环境中所有遗传物质为研究对象；DNA的WGS (3) 以环境中所有转录本为主要研究对象的宏转录组研究(metatranscriptome) (4) 基于单细胞的宏基因组研究

  33. Metagenomics 16S

  34. 基于双向Index的策略(Dual-index sequencing strategy on MiSeq) 每个Lane的样品数目: Index3’ number X Index 5’ 推荐16S的引物设计区域：347F/803R

  35. 文库多样性评估

  36. Whole-metagenome shotgun全基因组测序策略 样本收集 文库构建 上机测序 文库构建与测序 加barcode Miseq 数据质量评估与筛选 序列拼接 基因注释 数据统计注释 BLAST，MetaGeneMark GS de novo assembler Perl, BMTagger 菌群结构 功能基因/代谢途径 菌群特性 菌群结构与功能分析 MEGAN/MG-RAST 临床信息、样本特性 基于KEGG、SEED

  37. Assembly soft 1 MetaVelvet 2 Meta-IDBA

  38. 基于单细胞的宏基因组 Standard metagenomics and sequence ‘binning’ to produce composite microbial genomes.

  39. 单细胞分离技术 微流体技术(microfluidics), 梯度稀释法( Serial dilution), 显微操作技术(micromanipulation), 荧光激活细 胞分类( FACS，fluorescence-activated cellsorting)

  40. 单细胞扩增技术 最广泛的MDA，能够忠实的复制整个基因组DNA，扩增出10 -100 kb; MALBAC, 扩增长度500-2000bp;

  41. Sorting of Single Cells by Flow Cytometry Candidate phylum TM6 genome recovered from a hospital sink biofilm provides genomic insights into this uncultivated phylum PNAS. 2013 25;110(26):E2390-9

  42. TM6 genome recovered from a hospital sink biofilm PNAS. 2013 25;110(26):E2390-9

  43. IMS-MDA Immunomagneticseparation (IMS) and multiple displacement amplification (MDA) --Chlamydia trachomatis (antibodies or aptamers) Analysis of sequencing data from DNA extracts from clinical samples, with and without MDA

  44. 高通量测序技术给微生物基因组学研究带来了一个高效的新平台和巨大的发展机遇.高通量测序技术给微生物基因组学研究带来了一个高效的新平台和巨大的发展机遇. 谢谢！

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