Omics
Name: 苗奔奔
Student ID: 2111901034
Email: [email protected]
Date: 2019-12-04
Github: https://github.com/benben-miao/
Catalog 2. 3.
Transcripto Microorga
1.
Genomics mics nism
4. 5. 6. 7.
Proteomics Metabolo Epigenomi Single Cell
mics cs
Whole Genome Sequencing
RAD/GBS
GWAS (Genome-wide Association Study)
1. mtDNA (Mitochondrial DNA)
Genomics cpDNA (Chloroplast DNA,)
Genetic map
WES-Seq (Whole Exome Sequencing)
eQTL-Seq (expression quantitative trait Loci)
Experimental WES-Seq (Whole Exome Sequencing)
design
1.
Samples-Tissues
2. 3. Enrichment of DNA sequences
DNA Library in exon regions using probe
hybridization
Total >= 0.4 μg
Region 180bp~280bp
Agilent SureSelect Platform
Illumina PE150 4. 5.
Q30>=80% Illumina PE150 Bioinformatics
Analysis
Analysis
Process
Fastq Reads(.fq/.fastq)
Quality Control FastQC
Clean Reads Trim-galore
Mapping Genome BWA
Mark PCR duplicates Samtools GATK4
SNP / InDel / CNV detection, GATK4
annotation and statistics vcftools
Disease Analysis
Software/
Database
Databases:
NCBI (The National Center for Biotechnology Information) : https://www.ncbi.nlm.nih.gov/
EMBL (The European Bioinformatics Institute ) :https://www.ebi.ac.uk/
DDBJ () :https://www.ddbj.nig.ac.jp/index-e.html
Plant Genome DB: http://ensembl.gramene.org/genome_browser/index.html
Human/Mouse/Zebrafish: http://asia.ensembl.org/index.html
Fungi(真菌) Genome Database: http://asia.ensembl.org/index.html
Bacteria Genome Database : http://bacteria.ensembl.org/index.html
Biodb Collection (benben-miao) : https://biodb.neocities.org/
Softwares:
GATK4: https://software.broadinstitute.org/gatk/
Samtools: http://samtools.sourceforge.net/
Vcftools : http://vcftools.sourceforge.net/
BWA : http://bio-bwa.sourceforge.net/
2.
Transcripto
mics
Eukaryotic reference transcriptome
Eukaryotic referenceless transcriptome
LncRNA-Seq (Long No Code RNA)
miRNA-Seq (Micro RNA)
ceRNA-Seq(competing endogenous RNAs)
circRNA-Seq(Circular RNA)
Experimental Eukaryotic reference transcriptome
design
1. Clipping 2. mRNA:
Samples Tissues 5’ UnTranslated Region, (UTR)
3’ Seq AAUAAA, (polyA)
Extracting
5.
3. PolyT
RNA Enrichment
(mRNA, rRNA, tRNA, ncRNA)
4.
1.mRNA: Message RNA Remove rRNA
2.tRNA: Translate RNA
3.rRNA: Ribosomal RNA rRNA is 82% in all RNA.
4.ncRNA: No Code RNA
6.
mRNA
Analysis Raw Reads
Process Quality Control
Trimmomatic Clean Reads FastQC /
MultiQC(Python)
Ref Seq
Hisat2 / Mapped Reads
Bowtie2
Transcript
Vocano Graph: DESeq2(R) / Gene expression New gene
ggplot2(R) edger(R) identification
Genetic structure GATK4
optimization
Sample relationship
analysis
Differential gene Mutation analysis
analysis
StringDB PPI analysis Enrichment clusterProfiler(R) RNA Edit
analysis
Fusion gene STARfusion
GO KEGG Reactome analysis rMATS
Variable shear
analysis
Software/
Database
FastQC (Quality Control) : http://www.bioinformatics.babraham.ac.uk/projects/fastqc/
Hisat2(Map to Complete Genome): http://www.psc.edu/user-resources/software/hisat2
Bowtie2(Map to Complete Genome): http://bowtie-bio.sourceforge.net/bowtie2/index.shtml
STRING(Interaction of genes or proteins): https://string-db.org/
R(A beautiful data computer and visual language): https://www.r-project.org/
Rstudio(IDE for R): https://rstudio.com/
DESeq2(Differential Expression Sequences): https://bioconductor.org/packages/release/bioc/html/DESeq2.html
clusterProfiler(GO and KEGG): https://bioconductor.org/packages/release/bioc/html/clusterProfiler.html
3. 16S、18S、ITS Microbial
Microorga Sequences transcriptome
nism "S" (settling speed) represents a measure of sequencing
the settling speed, not the mass. The
sedimentation speed of each subunit is Microbial transcriptome
affected by both its shape and its mass. sequencing technology is
a method for sequencing
Prokaryotic ribosomes have a total of 70S single colony microbial
containing 23S, 16S and 5S. transcripts.
