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by Mir Asif Iquebal, Ratan Kumar Choudhary, Chandra Sekhar Mukhopadhyay
Basic Applied Bioinformatics
Cover
Title Page
Preface
Acknowledgments
List of Abbreviations
SECTION I: Molecular Sequences and Structures
CHAPTER 1: Retrieval of Sequence(s) from the NCBI Nucleotide Database
1.1 INTRODUCTION
1.2 COMPONENTS OF THE NCBI NUCLEOTIDE DATABASE
1.3 OBJECTIVES
1.4 PROCEDURE
1.5 SOME USEFUL NUCLEOTIDE SEQUENCE DATABASES OF NCBI
1.6 QUESTIONS
CHAPTER 2: Retrieval of Protein Sequence from UniProtKB
2.1 INTRODUCTION
2.2 OBJECTIVE
2.3 PROCEDURE
2.4 QUESTIONS
CHAPTER 3: Downloading Protein Structure
3.1 INTRODUCTION
3.2 OBJECTIVE
3.3 PROCEDURE
3.4 QUESTIONS
CHAPTER 4: Visualizing Protein Structure
4.1 INTRODUCTION
4.2 OBJECTIVE
4.3 PROCEDURE
4.4 QUESTIONS
CHAPTER 5: Sequence Format Conversion
5.1 INTRODUCTION
5.2 OBJECTIVE
5.3 PROCEDURE
5.4 QUESTIONS
5.5 BRIEF DESCRIPTION OF SOME OF THE IMPORTANT MOLECULAR SEQUENCE FORMATS
CHAPTER 6: Nucleotide Sequence Analysis Using Sequence Manipulation Suite (SMS)
6.1 INTRODUCTION
6.2 OBJECTIVE
6.3 PROCEDURE
6.4 FORMAT CONVERSION
6.5 SEQUENCE ANALYSIS
6.6 SEQUENCE FIGURES
6.7 RANDOM SEQUENCES
6.8 MISCELLANEOUS
6.9 QUESTIONS
CHAPTER 7: Detection of Restriction Enzyme Sites
7.1 INTRODUCTION
7.2 OBJECTIVE
7.3 PROCEDURE (USING NEBCUTTER)
7.4 QUESTIONS
SECTION II: Sequence Alignment
CHAPTER 8: Dot Plot Analysis
8.1 INTRODUCTION
8.2 OBJECTIVE
8.3 PROCEDURE
8.4 PARAMETERS OF DOT PLOT ANALYSIS
8.5 INTERPRETATION
8.6 QUESTIONS
CHAPTER 9: Needleman–Wunsch Algorithm (Global Alignment)
9.1 INTRODUCTION
9.2 OBJECTIVE
9.3 PROCEDURE
9.4 QUESTIONS
CHAPTER 10: Smith–Waterman Algorithm (Local Alignment)
10.1 INTRODUCTION
10.2 OBJECTIVE
10.3 PROCEDURE
10.4 QUESTIONS
CHAPTER 11: Sequence Alignment Using Online Tools
11.1 INTRODUCTION
11.2 OBJECTIVE
11.3 PROCEDURE
11.4 INTERPRETATION OF RESULTS
11.5 COLOR SCHEME FOR AMINO ACID RESIDUES
11.6 QUESTIONS
SECTION III: Basic Local Alignment Search Tools
CHAPTER 12: Basic Local Alignment Search Tool for Nucleotide (BLASTn)
12.1 INTRODUCTION
12.2 OBJECTIVE
12.3 PROCEDURE
12.4 QUESTIONS
CHAPTER 13: Basic Local Alignment Search Tool for Amino Acid Sequences (BLASTp)
13.1 INTRODUCTION
13.2 OBJECTIVE
13.3 PROCEDURE
13.4 QUESTIONS
CHAPTER 14: BLASTx
14.1 INTRODUCTION
14.2 OBJECTIVE
14.3 PROCEDURE
14.4 INTERPRETATION OF BLASTx RESULTS
14.5 QUESTIONS
CHAPTER 15: tBLASTn
15.1 INTRODUCTION
15.2 OBJECTIVE
15.3 PROCEDURE
15.4 ALGORITHM PARAMETERS
15.5 INTERPRETATION OF tBLASTn RESULTS
15.6 QUESTIONS
CHAPTER 16: tBLASTx
16.1 INTRODUCTION
16.2 OBJECTIVE
16.3 PROCEDURE
16.4 ALGORITHM PARAMETERS
16.5 INTERPRETATION OF tBLASTx RESULTS
16.6 QUESTIONS
SECTION IV: Primer Designing and Quality Checking
CHAPTER 17: Primer Designing – Basics
17.1 INTRODUCTION
17.2 OTHER IMPORTANT FEATURES FOR DESIGNING “GOOD” PRIMERS
17.3 QUESTIONS
CHAPTER 18: Designing PCR Primers Using the Primer3 Online Tool
18.1 INTRODUCTION
18.2 OBJECTIVE
18.3 PROCEDURE
18.4 OUTPUT
18.5 SELECTION OF THE BEST PRIMER‐PAIRS BY COMPARATIVE EVALUATION OF THE DESIGNED PRIMERS
18.6 QUESTIONS
CHAPTER 19: Quality Checking of the Designed Primers
19.1 INTRODUCTION
19.2 OBJECTIVE
19.3 PROCEDURE
19.4 IDT UNAFOLD – CHECKING THE SECONDARY STRUCTURE FORMATION OF THE AMPLICON
19.5 PRIMER‐BLAST – TO DETECT POSSIBLE SPURIOUS AMPLIFICATION
19.6 QUESTIONS
CHAPTER 20: Primer Designing for SYBR Green Chemistry of qPCR
20.1 INTRODUCTION
20.2 QUESTIONS
SECTION V: Molecular Phylogenetics
CHAPTER 21: Construction of Phylogenetic Tree: Unweighted‐Pair Group Method with Arithmetic Mean (UPGMA)
21.1 INTRODUCTION
21.2 ASSUMPTIONS
21.3 OBJECTIVE
21.4 PROCEDURE
21.5 INTERPRETATION OF UPGMA TREE
21.6 QUESTIONS
CHAPTER 22: Construction of Phylogenetic Tree: Fitch Margoliash (FM) Algorithm
22.1 INTRODUCTION
22.2 OBJECTIVE
22.3 PROCEDURE
22.4 INTERPRETATION OF THE FM TREE
22.5 QUESTIONS
CHAPTER 23: Construction of Phylogenetic Tree: Neighbor‐Joining Method
23.1 INTRODUCTION
23.2 OBJECTIVE
23.3 PROCEDURE
23.4 INTERPRETATION OF NJ TREE
23.5 QUESTIONS
CHAPTER 24: Construction of Phylogenetic Tree: Maximum Parsimony Method
24.1 INTRODUCTION
24.2 OBJECTIVE
24.3 PROCEDURE
24.4 INTERPRETATION OF MP TREE
24.5 QUESTIONS
CHAPTER 25: Construction of Phylogenetic Tree: Minimum Evolution Method
25.1 INTRODUCTION
25.2 OBJECTIVE
25.3 PROCEDURE
25.4 INTERPRETATION OF THE ME TREE
25.5 QUESTIONS
CHAPTER 26: Construction of Phylogenetic Tree Using MEGA7
26.1 INTRODUCTION
26.2 OBJECTIVE
26.3 PROCEDURE
26.4 INTERPRETATION OF PHYLOGENETIC TREE
26.5 QUESTIONS
CHAPTER 27: Interpretation of Phylogenetic Trees
27.1 INTRODUCTION
27.2 UNDERSTANDING PHYLOGENETIC TREES
27.3 REPRESENTATION OF PHYLOGENETIC TREES
