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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. 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). - PowerPoint PPT Presentation
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Diversity of the microbial universe
Acquire genes from environmentConjugationTransformationPhage infection (transduction)
Microbiology in the post-genomic era. Nature Reviews Microbiology 6, 419-430 (June 2008)
Molecular evolutionary mechanisms that shape bacterial species diversity: one genome, pan-genomeand metagenome.
a. Intra-species b .inter-species c. population dynamic mechanisms manipulate the genomic diversity of bacterial speciesd. Metagenomics embraces The community as the unit of study
Illumina ABI Pacific Biosciences
HiSeq MiSeq Ion PGM™ Ion Proton™ PACBIO RS
read 长度 2X100/150 2X150/300 100-400 100-200 3000-20000
数据量 40-60G 15G 600M 64G 500M
高通量测序平台
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
(A) 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
Long read assembly (Pacbio)
1. Soapdenovo input data : Illumina
2. ALLPATHS-LG
input data: 180bp +Mate pair or Illumina + PacBio(hybrid assembly)
De novo assembly
A fill fragments-> unipaths-> Error correct
Multi-reference assisted chromosome assembly
Reference-assisted chromosome assembly. Korbinian et al. PNAS.2011: 10249–10254
GapClose based pair end/mate pair reads
Toward almost closed genomes with GapFiller. Boetzer et al. Genome Biology 2012, 13:R56
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
Hybrid Error Correction & Assembly1. Trim/correct SR sequence2. Compute an SR layout for each LR 1. map SRs to LRs 2. Trim LRs at coverage gaps 3. compute consensus for each LR3. Co-assembly corrected LRs and SRs -WGS assembler can suport 16Kb reads
Hybrid assembly
Hybrid error correction and de novo assembly of single-molecule sequencing reads. Nat Biotechnol. ; 30(7): 693–700.
Contig sizes for various combinations of sequencing technologiesAssemblies 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, PacBioread length, and coverage. Assemblies of only second-generation data are comparable andaverage N50 ≈ 100 Kbp. By comparison, adding 25X or 50X of PBcR to these data setsincreases N50 as much as 5 fold and pushes the maximum contig size greater than 1 Mbp(for the PBcR/CCS combination).
Assemblies from different strategy
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
Genome annotation
genome
recombination
KEGG
t/rRNA
sRNA
repeat
Prohpage
COG
InterPor
NR
Ka/Ks
Ortholog
Tree
Pangenome
Syteny
ORF
Pan-genome
Strain-specificgenes
Core genesDispensable
genes
Strain-specific genes: genes present in only one strain and absent from all the others
Core genes: genes shared by all the strains
Dispensable genes: genes shared by some but not all the strains
Pan-genome: the global gene repertoirePertaining to a species
Pan-genome
Mathematical definition of the Pan-genome
Open pan-genome:Continuously increasing In size.Examples:E.ColiStreptococcus
Close pan-genome:No continuously increasing In sizeexampleBacillus anthracis
The Core and Pan-Genomes of E. coli
20 completely sequenced genomesVary in size more than 1MbCore genome: 1976 genesPan-genome: 17838 genes
All pan-genome >80% similarity
Removing IS
The Phylogenetic History of the Strains
concatenated gene of core genome (1878 genes) and maximum likelihood approachFirst split group B2 and group D; Group A,B1,S1,S3 and SS emerged more recently.
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
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.
Strand specific library
3’UTR5’UTRAntisenseOperonsFPKMsRNATranscriptional Start Sites(TSS)
(A) The different cDNA libraries that were generatedand 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
RNA-seq and ChIP-chip–based strategy to identify promoters, transcribed regions, and ssRNAs
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. metallireducens GS-15. (a) three instances of methylated sequence regions. (b) coverage and kinetic score for all genomic positions. (c) MTase specificities determined from the genomicpositions detected as methylated. (d) Summary of detected methylated positions acrossthe genome.
Nat Biotechnol. 2012 Dec;30(12):1232-9
Ecoli methylation
MTases targeting motifs in genome(a) and plasmid(b)
Nat Biotechnol. 2012 Dec;30(12):1232-9
The RM system associated with M.EcoGIII regulates the expression of manygenes and pathways.
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
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
Coverage C 库容评估 C = 1− n1/N
Richness estimator (SChao1) 预测样品中微生物的种类
Shannon-Wiener index 物种丰富程度和均匀程度评估
Rarefaction curve 库容评估
文库多样性评估
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文库构建
数据质量评估与筛选
样本收集
临床信息、样本特性
MEGAN/MG-RAST
菌群特性菌群结构功能基因 /代谢途径
数据统计注释
菌群结构与功能分析
文库构建与测序 Miseq
Whole-metagenome shotgun全基因组测序策略
上机测序
GS de novo assembler
加 barcode
BLAST,MetaGeneMark
Perl, BMTagger
基于 KEGG、 SEED
序列拼接 基因注释
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)
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
Immunomagnetic separation (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
IMS-MDA