Eukaryotic ribosomes have a total of 80S,
including 28S, 18S, 5.8S and 5S.
Metagenomic
Sequencing
Metagenomic sequencing is a method that
uses high-throughput sequencing technology
to complete the detection and functional
analysis of the genomes of all species in the
microbial community.
Experimental
design
1. 1.
Microbial Microbial
community community
Concentration>=5ng/μL Total>=200ng 2.
Total>=150ng DNA
2.
DNA
PCR amplify 16S 3. 3.
Add adapter DNA Library DNA Library
16S Seq Metagenomics 1.DNA Fragments
2.Add adapter to two ends
3.350bp library
4. 4.
Sequences Sequences
Ion S5 XL Platform Illumina Sequences platform
NovaSeq 6000 Platform PE150
PE250/SE400 5.
Bioinformatics Analysis
5.
Bioinformatics Analysis
Analysis RAW Reads 16S、18S、ITS
Process Sequences
Quality Control FastQC
Cut Adapte Cutadapt
Cutting, DE chimerism, qiime dada2 denoise-paired Qiime2
splicing切割、去嵌合体、拼接
Taxonomy qiime feature-classifier classify-sklearn Qiime2
Annotation
Alpha Diversity Vegan/Boxplot(R)
Calculation and visualization Vegan/Dotplot(R)
Beta Diversity
Calculation and visualization
Find differential bacteria LEfSe:
between groups https://bitbucket.org/biobakery/biobakery/wiki/lefse
Analysis RAW Reads Metagenomic
Process Quality Control FastQC Sequencing
Clean Reads Cutadapt
Kraken2 Annotation
qiime taxa barplot classification
Sorting and
visualization
Hatmap Pheatmap(R)
KEGG Statistical analysis LEfSe LEfSe
GO Functional Analysis ANCOM
Qiime2(Analysis of
MetaCyc composition of microbiomes)
EggNOG
PCoA
CAZy NMDS Vegan(R)
Enzyme
ggplot2
RDA/CCA
CARD
Software/
Database
FastQC (Quality Control) : http://www.bioinformatics.babraham.ac.uk/projects/fastqc/
Cutadapt (Remove adapt at reads) : https://cutadapt.readthedocs.io/en/stable/
Qiime2 (microbiome bioinformatics platform) : https://qiime2.org/
LEfSe (Difference analysis) :https://bitbucket.org/biobakery/biobakery/wiki/lefse
Vegan (Statistics) : https://www.rdocumentation.org/packages/vegan/versions/2.4-2
Pheatmap (Heatmap) : https://www.rdocumentation.org/packages/pheatmap/versions/1.0.12/topics/pheatmap
kEGG (Kyoto Encyclopedia of Genes and Genomes): https://www.kegg.jp/
GO (Gene Ontology) : http://geneontology.org/
MetaCyc (Database of metabolic pathways): https://metacyc.org/
EggNOG (Clusters of Orthologous Groups of proteins) : http://eggnogdb.embl.de/
CAZy (Enzyme) :http://www.cazy.org/
ENZYME (Enzyme) : https://enzyme.expasy.org/
1.Identification of Protein Glue Strips
2. Identification of protein interactions
3. Proteome full spectrum identification
4. Quantitative 1.DDA(Data Dependent Acquisition):Label-free, iTRAQ/TMT
Proteomics Proteome 2.DIA(Data Independent Acquisition):DIA, SWATH
1.Phosphorylated (磷酸化) proteome
2.Acetylated (乙酰化) protein
3.Ubiquitinated (泛素化) proteome
4.N-glycosylated (糖基化) protein
5.Succinylated (琥珀酰化) protein
6.Propionylated (丙酰化) proteome
Experimental Quantitative
design Proteome
1.
Samples
2. 7.
Protein extraction Bioinformatics
Analysis
iTRAQ:Isobaric Tags for Relative and Absolute
Quantitation
TMT:Tandem Mass Tags
3. 5. 6.