27.4 METHODS FOR CONSTRUCTING EVOLUTIONARY TREES FROM INFERENCES
27.5 INFERRING PHYLOGENETIC TREES
27.6 QUESTIONS
SECTION VI: Protein Structure Prediction
CHAPTER 28: Prediction of Secondary Structure of Protein
28.1 INTRODUCTION
28.2 OBJECTIVE
28.3 SECONDARY STRUCTURE PREDICTION USING ONLINE TOOL PSIPRED
28.4 SECONDARY STRUCTURE PREDICTION USING THE ONLINE CDM TOOL
28.5 QUESTIONS
CHAPTER 29: Prediction of Tertiary Structure of Protein: Sequence Homology
29.1 INTRODUCTION
29.2 OBJECTIVE
29.3 PROCEDURE (SWISS‐MODEL PROGRAM)
29.4 OUTPUT
29.5 VISUALIZING THE PREDICTED STRUCTURE
29.6 INTERPRETATION OF RESULTS
29.7 QUESTIONS
CHAPTER 30: Protein Structure Prediction Using Threading Method
30.1 INTRODUCTION
30.2 OBJECTIVE
30.3 PROCEDURE
30.4 RESULTS AND INTERPRETATION
30.5 QUESTIONS
CHAPTER 31: Prediction of Tertiary Structure of Protein: Ab Initio Approach
31.1 INTRODUCTION
31.2 OBJECTIVE
31.3 PROCEDURE (RAPTORX)
31.4 JOB STATUS
31.5 OUTPUT AND INTERPRETATION OF RESULTS
31.6 QUESTIONS
CHAPTER 32: Validation of Predicted Tertiary Structure of Protein
32.1 INTRODUCTION
32.2 OBJECTIVE
32.3 PROCEDURE (WHAT IF TOOL FOR VALIDATING THE 3D STRUCTURE PREDICTION RESULTS)
32.4 INTERPRETATION OF RESULTS OF WHAT IF
32.5 MOLPROBITY TOOL FOR RAMACHANDRAN PLOT
32.6 INTERPRETATION OF RAMACHANDRAN PLOT ANALYSIS
32.7 QUESTIONS
SECTION VII: Molecular Docking and Binding Site Prediction
CHAPTER 33: Prediction of Transcription Binding Sites
33.1 INTRODUCTION
33.2 OBJECTIVE
33.3 TRANSFAC
33.4 BINDING SITES SEARCHING USING THE MATCH TOOL
33.5 QUESTIONS
CHAPTER 34: Prediction of Translation Initiation Sites
34.1 INTRODUCTION
34.2 OBJECTIVE
34.3 PROCEDURE
34.4 QUESTIONS
CHAPTER 35: Molecular Docking
35.1 INTRODUCTION
35.2 OBJECTIVE
35.3 PROCEDURE
35.4 RESULT AND INTERPRETATION
35.5 QUESTIONS
SECTION VIII: Genome Annotation
CHAPTER 36: Genome Annotation in Prokaryotes
36.1 INTRODUCTION
36.2 OBJECTIVE
36.3 PROCEDURE
36.4 INTERPRETATION OF GENEMARK OUTPUT
36.5 QUESTIONS
CHAPTER 37: Genome Annotation in Eukaryotes
37.1 INTRODUCTION
37.2 OBJECTIVE
37.3 PROCEDURE
37.4 INTERPRETATION OF GENSCAN OUTPUT
37.5 QUESTIONS
SECTION IX: Advanced Biocomputational Analyses
CHAPTER 38: Concepts of Real‐Time PCR Data Analysis
38.1 INTRODUCTION
38.2 GETTING STARTED WITH RT‐qPCR
38.3 PCR FLUORESCENCE CHEMISTRY
38.4 RT‐qPCR DATA ANALYSIS: GENE EXPRESSION ANALYSIS
38.5 QUESTIONS
CHAPTER 39: Overview of Microarray Data Analysis
39.1 CONCEPT
39.2 GETTING STARTED WITH MICROARRAY
39.3 MICROARRAY DATA ANALYSIS: GENE EXPRESSION ANALYSIS
39.4 STEPS INVOLVED IN MICROARRAY DATA ANALYSIS
39.5 FUNCTIONAL INFORMATION USING GENE NETWORKS AND PATHWAYS
39.6 LIVESTOCK RESEARCH THAT INVOLVED MICROARRAY ANALYSIS (SOME EXAMPLES)
39.7 APPLICATIONS OF MICROARRAY
39.8 QUESTIONS
CHAPTER 40: Single Nucleotide Polymorphism (SNP) Mining Tools
40.1 INTRODUCTION
40.2 OBJECTIVE
40.3 PROCEDURE
40.4 INTERPRETATION OF RESULTS
40.5 QUESTIONS
CHAPTER 41: In Silico Mining of Simple Sequence Repeats (SSR) Markers
41.1 INTRODUCTION
41.2 OBJECTIVE
41.3 MISA (MICROSATELLITE IDENTIFICATION TOOL)
41.4 RESULT
41.5 QUESTIONS
CHAPTER 42: Basics of RNA‐Seq Data Analysis
42.1 INTRODUCTION
42.2 AIM OF AN RNA‐SEQ EXPERIMENT
42.3 FAST SEQUENCE ALIGNMENT STRATEGIES
42.4 QUESTIONS
CHAPTER 43: Functional Annotation of Common Differentially Expressed Genes
43.1 INTRODUCTION
43.2 FUNCTIONAL ANNOTATION
43.3 QUESTIONS
CHAPTER 44: Identification of Differentially Expressed Genes (DEGs)
44.1 SECTION I. QUALITY FILTERING OF DATA USING PRINSEQ
44.2 SECTION II. IDENTIFICATION OF DIFFERENTIALLY EXPRESSED GENES – I (USING CUFFLINKS)
44.3 SECTION III. IDENTIFICATION OF DIFFERENTIALLY EXPRESSED GENES – II (USING RSEM‐DE PACKAGES EBSEQ, DESEQ2 AND EDGER)
44.4 USE OF DE PACKAGES FOR IDENTIFYING THE DIFFERENTIALLY EXPRESSED GENES
44.5 QUESTIONS
CHAPTER 45: Estimating MicroRNA Expression Using the miRDeep2 Tool
45.1 INTRODUCTION
45.2 PREPROCESSING OF READS
45.3 INPUT FORMATS OF THE DATA FILE
45.4 OUTPUT FORMATS THAT CAN BE GENERATED
45.5 PRELIMINARY FILES USED IN THE EXAMPLE
45.6 QUESTIONS
CHAPTER 46: miRNA Target Prediction
46.1 INTRODUCTION
46.2 miRNA TARGET PREDICTION BY TARGETSCAN (http://targetscan.org/)
46.3 miRNA TARGET PREDICTION BY TARGETSCAN IN HUMAN
46.4 miRNA TARGET PREDICTION BY psRNATARGET (http://plantgrn.noble.org/psRNATarget>/)
46.5 miRNA TARGET PREDICTION BY miRANDA (http://www.microrna.org)
46.6 QUESTIONS
Appendix A: Usage of Internet for Bioinformatics
Appendix B: Important Web Resources for Bioinformatics Databases and Tools
INTRODUCTION
Appendix C: NCBI Database: A Brief Account
Appendix D: EMBL Databases and Tools: An Overview
INTRODUCTION
THE EMBL DATABASES
THE EMBL TOOLS
Appendix E: Basics of Molecular Phylogeny
GEOLOGICAL CLOCK
