Bioinformatics: Methods and

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Bioinformatics
Methods and Applications
Bioinformatics
Methods and Applications

Edited by
Dev Bukhsh Singh
Department of Biotechnology, Institute of Biosciences and Biotechnology,
Chhatrapati Shahu Ji Maharaj University, Kanpur, India

Rajesh Kumar Pathak

School of Agricultural Biotechnology, Punjab Agricultural University,
Ludhiana, India
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Contents

List of contributors xvii 2. Biological databases and their

Preface xxi application 17
Parva Kumar Sharma and Inderjit Singh Yadav
1. Introduction to basics of
bioinformatics 1 2.1 Introduction 17
2.1.1 Relational databases 17
Rajesh Kumar Pathak, Dev Bukhsh Singh
2.1.2 Object-oriented databases 17
and Rahul Singh
2.2 Sequence databases 19
1.1 Introduction 1 2.2.1 GenBank 19
1.1.1 Concept behind bioinformatics, 2.2.2 European Nucleotide Archive 19
in silico biology, and 2.2.3 DNA Database of Japan 20
computational biology 3 2.2.4 GenPept 20
1.1.2 Scientific discipline and support 2.2.5 Protein information resources 20
systems for bioinformatics 5 2.3 Composite database 21
1.1.3 Needs of bioinformatics 5 2.3.1 Universal Protein Resource (UniProt) 21
1.2 Historical background of bioinformatics 5 2.3.2 Nonredundant database 22
1.3 Aim of bioinformatics 7 2.4 Secondary database 22
2.4.1 PROSITE 22
1.4 The recent development in the field of
2.4.2 PRINTS 22
bioinformatics 7
2.4.3 Pfam 23
1.5 Challenges in bioinformatics 8
2.4.4 InterPro 23
1.6 Application of bioinformatics 8
2.5 Structural databases 23
1.6.1 Sequence analysis 8
2.5.1 Research collaboratory for structural
1.6.2 Phylogenetic analysis 8
bioinformatics protein data bank 23
1.6.3 Prediction of protein secondary
2.5.2 SCOP 24
structure 9
2.5.3 CATH/Gene3D 24
1.6.4 Protein 3D structure prediction 9
2.6 Specialized database 24
1.6.5 Evaluation and validation of
2.6.1 Clustering databases 24
predicted protein model 10
2.6.2 Bibliographic databases 26
1.6.6 Discovery and designing of small
2.6.3 Expression databases 26
molecules leading to drugs/
2.6.4 Taxonomy databases 26
agrochemical development 10
2.6.5 Interaction databases 26
1.6.7 Next-generation sequencing data 2.6.6 Pathway databases 27
analysis 10 2.6.7 Enzyme databases 27
1.6.8 SNP and SSR identification 10 2.6.8 microRNA database 27
1.6.9 Pharmacogenomics 10 2.6.9 Small molecule database 27
1.6.10 Metabolomics and metabolic 2.6.10 Vaccine design database 28
flux analysis 11 2.7 Database searching and annotation 28
1.6.11 Systems biology and omics 2.7.1 Entrez 28
data integration 11 2.7.2 The sequence retrieval system 28
1.7 Future perspective 11 2.7.3 Annotation 28
1.8 Conclusion 11 2.8 Conclusions 29
Conflict of interest 12 Conflict of interest 29
References 12 References 29

v
vi Contents

3. Biological sequence analysis 33 4.3 Data preprocessing 52

4.3.1 Quality control of raw
Samvedna Shukla, Bhawana Mishra, sequencing data 52
Himanshu Avashthi and Muktesh Chandra 4.3.2 Trimming and filtering of raw reads 52
3.1 Introduction 33 4.4 Genome assembly approaches: types of
3.2 Sequence alignments: determining assembly 52
similarity and deducing homology 34 4.4.1 De novo assembly approach 52
3.2.1 Why construct sequence alignment? 34 4.4.2 Reference-based
3.2.2 Similarity of sequences 34 assembly approach 54
3.2.3 Homology of sequences 35 4.4.3 Hybrid assembly approach 54
3.2.4 Global sequence alignment 35 4.4.4 Meta-assembly approach 54
3.2.5 Local sequence alignment 35 4.5 Tools and software for
3.2.6 Working of alignment algorithm 36 genome assembly 56
3.3 Scoring matrices: construction and 4.5.1 Genome finishing/polishing 56
proper selection 36 4.5.2 Assembly quality assessment and
3.3.1 Scoring matrices 36 validation 58
3.3.2 PAM matrices 37 4.6 Pitfall in genome assemblies 58
3.3.3 BLOSUM matrices 37 4.6.1 DNA quality 58
3.3.4 PAM versus BLOSUM 37 4.6.2 Library preparation 59
3.4 Basic Local Alignment Search Tool 38 4.6.3 Data quality 59
3.4.1 Seeding step 39 4.6.4 Repetitive DNA 59
3.4.2 Ungapped extension step 39 4.7 A mathematical calculation for depth/
3.4.3 Gapped extension step 39 coverage 60
3.4.4 Types of BLAST 39 4.8 Genome annotation 60
3.4.5 BLAT versus BLAST 39 4.8.1 Structural annotation 61
3.5 Multiple sequence alignment (MSA) 40 4.8.2 Functional annotation 62
3.5.1 Need for MSA 40 4.9 Application and future prospects of
3.5.2 Multiple sequence alignment genome assembly 62
algorithm 41 4.10 Conclusion 63
3.5.3 ClustalW 41 Conflict of interest 63
3.6 Phylogenetic analysis 42 References 63
3.6.1 Types of trees 43
3.6.2 Algorithms for phylogenetic analysis 43 5. Computational molecular phylogeny:
3.6.3 Terminology of phylogenetic tree 43 concepts and applications 67
3.7 Application of sequence alignment 44 Krishna Kumar Ojha, Swapnil Mishra
3.8 Conclusion 44 and Vijay Kumar Singh
Conflict of interest 45
References 45 5.1 Introduction 67
5.2 Convergent and divergent evolution 67
4. Genome assembly and annotation 49 5.3 Concept of cladistics and systematics 68
5.4 Phylogenetic trees’ terminology 69
Pallavi Mishra, Ranjeet Maurya,
5.4.1 Phylogenetic tree 69
Himanshu Avashthi, Shikha Mittal,
5.4.2 Taxon 69
Muktesh Chandra and
5.4.3 Node 69
Pramod Wasudeo Ramteke
5.4.4 Leaf 69
4.1 Introduction 49 5.4.5 Internal node 70
4.1.1 How do you reassemble a 5.4.6 Edge 70
genome after sequencing? 50 5.4.7 Topology 70
4.1.2 Assembler technology: historical 5.4.8 Root 70
landscape 50 5.4.9 Rooted tree 70
4.2 Genome assembly algorithms 51 5.4.10 Unrooted tree 70
4.2.1 The overlap-layout-consensus/string 5.4.11 Binary tree 70
graph assemblers 51 5.4.12 Clade 71
4.2.2 De Bruijn graph assemblers 51 5.4.13 Monophyletic taxon 71
 Contents vii

5.4.14 Paraphyletic taxon 71 6.7 Applications of RNA-sequencing 100

5.4.15 Polyphyletic taxon 71 6.8 Advantages of transcriptome
5.4.16 Split 72 sequencing over microarray technology 100
5.4.17 Subtree 72 6.9 Limitations and future perspective of
5.4.18 Edge length 72 RNA-sequencing 100
5.4.19 Ultrametric tree 72 6.10 Conclusion 100
5.4.20 Cladogram 73 Conflict of interest 101
5.4.21 Phylogram 73 References 101
5.4.22 Dendrogram 73
5.5 Evolutionary inference of phylogenetic 7. RNA-seq for revealing the function
trees 73 of the transcriptome 105
5.5.1 Importance of shared derived
characters in phylogeny 74 Kavita Goswami and Neeti Sanan-Mishra
5.6 Tree construction methods 74 7.1 Introduction 105
5.6.1 UPGMA 74 7.2 Next-generation sequencing platforms
5.6.2 Neighbor-joining algorithm 76 and their technologies 105
5.6.3 Maximum parsimony 80 7.2.1 Illumina 106
5.6.4 Maximum likelihood phylogeny 82 7.2.2 Roche 454 106
5.6.5 Bayesian phylogeny 83 7.2.3 ION torrent 107
5.7 Estimating reliability of phylogenetic tree 84 7.2.4 SoLid ABI 107
5.7.1 Bootstrapping 85 7.2.5 Illumina Tru-seq SLR technology 108
5.8 Phylogenetic tools 85 7.2.6 Pacific Biosciences (PacBio)
5.9 Application of molecular phylogeny 86 SMRT sequencing 108
5.9.1 Classification 86 7.2.7 Oxford Nanopore 108
5.9.2 Identifying the origin of pathogens 87 7.3 Analyzing the RNA-seq data 109
5.9.3 Species conservation 87 7.3.1 Quality and depth of raw
5.10 Conclusion 87 sequencing data 109
Conflict of interest 87 7.3.2 Adapter removal 110
References 87 7.3.3 Level of alignment 110
7.3.4 Redundancy rate of reads 111
6. Applications and challenges of 7.4 RNA-seq applications 111
microarray and RNA-sequencing 91 7.4.1 Transcriptome assembly 111
7.4.2 Identification of novel
Ankita Negi, Abhimati Shukla, Akanksha Jaiswar,
protein-coding genes 112
Jatin Shrinet and Rahul Singh Jasrotia
7.4.3 Identification of other classes of
6.1 Introduction 91 RNAs 113
6.2 Evolution of microarray 92 7.4.4 Profiling expression patterns 115
6.2.1 Automated arrays and cDNA 7.4.5 Degradome sequencing 119
cloning to microarray technology 92 7.4.6 Variants detection and
6.2.2 Principle of microarray 92 allele-specific expression 120
6.2.3 List of microarray tools and their 7.4.7 Expression quantitative trait loci 120
utility 94 7.5 Databases and software for small
6.3 DNA sequencing 94 RNA analysis 121
6.3.1 First generation 94 7.5.1 miRBase 121
6.3.2 Second generation 95 7.5.2 PMRD 121
6.3.3 Third generation 96 7.5.3 Armour 121
6.4 RNA-sequencing 96 7.5.4 UEA sRNA workbench 121
6.4.1 Library preparation and sequencing 96 7.5.5 sRNAtoolbox 122
6.4.2 Pipeline and usage of RNA- 7.5.6 sRNAbench 122
sequencing 97 7.5.7 miRNAconsTarget 122
6.5 Biological databases for 7.5.8 Massively parallel signature
data submission 99 sequencing database 122
6.6 Applications of microarray 99 7.5.9 CLC Genomics Workbench 122
viii Contents

7.6 Conclusion 122 9.3.5 DEEPred 149

Conflict of interest 123 9.3.6 SDNGO 149
References 123 9.3.7 AmiGO 149
9.3.8 OBO-Edit 149
8. Analysis of SSR and SNP markers 131 9.3.9 Gene Ontology Functional
Enrichment Annotation Tool 149
Ankita Mishra, Pramod Kumar Singh,
9.4 GO enrichment analysis 149
Abhishek Bhandawat, Vinay Sharma, Vikas Sharma,
9.4.1 DAVID (Database for Annotation,
Pradeep Singh, Joy Roy and Himanshu Sharma
Visualization, and Integrated
8.1 Introduction 131 Discovery) 150
8.2 Analysis of SSR markers 132 9.4.2 PANTHER (Protein ANalysis
8.2.1 Benefits and limitations of THrough Evolutionary
microsatellite markers 132 Relationships) 150
8.2.2 SSR mining and primer designing 133 9.4.3 g:Profiler 150
8.2.3 Classification and genomic 9.4.4 clusterProfiler 151
localization 133 9.4.5 Enrichr 151
8.2.4 Functional annotations of SSR 9.4.6 ToppGene 151
containing sequences 133 9.4.7 QuickGo 152
8.2.5 SSR amplification and evaluation of 9.4.8 REVIGO (Reduce & Visualize
polymorphic potential 133 Gene Ontology) 152
8.2.6 Cross-transferability of SSR markers 134 9.4.9 WEGO (Web Gene Ontology) 152
8.2.7 Future perspective 135 9.4.10 ShinyGO 152
8.3 Analysis of SNP markers 135 9.4.11 ViSEAGO 152
8.3.1 Approaches for sequence data 9.4.12 PoGo (Prediction of
generation 135 Gene Ontology) 152
8.3.2 Sample preparation 135 9.4.13 GOrilla (Gene Ontology
8.3.3 Sequencing of complex genomes 135 enRIchment anaLysis and
8.3.4 Assessment of sequence quality 136 visuaLizAtion tool) 152
8.3.5 Reads assembly and mapping to 9.4.14 EasyGO 152
reference genome 136 9.4.15 GOEAST (Gene Ontology
8.3.6 Postprocessing of mapped reads 138 Enrichment Analysis
8.3.7 Variant calling and filtration 138 Software Toolkit) 153
8.3.8 Functional annotation of variant 138 9.4.16 GOAT (Gene Ontology
8.4 Conclusion 140 Annotation Tool) 153
Acknowledgments 140 9.4.17 GOLEM (Gene Ontology Local
Conflict of interest 140 Exploration Map) 153
References 140 9.4.18 GOssTo (Gene Ontology
Further reading 144 semantic similarity Tool) 153
9.4.19 NaviGO 153
9. Gene Ontology: application and 9.5 Applications 153
importance in functional annotation 9.6 Future prospects 154
of the genomic data 145 9.7 Conclusion 154
Acknowledgment 154
Reshu Saxena, Ritika Bishnoi and Deepak Singla
Conflict of interest 155
9.1 Background 145 Author contributions 155
9.2 Gene Ontology based classification 146 References 155
9.2.1 Cellular component 147
9.2.2 Molecular function 147 10. Metagenomics: the boon for
9.2.3 Biological process 147 microbial world knowledge
9.3 Annotation of unknown gene/genome 147 and current challenges 159
9.3.1 Blast2GO 148
J.K. Choudhari, J. Choubey, M.K. Verma,
9.3.2 IPRscan (InterProScan) 148
T. Chatterjee and B.P. Sahariah
9.3.3 Genome Assembly and Annotation
Package 149 10.1 Introduction: an overview of
9.3.4 GOnet 149 metagenomics 159
 Contents ix