Reductive iTRAQ/TMT LC-MS/MS
alkylation(还原
4. 1.LC-MS/MS:
烷基化) Proteolysis Liquid Chromatography - Mass
Spectrometry
2.TOF: Time of Flight
3. ESI: Electron Spray Ionization
4.MALDI:
Matrix-assisted Laser
Desorption/ionization
Analysis Raw File MaxQuant(offline)/
Process Protein DB Search SEQUEST /
Proteome Xchange: MASCOT(offline/online)
http://www.proteomex
change.org/
Vocano Graph: Different expression
ggplot2(R) Proteins
Protein GO Anotation GO Graph:
Function Annotation barplot/dotplot/ggplot2(R)
KEGG Anotaion
Proteom analysis KEGG Graph:
With transcriptom COG Anotation barplot/dotplot/ggplot2(R)
Protein structure
domains predictor SMART:
Interaction Network https://smart.emb
l-heidelberg.de/
STRING:
https://string-db.org/
Software/
Database
Mascot (Protide search engineer): http://www.matrixscience.com/
Uniprot (Most Protein Database): https://www.uniprot.org/
Maxquant (Protide search engineer): https://www.maxquant.org/
SEQUEST: http://www.thermo.com
pFind (Search protein): http://www.pfind.net/
ExPAsy(Expert Protein Analysis): https://www.expasy.org/
STRING(Interaction of genes or proteins): https://string-db.org/
R(A beautiful data computer and visual language): https://www.r-project.org/
Rstudio(IDE for R): https://rstudio.com/
5.
Metabolo
mics
Untargeted Targeted Broadly targeted
metabolomics metabolomics metabolome
Qualitative and quantitative Targeted metabolomics Combines advantages of non-
analysis of all small molecule targeted analysis of a single targeted and targeted
compounds (molecular weight or a small amount of target metabolite detection
less than 1000) in biological metabolites in biological technology。
samples。 samples, and the absolute
abundance information of AB Sciex QTRAP® 4500 LC-
target metabolites obtained MS/MS
by comparison analysis with
standards。 Database of primary and
secondary metabolites (more
than 2,200 substances)
Medical metabolite database
(more than 1500 substances)
Experimental Untargeted
design metabolomics
1. 2. all small molecule compounds
Samples Metabolites (molecular weight less than 1000) in
biological samples
LC-MS/MS: 3. 4. 5.
Liquid Chromatography - Mass Spectrometry LC-MS/MS Concentrate Bioinformatics
GC-MS
Gas Chromatography - Mass Spectrometry Analysis
Analysis RAW Data
Process Qualitative
metabolites
mzCloud: Xcms:
https://www.mzcloud.org/ Quality Control https://xcmsonline.scripps.edu/
MetaboAnalyst:
Metabolite https://www.metaboanalyst.ca/h
screening ome.xhtml
Differential QC Analysis Platform
metabolites
Metabolites of multivariate Partial Least PCA Prcomp(R)
statistical analysis screening criteria Squares Enrichment Heatmap Pheatmap(R)
(VIP> 1) and univariate statistical Analysis
analysis screening criteria (FC> 2.0 Discriminati Vocano ggplot2(R)
and P value <0.05) are considered on Analysis Result
as metabolites with significant Report
differences PLS-DA
MetaboDiff(R) VIP
OPLS-DA
MetaboAnalystR(R)
Orthogonal
PLS-DA
CorrPlot Corrplot(R)
KEGG Barplot Z-score Plot Base stats(R)
FELLA(R) Network Cytoscape
KEGG Dotplot
Software/
Database
MetaboAnalyst(statistical, functional and integrative analysis of metabolomics data)
: https://www.metaboanalyst.ca/home.xhtml
XCMX: https://xcmsonline.scripps.edu/
R : https://www.r-project.org/
MetaboDiff : https://github.com/andreasmock/MetaboDiff/
MetaboAnalystR : https://github.com/xia-lab/MetaboAnalystR
Cytoscape : https://cytoscape.org/
FELLA (Interpretation and enrichment for metabolomics data)
: http://www.bioconductor.org/packages/release/bioc/html/FELLA.html
Prcomp (Principle component analysis)
: https://www.rdocumentation.org/packages/twidlr/versions/0.0.0.9000/topics/prcomp
MetaCyc : https://metacyc.org/
BioCyc : https://biocyc.org/
Corrplot : https://cran.r-project.org/web/packages/corrplot/index.html
mzCloud: https://www.mzcloud.org/
ChIP-Seq ATAC-seq
Chromatin immunoprecipitation Assay for Transposase-Accessible Chromatin
BS-Seq RRBS-Seq
Bisulfite Sequencing Reduced Representation Bisulfite Sequencing
6.
Epigenomi
cs
RIP-seq
RNA Binding Protein Immunoprecipitation Assay
MeRIP m6A-Seq
Methylated RNA Immunoprecipitation
Single Cell
Transcript
omics
Single Cell 7. Single Cell
Genomics Single Cell ATAC
10X
Genomics
Single Cell Single Cell
VDJ Smart Seq
(Immune
Profiling)
Thanks!
Designer: Benben Miao
Copyright @benben-miao