MORPHOLOGICAL PHYLOGENY TO MOLECULAR PHYLOGENY
BASIS OF MOLECULAR PHYLOGENY
MUTATION RATE
COMPONENTS OF A PHYLOGENETIC TREE
TYPES OF PHYLOGENETIC TREES
Appendix F: Evolutionary Models of Molecular Phylogeny
INTRODUCTION
Glossary
References
Webliography
Index
End User License Agreement
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Previous Chapter
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Next Chapter
Title Page
Table of Contents
Cover
Title Page
Preface
Acknowledgments
List of Abbreviations
SECTION I: Molecular Sequences and Structures
CHAPTER 1: Retrieval of Sequence(s) from the NCBI Nucleotide Database
1.1 INTRODUCTION
1.2 COMPONENTS OF THE NCBI NUCLEOTIDE DATABASE
1.3 OBJECTIVES
1.4 PROCEDURE
1.5 SOME USEFUL NUCLEOTIDE SEQUENCE DATABASES OF NCBI
1.6 QUESTIONS
CHAPTER 2: Retrieval of Protein Sequence from UniProtKB
2.1 INTRODUCTION
2.2 OBJECTIVE
2.3 PROCEDURE
2.4 QUESTIONS
CHAPTER 3: Downloading Protein Structure
3.1 INTRODUCTION
3.2 OBJECTIVE
3.3 PROCEDURE
3.4 QUESTIONS
CHAPTER 4: Visualizing Protein Structure
4.1 INTRODUCTION
4.2 OBJECTIVE
4.3 PROCEDURE
4.4 QUESTIONS
CHAPTER 5: Sequence Format Conversion
5.1 INTRODUCTION
5.2 OBJECTIVE
5.3 PROCEDURE
5.4 QUESTIONS
5.5 BRIEF DESCRIPTION OF SOME OF THE IMPORTANT MOLECULAR SEQUENCE FORMATS
CHAPTER 6: Nucleotide Sequence Analysis Using Sequence Manipulation Suite (SMS)
6.1 INTRODUCTION
6.2 OBJECTIVE
6.3 PROCEDURE
6.4 FORMAT CONVERSION
6.5 SEQUENCE ANALYSIS
6.6 SEQUENCE FIGURES
6.7 RANDOM SEQUENCES
6.8 MISCELLANEOUS
6.9 QUESTIONS
CHAPTER 7: Detection of Restriction Enzyme Sites
7.1 INTRODUCTION
7.2 OBJECTIVE
7.3 PROCEDURE (USING NEBCUTTER)
7.4 QUESTIONS
SECTION II: Sequence Alignment
CHAPTER 8: Dot Plot Analysis
8.1 INTRODUCTION
8.2 OBJECTIVE
8.3 PROCEDURE
8.4 PARAMETERS OF DOT PLOT ANALYSIS
8.5 INTERPRETATION
8.6 QUESTIONS
CHAPTER 9: Needleman–Wunsch Algorithm (Global Alignment)
9.1 INTRODUCTION
9.2 OBJECTIVE
9.3 PROCEDURE
9.4 QUESTIONS
CHAPTER 10: Smith–Waterman Algorithm (Local Alignment)
10.1 INTRODUCTION
10.2 OBJECTIVE
10.3 PROCEDURE
10.4 QUESTIONS
CHAPTER 11: Sequence Alignment Using Online Tools
11.1 INTRODUCTION
11.2 OBJECTIVE
11.3 PROCEDURE
11.4 INTERPRETATION OF RESULTS
11.5 COLOR SCHEME FOR AMINO ACID RESIDUES
11.6 QUESTIONS
SECTION III: Basic Local Alignment Search Tools
CHAPTER 12: Basic Local Alignment Search Tool for Nucleotide (BLASTn)
12.1 INTRODUCTION
12.2 OBJECTIVE
12.3 PROCEDURE
12.4 QUESTIONS
CHAPTER 13: Basic Local Alignment Search Tool for Amino Acid Sequences (BLASTp)
13.1 INTRODUCTION
13.2 OBJECTIVE
13.3 PROCEDURE
13.4 QUESTIONS
CHAPTER 14: BLASTx
14.1 INTRODUCTION
14.2 OBJECTIVE
14.3 PROCEDURE
14.4 INTERPRETATION OF BLASTx RESULTS
14.5 QUESTIONS
CHAPTER 15: tBLASTn
15.1 INTRODUCTION
15.2 OBJECTIVE
15.3 PROCEDURE
15.4 ALGORITHM PARAMETERS
15.5 INTERPRETATION OF tBLASTn RESULTS
15.6 QUESTIONS
CHAPTER 16: tBLASTx
16.1 INTRODUCTION
16.2 OBJECTIVE
16.3 PROCEDURE
16.4 ALGORITHM PARAMETERS
16.5 INTERPRETATION OF tBLASTx RESULTS
16.6 QUESTIONS
SECTION IV: Primer Designing and Quality Checking
CHAPTER 17: Primer Designing – Basics
17.1 INTRODUCTION
17.2 OTHER IMPORTANT FEATURES FOR DESIGNING “GOOD” PRIMERS
17.3 QUESTIONS
CHAPTER 18: Designing PCR Primers Using the
Primer3
Online Tool
18.1 INTRODUCTION
18.2 OBJECTIVE
18.3 PROCEDURE
18.4 OUTPUT
18.5 SELECTION OF THE BEST PRIMER‐PAIRS BY COMPARATIVE EVALUATION OF THE DESIGNED PRIMERS
18.6 QUESTIONS
CHAPTER 19: Quality Checking of the Designed Primers
19.1 INTRODUCTION
19.2 OBJECTIVE
19.3 PROCEDURE
19.4 IDT UNAFOLD – CHECKING THE SECONDARY STRUCTURE FORMATION OF THE AMPLICON
19.5 PRIMER‐BLAST – TO DETECT POSSIBLE SPURIOUS AMPLIFICATION
19.6 QUESTIONS
CHAPTER 20: Primer Designing for SYBR Green Chemistry of qPCR
20.1 INTRODUCTION
20.2 QUESTIONS
SECTION V: Molecular Phylogenetics
CHAPTER 21: Construction of Phylogenetic Tree: Unweighted‐Pair Group Method with Arithmetic Mean (UPGMA)
21.1 INTRODUCTION
21.2 ASSUMPTIONS
21.3 OBJECTIVE
21.4 PROCEDURE
21.5 INTERPRETATION OF UPGMA TREE
21.6 QUESTIONS
CHAPTER 22: Construction of Phylogenetic Tree: Fitch Margoliash (FM) Algorithm
22.1 INTRODUCTION
22.2 OBJECTIVE
22.3 PROCEDURE
22.4 INTERPRETATION OF THE FM TREE
22.5 QUESTIONS
CHAPTER 23: Construction of Phylogenetic Tree: Neighbor‐Joining Method
23.1 INTRODUCTION
23.2 OBJECTIVE
23.3 PROCEDURE
23.4 INTERPRETATION OF NJ TREE
23.5 QUESTIONS
CHAPTER 24: Construction of Phylogenetic Tree: Maximum Parsimony Method
24.1 INTRODUCTION
24.2 OBJECTIVE
24.3 PROCEDURE
24.4 INTERPRETATION OF MP TREE
24.5 QUESTIONS
CHAPTER 25: Construction of Phylogenetic Tree: Minimum Evolution Method
25.1 INTRODUCTION
25.2 OBJECTIVE
25.3 PROCEDURE
25.4 INTERPRETATION OF THE ME TREE
25.5 QUESTIONS