10.2 Resources in metagenomics 161 11.5.1 Mutation studies 185

10.3 Challenges in metagenomics 161 11.5.2 Unfolding studies 185
10.4 The workflow in metagenome analysis 162 11.5.3 Binding site prediction 185
10.5 Dataset acquire and processing 162 11.5.4 Protein docking and virtual
10.6 Quality control analysis 162 screening 185
10.6.1 Base quality score 162 11.5.5 Understanding the dynamics of
10.6.2 Sequence quality score 162 protein or protein ligand
10.6.3 Overrepresented sequences 163 complex 186
10.6.4 Per base N content 163 11.5.6 Structure evolution analysis 186
10.6.5 Duplicate sequences 163 11.6 Conclusion 186
10.6.6 Read length distribution 163 Conflict of interest 186
10.6.7 Per base sequence content 163 References 186
10.6.8 Guanine cytosine content 163 Further reading 188
10.6.9 Overrepresented k-mers 163
10.6.10 Quality analysis and improving 12. Structural and functional analysis
software tools 163 of protein 189
10.7 Genome assembly tools in
metagenomics 164 Neetu Singh Yadav, Pawan Kumar
10.8 Binning tools in metagenomics 164 and Indra Singh
10.8.1 Statistical analysis 166 12.1 Protein preliminaries 189
10.9 Data storage and sharing 166 12.2 Growth of the protein
10.10 Metagenomics analysis: a case study 166 structural database 189
10.11 Material, methodology, and outcome 166 12.3 Structural topology and fold
10.11.1 Metagenome dataset 166 classification scheme 190
10.11.2 Sequencing quality analysis 166 12.4 D-Structure quality
10.11.3 Hits distribution of metagenome assessment protocol 191
from the database sources 168 12.5 Protein 3D structure prediction 191
10.11.4 Hits distribution of functional 12.5.1 Energy functions 192
group 168 12.6 Machine learning in PSP 197
10.11.5 Taxonomic hits distribution 170 12.6.1 Feature engineering and
10.11.6 Rarefaction curve 170 representation 197
10.11.7 Alpha diversity 170 12.6.2 Feature selection 198
10.12 Advantages of metagenomics study 172 12.6.3 ML algorithms 198
10.13 Limitations and future perspective 172 12.6.4 ML models’ implementation
10.14 Conclusion 172 and evaluation 199
Conflict of interest 173 12.7 Conclusion 200
References 173 Conflict of interest 201
References 201
11. Protein structure prediction 177
13. Computational approaches in
Shikha Agnihotry, Rajesh Kumar Pathak,
Dev Bukhsh Singh, Apoorv Tiwari
drug designing 207
and Imran Hussain Anshul Tiwari and Sakshi Singh
11.1 Introduction 177 13.1 Introduction 207
11.2 Protein structure prediction 177 13.2 Computer-aided drug designing 207
11.3 Method of protein structure prediction 178 13.2.1 Structure-based drug design 207
11.3.1 Homology modeling 13.2.2 Ligand-based drug design 209
(comparative modeling) 178 13.3 Computational approaches 209
11.3.2 Threading or fold recognition 180 13.3.1 Molecular modeling 209
11.3.3 Ab initio approach 182 13.3.2 Binding site and cavity
11.4 Evaluation of predicted prediction 211
protein structure 184 13.3.3 Computational ligand
11.5 Applications of structure prediction 185 designing and searching 211
x Contents

13.3.4 Pharmacophore modeling 211 15.3.2 Conformational flexibility 236

13.3.5 Molecular docking 211 15.3.3 Scaffold hopping 236
13.3.6 Molecular dynamics simulation 212 15.3.4 Fragment-based drug design 236
13.3.7 Quantitative structure activity 15.4 Pharmacophore mapping 236
relationship 213 15.4.1 Diverse conformation
13.3.8 Lead optimization 214 generation 236
13.4 Limitations 214 15.4.2 Generation of 3D
13.5 Recent trends in drug designing 214 pharmacophore 237
13.6 Conclusion 215 15.4.3 Validation of the
Conflict of interest 215 pharmacophore model 237
References 215 15.5 Pharmacophore classifications 238
15.5.1 Ligand-based pharmacophore
14. Structure-based drug designing 219 modeling 238
Shubham Pant, Shivani Verma, 15.5.2 Structure-based pharmacophore
Rajesh Kumar Pathak and Dev Bukhsh Singh modeling 239
15.6 Application of pharmacophore in
14.1 Introduction 219 virtual screening and de novo design 239
14.2 Background of structure-based drug 15.6.1 Virtual screening 239
design 220 15.6.2 De novo pathway 240
14.3 Process of SBDD 221 15.7 Advancement in exploring 3D
14.3.1 Target identification 221 pharmacophore principles over
14.3.2 Target structure the above limitations 240
determination/prediction 221 15.8 Quantitative structure activity
14.3.3 Cavity/binding site prediction 223 relationship 240
14.3.4 Ligand structure preparation/ 15.8.1 Designation of QSAR 241
retrieval 223 15.8.2 Backbone of chemical
14.3.5 Molecular docking and virtual similarity 241
screening 224 15.8.3 QSAR data 242
14.3.6 ADMET analysis 226 15.8.4 Classification of 3D QSAR
14.3.7 Molecular dynamics simulation 226 approaches 243
14.3.8 Binding-free-energy 15.8.5 Molecular interactions and
calculation-MM-PBSA 227 energies 243
14.4 Recent development in SBDD 227 15.8.6 QSAR modeling 245
14.5 Challenges and limitations 228 15.8.7 Concept of applicability
14.6 Future prospective 228 domain and QSAR approaches 246
14.7 Conclusion 229 15.9 Development of new QSAR: HQSAR 248
Conflict of interest 229 15.10 Application of QSAR/SAR 248
References 229 15.10.1 Synthetic organic chemistry
and QSAR 248
15. Ligand-based drug designing 233 15.10.2 Prediction of kinetic and
thermodynamic parameters 249
Suchitra M. Ajjarapu, Apoorv Tiwari,
15.10.3 Drug development and other
Pramod Wasudeo Ramteke, Dev Bukhsh Singh
applications 249
and Sundip Kumar
15.11 Conclusion 250
15.1 Introduction 233 Conflict of interest 250
15.2 Pharmacophore 234 References 250
15.2.1 Build pharmacophore
hypothesis 234
15.2.2 Alignment of molecules 235 16. Discovery and optimization of lead
15.2.3 Similarity search methods 235 molecules in drug designing 253
15.2.4 2D finger prints 235
Shivani Verma and Rajesh Kumar Pathak
15.3 3D fingerprints 235
15.3.1 3D similarities depend on the 16.1 Introduction 253
alignment 236 16.2 Principles of CADD 254
 Contents xi

16.2.1 Case study: inhibition of 17.5.2 Prediction of pharmacokinetic

lipoxygenases 255 properties 279
16.3 Discovery of the lead molecule 256 17.5.3 Target identification and de novo
16.4 Types of lead molecules 256 ligand design using
16.4.1 Natural lead compounds 256 pharmacophore approaches 279
16.4.2 Synthetic lead molecules 257 17.5.4 Protein functionality studies 279
16.4.3 Semisynthetic drugs 259 17.5.5 3D pharmacophore modeling
16.5 Lead optimization and strategies 259 using a web platform 279
16.5.1 By using organic synthetic 17.5.6 Pharmacophore methods in
chemistry 259 light of molecular dynamics
16.5.2 Structure simplification 261 simulations 280
16.5.3 Structure modification 261 17.6 Applications of pharmacophore
16.5.4 Functional group interconversion 261 modeling 281
16.5.5 Bonding strength and selectivity 262 17.6.1 Generation of e-pharmacophore
16.5.6 Using thermodynamic, for virtual screening of drug
pharmacodynamics, and molecules 281
pharmacokinetic parameters 262 17.6.2 ADME-Tox analysis 282
16.6 Computational lead optimization 263 17.6.3 Generation of a multitarget
16.7 Advantages of computational lead ligand 282
designing 263 17.6.4 Modulation of the immune
16.8 Future perspectives 263 system 282
16.9 Conclusion 264 17.6.5 Pharmacophore-guided drug
Conflict of interest 265 target identification 282
References 265 17.7 Future perspectives of pharmacophore
models 283
17. Pharmacophore modeling and its 17.7.1 Fragment-based drug design 283
applications 269 17.7.2 Protein protein interaction
inhibition 283
Rashmi Tyagi, Amisha Singh, Kamal Kumar
17.7.3 A potential role in protein design 283
Chaudhary and Manoj Kumar Yadav
17.8 Conclusion 284
17.1 Introduction 269 Conflict of interest 284
17.2 Basics of pharmacophore modeling 271 References 284
17.2.1 Division of initial data into
diverse datasets 271 18. Molecular docking and molecular
17.2.2 Conformational analysis with dynamics simulation 291
three-dimensional structures 272
Sakshi Singh, Qanita Bani Baker
17.3 Different methods of pharmacophore
and Dev Bukhsh Singh
generation 273
17.3.1 Ligand-based pharmacophore 18.1 Introduction 291
modeling 273 18.2 Molecular docking 292
17.3.2 Structure-based pharmacophore 18.2.1 Algorithms 292
modeling 275 18.2.2 Scoring functions 294
17.4 Validation of pharmacophore models 276 18.2.3 Knowledge-based SFs 294
17.4.1 Receiver operating characteristic 18.3 Docking methodologies 295
curve 276 18.3.1 Flexible docking 295
17.4.2 Structure-based pharmacophore 18.3.2 Semiflexible docking 295
modeling approach for the design 18.3.3 Virtual screening of
of azaindole derivatives as DprE1 high-throughput docking 295
inhibitors for tuberculosis: 18.3.4 Fragment docking 296
a case study 277 18.3.5 Machine learning in docking 296
17.5 Recent trends in pharmacophore 18.3.6 Docking tools and their features 297
generation 278 18.4 Molecular dynamics simulation 297
17.5.1 Machine-learning models 18.4.1 Postdocking refinement 298
incorporated with pharmacophore 18.4.2 Binding-free energy calculations:
descriptors 278 MM-GBSA/MM-PBSA 299
xii Contents

18.5 Challenges in molecular docking and 20.3.2 Prediction of linear B-cell

MD simulation techniques 300 epitopes 321
18.6 Conclusion 301 20.4 In silico method for T-cell epitope
Conflict of interest 301 prediction 322
References 301 20.4.1 MHC class I binder prediction 323
20.4.2 MHC class II binder prediction 324
19. Pharmacokinetics and 20.4.3 MHC gene diversity and its
pharmacodynamics analysis of importance in T-cell epitope
drug candidates 305 prediction 325
20.5 Adjuvant and linker selection 325
Satendra Singh, Dev Bukhsh Singh, 20.6 Building 3D model and validation of
Budhayash Gautam, Anamika Singh fusion vaccine construct 326
and Namrata Yadav 20.7 Miscellaneous properties 326
19.1 Introduction 305 20.7.1 Peptide toxicity 326
19.2 Postgenomic era and drug discovery 306 20.7.2 Half-life or stability 327
19.3 Pharmacokinetics 307 20.7.3 Delivery methodology 327
19.3.1 Drug absorption 308 20.8 Role of next-generation sequencing
19.3.2 Drug distribution (binding/ technology in vaccine design 328
localization/storage) 308 20.9 Computer-aided vaccine
19.3.3 Drug metabolism 309 development example 328
19.4 Pharmacodynamics 311 20.10 Conclusion 329
19.4.1 Drug toxicity 311 Acknowledgments 329
19.5 Computational approaches for Conflict of interest 330
ADMET prediction 311 References 330
19.6 Translational bioinformatics 313
19.7 Drug repurposing 313 21. Metabolomics and flux balance
19.7.1 Benefits of drug repurposing 313 analysis 337
19.7.2 Computational drug repurposing 314
19.8 Role of pharmacogenomics in Priyanka Narad, G. Naresh
precision medicine 314 and Abhishek Sengupta
19.9 Chemical diversity of natural 21.1 Introduction 337
products: a source for computer-aided 21.2 Definition of metabolomics 338
drug discovery 315 21.3 MS- and NMR-based techniques in
19.10 Conclusion 315 metabolomics 338
Conflict of interest 315 21.4 Data processing in metabolomics 339
References 315 21.4.1 Nuclear magnetic resonance
Further reading 316 spectroscopy 339
21.4.2 Workflow of MS-based
20. Computational approaches for metabolomics 339
vaccine designing 317 21.4.3 Limitations of NMR and MS
methods 340
Animesh Awasthi, Gaurav Sharma
21.4.4 Recent advances in MS- and
and Piyush Agrawal
NMR-based metabolomics 341
20.1 Introduction 317 21.4.5 Challenges and affecting factors 341
20.2 Antigen selection and immunological 21.5 Applications of metabolomics 341
databases 318 21.5.1 Microbial science 341
20.2.1 Exogenous antigens 318 21.5.2 Plant science 341
20.2.2 Endogenous antigens 319 21.5.3 Animal science 342
20.2.3 Autoantigens 319 21.5.4 Medical science 342
20.3 In silico method for B-cell epitope 21.5.5 Food and herbal medicines 343
prediction 319 21.6 Flux balance analysis 343
20.3.1 Prediction of conformational 21.7 Metabolic networks and model
B-cell epitopes 320 construction 344
 Contents xiii

