Next Generation DNA Sequencing and PCR Protocols & Manipulation

Nyhet

Inbunden, Engelska, 2026

Av Akbar S. Khan, Helen Cui, Akbar S Khan

1 829 kr

Kommande

Produktinformation

Utgivningsdatum2026-05-05
Vikt666 g
FormatInbunden
SpråkEngelska
Antal sidor300
FörlagJohn Wiley & Sons Inc
ISBN9781119100362

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Biologi inom Naturvetenskap och teknik

Block One: Brief history of PCR and DNA sequencing techniques, and their roles in advancing molecular biology and life science at large; the guiding principles and objectives of the book. Chapter I. General Introductiona.) A short history in PCR and DNA sequencing techniquesb.) Landmark molecular biology and life science advancement resulted from DNA sequencing and PCR techniques Chapter II. Guiding principle and organization of the booka.) Purpose, Objective, Scopeb.) Intended audiencec.) Flow and logic of the bookd.) Inclusions and exclusions, limitations Block Two: Chapters three through five will first describe the current broadly used next generation sequencing platforms, PCR instrument application, followed by a comparative overview of their utilities and applications, particularly in microorganism genomic analysis. Chapter III. Next Generation Sequencing Principles and Platforma.) Principle chemistry: sequencing by ligationb.) Principle chemistry: sequencing by synthesisc.) Leading NGS platformsi. Ion Torrent PGM (Personal Genome Machine)ii. Illumina HiSeq and MiSeqiii. PacBio (Pacific Biosciences) RSIIiv. Other platforms: SOLiD, illumina GA (Genome Analyzer)d.) Technical specifications to targeted utilities Chapter IV. PCR instrument and applications in NGSa.) Principles and instrumentb.) Basic chemistriesc.) Utility of NGS Chapter V. Overview of utilities and comparative applicationa.) Targeted analytic requirementsb.) Source material determinationc.) Microorganism genome sequencingd.) Genome sequencinge.) Transcriptome sequencingf.) Chromatin immunoprecipitation sequencingg.) Metagenome sequencingh.) Epigenomic variation analysis Block Three: Chapters six through ten describe the sequencing methodologies using each type of the NGS instruments, selection of platforms and reagent kits for targeted applications, metagenomic sequencing, single cell sequencing, epigenetic analysis, procedure modification for adaptation and optimization, and sample specific treatment. These chapters will also discuss workflow logic, sequencing chemistry, selection of methodologies, system errors and errors generated by other causes. Chapter VI. Methodologiesa.) Source material specific sample preparationi. Infectious material inactivationii. Chemical and physical approachesiii. Storageb.) DNA/RNA Extractioni. Instrument specification evaluation––Quantity estimate (amount)––Quality evaluation (fragment length)ii. DNA extraction quality control––Concentration measurement––Quality evaluationiii. RNA extraction quality control––Concentration measurement––Quality and sizing evaluationc.) NGs cDNA Library Prep i. Amplification–based library preparationii. Amplification–free library preparationiii. DNA fragmentationiv. Enzymatic treatment for DNA repairv. Poly–A tailing of fragmentvi. Library validation Chapter VII. NGS Platform specific operational protocolsa.) Illuminai. cDNA Library preparation from DNA extraction––DNA fragmentation––Adapter ligation––PCR enrichment––Comparison of commercial kitsii. cDNA library preparation from RNA extraction––mRNA selection––polyA tail selection––ribosome RNA removal––DNA Fragmentation––cDNA synthesis––Ligation of adapters––PCR enrichmentiii. Library validation––Concentration measurement––Sizing estimate––Sequenceable concentration estimationiv. Sequencing––Instrument dependent condition––Read lengths, number of reads and time requirement b.) PacBioi. DNA extractionii. Source material specific notationiii. Library preparation of 10Kb template––DNA Fragmentation––Insert size determination––Repair of DNA damage––Repair of insert ends––Blunt ligation––Inactivation of ligate––Digest of misligate product c.) Library preparation of 20Kb template––Fragmentation of DNA––Insert size determination––Repair of DNA damage––Insert ends repair––Blunt ligation––Inactivation of ligate––Digest of misligate product––Size selection d.) Sequencing primer annealinge.) Binding Reactionf.) Sequencing––Binding of DNA polymerase to SMRT template iii. Ion Torrent PGM, Ion Proton––Library preparation––Emulsion PCR––Enrichment––Quality control––Sequencing vii. SOLiD Chapter VIII. Choice of platform and reagent kit selectiona.) Platform specific considerationsi. Nucleic acid qualityii. Quantity availabilityiii. Read number requirementiv. Read length requirementv. Timevi. Sequencing cost per Gb b.) Sample Source material based selection i. Microbial isolates, viral samples, animal, human, plant, environmentalii. Formalin–fixed, paraffin–embedded samplesiii. Trace amount analysisiv. Metagenomicv. Single cell c.) Research objective based selectiond.) Throughput based consideration Chapter IX. Targeted application areasa.) SNP analysis from draft sequencing outputb.) Large scale small RNA studyc.) Landscape of human genome researchd.) Forensics application Chapter X. Enhancement and Optimizationa.) Barcoding, multiplexingb.) Fragment sizingc.) Kit selection and modification Block Four: Chapter eleven provides a prospect of future generations of techniques near release and coming into horizon. Chapter XI. Future techniquesa.) Close to release: Oxford Nanoporeb.) In the horizon: Thumb–drive sequencerc.) Prospect Appendices:CitationsReferencessNotes