CHAPTER 26: Construction of Phylogenetic Tree Using MEGA7
26.1 INTRODUCTION
26.2 OBJECTIVE
26.3 PROCEDURE
26.4 INTERPRETATION OF PHYLOGENETIC TREE
26.5 QUESTIONS
CHAPTER 27: Interpretation of Phylogenetic Trees
27.1 INTRODUCTION
27.2 UNDERSTANDING PHYLOGENETIC TREES
27.3 REPRESENTATION OF PHYLOGENETIC TREES
27.4 METHODS FOR CONSTRUCTING EVOLUTIONARY TREES FROM INFERENCES
27.5 INFERRING PHYLOGENETIC TREES
27.6 QUESTIONS
SECTION VI: Protein Structure Prediction
CHAPTER 28: Prediction of Secondary Structure of Protein
28.1 INTRODUCTION
28.2 OBJECTIVE
28.3 SECONDARY STRUCTURE PREDICTION USING ONLINE TOOL PSIPRED
28.4 SECONDARY STRUCTURE PREDICTION USING THE ONLINE CDM TOOL
28.5 QUESTIONS
CHAPTER 29: Prediction of Tertiary Structure of Protein: Sequence Homology
29.1 INTRODUCTION
29.2 OBJECTIVE
29.3 PROCEDURE (SWISS‐MODEL PROGRAM)
29.4 OUTPUT
29.5 VISUALIZING THE PREDICTED STRUCTURE
29.6 INTERPRETATION OF RESULTS
29.7 QUESTIONS
CHAPTER 30: Protein Structure Prediction Using Threading Method
30.1 INTRODUCTION
30.2 OBJECTIVE
30.3 PROCEDURE
30.4 RESULTS AND INTERPRETATION
30.5 QUESTIONS
CHAPTER 31: Prediction of Tertiary Structure of Protein:
Ab Initio
Approach
31.1 INTRODUCTION
31.2 OBJECTIVE
31.3 PROCEDURE (RAPTORX)
31.4 JOB STATUS
31.5 OUTPUT AND INTERPRETATION OF RESULTS
31.6 QUESTIONS
CHAPTER 32: Validation of Predicted Tertiary Structure of Protein
32.1 INTRODUCTION
32.2 OBJECTIVE
32.3 PROCEDURE (WHAT IF TOOL FOR VALIDATING THE 3D STRUCTURE PREDICTION RESULTS)
32.4 INTERPRETATION OF RESULTS OF WHAT IF
32.5 MOLPROBITY TOOL FOR RAMACHANDRAN PLOT
32.6 INTERPRETATION OF RAMACHANDRAN PLOT ANALYSIS
32.7 QUESTIONS
SECTION VII: Molecular Docking and Binding Site Prediction
CHAPTER 33: Prediction of Transcription Binding Sites
33.1 INTRODUCTION
33.2 OBJECTIVE
33.3 TRANSFAC
33.4 BINDING SITES SEARCHING USING THE MATCH TOOL
33.5 QUESTIONS
CHAPTER 34: Prediction of Translation Initiation Sites
34.1 INTRODUCTION
34.2 OBJECTIVE
34.3 PROCEDURE
34.4 QUESTIONS
CHAPTER 35: Molecular Docking
35.1 INTRODUCTION
35.2 OBJECTIVE
35.3 PROCEDURE
35.4 RESULT AND INTERPRETATION
35.5 QUESTIONS
SECTION VIII: Genome Annotation
CHAPTER 36: Genome Annotation in Prokaryotes
36.1 INTRODUCTION
36.2 OBJECTIVE
36.3 PROCEDURE
36.4 INTERPRETATION OF GENEMARK OUTPUT
36.5 QUESTIONS
CHAPTER 37: Genome Annotation in Eukaryotes
37.1 INTRODUCTION
37.2 OBJECTIVE
37.3 PROCEDURE
37.4 INTERPRETATION OF GENSCAN OUTPUT
37.5 QUESTIONS
SECTION IX: Advanced Biocomputational Analyses
CHAPTER 38: Concepts of Real‐Time PCR Data Analysis
38.1 INTRODUCTION
38.2 GETTING STARTED WITH RT‐qPCR
38.3 PCR FLUORESCENCE CHEMISTRY
38.4 RT‐qPCR DATA ANALYSIS: GENE EXPRESSION ANALYSIS
38.5 QUESTIONS
CHAPTER 39: Overview of Microarray Data Analysis
39.1 CONCEPT
39.2 GETTING STARTED WITH MICROARRAY
39.3 MICROARRAY DATA ANALYSIS: GENE EXPRESSION ANALYSIS
39.4 STEPS INVOLVED IN MICROARRAY DATA ANALYSIS
39.5 FUNCTIONAL INFORMATION USING GENE NETWORKS AND PATHWAYS
39.6 LIVESTOCK RESEARCH THAT INVOLVED MICROARRAY ANALYSIS (SOME EXAMPLES)
39.7 APPLICATIONS OF MICROARRAY
39.8 QUESTIONS
CHAPTER 40: Single Nucleotide Polymorphism (SNP) Mining Tools
40.1 INTRODUCTION
40.2 OBJECTIVE
40.3 PROCEDURE
40.4 INTERPRETATION OF RESULTS
40.5 QUESTIONS
CHAPTER 41:
In Silico
Mining of Simple Sequence Repeats (SSR) Markers
41.1 INTRODUCTION
41.2 OBJECTIVE
41.3 MISA (MICROSATELLITE IDENTIFICATION TOOL)
41.4 RESULT
41.5 QUESTIONS
CHAPTER 42: Basics of RNA‐Seq Data Analysis
42.1 INTRODUCTION
42.2 AIM OF AN RNA‐SEQ EXPERIMENT
42.3 FAST SEQUENCE ALIGNMENT STRATEGIES
42.4 QUESTIONS
CHAPTER 43: Functional Annotation of Common Differentially Expressed Genes
43.1 INTRODUCTION
43.2 FUNCTIONAL ANNOTATION
43.3 QUESTIONS
CHAPTER 44: Identification of Differentially Expressed Genes (DEGs)
44.1 SECTION I. QUALITY FILTERING OF DATA USING PRINSEQ
44.2 SECTION II. IDENTIFICATION OF DIFFERENTIALLY EXPRESSED GENES – I (USING CUFFLINKS)
44.3 SECTION III. IDENTIFICATION OF DIFFERENTIALLY EXPRESSED GENES – II (USING RSEM‐DE PACKAGES EBSEQ, DESEQ2 AND EDGER)
44.4 USE OF DE PACKAGES FOR IDENTIFYING THE DIFFERENTIALLY EXPRESSED GENES
44.5 QUESTIONS
CHAPTER 45: Estimating MicroRNA Expression Using the
miRDeep2
Tool
45.1 INTRODUCTION
45.2 PREPROCESSING OF READS
45.3 INPUT FORMATS OF THE DATA FILE
45.4 OUTPUT FORMATS THAT CAN BE GENERATED
45.5 PRELIMINARY FILES USED IN THE EXAMPLE
45.6 QUESTIONS
CHAPTER 46: miRNA Target Prediction
46.1 INTRODUCTION
46.2 miRNA TARGET PREDICTION BY TARGETSCAN (http://targetscan.org/)
46.3 miRNA TARGET PREDICTION BY TARGETSCAN IN HUMAN
46.4 miRNA TARGET PREDICTION BY psRNATARGET (http://plantgrn.noble.org/psRNATarget>/)
46.5 miRNA TARGET PREDICTION BY miRANDA (http://www.microrna.org)
46.6 QUESTIONS
Appendix A: Usage of Internet for Bioinformatics