21.7.1 Metabolic model: construction 22.4.1 Graph theory 371

and refinement 345 22.4.2 Adjacency matrices 373
21.7.2 Mass balance 348 22.5 Topological parameters 373
21.7.3 Steady state 349 22.5.1 Node degree 374
21.7.4 Types of constraints 349 22.5.2 The average of shortest
21.7.5 Optimization 350 path length 374
21.7.6 Steps in FBA 351 22.5.3 Clustering coefficient 375
21.8 Metabolic control analysis and isotopic 22.5.4 Betweenness centrality 375
steady state/carbon flux analysis 352 22.5.5 Statistical comparison 375
21.8.1 Metabolic control analysis 352 22.6 Biological significance of
21.8.2 Carbon flux analysis 352 network motifs 375
21.8.3 Different types of flux balance 22.7 Applications of network biology 376
analysis at different conditions 353 22.8 Limitations and challenges 376
21.9 Some important tools of flux balance 22.9 Conclusion 376
analysis 355 Conflict of interest 377
21.9.1 OptKnock 355 References 377
21.9.2 OptGene 355
21.9.3 OptStrain 356 23. Network biology and applications 381
21.9.4 COBRA Toolbox 356
21.9.5 MetaboAnalyst 4.0 356 Neeru Redhu and Zoozeal Thakur
21.9.6 OptFlux 356 23.1 Introduction to biological networks 381
21.9.7 OpenFlux 357 23.2 Biological networks properties 381
21.9.8 CellNetAnalyzer 357 23.2.1 Path, average path length,
21.9.9 SBRT 357 and diameter 381
21.9.10 Escher-FBA 357 23.2.2 Degree aka connectivity 382
21.9.11 MetaFluxNet 357 23.2.3 Scale free 382
21.10 Applications, challenges, and future 23.2.4 Small world network 382
perspectives of FBA 358 23.2.5 Date and party hub 382
21.10.1 Applications 358 23.2.6 Network motifs 382
21.10.2 Challenges and future 23.3 Types of biological networks 382
perspectives 360 23.3.1 Ecological networks 383
21.11 Case study: applications of 23.3.2 Gene (genetic) regulatory
metabolomics and flux balance network 383
analysis in industrially important 23.3.3 Protein protein interaction
microorganisms 361 network 384
21.11.1 Lactococcus lactis 361 23.3.4 Metabolic networks 385
21.11.2 Saccharomyces cerevisiae 361 23.3.5 Cellular signaling network 385
21.11.3 Escherichia coli 362 23.3.6 Gene coexpression network 387
21.12 Conclusion 363 23.4 Experimental methods in network
References 363 biology 387
23.4.1 Microarray 387
22. Topological parameters, patterns, 23.4.2 Deep mRNA sequencing 388
and motifs in biological networks 367 23.4.3 Exome sequencing 388
23.4.4 ChIP-seq 389
Arvind Kumar Yadav, Rohit Shukla
23.4.5 Genome-wide bisulfite
and Tiratha Raj Singh
sequencing 389
22.1 Introduction 367 23.4.6 Yeast two-hybrid 390
22.2 Biological networks 368 23.4.7 Mass spectrometry-based
22.2.1 Construction of biological proteomics 391
networks 368 23.4.8 Flow and mass cytometry 392
22.3 Network motifs and patterns 369 23.4.9 Live cell imaging 392
22.3.1 Motif discovery and counting 369 23.5 Resources for biological
22.4 Analysis of biological network 370 network-based studies 393
xiv Contents

23.5.1 Kyoto Encyclopedia of Genes 24.5 Platforms used for modeling and
and Genomes 393 simulations 415
23.5.2 BioCyc Database Collection 393 24.5.1 Pathway designing tools 415
23.5.3 ENZYME 393 24.5.2 Pathway Tools 415
23.5.4 ExplorEnz: the enzyme database 394 24.5.3 Simulation tools 416
23.5.5 Biochemical Genetic and 24.6 Applications of pathway modeling and
Genomic/Biochemical Genetic simulations 418
and Genomic models 394 24.6.1 Metabolic engineering 418
23.5.6 STRING 394 24.6.2 Drug designing 418
23.5.7 metaTIGER 394 24.6.3 Study of phenomics 419
23.6 Tools for network pathway analysis 394 24.6.4 Flux balance analysis 419
23.6.1 Pathway tools 395 24.7 Challenges 420
23.6.2 ERGO 395 24.7.1 Knowledge gaps between
23.6.3 KEGGtranslator 395 computationalists and
23.6.4 ModelSEED 395 experimentalists 420
23.6.5 Network Analysis Tools 396 24.7.2 Theory development 420
23.6.6 BioNetStat 396 24.7.3 Miscellaneous computational
23.6.7 OmicsNet 396 challenges 420
23.7 Applications of network biology 396 24.8 Conclusion 420
23.7.1 Applications in rare diseases 396 Conflict of interest 421
23.7.2 Determination of References 421
protein function 398
23.7.3 Pathway determination 398 25. Systems biology and big data
23.7.4 Essential protein identification 398 analytics 425
23.7.5 Functional modules’
identification 399 Rohit Shukla, Arvind Kumar Yadav,
23.8 Challenges and future perspective 399 William O. Sote, Moacyr Comar Junior
23.8.1 Pseudo temporal ordering 399 and Tiratha Raj Singh
23.8.2 Multiple data sources 400 25.1 Introduction 425
23.8.3 Combination of algorithms 400 25.2 Big data in general and in the context
23.9 Conclusion 400 of biology 425
Conflict of interest 401 25.3 Types of data in systems biology 426
References 401 25.3.1 Biological sequences 428
25.3.2 Molecular structure 428
24. Pathway modeling and simulation 25.3.3 Gene expression 428
analysis 409 25.3.4 Binding sites and domains 429
25.3.5 Protein protein interaction 429
Gitanjali Tandon, Sunita Yadav
25.3.6 Mass spectroscopy 429
and Sukhdeep Kaur
25.3.7 Metabolic pathways 429
24.1 Introduction 409 25.4 Biological big data resources 429
24.2 Computational modeling of 25.4.1 Genomics and transcriptomics
a pathway 409 resources 430
24.2.1 Type of modeling 410 25.4.2 Proteomics resources 430
24.2.2 Approaches of modeling 410 25.4.3 Cellular metabolome 430
24.3 General diagram and language used in 25.4.4 Protein protein interaction
pathway modeling 411 databases 430
24.3.1 Systems Biology Graphical 25.4.5 Drug and chemical compound
Notation 411 databases 432
24.3.2 Systems Biology Markup 25.4.6 Different other databases 432
Language 412 25.5 Network generation and its analysis
24.4 Pathway simulations analysis 413 from various sources of data 432
24.4.1 Ordinary differential equations 413 25.6 Big data in drug repurposing and
24.4.2 Stochastic simulation 414 systems pharmacology 434
 Contents xv

25.6.1 Network-based approaches for 27.3.2 Estimation 458

systems pharmacology 435 27.3.3 Prediction 459
25.7 Case study related to transcriptome 27.3.4 Association 459
data analysis 435 27.3.5 Clustering 459
25.8 Limitations in big data analysis 437 27.3.6 Description and visualization 460
25.9 Conclusion 438 27.3.7 Case studies using Waikato
Acknowledgment 438 environment for knowledge
Conflict of interest 438 analysis 461
References 438 27.4 Feature selection technique in data
mining 461
26. Machine learning in bioinformatics 443 27.4.1 Objective of feature selection 462
27.5 Major data mining algorithms
Indrajeet Kumar, Surya Pratap Singh applicable to biological data 462
and Shivam 27.5.1 C4.5 algorithm 462
26.1 Introduction 443 27.5.2 K-means algorithm 462
26.2 Supervised learning 443 27.5.3 Support vector machine 463
26.2.1 Classification 444 27.6 Biological data evolution and related
26.2.2 Supervised machine learning in issues 463
bioinformatics 444 27.6.1 Biological data availability 463
26.3 Unsupervised machine learning 445 27.6.2 Biological data availability in
26.4 Problems to understand supervised computer-readable form 464
learning and unsupervised learning 446 27.6.3 Biological data cleaning 464
26.5 Regression 446 27.6.4 Biological data quality 464
26.5.1 Hypothesis 447 27.6.5 Biological data dimensionality 464
26.6 Clustering 450 27.6.6 Biological data knowledge
26.6.1 K-means clustering 450 discovery 466
26.6.2 Density-based clustering 450 27.7 Bioinformatics research areas and tools 466
26.6.3 Distribution-based clustering 451 27.7.1 Sequence searching, comparison,
26.6.4 Fuzzy clustering 451 and evolutionary analysis 467
26.7 Unsupervised learning in 27.7.2 Annotation of gene/protein
bioinformatics 452 structure and function 467
26.8 Application of machine learning 452 27.7.3 Gene and protein expression
26.8.1 Genome annotation 452 analysis 468
26.8.2 Protein structure prediction 452 27.7.4 Mutation and disease association
26.8.3 Research area in bioinformatics study 468
with deep learning 453 27.7.5 Protein structure prediction,
26.9 Discussion 453 docking, and protein protein
26.10 Conclusion 454 interaction analysis 468
Conflict of interest 454 27.7.6 Biological systems modeling
References 454 and network analysis 468
27.7.7 Expressed sequence tag analysis 469
27. Bioinformatics and biological 27.7.8 MicroRNA and target prediction 469
27.7.9 Medical and health data analysis
data mining 457
and clinical decision support
Aditya Harbola, Deepti Negi, system 469
Mahesh Manchanda and 27.8 Limitations 469
Rajesh Kumar Kesharwani 27.9 Conclusion 470
Conflict of interest 470
27.1 Biological data mining 457
References 470
27.2 Data mining applications 457
27.3 Data mining process 458
Index 473
27.3.1 Classification 458
List of contributors

Shikha Agnihotry Department of Computational Biology and Bioinformatics, Jacob Institute of Biotechnology and
Bio-Engineering, Sam Higginbottom University of Agriculture, Technology and Sciences, Allahabad, India
Piyush Agrawal Centre for Computational Biology, Indraprastha Institute of Information Technology Delhi (IIITD),
Okhla Phase III, New Delhi, India
Suchitra M. Ajjarapu Bioinformatics Sub-DIC, Molecular Biology & Genetic Engineering, College of Basic Science
and Humanities, G.B. Pant University of Agriculture and Technology, India; Department of Biotechnology, Andhra
University, India
Himanshu Avashthi Department of Computational Biology & Bioinformatics, Sam Higginbottom University of
Agriculture Technology & Sciences, India; Division of Genomic Resources, ICAR National Bureau of Plant Genetic
Resources, Pusa Campus, New Delhi, India
Animesh Awasthi Institute of Bioinformatics and Applied Biotechnology (IBAB), Bengaluru, India; Department of
Biotechnology, Indian Institute of Technology Kharagpur, Kharagpur, India
Qanita Bani Baker Department of Computer Science, Jordan University of Science and Technology, Irbid, Jordan
Abhishek Bhandawat Agri-Biotechnology Division, National Agri-Food Biotechnology Institute, Mohali, India
Ritika Bishnoi School of Agricultural Biotechnology, Punjab Agricultural University, Ludhiana, India
Muktesh Chandra Centre of Bioinformatics, Institute of Interdisciplinary Sciences, University of Allahabad,
Prayagraj, India
T. Chatterjee Raipur Institute of Technology, Raipur, India
Kamal Kumar Chaudhary School of Biosciences, IMS Ghaziabad University Courses Campus, India
J. Choubey Raipur Institute of Technology, Raipur, India
J.K. Choudhari Chhattisgarh Swami Vivekanand Technical University, Bhilai, India
Budhayash Gautam Department of Computational Biology and Bioinformatics, Jacob Institute of Biotechnology and
Bioengineering, SHUATS, Prayagraj, India
Kavita Goswami Plant RNAi Biology Group, International Centre for Genetic Engineering and Biotechnology, New
Delhi, India
Aditya Harbola School of Computing, Graphic Era Hill University, Dehradun, India
Imran Hussain School of Life and Allied Health Sciences, Glocal University, Saharanpur, India
Akanksha Jaiswar Mossakowski Medical Research Centre, Polish Academy of Sciences, Warsaw, Poland
Rahul Singh Jasrotia Department of Biological Sciences, Florida State University, FL, United States
Moacyr Comar Junior Campus Centro-Oeste, Department of Biochemistry, Federal University of Sao Joao Del Rei,
Minas Gerais, Brazil
Sukhdeep Kaur Department of Computational Biology and Bioinformatics, Sam Higginbottom University of
Agriculture, Technology and Sciences (SHUATS), Prayagraj, India
Rajesh Kumar Kesharwani Nehru Gram Bharati Deemed University, Prayagraj, India
Indrajeet Kumar Graphic Era Hill University, Dehradun, India
Pawan Kumar Bioinformatics Center, National Institute of Immunology, New Delhi, India

