3.17 De Novo Genome Sequencing . . . . . .. . . . . . . . . . . . . . .. . . . . . . . . . . . . .. . . . . . . . . . . . . .. . . . . . 139
3.18 Resequencing . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . 140
3.19 N50 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140
3.20 Paired-End Sequencing and Unidirectional Sequencing . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140
3.21 Mate-Pair Sequencing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142
3.22 Multiplexing, Indexing, and Bar Coding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142
3.23 Phasing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143
3.24 Hi-C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143
3.25 mRNA/cDNA/EST/RNA-Seq Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145
3.26 Proteogenomic Mapping . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145
3.27 Structural Annotation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145
3.28 Functional Annotation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 146
4 Bioinformatics and Computer Concerns . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147
5 Sequencing, Assembly, and Annotation of Genomes De Novo . . . . . . . . . . . . . . . . . . . . . . . . . . . 151
5.1 The Human Genome Project (HGP) Sets the Stage for Plant Genome Sequencing . 151
5.2 BAC-by-BAC Sequencing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152
5.3 Shotgun Sequencing with Aspects of BAC-Based Physical Mapping . . . . . . . . . . . . . . . 154
5.4 Second Generation + Sanger . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 154
5.5 Second Generation Alone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 164
5.6 Third- + Second-Generation Sequencing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 164
5.7 The Present . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 165
6 The Best Plant Genome Sequences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 165
6.1 A Different Perspective . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 165
6.2 The Myth of Plant Genome Sequences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 166
6.3 The Un- and Underrepresented . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 166
7 What Genome Sequencing Has Revealed About Plant Genome Structure
and Function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 168
7.1 Whole Genome Duplications Are Very Common . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 168
7.2 The Trouble with Genes Is . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 170
7.3 Long Terminal Repeat (LTR) Retrotransposons Account for Much Genome Size
Variation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 172
8 Genomes as Maps . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175
Abstract The first decade of the twenty-first century witnessed the development
and commercialization of the so-called “second-generation” sequencing techniques.
These short-read sequencing methods produced such large amounts of sequence data
at such low prices that the costly and time-consuming physical map-based sequencing techniques, used to generate the human and the Arabidopsis genomes, were
largely abandoned. The rise of second-generation sequencing at the cost of physical
mapping resulted in a tremendous increase in the number and diversity of plant
genome projects, a proliferation in the number of individuals and institutions
involved in genome sequencing endeavors, and an overall decrease in the quality
of resulting genome assemblies. Single-molecule (“third-generation”) sequencing
techniques, which came onto the scene more recently, provide much longer (and
currently lower quality) reads than second-generation techniques, and a combination
of second- and third-generation technologies is resulting in higher quality, more
complete genome assemblies than second-generation techniques alone. At present,
excellent results are being obtained by assembling genomes with third-generation
reads, polishing the resulting contigs/scaffolds with second-generation (Illumina)
110
D. G. Peterson and M. Arick
3.18 Resequencing . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . 140
3.19 N50 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140
3.20 Paired-End Sequencing and Unidirectional Sequencing . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140
3.21 Mate-Pair Sequencing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142
3.22 Multiplexing, Indexing, and Bar Coding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142
3.23 Phasing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143
3.24 Hi-C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143
3.25 mRNA/cDNA/EST/RNA-Seq Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145
3.26 Proteogenomic Mapping . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145
3.27 Structural Annotation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145
3.28 Functional Annotation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 146
4 Bioinformatics and Computer Concerns . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147
5 Sequencing, Assembly, and Annotation of Genomes De Novo . . . . . . . . . . . . . . . . . . . . . . . . . . . 151
5.1 The Human Genome Project (HGP) Sets the Stage for Plant Genome Sequencing . 151
5.2 BAC-by-BAC Sequencing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152
5.3 Shotgun Sequencing with Aspects of BAC-Based Physical Mapping . . . . . . . . . . . . . . . 154
5.4 Second Generation + Sanger . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 154
5.5 Second Generation Alone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 164
5.6 Third- + Second-Generation Sequencing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 164
5.7 The Present . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 165
6 The Best Plant Genome Sequences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 165
6.1 A Different Perspective . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 165
6.2 The Myth of Plant Genome Sequences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 166
6.3 The Un- and Underrepresented . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 166
7 What Genome Sequencing Has Revealed About Plant Genome Structure
and Function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 168
7.1 Whole Genome Duplications Are Very Common . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 168
7.2 The Trouble with Genes Is . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 170
7.3 Long Terminal Repeat (LTR) Retrotransposons Account for Much Genome Size
Variation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 172
8 Genomes as Maps . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175
Abstract The first decade of the twenty-first century witnessed the development
and commercialization of the so-called “second-generation” sequencing techniques.
These short-read sequencing methods produced such large amounts of sequence data
at such low prices that the costly and time-consuming physical map-based sequencing techniques, used to generate the human and the Arabidopsis genomes, were
largely abandoned. The rise of second-generation sequencing at the cost of physical
mapping resulted in a tremendous increase in the number and diversity of plant
genome projects, a proliferation in the number of individuals and institutions
involved in genome sequencing endeavors, and an overall decrease in the quality
of resulting genome assemblies. Single-molecule (“third-generation”) sequencing
techniques, which came onto the scene more recently, provide much longer (and
currently lower quality) reads than second-generation techniques, and a combination
of second- and third-generation technologies is resulting in higher quality, more
complete genome assemblies than second-generation techniques alone. At present,
excellent results are being obtained by assembling genomes with third-generation
reads, polishing the resulting contigs/scaffolds with second-generation (Illumina)
110
D. G. Peterson and M. Arick