Appendix B: Important Web Resources for Bioinformatics Databases and Tools
INTRODUCTION
Appendix C: NCBI Database: A Brief Account
Appendix D: EMBL Databases and Tools: An Overview
INTRODUCTION
THE EMBL DATABASES
THE EMBL TOOLS
Appendix E: Basics of Molecular Phylogeny
GEOLOGICAL CLOCK
MORPHOLOGICAL PHYLOGENY TO MOLECULAR PHYLOGENY
BASIS OF MOLECULAR PHYLOGENY
MUTATION RATE
COMPONENTS OF A PHYLOGENETIC TREE
TYPES OF PHYLOGENETIC TREES
Appendix F: Evolutionary Models of Molecular Phylogeny
INTRODUCTION
Glossary
References
Webliography
Index
End User License Agreement
List of Tables
Chapter 04
TABLE 4.1 Computer short‐cuts to work on the image displayed by RasMol (http://www.openrasmol.org/doc/).
Chapter 07
TABLE 7.1 Meaning of different terminologies used in
NEBCutter
(Vince
et al
., 2003).
Chapter 09
TABLE 9.1
TABLE 9.2
Chapter 10
TABLE 10.1 Similarities and differences between NW and SW algorithms.
Chapter 12
TABLE 12.1 Overview of various types of BLAST algorithms available at the National Center for Biotechnology Information (NCBI) website, with their applications.
TABLE 12.2 Optional BLASTn parameters. Numbered arrows refer to the serial number (SN) of discussion in Table 12.3.
TABLE 12.3 Databases against which a query can be searched in BLASTn (http://www.ncbi.nlm.nih.gov/books/NBK153387/).
Chapter 13
TABLE 13.1 Algorithm parameters of BLASTp: Numbered arrows in Figure 13.2 refer to the serial number (SN) of discussion in this table.
Chapter 17
TABLE 17.1 Important parameters to be considered for designing “good” primers (http://www.premierbiosoft.com/tech_notes/PCR_Primer_Design.html).
TABLE 17.2 The acceptable values of Gibb’s free energy for various secondary structures of primer (http://ls23l.lscore.ucla.edu/Primer3/primer3web_help.htm).
Chapter 18
TABLE 18.1 Primer3 parameters, description and their optimal values/options (http://ls23l.lscore.ucla.edu/Primer3/primer3web_help.htm).
TABLE 18.2 Important parameters based on which primer is selected.
TABLE 18.3
Chapter 19
TABLE 19.1
TABLE 19.2
Chapter 20
TABLE 20.1 Optimal and permissible ranges of parameters of qPCR primers (SYBR green chemistry).
Chapter 21
TABLE 21.1
TABLE 21.2
TABLE 21.3
TABLE 21.4
TABLE 21.5
Chapter 22
TABLE 22.1
TABLE 22.2
TABLE 22.3
TABLE 22.4
TABLE 22.5
TABLE 22.6
Chapter 23
TABLE 23.1
TABLE 23.2
TABLE 23.3
TABLE 23.4
TABLE 23.5
TABLE 23.6
TABLE 23.7
TABLE 23.8
TABLE 23.9
TABLE 23.10
TABLE 23.11
TABLE 23.12
TABLE 23.13
TABLE 23.14
TABLE 23.15
TABLE 23.16
TABLE 23.17
TABLE 23.18
TABLE 23.19
TABLE 23.20
TABLE 23.21
TABLE 23.22
TABLE 23.23
TABLE 23.24
Chapter 24
TABLE 24.1
TABLE 24.2
TABLE 24.3
TABLE 24.4
TABLE 24.5
Chapter 25
TABLE 25.1
TABLE 25.2
Chapter 27
Table 27.1
TABLE 27.2 Comparison between the features of the trees generated from the following important phylogenetic algorithms (Desper and Gascuel, 2005).
TABLE 27.3 Pairwise distances (calculated by maximum composite likelihood model, using MEGA7) between the input sequences are shown in the lower triangular matrix.
Chapter 42
TABLE 42.1 Example and purpose of short read aligners.
TABLE 42.2 Example and purpose of long read aligners.
TABLE 42.3 Information about total reads of samples 1, 2, and 3, and values obtained by dividing the reads for each gene in each sample with the corresponding total reads.
TABLE 42.4 Calculation of RPKM by dividing the reads obtained after step 1 for each gene with gene length.
TABLE 42.5 Total reads of samples 1, 2, 3, and 4.
TABLE 42.6 Total reads per kb (RPK) of gene for sample 1, 2, and 3 (millions of reads equated to a scale of tens of reads).
TABLE 42.7 Calculation of TPM by dividing the total reads obtained in step 1 sample, with total reads per kb of gene.
TABLE 42.8 Comparison of reads of RPKM.
TABLE 42.9 Comparison of reads of TPM.
Appendix D
TABLE 1 Features and links of various EMBL databases.
TABLE 2 Description of various EMBL tools.
List of Illustrations
Chapter 01
FIGURE 1.1 Main search window of NCBI Nucleotide page and list of hits for nucleotide sequences of taurine
Drosha
(gene/mRNA).
FIGURE 1.2 Click on the “Send to” button to download and save (in a text file) the first three Drosha mRNA sequences in “Summary” format.
Chapter 02
FIGURE 2.1 Homepage of ExPASy server: select the “proteomics” option from the drop‐down menu for databases, and enter your protein name along with other keywords to begin search.
FIGURE 2.2 Click on the specific entry to open it in a separate window.
FIGURE 2.3 Peptide sequence of taurine SRY in FASTA format.
Chapter 03
FIGURE 3.1 Homepage of RCSB‐PDB. Specify the name of the protein and the species in the given box, and click on the search button (denoted by the symbol of a lens).