xvii
xviii List of contributors

Sundip Kumar Bioinformatics Sub-DIC, Molecular Biology & Genetic Engineering, College of Basic Science and
Humanities, G.B. Pant University of Agriculture and Technology, India
Mahesh Manchanda School of Computing, Graphic Era Hill University, Dehradun, India
Ranjeet Maurya CSIR-Institute of Genomics and Integrative Biology, India
Ankita Mishra Agri-Biotechnology Division, National Agri-Food Biotechnology Institute, Mohali, India
Bhawana Mishra Department of Chemistry, Central University of Haryana, Jant-Pali, Mahendergarh, Haryana, India
Pallavi Mishra Centre for Agriculture Bioinformatics, ICAR-Indian Agricultural Statistics Research Institute, India;
Department of Computational Biology & Bioinformatics, Sam Higginbottom University of Agriculture Technology
& Sciences, India
Swapnil Mishra Center for Bioinformatics, University of Allahabad, India
Shikha Mittal Division of Genomic Resources, ICAR National Bureau of Plant Genetic Resources, Pusa Campus,
New Delhi, India
Priyanka Narad Amity Institute of Biotechnology, Amity University Uttar Pradesh, Noida, India
G. Naresh Amity Institute of Biotechnology, Amity University Uttar Pradesh, Noida, India
Ankita Negi Centre for Agricultural Bioinformatics (CABin), ICAR-Indian Agricultural Statistics Research Institute
(IASRI), New Delhi, India
Deepti Negi School of Computing, Graphic Era Hill University, Dehradun, India
Krishna Kumar Ojha Department of Bioinformatics, Central University of South Bihar, India
Shubham Pant Electrochemical Process Engineering Division, CSIR-Central Electrochemical Research Institute,
Karaikudi, India
Rajesh Kumar Pathak School of Agricultural Biotechnology, Punjab Agricultural University, Ludhiana, India
Pramod Wasudeo Ramteke Department of Computational Biology and Bioinformatics, Jacob Institute of
Biotechnology and Bio-Engineering, Sam Higginbottom University of Agriculture, Technology and Sciences, India;
Department of Molecular Biology & Genetic Engineering, RTM Nagpur University, India; Department of Life
Sciences, Mandsaur University, India; Department of Biological Sciences, Sam Higginbottom University of
Agriculture Technology & Sciences, India; Faculty of Life Sciences, Mandsaur University, India
Neeru Redhu Department of Molecular Biology, Biotechnology & Bioinformatics, College of Basic Sciences &
Humanities, CCS Haryana Agricultural University, Hisar, India
Joy Roy Agri-Biotechnology Division, National Agri-Food Biotechnology Institute, Mohali, India
B.P. Sahariah Chhattisgarh Swami Vivekanand Technical University, Bhilai, India
Neeti Sanan-Mishra Plant RNAi Biology Group, International Centre for Genetic Engineering and Biotechnology,
New Delhi, India
Reshu Saxena Fels Institute for Cancer Research & Molecular Biology, Lewis Katz School of Medicine, Temple
University, Philadelphia, PA, Unites States; Current address: Department of Biological Sciences and Biotechnology,
Institute of Advanced Research (IAR), Gandhinagar, India
Abhishek Sengupta Amity Institute of Biotechnology, Amity University Uttar Pradesh, Noida, India
Gaurav Sharma Institute of Bioinformatics and Applied Biotechnology (IBAB), Bengaluru, India
Himanshu Sharma Agri-Biotechnology Division, National Agri-Food Biotechnology Institute, Mohali, India
Parva Kumar Sharma Indian Council of Agricultural Research—Indian Agricultural Statistics Research Institute,
New Delhi, India
Vikas Sharma Department of Botany, Sant Baba Bhag Singh University, Khiala, India
Vinay Sharma Agri-Biotechnology Division, National Agri-Food Biotechnology Institute, Mohali, India
Shivam Graphic Era Hill University, Dehradun, India
Jatin Shrinet Department of Biological Sciences, Florida State University, FL, United States
 List of contributors xix

Abhimati Shukla Department of Biochemical Engineering, Harcourt Butler Technical University, Kanpur, India
Rohit Shukla Department of Biotechnology and Bioinformatics, Jaypee University of Information Technology (JUIT),
Solan, India
Samvedna Shukla Molecular and Bioprospection Division, CSIR-Central Institute of Medicinal and Aromatic Plants,
Lucknow, India
Amisha Singh Department of Biotechnology, JAIN University, India
Anamika Singh Department of Botany, Maitreyi College, University of Delhi, New Delhi, India
Dev Bukhsh Singh Department of Biotechnology, Institute of Biosciences and Biotechnology, Chhatrapati Shahu Ji
Maharaj University, Kanpur, India
Indra Singh School of Biotechnology, Banaras Hindu University, Varanasi, India
Pradeep Singh Department of Biotechnology, Guru Nanak Dev University, Amritsar, India
Pramod Kumar Singh Department of Biosciences, Christian Eminent College, Indore, India
Rahul Singh Department of Basic and Translational Sciences, School of Dental Medicine, University of Pennsylvania,
Philadelphia, PA, United States
Sakshi Singh Department of Molecular and Human Genetics, Institute of Science, Banaras Hindu University,
Varanasi, India
Satendra Singh Department of Computational Biology and Bioinformatics, Jacob Institute of Biotechnology and
Bioengineering, SHUATS, Prayagraj, India
Surya Pratap Singh Graphic Era Hill University, Dehradun, India
Tiratha Raj Singh Centre of Excellence in Healthcare Technologies and Informatics (CHETI), Department of
Biotechnology and Bioinformatics, Jaypee University of Information Technology (JUIT), Solan, India
Vijay Kumar Singh Department of Bioinformatics, Central University of South Bihar, India
Deepak Singla School of Agricultural Biotechnology, Punjab Agricultural University, Ludhiana, India
William O. Sote Campus Centro-Oeste, Department of Biochemistry, Federal University of Sao Joao Del Rei, Minas
Gerais, Brazil
Gitanjali Tandon Centre for Agricultural Bioinformatics (CABin), ICAR—Indian Agricultural Statistics Research
Institute (IASRI), New Delhi, India
Zoozeal Thakur Bacteriology Lab, National Research Centre on Equines, Hisar, India
Anshul Tiwari Channing Division of Network Medicine, Brigham and Woman Hospital, Harvard Medical School,
Boston, MA, United States; Dept. of Ophthalmology, King George’s Medical University, Lucknow, India
Apoorv Tiwari Department of Computational Biology and Bioinformatics, Jacob Institute of Biotechnology and Bio-
Engineering, Sam Higginbottom University of Agriculture, Technology and Sciences, India; Bioinformatics Sub-
DIC, Molecular Biology & Genetic Engineering, College of Basic Science and Humanities, G.B. Pant University of
Agriculture and Technology, Pantnagar, India
Rashmi Tyagi Centre for Drug Design Discovery and Development (C4D), SRM University, India
M.K. Verma Chhattisgarh Swami Vivekanand Technical University, Bhilai, India; National Institute of Technology
Raipur, Raipur, India
Shivani Verma Department of Chemistry, College of Basic Sciences & Humanities, G. B. Pant University of
Agriculture and Technology, Pantnagar, India
Arvind Kumar Yadav Department of Biotechnology and Bioinformatics, Jaypee University of Information
Technology (JUIT), Solan, India
Inderjit Singh Yadav School of Agricultural Biotechnology, Punjab Agricultural University, Ludhiana, India
Manoj Kumar Yadav Centre for Drug Design Discovery and Development (C4D), SRM University, India;
Department of Bioinformatics, SRM University, India
Namrata Yadav Department of Molecular and Human Genetics, Banaras Hindu University, Varanasi, India
xx List of contributors

Neetu Singh Yadav Department of Biochemical Engineering & Biotechnology, Indian Institute of Technology Delhi,
New Delhi, India; Department of Chemical & Biomolecular Engineering, Clemson University, Clemson, SC, United
States
Sunita Yadav Centre for Agricultural Bioinformatics (CABin), ICAR—Indian Agricultural Statistics Research
Institute (IASRI), New Delhi, India
Preface

Bioinformatics is recognized as the science of the 21st century, and a lot of research has been conducted and on-going
throughout the world in this field. An updated resource with respect to time is required for a better understanding of the
tremendous power of computational approaches and their application in solving complex biological problems. This
book covers the basic understanding of the bioinformatics topics as well as focuses on recent developments in the meth-
ods, tools, and approaches related to bioinformatics, which includes biological databases and their application, sequence
analysis and comparison, phylogeny, NGS data analysis, genome, and transcriptome assembly and annotation, gene
ontology, metagenomics studies, SNP and SSR identification and analysis, transcriptome analysis; microarray studies,
RNA-Seq data analysis, protein structure prediction, visualization, analysis and validation; pharmacophore modeling,
structure-based and ligand-based drug design, molecular docking, molecular dynamics simulation, optimization of lead
compounds, pharmacokinetics and pharmacodynamics modeling, in silico vaccine designing, pathway modeling and
simulation, network biology, metabolomics, and flux balance analysis, systems biology and big data analysis, machine
learning, and data mining approaches.
This book also covers the broad spectrum of computational analysis and case studies and enables the reader to find
information about various bioinformatics methods, tools, and their applications in a single resource. Bioinformatics has
a very broad range of applications, and this field is upgrading very rapidly as many new resources and approaches are
being developed day by day. The chapters of this book have been compiled considering the diverse applications of this
field. This book can serve as a very useful learning source for undergraduate, postgraduate, and research students of
bioinformatics, biotechnology, life sciences, and agricultural sciences, chemical, pharmaceutical, and medical sciences
who have no computational background. Besides, it is also useful for bioinformatics and computer science students,
research scientists, and pharmaceutical persons to understand the fundamental concepts of bioinformatics and utilize
this knowledge to tackle research projects. However, in spite of the best efforts, the first version of a book always has
some opportunities to improve. We will be happy to receive the important suggestions for further improvements in the
content coverage.

xxi
Chapter 1

Introduction to basics of bioinformatics

Rajesh Kumar Pathak1, Dev Bukhsh Singh2 and Rahul Singh3
1
School of Agricultural Biotechnology, Punjab Agricultural University, Ludhiana, India, 2Department of Biotechnology, Institute of Biosciences and
Biotechnology, Chhatrapati Shahu Ji Maharaj University, Kanpur, India, 3Department of Basic and Translational Sciences, School of Dental
Medicine, University of Pennsylvania, Philadelphia, PA, United States

1.1 Introduction
Researchers are now constantly making efforts to explore the function of the biological system. Efforts are only at a
stage of unprecedented development and growth, expressed in the amount of data produced from each experiment
(Avashthi et al., 2014; Kumar, Pathak, Gupta, Gaur, & Pandey, 2015; Mount, 2001). Via bioinformatics, these huge
datasets from the experiments are turned into usable information (Mount, 2001; Wang, Zaki, Toivonen, & Shasha,
2005). Bioinformatics is recognized as the science of the 21st century and has tremendous potential for decoding com-
plex biological systems via analysis and integration of multiomics data. It uses information technology (IT) to allow
various types of biological data to be analyzed, linked, and manipulated to understand new biological insights
(Avashthi et al., 2020; Kumar et al., 2015; Pathak, Taj, Pandey, Arora, & Kumar, 2013).
In other words, bioinformatics is a data management and manipulation method for molecular biology, biochemistry,
the health sector, environmental biology, and agriculture, addressing the storage of data sets, data mining and proces-
sing, structural and functional annotations of gene and protein, system modeling, and drugs’ discovery (Avashthi et al.,
2018; Jayaram & Priyanka, 2010; Verma, Pathak, Kasana, & Kumar, 2017). It is used to predict the structure and func-
tion of a newly examined protein and protein sequences to create a cluster of similar family sequences and construct
phylogenetic trees for the study of evolutionary relationships (Jayaram & Priyanka, 2010; Mount, 2001; Wang et al.,
2005). Bioinformatics has a very important role to play in agriculture-dependent countries, where it can be used to boost
nutritional content, increase agricultural produce yields, and implant resistance to many biotic and abiotic stresses
(Jayaram & Priyanka, 2010; Meidanis, 2003; Pathak, Giri, Taj, & Kumar, 2013).
For the agricultural science sector, plant and animal genome sequencing should have an enormous yield. In both the
integration and analysis of genomics, transcriptomics, and other high-throughput sequencing results, bioinformatics plays
a vital role, with great potential in redesigning to boost productivity. To understand the function and interaction of many
genes, there has been a paradigm change from the single-gene approach (i.e., gene-by-gene approach) (Kumar et al.,
2015). This change has resulted from the discovery that cross-talking of several biomolecules acting in an interdependent
manner and results in any biological answer. As a consequence, several high-throughput technologies have been developed
that offer insight into all the molecules involved in a process (Kumar et al., 2015). Study in the field of genomics has
accelerated the process. There is, however, a large difference in the expression of a trait between the genotype and the
phenotype (Kumar et al., 2015; Pathak & Singh, 2020b). Studies are performed at various levels to fill this gap: the whole
system, organism, biochemical, gene, and protein levels. All these fields have contributed to the collection of vast amounts
of biological knowledge due to unprecedented research efforts. Bioinformatics, which culminates in biology and computa-
tional technology, aims to develop novel strategies for wide-scale analysis of biological system (Pathak & Singh, 2020a).
Bioinformatics techniques, such as simulation, docking, protein protein interaction, and analysis of next-generation
sequencing (NGS) data, may be used to investigate or modify the sequence for better fitting of essential genes for a par-
ticular function and to study the function of these genes or proteins at the system level. It was then possible to use this
specified genetic, genomic, and proteomic information to grow resistant, nutritionally improved, and profitable crops and
also discover therapeutic drugs (Agnihotry, Pathak, Srivastav, Shukla, & Gautam, 2020; Singh & Pathak, 2020). Some
important tools and databases along with their application and link of availability are highlighted in Tables 1.1 and 1.2.

