Tag-based Next Generation Sequencing; Contents; Preface; List of Contributors; Part One: Tag-Based Nucleic Acid Analysis; 1 DeepSuperSAGE: High-Throughput Transcriptome Sequencing with Now- and Next-Generation Sequencing Technologies; 1.1 Introduction; 1.2 Overview of the Protocols; 1.2.1 Principle of the SuperSAGE Method; 1.2.2 Power of the SuperSAGE Tag; 1.2.3 Development of DeepSuperSAGE; 1.2.4 Ditag-Based DeepSuperSAGE (for 454 Pyrosequencing); 1.2.5 Single-Tag-Based DeepSuperSAGE (HT-SuperSAGE); 1.3 Methods and Protocols; 1.3.1 Linker or Adapter Preparation; 1.3.2 RNA Samples.

1.3.3 cDNA Synthesis and NlaIII Digestion; 1.3.4 Tag Extraction from cDNA; 1.3.5 Tag Extraction from cDNA; 1.3.6 Purification of Linker-Tag Fragments; 1.3.7 Ditag or Adapter-Tag Formation and Amplification; 1.3.8 Preparation of Templates for Sequencing; 1.4 Applications; 1.4.1 Applications of DeepSuperSAGE in Combination with 454 Pyrosequencing; 1.4.2 Practical Analysis of HT-SuperSAGE; 1.5 Perspectives; References; 2 DeepCAGE: Genome-Wide Mapping of Transcription Start Sites; 2.1 Introduction; 2.2 What is CAGE?; 2.3 Why CAGE?; 2.4 Methods and Protocols; 2.4.1 Key Reagents and Consumables.

2.4.2 Precautions; 2.4.3 RNA Samples Used for DeepCAGE Library Preparation; 2.4.4 DeepCAGE Library Preparation; 2.5 Applications; 2.6 Perspectives; References; 3 Definition of Promotome-Transcriptome Architecture Using CAGEscan; 3.1 Introduction; 3.2 What is CAGEscan?; 3.3 Why CAGEscan?; 3.4 Methods and Protocols; 3.4.1 Key Reagents and Consumables; 3.4.2 Precautions; 3.4.3 RNA Samples Used for CAGEscan Library Preparation; 3.4.4 Considerations on Pooling CAGEscan Libraries; 3.4.5 CAGEscan Library Preparation; 3.5 Applications and Perspectives; References.

4 RACE: New Applications of an Old Method to Connect Exons; 4.1 Introduction; 4.2 Deep-RACE; 4.2.1 Choice of the Sequencer; 4.2.2 Validation of Promoter Studies; 4.2.3 Other Applications of Deep-RACE; 4.2.4 Limitations of Deep-RACE; 4.3 Methods Outline; 4.3.1 Primer Design; 4.3.2 Molecular Biology of Deep-RACE Library Preparation; 4.3.3 Sequencing of Deep-RACE Libraries; 4.3.4 Analysis; 4.4 Perspectives; References; 5 RNA-PET: Full-Length Transcript Analysis Using 5'- and 3'-Paired-End Tag Next-Generation Sequencing; 5.1 Introduction; 5.2 Methods and Protocols.

5.2.1 Key Reagents and Consumables; 5.2.2 Protocol; 5.3 Applications; 5.3.1 PET Sequencing with SOLiD; 5.3.2 Mapping of the PETs; 5.3.3 PET Clustering, Annotation, and Genome Browser Visualization; 5.4 Perspectives; References; 6 Stranded RNA-Seq: Strand-Specific Shotgun Sequencing of RNA; 6.1 Introduction; 6.1.1 Before Starting; 6.2 Methods and Protocols; 6.2.1 Preface; 6.2.2 Materials and Consumables; 6.2.3 Protocol; 6.3 Bioinformatic Considerations; 6.4 Applications; 6.5 Perspectives; References.

7 Differential RNA Sequencing (dRNA-Seq): Deep-Sequencing-Based Analysis of Primary Transcriptomes.

Tag-based approaches were originally designed to increase the throughput of capillary sequencing, where concatemers of short sequences were first used in expression profiling. New Next Generation Sequencing methods largely extended the use of tag-based approaches as the tag lengths perfectly match with the short read length of highly parallel sequencing reactions. Tag-based approaches will maintain their important role in life and biomedical science, because longer read lengths are often not required to obtain meaningful data for many applications. Whereas genome re-sequencing and de novo sequ.

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