FIGURE 3.2 Visualization of 3D peptide structure obtained following PDB search.
Chapter 04
FIGURE 4.1 Graphical user interface (GUI) of RasMol and the drop‐down menu to open, modify or alter the display of the peptide.
FIGURE 4.2 A single peptide, displayed in ‘Wireframe’, ‘Backbone’, ‘Sticks’, ‘Spacefill’, ‘Ball and Stick’, ‘Ribbons’, ‘Strands’, ‘Cartoons’ and ‘Molecular surface’ patterns.
Chapter 05
FIGURE 5.1 Homepage of the
ReadSeq
biosequence format conversion tool.
FIGURE 5.2 Three sequence formats – namely, FASTA, Phylip and Clustal.
Chapter 06
FIGURE 6.1 “Combine FASTA” input page to provide input data, and the corresponding output page with the result.
FIGURE 6.2 “EMBL Trans Extractor” input page, and the corresponding output page with extracted results.
FIGURE 6.3 “Filter DNA” input page, along with various options as control parameters.
FIGURE 6.4 “Range Extractor Protein” input page and the corresponding output page with extracted sequences.
FIGURE 6.5 “Reverse Complement” input page and the corresponding output pages for “Complement”, “Reverse” and “Reverse Complement”, respectively (from left to right), of the input sequences.
FIGURE 6.6 “Protein Isoelectric Point” input page and the corresponding output page with results, with respect to the parameters.
Chapter 07
FIGURE 7.1 A short nucleotide sequence (oligo) can be searched in the input sequence for determining specific RE sites present in the oligos.
FIGURE 7.2 More options enable the user to make stringent selection of RE sites.
FIGURE 7.3 Result output window of
NEBCutter
. Details are discussed in the text under the sub‐heading “Inferring the output”.
Chapter 08
FIGURE 8.1 Depiction of plotting the straight line based on the runs of dots obtained from matches between residues along the
X
‐ and
Y
‐axes. Insertion in any of the sequences will distort the run of the straight line.
FIGURE 8.2 Interpretation of dot plot based on the same repeat sequence (shown above) which has been placed along both axes. The four different colors (yellow, green, blue and gray) have been shown to indicate the 1st, 2nd, 3rd and 4th repeat of “TACGGCTACAGTCACG”.
Chapter 09
FIGURE 9.1 Three types of movement along the matrix in dynamic programming.
FIGURE 9.2 Increment in the respective indexes of the cells (denoting row and column numbers, respectively) of the matrix, to indicate the movement along the cells.
FIGURE 9.3 Each cell is assigned three scores obtained from three possible movements – namely, horizontal, diagonal and vertical. The arrows indicate back‐tracing based on the highest score out of the three scores.
FIGURE 9.4 Trace‐back starts from the bottom right cell towards the top left cell, according to the highest score(s) obtained in the previous step. There could be more than one path at a point (i.e., cell), if that cell has been awarded more than one highest score, due to two or three movements in the previous step.
FIGURE 9.5 Global alignment (by NWA) has yielded seven equally good (same alignment score of 4) alignments.
Chapter 10
FIGURE 10.1 The scores in each cell are obtained from the movements from three directions – namely, horizontal, diagonal and vertical. The arrows indicate back‐tracing based on the highest score out of the three scores.
FIGURE 10.2 Trace‐back step: starting with the highest score in the matrix, moving towards the top left and stopping at the last positive score.
Chapter 11
FIGURE 11.1 The output of multiple sequence alignment using Clustal Omega is obtained in different tabs – “Alignments”, “Result Summary”, “Submission Details”.
Jalview
is the Java alignment viewer that displays the alignment, along with the consensus sequence.
Chapter 12
FIGURE 12.1 Main page for BLASTn search at NCBI. The sequence can be entered into the box as query sequences with either accession number or sequence in FASTA format. The gene identity number (i.e., the gi mentioned in this figure) is not currently used as sequence identifier in the NCBI nucleotide database.
FIGURE 12.2 Optional BLASTn parameters. Numbered arrows refer to the serial number of discussion in Table 12.3.
FIGURE 12.3 The result page of BLASTn contains the color key‐based alignment display, followed by a tabular description of sequence alignments and, finally, alignments of each of the sequence pairs (query vs. database sequence).
FIGURE 12.4
FIGURE 12.5
Chapter 13
FIGURE 13.1 Setting the parameters for BLASTp search at NCBI. The sequence(s) can be entered into the box as query sequence(s), with either NCBI Protein accession number or sequence(s) in FASTA format.
FIGURE 13.2 Optional BLASTp parameters. The numbered arrows refer to the serial number of discussion in Table 13.1.
FIGURE 13.3 Different sections of the result page of BLASTp. “A” indicates the putative conserved domain(s) detected by BLASTp search. Clicking on this image will open the graphical summary of the conserved domain(s) of that protein. “B” indicates the alignment and the scores in terms of color key, for each of the alignments. “C” indicates the table of alignment detail (Description, Max score, Total score, Query coverage, E‐value, Identity, and Accession). “D” shows the detail of the alignment residue‐wise.
FIGURE 13.4 Results of PSI‐BLAST. ‘E’ indicates the “Select for PSI blast” column, and “F” indicates the detailed result for each alignment.
FIGURE 13.5 Result of PHI‐BLAST. ‘G’ indicates the detailed result of each alignment. The asterisks in the second row of alignment indicate the pattern which has been given for PHI‐BLAST analysis.
FIGURE 13.6 The result page of DELTA‐BLAST. The components and parameters are similar to PSI‐BLAST.
Chapter 14
FIGURE 14.1 Homepage of BLASTx at NCBI. The sequence can be entered into the box (angled arrow) as query sequences, either with accession number(s) or as sequence(s) in FASTA format.
FIGURE 14.2 The results page of BLASTx contains a color key‐based alignment display, followed by a tabular description of sequence alignments and, finally, alignment of each of the sequence pairs (a query versus database sequence, called a subject sequence).
Chapter 15
FIGURE 15.1 Homepage for tBLASTn at NCBI. The query sequence(s) can be entered with either accession numbers or sequence(s) in FASTA format.
FIGURE 15.2 The results page of tBLASTn contains the color key‐based alignment display, followed by a tabular description of sequence alignments and, finally, alignment of each of the sequence pairs (query versus database sequences).
Chapter 16
FIGURE 16.1 Main page for tBLASTx search at NCBI. The sequence can be entered into the box as query sequences, with either accession no. or sequence, in FASTA format.
FIGURE 16.2 The result page of tBLASTx contains the color key‐based alignment display, followed by tabular description of sequence alignments and, finally, alignment of each of the sequence pairs (query versus database subject sequences).
Chapter 18
FIGURE 18.1 Setting the parameters of the Primer3 online tool for primer designing.
FIGURE 18.2 Output page of Primer3 online tool, displaying one pair of primers and their position in the input target sequence (asterisks below the bases).
Chapter 19
FIGURE 19.1 Homepage of Oligoanalyzer 3.1, indicating different parameters and functions for the output of the function “Analyze”.
FIGURE 19.2 Output of the function “Hairpin” of the Oligoanalyzer 3.1 tool, displaying the possible hairpins and the related thermodynamic values.