Bioinformatics. DOI: https://doi.org/10.1016/B978-0-323-89775-4.00006-7

© 2022 Elsevier Inc. All rights reserved. 1
2 Bioinformatics

TABLE 1.1 List of important and popular database resources for bioinformatics.

S. no. Database Application Availability References

1. National Centre for It offers access to biomedical and https://www.ncbi.nlm.nih.gov/ Benson, Boguski,
Biotechnology genomic information to boost research Lipman, and Ostell
Information (NCBI) activity. (1990)
2. GenBank It is a nucleotide database available at https://www.ncbi.nlm.nih.gov/ Benson et al.
NCBI. It is used for the retrieval of genbank/ (2012)
nucleotide sequences.
3. European It is a nucleotide database available at https://www.ebi.ac.uk/ena/ Leinonen,
Nucleotide Archive EBI-EMBL. It provides a detailed record of browser/home Sugawara, and
nucleotide sequencing data, covering raw Shumway (2010)
sequencing data, information about
sequence assembly, and functional
annotation.
4. DDBJ It is a nucleotide database available at https://www.ddbj.nig.ac.jp/index- Tateno et al. (2002)
NIG, Japan. It is used for the retrieval of e.html
nucleotide sequences.
5. Protein Information PIR is an integrated database to support https://proteininformationresource. Wu et al. (2003)
Resource (PIR) research and scientific studies in org/
genomics, proteomics, and systems
biology.
6. UniProt UniProt aims to provide a complete, https://www.uniprot.org/ Apweiler et al.
high-quality, and freely available protein (2004)
sequence and functional information
resource to the scientific community.
7. Pfam It is a broad set of protein families https://pfam.xfam.org/ Bateman et al.
database used for domain analysis. (2002)
8. CATH (Class, The CATH database offers information on https://www.cathdb.info/ Orengo et al.
Architecture, the evolutionary relationships of protein (1997)
Topology, and domains as a free, publicly accessible
Homology) online database resource.
9. SCOP The purpose of the SCOP database is to http://scop.mrc-lmb.cam.ac.uk/ Murzin, Brenner,
provide a detailed and systematic Hubbard, and
explanation of the structural and Chothia (1995)
evolutionary relationships between
proteins deposited in the RCSB Protein
Data Bank.
10. Protein Data Bank It is a structural database that contains the https://www.rcsb.org/ Berman et al.
(PDB) 3D structure of macromolecules. It is (2000)
crucial for research in the area of
structural bioinformatics, drug discovery
and protein structure prediction, etc.
11. PubChem It is the largest database of chemical https://pubchem.ncbi.nlm.nih.gov/ Bolton, Wang,
information. Here, we search chemical Thiessen, and
molecules and retrieve their structure for Bryant (2008)
molecular docking/virtual screening.
12. ZINC It is a database that contains https://zinc.docking.org/ Sterling and Irwin
commercially available molecules for (2015)
computational screening. We can search
and retrieve analogs for any molecule
based on similarity. It plays a key role in
the discovery of lead molecules for drug
development.

(Continued )
 Introduction to basics of bioinformatics Chapter | 1 3

TABLE 1.1 (Continued)

S. no. Database Application Availability References

13. GEO GEO is a database for functional https://www.ncbi.nlm.nih.gov/ Edgar, Domrachev,
genomics. It contains gene expression/ geo/ and Lash (2002)
microarray data. Here, we can download
gene expression profiles submitted by the
scientific community throughout the
world for further investigation as per
need.
14. Sequence Read It is the largest publicly available https://www.ncbi.nlm.nih.gov/sra Leinonen et al.
Archive repository of high-throughput sequencing (2010)
data. Here, we can download NGS data
submitted by the scientific community
throughout the world for further
investigation as per need.
15. The Arabidopsis It is a database of the model plant https://www.arabidopsis.org/ Rhee et al. (2003)
Information Arabidopsis thaliana provides genetic and
Resource genomic information to the scientific
community.
16. Rice Genome It provides sequence and annotation data http://rice.plantbiology.msu.edu/ Kawahara et al.
Annotation Project for the rice genome. (2013)
17. Gramene It is a curated, open-source, integrated https://www.gramene.org/ Ware et al. (2002)
database platform for research in the area
of comparative functional genomics of
crop plant species.
18. Kyoto Encyclopedia Database of gene/genome sequencing https://www.genome.jp/kegg/ Kanehisa and Goto
of Genes and information for the understanding (2000)
Genomes (KEGG) function of biological system
19. Search Tool for the It is a database resource for known and https://string-db.org/ Szklarczyk et al.
Retrieval of predicted protein protein interactions (2019)
Interacting Genes/ derived from experimental,
Proteins (STRING) computational methods, and text mining.
20. BioModel It is a repository of mathematical models. https://www.ebi.ac.uk/biomodels/ Le Novere et al.
It provides a wide variety of current (2006)
physiologically and pharmaceutically
applicable mechanistic models based on
literature in standard file formats.

1.1.1 Concept behind bioinformatics, in silico biology, and computational biology

There are many definitions are available for bioinformatics in different books and the Internet. We can say that
“Decoding problems arising in the field of biological sciences via computation” is known as bioinformatics. It handles
biological data obtained through several experimental techniques for their documentation in the form of databases for
further availability to the scientific community. This dataset is analyzed for dissecting the complexity of biological sys-
tems, and acquired knowledge accelerates research activity and helps scientists to get fruitful results and novel insight.
The term “in silico biology” is concerned with bioinformatics. Generally, we can say that performing any work using a
computer for biology is known as in silico biology, for example, retrieval of biological sequences from databases.
Computational biology is different from bioinformatics and in silico biology. Here, we are talking about the devel-
opment of algorithms and theoretical methods for solving biological problems. These developed algorithms and meth-
ods are further utilized in the design and development of biological software or tools that help in analyzing biological
data using the computer to create knowledge, that is, bioinformatics. Therefore these disciplines are linked to each other
and use several disciplines of biology and natural sciences along with the computer, IT, mathematics, and statistics.
That is why it is called interdisciplinary science.
4 Bioinformatics

TABLE 1.2 A list of commonly used bioinformatics software.

S. no. Software/tool Application Availability References

1. Basic Local It is a very common tool in bioinformatics used for https://blast.ncbi.nlm.nih. Altschul, Gish,
Alignment Search similarity searching and identification of homologs gov/Blast.cgi Miller, Myers, and
Tool (BLAST) and paralogous sequences. Lipman (1990)
2. CLUSTAL It is a widely used program for multiple sequence http://www.clustal.org/ Chenna et al.
alignment. Many versions of the Clustal program omega/ (2003) and Sievers
are available for sequence analysis. et al. (2011)
3. Molecular It is a software program for performing statistical https://www.megasoftware. Kumar, Tamura,
Evolutionary analysis of molecular evolution and for building net/ and Nei (1994)
Genetics Analysis phylogenetic trees.
(MEGA)
4. Modeller It is a well-known program for modeling protein https://salilab.org/modeller/ Eswar et al. (2006)
3D structure based on sequence information.
5. PyMOL PyMOL is a comprehensive software package for https://pymol.org/2/ DeLano (2002)
the visualization and animation of 3D structures.
6. Swiss PDB Swiss PDB Viewer is a bioinformatics program that https://spdbv.vital-it.ch/ Guex and Peitsch
Viewer offers a user-friendly interface for the simultaneous (1997)
study of many proteins. It is used for structural
alignments and comparison, calculation of H-
bonds, angles, and distances between atoms.
7. Chimera It is an interactive visualization and analysis https://www.cgl.ucsf.edu/ Pettersen et al.
software for molecular structures and related data, chimera/ (2004)
including maps of density, trajectories, and
alignments of sequences. It is possible to create
high-quality images and animations.
8. MarvinSketch It is used to draw, edit, import, and export https://chemaxon.com/ Bunin, Siesel,
chemical structures, and also for the conversion of products/marvin Morales, and
structural file formats. Bajorath (2007)
9. AutoDock It is a computer program used for the identification http://autodock.scripps.edu/ Goodsell, Morris,
of lead compounds through molecular docking. and Olson (1996)
10. Gromacs It is a molecular dynamics package developed http://www.gromacs.org/ Van Der Spoel
specifically for protein, lipid, and nucleic acid et al. (2005)
simulations.
11. Velvet It is a de novo based genome assembly program https://www.ebi.ac.uk/ Zerbino and
developed especially for short-read sequencing Bzerbino/velvet/ Birney (2008)
data.
12. Trinity It is a transcriptome/RNA-seq data assembly https://github.com/ Grabherr et al.
program generated by the Illumina NGS platform trinityrnaseq/trinityrnaseq/ (2011)
based on the de novo algorithm. wiki
13. FastQC It provides a quick way to check the quality control https://www.bioinformatics. Andrews (2010)
of raw sequencing data generated from NGS babraham.ac.uk/projects/
platforms. fastqc/
14. Trimmomatic It is a widely used flexible read trimming tool for http://www.usadellab.org/ Bolger, Lohse, and
NGS data coming from Illumina. cms/?page 5 trimmomatic Usadel (2014)
15. Cutadapt It detects and removes adapter sequences, poly-A https://cutadapt. Martin (2011)
tails, primer sequences, and other regions from readthedocs.io/en/stable/
sequencing reads.
16. DIAMOND It is a sequence aligner for protein or translated http://www.diamondsearch. Buchfink, Xie, and
DNA searches, developed for big sequence data org/index.php Huson (2015)
analysis. It is very fast as compare to BLAST.

(Continued )
 Introduction to basics of bioinformatics Chapter | 1 5

TABLE 1.2 (Continued)

S. no. Software/tool Application Availability References

17. Blast2GO Used for gene ontology, and annotation of https://www.blast2go.com/ Conesa et al.
genomic data. (2005)
18. EffectorP It is a tool used for the prediction of fungal effector http://effectorp.csiro.au/ Sperschneider
proteins. It is trained in plant-pathogenic fungi to et al. (2016)
differentiate secreted proteins from secreted
effectors.
19. CellDesigner It is a computational systems biology program used http://celldesigner.org/ Funahashi,
for pathway modeling and simulation analysis. Morohashi, Kitano,
and Tanimura
(2003)
20. Cytoscape It is a widely used and well-known software https://cytoscape.org/ Shannon et al.
platform in the field of complex network science (2003)
and network biology for visualization and analysis.
Different types of Cytoscape Apps are available for
different types of analysis.

NGS, Next-generation sequencing.

1.1.2 Scientific discipline and support systems for bioinformatics

Bioinformatics is a central discipline that is linked with several disciplines of science. These disciplines of science and
technology provide infrastructure and interdisciplinary nature to bioinformatics. In the sciences discipline, several tradi-
tional and advanced subjects are associated with bioinformatics, such as plant sciences, animal sciences, molecular biol-
ogy, genetics, and evolutionary biology, pharmaceuticals, mathematical, and statistical sciences, and omics. Besides, in
the support system, bioinformatics is closely associated with computer science, IT, and computational resources because
they work as a backbone for bioinformatics. Therefore these scientific disciplines and support systems are combined to
build a new discipline called bioinformatics for decoding intricate problems linked to biological systems via innovative
manner with fast processing due to the involvement of computer science and IT (Fig. 1.1).

1.1.3 Needs of bioinformatics

Bioinformatics is a need of time because due to advances in several omics platforms have produced big data in biology
related to genomes, transcriptomes, genotyping by sequencing, proteome, metabolome, etc. The management and analy-
sis of these high-throughput data need bioinformatics. In the era of omics and availability of organism and discipline/
subject-specific data, that is, genomic data, proteomic data, etc., there is a need to develop specific databases for their
documentation. This will further accelerate the research and help scientists to integrate these available data with novel
discoveries. Besides, these datasets will help in improving the accuracy of available tools and enable scientists to
trained data for the development of new tools via machine and deep learning approaches.
Thus in view of the above facts and contiguous process of experimental data generation needs bioinformatics profes-
sionals to manage and analyze these big data of biology to boosting research and development in a smart way for min-
ing useful information. This will deferentially help society.

