Omics

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