FIGURE 19.3 Prediction of secondary structure in the amplicon using the UNAFold tool of IDT.
FIGURE 19.4 Output result of Primer‐BLAST and selection of primers from the list displayed.
Chapter 21
FIGURE 21.1
FIGURE 21.2
FIGURE 21.3
FIGURE 21.B1
FIGURE 21.B2
FIGURE 21.4
FIGURE 21.5
Chapter 22
FIGURE 22.1
FIGURE 22.2
FIGURE 22.3
Chapter 23
FIGURE 23.1
FIGURE 23.2
FIGURE 23.3
FIGURE 23.4
FIGURE 23.5
Chapter 24
FIGURE 24.1
FIGURE 24.2
FIGURE 24.3
FIGURE 24.4
FIGURE 24.5
FIGURE 24.6
Chapter 25
FIGURE 25.1 Comparative depiction of the phylogenetic tree constructed from the same data set, using the MP method.
Chapter 26
FIGURE 26.1 Compile the unaligned, homologous molecular sequences in FASTA format in a text file.
FIGURE 26.2 Aligning the input sequences using either ClustalW or Muscle available in MEGA7 interface.
FIGURE 26.3 Exporting the alignment file and saving the alignment session for further use.
FIGURE 26.4 Selection of the best evolutionary model for further analyses.
FIGURE 26.5 Setting the parameters for phylogenetic analysis.
FIGURE 26.6 Controlling the display parameters using the menu bar parameters.
FIGURE 26.7 Controlling the tree display parameters using the left‐hand‐side buttons.
FIGURE 26.8 Insertion of figures for the external nodes (species name).
FIGURE 26.9 Saving the output phylogenetic tree as a PNG file.
FIGURE 26.10
Chapter 27
FIGURE 27.1 This dendrogram represents the evolutionary relationship among the taxa. The horizontal axis represents the evolutionary changes over time.
FIGURE 27.2 Swapping of the branches of the sub‐tree of the main tree does not change any meaning represented by the tree. The evolutionary distances between the OTUs remain unchanged.
FIGURE 27.3 Representing the same phylogenetic tree as circular, radiation, rectangular and straight orientations.
FIGURE 27.4 Converting a straight tree to a radiation tree by eliminating the depiction of divergence from common ancestor.
FIGURE 27.5 Phylogenetic trees constructed from nine sequences of 18s rRNA gene belonging to divergent species using various algorithms.
Chapter 28
FIGURE 28.1 Graphical user interface (GUI) of PSIPRED and filling the inputs in the Input tab.
FIGURE 28.2 The output tabs of PSIPRED shown in three sections.
FIGURE 28.3 GUI of the online CDM tool for prediction of protein secondary structures.
Chapter 29
FIGURE 29.1 Pairwise sequence alignment to determine the extent of sequence identity between the query and template sequences.
FIGURE 29.2 Open the page to initiate homology modeling using the SWISS‐MODEL workspace
FIGURE 29.3 Window of SWISS‐MODEL workspace for providing the input parameters and starting homology modeling.
FIGURE 29.4 Important sections of the SWISS‐MODEL output. One can download the complete result in PDF format, or can specifically download the sections as required.
Chapter 30
FIGURE 30.1 Home window of RaptorX for job submission.
FIGURE 30.2 (A) The modeled and non‐modeled residues; (B) 3D cartoon view of selected template; (C) the target‐template alignment view.
FIGURE 30.3 3D cartoon view of tertiary structure predicted by RaptorX server.
FIGURE 30.4 3 Class SS3 and 8 Class SS8 secondary structural element contribution to the 3D structure.
FIGURE 30.5 Conformationally ordered and disordered contribution of the residues in the 2D and 3D structure (C). Contribution of each residue in solvent accessibility (D).
Chapter 31
FIGURE 31.1 Job progress box of RaptorX after job submission.
FIGURE 31.2 Results windows of RaptorX, indicating assignment of protein domain and 3D prediction results.
Chapter 32
FIGURE 32.1 Homepage of WHAT IF web interface; click on “Build/check/repair model” link (in the left‐hand pane) to initiate validation of the predicted tertiary structure of the peptide.
FIGURE 32.2 Click on the “Upload your file” button to browse the input file and then click on “Send” button to upload the file to the server.
FIGURE 32.3 Output of the WHAT IF analysis of the predicted tertiary structure of the peptide.
FIGURE 32.4 Ramachandran plot for a typical protein structure. The different regions were taken from the observed phi‐psi distribution for 121 870 residues from 463 known X‐ray protein structures.
FIGURE 32.5 The homepage of MOLProbity tool for Ramachandran plot analysis.
Chapter 33
FIGURE 33.1 (a) TRANSFAC database search; (b) FACTOR table search; (c) TRANSFAC Factor entries; (d) output of TRANSFAC Factor table.
FIGURE 33.2 Creating sequences logos using the web interface.
FIGURE 33.3 (a) Searching Transfac matrix table; (b) TRANSFAC Matrix entries; (c) output of TRANSFAC Matrix table.
FIGURE 33.4 (a) MATCH user interface; (b) results page of MATCH output; (c) a simple visual representation of locations of the found matches.
Chapter 34
FIGURE 34.1 File format of inserted nucleotide sequence in NetStart 1.0.
FIGURE 34.2 Output format for translation start predictions for a vertebrate sequence.
FIGURE 34.3 File format of inserted nucleotide sequence in TIS Miner.
FIGURE 34.4 Output format for TIS Miner
Chapter 35
FIGURE 35.1 The identified active site or cavity within the receptor is marked as a star.
FIGURE 35.2 The “Submit Docking” tab at the top of the homepage of the SwissDock online tool takes you to this page. Upload the target and ligand files by clicking on the appropriate buttons.
FIGURE 35.3 Fitness of ligand and free energy of docked complex of the first and second binding poses, shown as “A” and “B”.
FIGURE 35.4 Fitness of ligand and free energy of docked complex of the third, fourth and fifth binding poses, shown as “C”, “D” and “E”.
Chapter 36
FIGURE 36.1 Homepage of the GeneMark online tool.
FIGURE 36.2 Specifying the parameters in GeneMark.hmm for prokaryotes for gene finding and annotation.
FIGURE 36.3 The output pages of the GeneMark online tool for prokaryotic gene prediction.
Chapter 37
FIGURE 37.1 Homepage of the online GENSCAN software.
FIGURE 37.2 Output page of the GENSCAN software.
Chapter 38
FIGURE 38.1 Amplification plot of RT‐qPCR.
FIGURE 38.2 Construction of standard curve. Construct standard curves for both target and reference genes individually, by plotting
C
t
values (through the
Y
‐axis) against the log
10
(template amount or dilution)
along the
X
‐axis.
FIGURE 38.3 SYBR Green fluorophore binds with double‐stranded DNA (PCR amplicon). The amount of DNA amplified is proportional to fluorescence intensity.
FIGURE 38.4 Absolute quantification of the transcript using the standard curve method. Using a known amount of DNA, a standard curve is made, and unknown samples are plotted on a regression line of known samples.