1.2 Historical background of bioinformatics

The term “bioinformatics” was primarily used from the late 1980s onward to refer to computational analysis of genome
data and described more broadly as the “study of informatic processes in biotic systems” (Hogeweg, 2011). Paulien
Hogeweg and Ben Hesper first used the term “bioinformatics” at the beginning of the 1970s, and the term was pub-
lished by him in 1978 (Hogeweg, 1978, 2011; Hogeweg & Hesper, 1978).
More than 50 years ago, bioinformatics took place when desktop computers and deoxyribonucleic acid (DNA)
sequencing were a dream. Not much was known about deoxyribonucleic acid (DNA) in the early 1950s. At that
moment, its role as the carrier molecule of genetic information was still controversial (Gauthier, Vincent, Charette, &
6 Bioinformatics

FIGURE 1.1 Scientific disciplines associated with bioinformatics and their support systems.

Derome, 2019). Avery, Colin, and McCarty (1944) demonstrated that the absorption of pure DNA from a virulent bac-
terial strain could give a nonvirulent strain virulence, but their findings were not immediately recognized by the scien-
tific community. Many assumed that proteins were worked as genetic information carriers. Hershey and Chase
confirmed the role of DNA as a genetic information encoding molecule in 1952 when they proved beyond a reasonable
doubt that it was DNA, not protein, which was absorbed and transmitted by bacterial cells infected by a bacteriophage
(Gauthier et al., 2019; Hershey & Chase, 1952). So, the information about the function of DNA is known but its struc-
ture was not determined. In 1953 Watson, Crick, and Franklin finally resolved the double-helix structure of DNA.
Despite this major discovery, it would take 13 more years before the genetic code was deciphered and 25 more years
before the first methods for DNA sequencing were available. Consequently, in DNA analysis, the use of bioinformatics
lagged almost two decades behind the analysis of proteins whose chemical nature was already better understood than
that of DNA (Maxam & Gilbert, 1977; Nirenberg & Leder, 1964; Sanger, Nicklen, & Coulson, 1977; Tamm, Shapiro,
& Lipshitz, 1953; Watson & Crick, 1953). Therefore the analysis of protein was the starting point in bioinformatics
(Gauthier et al., 2019).
The major progress in solving protein structure through crystallography during the late 1950s, the first sequence of a
protein, insulin, was published. Furthermore, many advancements in the determination of protein sequence and their
structure were reported. Margaret Dayhoff (1925 1983), the first bioinformatician, was an American physical chemist,
known for her significant contribution and application of computational methods in the field of biochemistry and pro-
tein sciences. Therefore she is known as the mother and father of bioinformatics (Jaskolski, Dauter, & Wlodawer, 2014;
Moody, 2004; Sanger & Thompson, 1953).
The first dynamic programming algorithm for pairwise alignments of protein sequence was developed by
Needleman and Wunsch in 1978 and the first multiple sequence alignment (MSA) algorithms emerged in the 1980s.
The software CLUSTAL was introduced in 1988 for MSA. Then, the concept of a mathematical framework for amino
acid substitutions was introduced with the development of a point accepted mutation matrix by Dayhoff. In 1970 80
the paradigm has been shifted from protein to DNA analysis. Between 1980 and 1990 the parallel advances in biology
and computer science occurred. In the establishment of the National Centre for Biotechnology Information (NCBI)
database in 1988 and at NIH and launched of Human Genome Project in 1990, bioinformatics gains huge importance
due to its application in bioinformatics for the management and analysis of biological data, which lead to the develop-
ment of genomics and structural bioinformatics between 1990 and 2000. In 2000 10 the concepts of high-throughput
Another random document with
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 “Your reverend love should know that the lord Ep. 58. a.d. 796.
King Charles has often spoken to me of you in a
loving and trusting manner. You have in him an entirely most faithful
friend. Thus he sent messengers to Rome for the judgement of the
lord apostolic and Ethelhard the archbishop. To your love he sent
gifts worthy. To the several episcopal sees he sent gifts in alms for
himself and the lord apostolic, that you might order prayers to be
offered for them. Do you act faithfully, as you are wont to do with all
your friends.
“In like manner he sent gifts to King Æthelred and his episcopal
sees. But, alas for the grief! when the gifts and the letters were in the
hands of the messengers, the sad news came from those who had
returned from Scotia[111] by way of you, that the nation had revolted
and the king [Æthelred] was killed. King Charles withdrew his gifts,
so greatly was he enraged against the nation—‘that perfidious and
perverse nation,’ as he called them, ‘murderers of their own lords,’
holding them to be worse than pagans. Indeed, if I had not
interceded for them, whatever good thing he could have taken away
from them, whatever bad thing he could have contrived for them, he
would have done it.
“I was prepared to come to you with the king’s gifts, and to go
back to my fatherland.” This was from three to four years later than
his latest visit to our shores. “But it seemed to me better, for the sake
of peace for my nation, to remain abroad. I did not know what I could
do among them, where no one is safe, and no wholesome counsel is
of any avail. Look at the very holiest places devastated by pagans,
the altars fouled by perjuries, the monasteries violated by adulteries,
the earth stained with the blood of lords and princes. What else
could I do but groan with the prophet,[112] ‘Woe to the sinful nation, a
people laden with iniquity, a seed of evildoers; they have forsaken
the Lord, and blasphemed the holy Saviour of the world in their
wickedness.’ And if it be true, as we read in the letter of your dignity,
that the iniquity had its rise among the eldermen, where is safety and
fidelity to be hoped for if the turbid torrent of unfaithfulness flowed
forth from the very place where the purest fount of truth and faith
was wont to spring?
 “But do thou, O most wise ruler of the people of God, most
diligently bring thy nation away from perverse habits, and make them
learned in the precepts of God, lest by reason of the sins of the
people the land which God has given us be destroyed. Be to the
Church of Christ as a father, to the priests of God as a brother, to all
the people pious and fair; in conversation and in word moderate and
peaceable; in the praise of God always devout; that the divine
clemency may keep thee in long prosperity, and may of the grace of
its goodness deign to exalt, dilate, and crown to all eternity, with the
benefaction of perpetual pity, thy kingdom—nay, all the English.
“I pray you direct the several Churches of your reverence to
intercede for me. Into my unworthy hands the government of the
Church of St. Martin has come. I have taken it not voluntarily but
under pressure, by the advice of many.”
Offa died in the year in which this letter was written, and his death
brought great changes in Mercia. Excellent as Offa had in most ways
been, we have evidence that the Mercian people were by no means
worthy of the fine old Mercian king. In reading the letter which
contains this evidence, we shall see that Offa had a murderous side
of his character. In those rude days, chaos could not be dealt with
under its worse conditions by men who could not at a crisis strike
with unmitigated severity.
 CHAPTER VI
Grant to Malmesbury by Ecgfrith of Mercia.—Alcuin’s letters to Mercia.—Kenulf
and Leo III restore Canterbury to its primatial position.—Gifts of money to the
Pope.—Alcuin’s letters to the restored archbishop.—His letter to Karl on the
archbishop’s proposed visit. Letters of Karl to Offa (on a question of discipline) and
Athelhard (in favour of Mercian exiles).

