Next-generation sequencing. Current technologies and applications (Q2871884)

The volume is built as a series of chapters on different aspects of genome analysis that make use of Next Generation Sequencing (NGS). The first chapter commences with an overview of second generation sequencing platforms, like Roche pyrosequencing, Illumina (Solexa) and SOLiD. For each of these technologies the general workflow is presented together with details of the run time, yield, reagent costs and error rates. The chapter also contains an overview of third generation sequencing platforms including the Ion Torrent semiconductor TGS platform and single molecule sequencing (SMS) platforms. A brief general description of Helicos tSMS and PacBio RS SMART technologies is also included. The second chapter continues the presentation of Helicos single molecule DNA sequencing technology outlining the low quantity DNA/ cDNA sequencing capabilities and applications. The technical details of the approach are included and some brief indications for data analysis conclude the chapter. The third chapter focuses on the bioinformatics side of the analysis presenting methods for single nucleotide polymorphisms (SNP) assessment on draft genomes. Two methods, ACCUSA and ACCUSA2, are presented in detail (including a step by step description with examples) using an in silico benchmark. The fourth chapter presents yet another use of NGS for understanding the regulatory functions encoded in the genomes. Tools for processing several classes of small RNAs (sRNAs) like microRNA, heterochromatin siRNAs and general sRNAs are presented. The chapter commences with a brief description of sRNA library preparation and sequencing, followed by a detailed overview of helper tools such as adaptor removal tools, filtering tools, solutions for the mapping of the reads on the genome and normalization approaches. Next, the authors present sRNA quantitative statistics and methods to identify particular classes of sRNAs. The chapter concludes with an overview of target prediction and normalization tools.NEWLINENEWLINE The fifth chapter discusses the utility of NGS in the context of rare variant detection, i.e., sequences present in less than \(1\%\) of the sequence data. The chapter commences with a description of rare variants and a motivation for detection methods. Next, the contributers discuss the status of rare variant detection and how much HTS data are needed to accurately detect rare variants in relation to the obtained coverage. The chapter concludes with an overview of methods used for testing the feasibility of using HTS for rare SNP detection. A set of experimental validation approaches is also included. The sixth chapter focuses on another type of genomic variants, breakpoints of insertions and deletions. Using Pindel and other split read approaches to exemplify the phenomenon, the authors overview in detail the causes that lead to this and the proposed solutions. The chapter commences with a presentation of read depth methods. Next, the Pindel methods, pattern growth methods to identify precise breakpoints of indels and SVs are discussed in detail. The performance on real data and some recent developments are also included. The chapter concludes with a brief overview of other similar methods and future perspectives.NEWLINENEWLINE The next chapter presents novel insights from resequencing of human exons through NGS. First, the protocol is described in detail. Next, the authors compare exome capture platforms and available kits, i.e., NimbleGen, Aglient and TruSeq. The authors continue with an overview of quality control metrics and performance evaluations such as the summary of exome capture kits. Next, the bioinformatics analysis is briefly overviewed using the Mendelian disease analysis as a demo case. The chapter concludes with examples of applications in human disease research for Mendelian disorders, complex diseases and cancer. The eight chapter offers a different perspective for NGS technologies presenting new methods to better understand plant transcriptional networks. The authors show how RNAseq can be used to dissect transcription at cellular resolution and use an example of ChipSeq for dissecting plant transcriptional networks and better understanding the epigenomes. The last two chapters focus on microbial metagenomics. In chapter 9 the authors present the challenges of assembling multiple microbial genomes for the study of microbial ecology. First, methods to track microbial diversity are overviewed. Next, the authors discuss how to apply omics technologies and how to efficiently design experiments for this field. The tenth chapter focuses on marker specific microbial community surveys and its applications for a better understanding of human health and disease. The chapter concludes with systems biology views onto the host-microbiome interactions and on potential cloud based informatics solutions.NEWLINENEWLINE Although written in an accessible style, the book is aimed at specialists in the NGS field, its range of prerequisites varying from advanced molecular biology to a good understanding of bioinformatics techniques.