FIGURE 38.5 Relative quantification of RT‐qPCR transcript measurement, always expressed in terms of two samples (say, sample A in comparison to Sample B). Relative expression is measured in terms of fold change (either positive or negative fold change). Positive fold change indicates upregulation of genes in the A vs. B sample, whereas negative fold change indicates downregulation of genes in the A vs. B sample.
FIGURE 38.6 Analytical flow diagram of the use of real‐time PCR.
Chapter 39
FIGURE 39.1 Reference design (a) and loop design (b) of a two‐color microarray. Different colors (red and green here) represent microarray chips. In order to avoid dye bias, the same samples are used twice, with opposing labeling schemes, such as array 1: sample a (labeled with red dye) vs. Sample b (labeled with green dye) and array 2: sample b (labeled with red dye) vs. sample a (labeled with green dye).
FIGURE 39.2 Application of microarray for gene expression analysis. Fluorescently labeled cDNA or cRNA is hybridized with probes, and the image is scanned through a scanner. Based upon the intensity of the signal, up regulated (red dots) and down regulated (green dots) genes are detected.
FIGURE 39.3 Data transformation converts the raw signal intensity of each probe‐target hybridization into a log scale. Transformation of the data brings values in a normal distribution.
Chapter 40
FIGURE 40.1 Screenshot of Stacks software: http://creskolab.uoregon.edu/stacks/
FIGURE 40.2 Image of denovo_map.pl script of Stacks to call SNPs
de novo
from RADSeq data.
FIGURE 40.3 Image of ref_map.pl script of STACKS to call SNPs reference based from RAD‐Seq data.
FIGURE 40.4 Screenshot of GATK software website: https://www.broadinstitute.org/gatk/index.php.
FIGURE 40.5 Image of GATK command used to mine SNPs from an example dataset.
FIGURE 40.6 Result of GATK SNPs mining from an example dataset.
Chapter 41
FIGURE 41.1 MISA homepage.
FIGURE 41.2 Download misa.pl.
FIGURE 41.3 Download misa.ini.
FIGURE 41.4 The command prompt where code is written.
FIGURE 41.5 The output, as seen in testfile.misa.
FIGURE 41.6 The output, as seen in testfile.statistics.
Chapter 42
FIGURE 42.1
FIGURE 42.2
Chapter 43
FIGURE 43.1
FIGURE 43.2
FIGURE 43.3
FIGURE 43.4
FIGURE 43.5
FIGURE 43.6
FIGURE 43.7
FIGURE 43.8
FIGURE 43.9
FIGURE 43.10
FIGURE 43.11
FIGURE 43.12
FIGURE 43.13
FIGURE 43.14
Chapter 44
FIGURE 44.1 The basic command for running PRINSEQ‐lite.
FIGURE 44.2 Summary statistics after running prinseq‐lite.pl.
FIGURE 44.3 Six files generated after running prinseq‐lite.pl.
FIGURE 44.4 Workflow for identifying DEGs using Cufflinks.
FIGURE 44.5 UCSC genome browser.
FIGURE 44.6 Click on downloads, genomics data and then select “cow”.
FIGURE 44.7 Zip file and FASTA file of the cow genome.
FIGURE 44.8 Downloading the GTF file.
FIGURE 44.9 Indexing the genome using GMAP.
FIGURE 44.10 Indexing files generated after indexing.
FIGURE 44.11 Files generated after running cufflinks on control BAM file.
FIGURE 44.12 Files generated after running cufflinks on infected BAM file.
FIGURE 44.13 The assemblies.txt file.
FIGURE 44.14 gene_exp.diff file giving the fold change of the genes, along with significance.
FIGURE 44.15 Workflow for identifying DEGs using RSEM and DE packages.
FIGURE 44.16 .bash_profile with the path added.
FIGURE 44.17 Echo $PATH indicating that the path is added.
FIGURE 44.18 wget command downloading the genome from the ensemble genome browser.
FIGURE 44.19 Folder ftp.ensembl.org created after the download.
FIGURE 44.20 The chromosome gunzip files in the folder ftp.ensembl.org.
FIGURE 44.21 Direct download from the.ftp site.
FIGURE 44.22 Index files created after indexing using bowtie 2.0.
FIGURE 44.23 Six files generated after running the calculate expression command.
FIGURE 44.24 Expected counts, TPM and FPKM of each of the ensemblIDs.
FIGURE 44.25 Combining the counts of all the files and rounding them to the nearest integer.
FIGURE 44.26 Loading the EBSeq package in R.
FIGURE 44.27 Input file for EBSeq.
FIGURE 44.28 Running iterations of EBSeq.
FIGURE 44.29 Identifying DEGs in EBSeq.
FIGURE 44.30 Fold change of all the ensemblIDs.
FIGURE 44.31 Significant DE genes.
FIGURE 44.32 Loading DESeq2 package.
FIGURE 44.33 Example input data set.
FIGURE 44.34 Fold change and significance of ensemblIDs.
FIGURE 44.35 Running the DESeq2 package.
FIGURE 44.36 Fold change and significance of ensemble IDs in the file DEDESeq2.txt.
FIGURE 44.37 Significant DEGs in DEDEseq2.txt.
FIGURE 44.38 reOrdered command ouput and the various column IDs generated.
FIGURE 44.39 Loading the edgeR package in R.
FIGURE 44.40 Input file for edgeR.
FIGURE 44.41 Running edgeR in R
FIGURE 44.42 Fold change and significance of ensemblIDs in DEEdgeR.txt.
Chapter 45
FIGURE 45.1
FIGURE 45.2
FIGURE 45.3
FIGURE 45.4
FIGURE 45.5
FIGURE 45.6
FIGURE 45.7
Chapter 46
FIGURE 46.1 Home page of TargetScan tool.
FIGURE 46.2 Output page showing multiple transcripts in the TargetScan tool.
FIGURE 46.3 Output page of the TargetScan tool, showing conserved sites for miRNA families.
FIGURE 46.4 Detailed table of all the conserved sites.
FIGURE 46.5 Input page of the TargetScan tool.
FIGURE 46.6 Detailed information about the target gene symbol in the TargetScan tool.
FIGURE 46.7 Input page of the psRNATarget tool.
FIGURE 46.8 Input page of the psRNATarget tool for other parameters.
FIGURE 46.9 Result page of the psRNATarget tool.
FIGURE 46.10 Results file of the miRanda tool.
Appendix E
FIGURE E1 Different types of point mutations leading to codon change.
FIGURE E2 The components of a rooted phylogenetic tree.
FIGURE E3 Diagrammatic representation of monophyletic, paraphyletic and polyphyletic groups of taxa.
Appendix F
FIGURE F1 Substitution of nucleotides leading to transition and transversion.
FIGURE F2 Jukes–Cantor one‐parameter substitution model
FIGURE F3 Rates of transition and transversion are the same (
α
).
FIGURE F4 Amount of base‐substitution in a period of time “
ζ
” and equal rates of transition and transversion (
α
).
FIGURE F5 K80 model: amount of base‐substitution in unit time period (
t
= 1), assuming different rates of transition (
α
) and transversion (
β
).
FIGURE F6 F81 model: rate of base‐substitution is different for four bases: adenine (
ρ
A
), guanine (
ρ
G
), cytosine (
ρ
C
) and thymine (
ρ
T
).
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