Before proceeding to examine Alcuin’s letter to a Mercian

nobleman on the death of Offa and his son Ecgfrith, it should be
remarked that we of the diocese of Bristol must not allow the
mention of this poor young king Ecgfrith to pass without our
acknowledgement for a deed of justice done. When Offa defeated
the West Saxon king at Bensington, he took possession of a good
deal of the border land, including two tracts of land which King
Cadwalla of Wessex had given to Malmesbury, namely Tetbury in
Gloucestershire and Purton in Wilts. William of Malmesbury naturally
reports the iniquity of Offa in thus pillaging the abbey which was the
home of William’s life and studies. Offa gave Tetbury to the Bishop of
Worcester. Purton was the subject of a deed by Ecgfrith during his
reign of a few months. The deed has remarkable interest for us in
this diocese, in that it is doubly dated; first as in the seven hundred
and ninety-sixth year from the Incarnation, and next, with a very
interesting recognition of our own Aldhelm, due to the fact that the
theft had been from Aldhelm’s own Malmesbury, “in the eighty-
seventh year from the passing of father Aldhelm.” The deed restores
land of thirty-five families at Piritune, on the east side of Braden
Wood, to the abbat and brethren of Malmesbury, for the repose of
the soul of his father Offa who had taken it from them, and in order
that the memory of Ecgfrith might always be preserved in their
prayers. As a sort of unimportant afterthought he adds that the abbat
and brethren have given him two thousand shillings of pure silver,
probably as many pounds of our money. The deed was signed by
Athelhard of Canterbury, not by Lichfield. The reason no doubt is that
Tetbury and Purton are south of the Thames, and so outside the
Province of Lichfield and within the diminished Province of
Canterbury.
When the death of Offa’s son, the youthful Ecgfrith, king of Mercia,
occurred in this same year 796 in which year his father Offa had
died,[113] and a distant cousin Kenulf succeeded, Alcuin, as has
been said, wrote a very serious letter to one of the chief officers of
Mercia.
“These are times of tribulation everywhere in the Ep. 79. a.d. 797.
land; faith is failing; truth is dumb; malice
increases; and arrogance adds to your miseries. Men are not content
to follow in the steps of our early fathers, in dress, or food, or honest
ways. Some most foolish man thinks out something unsuited to
human nature, and hateful to God; and straightway almost the whole
of the people set themselves busily to follow this above all.
“That most noble youth [Ecgfrith] is dead; not, as I think, because
of his own sins alone, but also because the vengeance of his father’s
bloodshedding has reached the son. For you know best of all how
much blood the father shed that the kingdom might be safe for the
son. It proved to be the destruction, not the confirmation, of his reign.
“Admonish the more diligently your new king [Kenulf], yes, and the
king of Northumbria [Ardwulf] too, that they keep in touch with the
divine piety, avoiding adulteries; that they do not neglect their early
wives[114] for the sake of adulteries with women of the nobility, but
under the fear of God have their own wives, or by consent live in
chastity. I fear that Ardwulf, the king of my part of the country, will
soon[115] have to lose the kingdom because of the insult which he
has offered to God in sending away his own wife, and, it is said,
living openly with a concubine. It seems that the prosperity of the
English is nearly at an end; unless indeed by assiduous prayers, and
honest ways, and humble life, and chaste conversation, and keeping
the faith, they win from God to keep the land which God of His free
gift gave to our forefathers.”
 With this letter we may fitly compare the letter which Alcuin wrote
to the king himself, Kenulf, who had thus unexpectedly succeeded. It
begins in a complimentary manner, but it is a very faithful letter. It
carefully recognizes the inconsistencies of Offa’s life, inconsistencies
which appear to have characterized the best rulers in those times,
very rude and violent times, when one occasion and another seemed
to demand ruthless treatment.
“To the most excellent Coenulf, King of the Ep. 80. a.d. 797.
Mercians, the humble levite Albinus wishes health.
“Your goodness, moderation, and nobility of conduct, are a great
joy to me. They are befitting to the royal dignity, which excels all
others in honour, and ought to excel also in perfectness of conduct,
in fairness of justice, in holiness of piety. The royal clemency should
go beyond that of ordinary men, as we read in ancient histories, and
in holy Scripture where it is said[116]—Mercy and truth exalt a throne;
and in the Psalms it is said[117] of Almighty God—All the paths of the
Lord are mercy and truth. The more a man shines forth in works of
truth and mercy, the more has he in him of the image of the divine.
“Have always in mind Him who raised thee from a poor position
and set thee as a ruler over the princes of His people. Know that
thou art rather a shepherd, and a dispenser of the gifts of God, than
a lord and an exactor.
“Have always in mind the very best features of the reign of your
most noble predecessor Offa; his modest conversation; his zeal in
correcting the life of a Christian people. Whatever good
arrangements he made in the kingdom to thee by God given, let your
devotion most diligently carry out; but if in any respect he acted with
greed, or cruelty, know that this you must by all means avoid. For it
is not without cause that that most noble son of his survived his
father for so short a time. The deserts of a father are often visited on
a son.
“Have prudent counsellors who fear God; love justice; seek peace
with friends; show faith and holiness in pious manner of life.
 “For the English race is vexed with tribulations by reason of its
many sins. The goodness of kings, the preaching of the priests of
Christ, the religious life of the people, can raise it to the height of its
ancient honour; so that a blessed progeny of our fathers may
deserve to possess perpetual happiness, stability of the kingdom,
and fortitude against any foe; that the Church of Christ, as ordained
by holy fathers, may grow and prosper. Always have in honour, most
illustrious ruler, the priests of Christ; for the more reverently you are
disposed to the servants of Christ, and the preachers of the word of
God, the more will Christ, the King pious and true, exalt and confirm
your honour, on the intercession of His saints.”
When Kenulf, this distant cousin of Ecgfrith, came to the throne,
he looked into the matter of the archbishopric of Lichfield, and he
took a view adverse to Offa’s action. He wrote to Pope Leo III a
letter,[118] in which he put the points very clearly. His bishops and
learned men had told him that the division of the Province of
Canterbury into two provinces was contrary to the canons and
apostolical statutes of the most blessed Gregory, who had ordered
that there should be twelve bishops under the archbishop of the
southern province, seated at London. On the death of Augustine of
Canterbury, it had seemed good to all the wise men of the race, the
Witangemote, that not London but Canterbury should be the seat of
the Primacy, where Augustine’s body lay. King Offa, by reason of his
enmity with the venerable archbishop Jaenbert and the people of
Kent, set to work to divide the province into two. The most pious
Adrian, at the request of the said king, had done what no one before
had presumed to do, had raised the Mercian prelate to the dignity of
the pallium. Kenulf did not blame either of them; but he hoped that
the Pope would look into the matter and make a benign and just
response. He had sent an embassy on the part of himself and the
bishops in the previous year by Wada the Abbat; but Wada, after
accepting the charge, had indolently—nay foolishly—withdrawn. He
now sent by the hands of a presbyter, Birine, and two of his officers,
Fildas and Cheolberth, a small present, out of his love for the Pope,
namely, 120 mancuses,[119] some forty to fifty pounds, say not far off
£1000 of our time.
 Pope Leo addressed his reply to king Kenulf, his most loved
bishops, and most glorious dukes. It was a difficult letter to write, for
Kenulf had been very frank about the uncanonical action of Hadrian
the Pope. Leo answered this part of Kenulf’s letter by stating that his
predecessor had acted as he had done (1) because Offa had
declared it to be the universal wish, the petition of all, that the
archbishopric should be divided into two; (2) because of the great
extension of the Mercian kingdom; (3) for very many causes and
advantages. He, Leo, now authorized the departure from Pope
Gregory’s order in so far as this, that he recognized Canterbury, not
London, as the chief seat of archiepiscopal authority. He declared
that Canterbury was the primatial see, and must continue and be
viewed as such. I cannot find in his letter a definite declaration that
he annuls the act of his predecessor, but that is the effect of the
letter; nor does he declare that Lichfield is no longer an
archbishopric. Kenulf, as we have seen, had sent him, out of his
affection for him, a gift of 120 mancuses. But he reminded the king
that Offa had bound his successors to maintain the gift to the Pope,
in each year, of as many mancuses as there are days in the year,
namely, he says, 365, as alms to the poor, and as an endowment for
keeping in order the lamps [in the churches]. This is much more
likely than the shadowy gifts of Ina, king of Wessex, to have been
the origin of Peter’s Pence, a sum of money collected in England, at
first fitfully and eventually year by year, and sent out to the Pope.
The money was collected in the parishes of each diocese down to
the time of the Reformation. It is a regular item in the
churchwardens’ accounts of the earlier years of Henry VIII. Only a
fixed amount of the whole sum collected was sent to the Pope, the
balance being used for repairs in the several dioceses. We have a
list prepared by a representative of a late mediaeval Pope giving
£190 6s. 8d. as the amount received by him for the year,
corresponding roughly to a normal 300 marks a year.
Offa’s money for the Pope went of course from Mercia. When
Wessex became predominant, Ethelwulf, the son of Ecgbert and
father of Alfred, made large gifts to Rome, and left by will 300
mancuses, 100 in honour of St. Peter, specially for filling with oil all
the lamps of his apostolic church on Easter Eve and at cock crow,
100 in honour of St. Paul, in the same terms and for the same
purpose in respect of the basilica of St. Paul, and 100 for the Pope
himself. King Alfred also sent presents to Rome. From 883 to 890
there are four records of gifts from Wessex. After 890 we have no
such record in Alfred’s reign; and in Alfred’s will there is no mention
of the spiritual head of the Church of the West.
We learn from our own great historian, William of Malmesbury, that
Kenulf wrote two later letters to Leo on this subject, and he gives us
Leo’s reply.[120] Athelhard, the Pope says, has come to the holy
churches of the blessed apostles Peter and Paul, to fulfil his vow of
prayer and to inform the Pope of his ecclesiastical mission. He tells
the king that by the authority of St. Peter, the chief of the apostles,
whose office though unworthily he fills, he gives to Athelhard such
prelatical authority that if any in the province, whether kings, princes,
or people, transgress the commands of the Lord, he shall
excommunicate them till they repent. Concerning the jurisdiction
which the archbishops of Canterbury had held, as well over bishops
as over monasteries, of which they had been unjustly deprived, the
Pope had made full inquiry, and now placed all ordinations and
confirmations on their ancient footing, and restored them to him
entire. Thus did Pope Leo III condemn the injustice of Pope Hadrian
I. We had better have managed our own affairs, instead of paying to
foreigners infinite sums of money to mismanage them.
Before we leave this strange episode of the creation of an
archbishopric of Lichfield, it is of special local interest to us in Bristol,
and to the deanery of Stapleton, that the chief Mercian prelate,
Higbert of Lichfield, signed deeds relating to Westbury upon Trym
and Aust on Severn, above the archbishop of Canterbury. This was
in 794. Offa the king signed first, Ecgferth, the king’s young son,
second, and then Hygeberht; Ethelhard of Canterbury coming fifth in
one and fourth in the other. The first deed gave from the king to his
officer Ethelmund, in 794, four cassates of land at the place called
Westbury, in the province of the Huiccians, near the river called
Avon, free of all public charges except the three which were common
to all, namely, for the king’s military expeditions, for the building of
bridges, and for the fortification of strongholds.[121] The other deed
restores to the see of Worcester (Wegrin) the land of five families at
Aust, which the duke Bynna had taken without right, it being the
property of the see of Worcester. To make all safe, six dukes made
the sign of the cross at the foot of this deed, which is, as we all know,
the origin of the modern phrase ‘signing’ a deed or a letter. The
dukes included Bynna himself.
Alcuin wrote a very wise letter to Athelhard of Ep. 85. a.d. 797.
Canterbury on the occasion of the restoration of the
primacy. He advised that penance should be done. Athelhard and all
the people should keep a fast, he for having left his see, they for
having accepted error. There should be diligent prayers, and alms,
and solemn masses, everywhere, that God might wipe out what any
of them had done wrong. The archbishop was specially urged to
bring back study into the house of God, that is, the conventual home
of the monks and the archbishop, with its centre, the cathedral
church. There should be young men reading, and a chorus of
singers, and the study of books, in order that the dignity of that holy
see might be renewed, and they might deserve to have the privilege
of electing to the primacy.
“The unity of the Church, which has been in part cut asunder, not
as it seems for any reasonable cause but from grasping at power,
should, if it can be done, be restored in peaceful ways; the rent
should be stitched up again. You should take counsel with all your
bishops, and with your brother of York, on this principle, that the
pious father Higbert of Lichfield be not deprived of his pall during his
lifetime, but the consecration of bishops must come back to the holy
and primal see. Let your most holy wisdom see to it that loving
concord exist among the chief shepherds of the churches of Christ.”
With regard to the remark of Alcuin that Ep. 171. a.d. 801.
Athelhard should do penance for having left his
see, it may be explained that Alcuin had in vain advised Athelhard
not to leave England on the restoration of the primacy to Canterbury.
Athelhard persisted in visiting Rome, and informed Alcuin that he
had commenced the journey. Alcuin thereupon wrote this:—“Return,
return, holy father, as soon as your pious embassy is finished, to
your lost sheep. As there are two eyes in the body, so I believe and
desire that you two, Canterbury and York, give light throughout the
breadth of all Britain. Do not deprive your country of its right eye.”
Then Alcuin gives a very significant hint that the ways of the clergy
of England are not good enough for France, and they had better not
let Charlemagne see anything of that kind.
“If you come to the lord king, warn your companions, and
especially the clergy, that they acquit themselves in an honourable
manner, in all holy religion, in dress, and in ecclesiastical order; so
that wherever you go you leave always an example of all goodness.
Forbid them to wear in the presence of the lord king ornaments of
gold or robes of silk; let them go humbly clad, after the manner of
servants of God. And through every district you must pass with
peace and honest conversation, for you know the manner and
custom of this Frankish race.”
Nothing could make more clear the commanding position held by
Alcuin than this exceedingly free counsel from a deacon to the
Primate of England. We may quote portions of yet another letter
giving the same impression.
In a letter to Athelhard after his safe return to Ep. 190. a.d. 802.
England and a favourable reception which he had
reported to Alcuin, Alcuin congratulated the archbishop on the
restoration to its ancient dignity of the most holy see of the first
teacher of our race. By divine favour, the members now once more
cohered in unity with the proper head, and natural peace shone forth
between the two chief prelates of Britain, and one will of piety and
concord was vigorous under the two cities of metropolitans. “And
now,” he writes, “now that you have received the power to correct
and the liberty to preach, fear not, speak out! The silence of the
bishop is the ruin of the people.”
It is an interesting fact that we have a letter which Alcuin wrote to
Karl, introducing to him this same archbishop on the very journey of
which he so decidedly disapproved.
“To the most greatly desired lord David the king, Ep. 172. a.d. 801.
Flaccus his pensioner wishes eternal health in
Christ.
“The sweetness of your affection, and the assurance of your
approved piety, very often urge me to address letters to your
authority, and by the office of syllables to trace out that which bodily
frailty prevents my will from accomplishing. But novel circumstances
compel me now to write once more, that the paper may bring the
affection of the heart, and may pour into the ears of your piety the
prayers which never have been in vain in the presence of your pity.
Nor do I believe that my prayers for your stableness and safety are
vain in the sight of God, for the divine grace gladly receives the tears
which flow forth from the fount of love[122].
“I have been informed that certain of the friends of your Flaccus,
Edelard to wit, Metropolitan of the See of Dorobernia and Pontiff of
the primatial see in Britain, and Ceilmund[123] of the kingdom of the
Mercians, formerly minister of king Offa, and Torhcmund[124] the
faithful servant of king Edilred, a man approved in faith, strenuous in
arms, who has boldly avenged the blood of his lord, desire to
approach your piety[125]. All of these have been very faithful to me,
and have aided me on my journey; they have also aided my boys as
they went about hither and thither. I pray your best clemency to
receive them with your wonted kindness, for they have been close
friends to me. I have often known bishops religious and devoted in
Christ’s service, and men strong and faithful in secular dignity, to be
laudable to your equity; for there is no doubt that all the best men,
approved by their own conscience, love good men, being taught by
the example of the omnipotent God who is the highest good. And it is
most certain that every creature that has reason has by His
goodness whatever of good it has, the Very Truth saying, ‘I am the
light of the world. He that followeth me walketh not in darkness but
shall have the light of life.’ John viii. 12.”
Before we leave Mercian affairs and the relations between Karl
and Offa, it may be of interest to give a letter[126] from Karl to Offa
which will serve to show the extreme care he took in order to
maintain ecclesiastical discipline, and the severity of that discipline.
That a man with all the affairs of immense dominions on his hands
should have made time to produce such a letter on such a point
seems very worthy of note. Karl’s statement of his titles shows that
this is an early letter.
“Karl, by the grace of God king of the Franks and Defender of the
Holy Church of God, to his loved brother and friend Offa greeting.
“That priest who is a Scot[127] has been living among us for some
time, in the diocese of Hildebold, Bishop[128] of Cologne. He has
now been accused of eating meat in Lent. Our priests refuse to
judge him, because they have not received full evidence from the
accusers. They have, however, not allowed him to continue to reside
there, on account of this evil report, lest the honour in which the
priesthood is held should be diminished among ignorant folk, or
others should be tempted by this rumour to violate the holy fast. Our
priests are of opinion that he should be sent to the judgement of his
own bishop, where his oath was taken.
“We pray your providence to order that he transfer himself as soon
as conveniently may be to his own land, that he may be judged in
the place from which he came forth. For there also it must be that the
purity in manners and firmness in faith and honesty of conversation
of the Holy Church of God are diligently kept according to canonical
sanction, like a dove perfect and unspotted, whose wings are as of
silver and the hinder parts should shine as gold.
“Life, health, and prosperity be given to thee and thy faithful ones
by the God Christ for ever.”
A letter which Karl wrote to Athelhard of Canterbury begging him
to intercede for some exiles, sets forth his style and title very
differently[129], evidently at a later date.
It bears very directly upon one of the complaints which, as we
have seen, Offa had made in letters to Karl; namely, the shelter
afforded at Karl’s court to fugitives from Mercia.
“Karl, by the grace of God king of the Franks and Lombards and
Patrician of the Romans, to Athilhard the archbishop and Ceolwulf
his brother bishop, eternal beatitude.
 “In reliance on that friendship which we formed in speech when we
met, we have sent to your piety these unhappy exiles from their
fatherland; praying that you would deign to intercede for them with
my dearest brother king Offa, that they may be allowed to live in their
own land in peace, without any unjust oppression. For their lord
Umhrinsgstan[130] is dead. It appeared to us that he would have
been faithful to his own lord if he had been allowed to remain in his
own land; but, as he used to say, he fled to us to escape the danger
of death, always ready to purge himself of any unfaithfulness. That
reconciliation might ensue we kept him with us for a while, not from
any unfriendliness.
“If you are able to obtain peace for these his fellow tribesmen, let
them remain in their fatherland. But if my brother gives a hard reply
about them, send them back to me uninjured. It is better to live
abroad than to perish, to serve in a foreign land than to die at home.
I have confidence in the goodness of my brother, if you plead
strenuously with him for them, that he will receive them benignantly
for the love that is between us, or rather for the love of Christ, who
said, Forgive and it shall be forgiven you.
“May the divine piety keep thy holiness, interceding for us, safe for
ever.”
It was a skilful stroke of business on Karl’s part to send the men
over to the charge of the archbishop, which amounted to putting
them in sanctuary. If he had kept them in France and written to beg
that they might be allowed to return, it would have been much easier
for Offa to say no. And if he had sent them direct to Offa in the first
instance, they would probably never have got out of his clutches at
all.
 CHAPTER VII
List of the ten kings of Northumbria of Alcuin’s time.—Destruction of Lindisfarne,
Wearmouth, and Jarrow, by the Danes.—Letters of Alcuin on the subject to King
Ethelred, the Bishop and monks of Lindisfarne, and the monks of Wearmouth and
Jarrow.—His letter to the Bishop and monks of Hexham.

We must now turn to Alcuin’s native kingdom of Northumbria, over

whose evil fortunes he grieved so greatly in the home of his
adoption.
I do not know how better some idea can be formed of the political
chaos to which Northumbria was reduced in the time of Alcuin than
by reading a list of the kings of that time. It is a most bewildering list.
All went well so long as Eadbert, the brother of Archbishop
Ecgbert, reigned. He was the king of Alcuin’s infancy and boyhood
and earliest manhood. His reign lasted from 737 to 758, when he
retired into a monastery. He was the 21st king, beginning with Ida
who created the kingdom in 547. He was succeeded by (22) Oswulf
his son, who was within a year slain by his household officers, July
24, 759, and was succeeded on August 4 by (23) Ethelwald, of
whose parentage we do not know anything. In 765 he was deprived
by a national assembly, and (24) Alchred was placed on the throne,
a fifth cousin of the murdered Oswulf, and therefore of the royal line.
In 774 he was banished, and went in exile to the king of the Picts,
being succeeded by (25) Ethelred, the son of his deprived
predecessor Ethelwald. Ethelred reigned from 774 to 779, when in
consequence of cruel murders ordered by him he was driven out,
and (26) Alfwold, son of (22) Oswulf, and therefore of the old royal
line, succeeded. Alfwold was murdered in 788, and was succeeded
by (27) Osred, the son of (24) Alchred, sixth cousin of his
predecessor, and therefore of the royal line. After a year he was
deposed and tonsured, and was eventually put to death in 792 by
(25) Ethelred, who had recovered the throne lost by his expulsion in
779. He was killed in 796 in a faction fight, after he had put to death
the last two males, so far as we know, of the royal line of Eadbert,
Ælf and Ælfwine, sons of (26) Alfwold. Simeon of Durham tells us
(a.d. 791) that they were persuaded by false promises to leave
sanctuary in the Cathedral Church of York; were taken by violence
out of the city; and miserably put to death by Ethelred in
Wonwaldrenute. He was succeeded by (28) Osbald, of unknown
parentage, but a patrician of Northumbria; he only reigned twenty-
seven days, fled to the king of the Picts, and died an abbat three
years later, in 799. He was succeeded by (29) Eardulf, a patrician of
the blood royal,[131] who had been left for dead by (25) Ethelred, but
had recovered when laid out for burial by the monks of Ripon. He
had the fullest recognition as king; was consecrated at the great altar
of St. Paul in York Minster on May 26, 796, by Archbishop Eanbald.
In his reign Alcuin died. In 806 he was driven out by (30) Elfwald, of
unknown parentage, but by the help of the Emperor Charlemagne he
was restored in 808. He died in 810, and was succeeded by his son
(31) Eanred, who was the last king but one of the royal house, and
the last independent king of Northumbria, dying in 840, and being
succeeded by his son (32) Ethelred II, expelled in 844, restored in
the same year, and killed sine prole in 848.
This, as has been said, is a most bewildering list. It is, however,
convenient to have it stated at length, inasmuch as several of these
kings are named in a noteworthy manner in the letters of Alcuin. To
emphasize the view that Alcuin took of the state of Northumbria, the
list just given may be summarized thus, it being borne in mind that
every king who reigned in Alcuin’s time after Eadbert’s death in 758
is included in the summary. Oswulf, murdered 759; Ethelwald,
deprived 765; Alchred, banished 774; Ethelred, expelled 779;
Alfwold, murdered 788; Osred, deposed 789; Ethelred, killed by his
own people, 796; Osbald, expelled 797; Eardulf, expelled 806.
The Venerable Bede had said in his letter to Archbishop Ecgbert in
735 that unless some very great change for the better was made in
all walks of life in Northumbria, that country would find its men quite
unable to defend it successfully if an invasion took place. We have
seen that so far as the reigning persons were concerned, the change
was for the worse; we have now to see how bitterly true Bede’s
prophecy, or rather his calculation of the necessary consequences,
proved to be. We are taken in thought to the year 793, not quite sixty
years after Bede’s letter. One excellent reign had lasted twenty-one
years, the next eight reigns averaged four and a half years, and all
ended in violence.
Higbald, the eleventh Bishop of Lindisfarne, 780-803, takes us
back nearly to the best times of that specially Holy Isle. Ethelwold,
724-40, his next predecessor but one, was the bishop under whom
King Ceolwulf, to whom Bede dedicated his famous work the
Ecclesiastical History of the English Race, became a monk. It was
this king-monk that taught the monks of Lindisfarne to drink wine and
ale instead of the milk and water prescribed by their Scotic founder,
Aidan. His head was preserved in St. Cuthbert’s coffin. Ethelwold’s
immediate predecessor was Eadfrith, 698-721, who wrote that
glorious Evangeliarium which is a chief pride of England, the
Lindisfarne Gospels. To Bishop Eadfrith and his monks Bede
dedicated his Life of St. Cuthbert, between whom and Eadfrith only
one bishop had intervened. The entry at the end of the Lindisfarne
Gospels connects Ethelwold and Eadfrith with the production and
binding of that noble specimen of the earliest Anglian work. Put into
modern English it runs thus:—
“Eadfrith, bishop of the church of Lindisfarne, he wrote this book at
first, for God and St. Cuthbert and all the saints that are in the island,
and Ethelwald, the bishop of Lindisfarne island, he made it firm
outside and bound it as well as he could.”
The entry proceeds to tell that Billfrith, the anchorite, wrought in
smith’s work the ornaments that were on the outside with gold and
gems and silver overlaid, a treasure without deceit. And Aldred, the
presbyter, unworthy and most miserable, glossed it in English, and
made himself at home with the three parts, the Matthew part for God
and St. Cuthbert, the Mark part for the bishop—unfortunately it is not
said for which of the bishops, the Luke part for the brotherhood. Only
one bishop came between Ethelwold, who bound this priceless
treasure, and Higbald, to whom we now turn.
 The Saxon Chronicle has under the year 787 this entry:—“In this
year King Beorhtric [of Wessex] took to wife Eadburg, daughter of
King Offa. In his days came three ships of the Northmen from
Haurthaland [on the west coast of Norway]. And the sheriff rode to
meet them there, and would force them to the king’s residence, for
he knew not what they were. And there they slew him. These were
the first ships of Danish men that sought the land of the English
race.”
They soon came again, this time not to the coast of Wessex, but to
the coast easiest of access from their own land. In 793 this is the
entry in the Saxon Chronicle:—
“In this year dire forewarnings came over the land of Northumbria
and pitifully frightened the people, violent whirlwinds and lightnings,
and fiery dragons were seen flying in the air. These tokens mickle
hunger soon followed, and a little after that, in this same year, on the
sixth of the ides of January [January 8] the harrying of heathen men
pitifully destroyed God’s church in Lindisfarne through rapine and
manslaughter.”
In the next year, 794, it is said:—
“The heathen ravaged among the Northumbrians, and plundered
Ecgferth’s minster at Donmouth [Wearmouth]; and there one of their
leaders was slain, and also some of their ships were wrecked by a
tempest, and many of them were there drowned, and some came to
shore alive and men soon slew them off at the river mouth.”
Wattenbach and Dümmler make the ruin of Lindisfarne take place
not on January 8 but on June 8. The Saxon Chronicle has Ianr. in
both of the MSS. which name the month. There is only one other
entry in the year 793, and it follows this,—“And Sicga [who had
murdered King Alfuold] died[132] on the 8th of the Kalends of March,”
that is, February 22. It is clear that these two events took place at the
end of 793, the years at that time ending with March, and January,
not June, was the month of ruin.
The twin monasteries of Wearmouth and Jarrow are described as
Ecgferth’s minster, because King Ecgfrith of Northumbria, 670-85,
gave land to Benet Biscop to found a monastery at the mouth of the
Don, now called the Wear, and some years later another portion of
land for the twin monastery of St. Paul, Jarrow. Later in Biscop’s life
he purchased two additional pieces of land from the next king,
Aldfrith, giving for the first two royal robes, or palls, made all of silk,
worked in an incomparable manner, which he had bought in Rome.
For the second, a much larger piece, he gave to the king a
manuscript collection of geographical writings, of beautiful
workmanship. We in the south-west must always remember that
Benedict Bishop first brought his vast ecclesiastical treasures to the
court of Wessex, but finding his royal patron dead went up north with
them. But for the death of the King of Wessex, we should have had
Wearmouth and Jarrow here as well as Malmesbury, Bede as well as
Aldhelm, and it may be Alcuin too.
We have letters of Alcuin to King Ethelred, to Higbald the Bishop
of Lindisfarne, and to the monks of the twin monastery of
Monkwearmouth and Jarrow, on this catastrophe. The letter to
Ethelred comes first:—
“To my most loved lord King Ethelred and all his Ep. 22. a.d. 793.
chief men the humble levite Alchuine sends
greeting.
“Mindful of your most sweet affection, my brothers and fathers and
lords honourable in Christ; deeply desiring that the divine mercy may
preserve to us in long-lived prosperity the fatherland which that
mercy long ago gave to us with gratuitous freedom; I therefore,
comrades most dear, whether present, if God allow it, by my words,
or absent by my writings under the guidance of the divine spirit, do
not cease from admonishing you, and by frequent repetition to
convey to your ears, you who are citizens of the same fatherland,
those things which are known to pertain to the safety of this earthly
realm and to the blessedness of the heavenly home; so that things
many times heard may grow into your minds with good result. For
what is love to a friend if it keeps silence on matters useful to the
friend? To what does a man owe fidelity if not to his country? To
whom does a man owe prosperity if not to its citizens? By a double
relationship we are fellow-citizens of one city in Christ, that is as
sons of Mother Church and of one native country. Let not therefore
your humanity shrink from accepting benignly what my devotion
seeks to offer for the safety of our land. Think not that I am charging
faults against you: take it that I aim at warding off penalties.”
We should here bear in mind that Ethelred had fourteen years
before this been expelled for cruel murders, and that he was now in
the first year of his restored reign and had already sent away his first
wife and taken another, a scandal so great in those days—bad as
they were—that the Saxon Chronicle with remarkable particularity
gives the month and the day of the gross offence, September 29. He
afterwards murdered the two surviving members of the royal house.
Alcuin’s letter to the king proceeds:—
“It is now nearly 350 years that we and our fathers have dwelt in
this most fair land, and never before has such a horror appeared in
Britain as we now have suffered at the hands of pagans. And it was
not supposed that such an attack from the sea was possible.[133]
Behold, the church of the holy Cuthbert is deluged with the blood of
the priests of God, is spoiled of all its ornaments; the place more
venerable than any other in Britain is given as a prey to pagan races.
From the spot where, after the departure of the holy Paulinus from
York, the Christian religion took its beginning amongst us, from that
spot misery and calamity have begun. Who does not fear? Who
does not mourn this as if his fatherland itself was captured?”
We should note Alcuin’s recognition of the fact that the restoration
of Christianity in Northumbria was due not to persons of the Anglo-
Saxon race and Church, but to Aidan and his monks of the Irish race
and Church.
“My brethren, give your most attentive consideration, your most
diligent investigation, to this question,—is this most unaccustomed,
most unheard-of evil, brought upon us by some unheard-of evil
custom? I do not say that there was not among the people of old the
sin of fornication. But since the days of King Alfwold[134] fornications,
adulteries, incests, have inundated the land to such an extent that
these sins are unblushingly perpetrated even among the handmaids
dedicated to God. What shall I say of avarice, rapine, and judicial
violence, when it is clearer than the light how these crimes have
increased, and a despoiled people are the evidence of it. He who
reads the Holy Scriptures, and revolves ancient history, and
considers the working of the world, will find that for sins of this nature
kings lose kingdoms, and peoples lose their father-land. He will find
that when men in power have unjustly seized the property of others,
they have justly lost their own....
“Consider the manner of dress, the manner of wearing the hair, the
luxurious habits of princes and of people. Look at the way in which
the pagan manner of trimming the beard and cutting the hair is
imitated. Do you not fear those whom you thus copy? Look at the
immoderate use of clothes, beyond any necessity of human nature.
This superfluity of the princes is the poverty of the people. Some are
loaded with garments, while others perish with cold. Some flow over
with luxuries and feasts like the rich man in purple, while Lazarus at
the gate dies of hunger. Where is brotherly love? Where is that pity
which we are bidden have for the wretched? The satiety of the rich
man is the hunger of the poor. That Scripture saying is to be
dreaded, ‘He shall have judgement without mercy that hath shewed
no mercy’[135]; and we have the words of the blessed Peter the
Apostle[136], ‘The time is come that judgement must begin at the
house of God.’ Judgement has begun, and with terrible force, at the
house of God where rest so many lights of the whole of Britain. What
is to be expected for other places, if the divine judgement has not
spared this most holy place? It is not for the sins of only those who
dwelled there that this has been sent.
“Would that the penalty that has come upon them could bring
others to amend their lives. Would that the many would fear what the
few have suffered, and each would say in his heart, groaning and
trembling, ‘if such men, if fathers so holy, did not save their own
habitation, the place of their own repose, who shall save mine?’
Save your country by assiduous prayers to God, by works of justice
and of mercy. Be moderate in dress and in food. There is no better
defence of a country than the equity and piety of princes, and the
prayers of the servants of God.”