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DNA sequencing: technologies, markets and companies

This report briefly reviews basics of human genome variations, development of sequencing technologies, and their applications. Current large and small sequencers are described as well as companies developing them. Various applications of sequencing are described including those for genetics, medical diagnostics, drug discovery and cancer. Next generation sequencing technologies, both second and third generations, are reviewed. Companies developing software for analysis of sequencing data are also included. Selected academic institutes conducting research in sequencing are also listed.

Current market is mostly for research applications and future markets will be other applications related to healthcare. The value of DNA sequencer market in 2013 is described with estimates for 2018 and 2023. Various methods and factors on which market estimates depend are described briefly. Markets are tabulated according to geographical areas as well as applications. Small sequencers form the basis of SWOT (strengths, weaknesses, opportunities, threats) analysis. Several marketing strategies have been outlined.

The report includes profiles of 126 companies involved in sequencing and their 137 collaborations. The report text is supplementd by 39 tables, 18 figures and 420 selected references to the literature.

Table of Contents

0. Executive Summary

1. Introduction

  • Definition and scope of sequencing
  • Historical aspects of sequencing
  • Basics of molecular biology
    • DNA
      • DNA polymerases
      • Restriction endonucleases
      • DNA methylation
    • RNA
      • RNA polymerases
      • Non-coding RNAs
    • DNA transcription
    • Chromosomes
      • Chromatin
      • Chromosome sequences
    • Telomeres
    • Mitochondrial DNA
    • Genes
      • The genetic code
      • Gene expression
    • The human genome
      • ENCODE
    • The epigenome
      • Epigenetics and epigenomics
      • DNA methylation
      • Human Epigenome Atlas
  • Variations in the human genome
    • Variations in DNA sequences
    • Single nucleotide polymorphisms
    • Haplotyping
    • Complex chromosomal rearrangements
    • Insertions and deletions in the human genome
    • Large scale variation in human genome
    • Variation in copy number in the human genome
    • Structural variations in the human genome
    • Transposons
      • Retrotransposon capture sequencing
    • Mapping and sequencing of structural variation from human genomes
  • Impact of sequencing on healthcare

2. DNA Sequencing Technologies

  • Introduction
    • DNA extraction and sample preparation
      • Apollo 300 System for next generation sequencing
      • Electrophoresis-based method
      • Ion OneTouch System
      • Microfluidics-based extraction and sample preparation
      • Pressure Cycling Technology
      • Selective immobilization of nucleic acids onto magnetic microparticles
      • Targeted and hybridization-based DNA capture
    • Sanger-sequencing technology
      • Dye-terminator sequencing
      • Large-scale sequencing
      • Automated DNA-sequencing
      • Enhancements of Sanger-sequencing
      • ABI PRISM® 310 Genetic Analyzer
      • Life Technologies' 3500 Dx genetic analyzer
  • Limitations of sequencing methods and measures to remedy them
    • Paired end transcriptome sequencing to overcome short read lengths
    • Long vs short read lengths
    • Validation of NGS data
  • Emerging sequencing technologies
  • Chemical DNA sequencing
    • Chemical affinity capture and massively parallel DNA sequencing
  • Second generation sequencers
    • 4300 DNA analyzer
    • Apollo 100
    • Applied Biosystems 3500 series Genetic Analyzer
    • "Color blind" approach to DNA sequencing
    • Cyclic array sequencing
    • CEQ™ 8000
    • DeepCAGE sequencing
    • Electron microscope-based DNA sequencing
    • GS-FLEX system (Roche/454)
      • Background of sequencing technology
      • 454 sequencing
    • IBS sequencing technology
    • Illumina Genome Analyzer System
      • MiSeqDx
      • NextSeq 500 desktop sequencer
    • Ion Torrent's sequencing technology
    • MegaBACE 500
    • Microdroplet-based PCR for large-scale targeted sequencing
    • Millikan sequencing
    • Multiplex amplification of human DNA sequences
    • Nanoscale sequencing
    • Polonator sequencer
    • RainStorm™ microdroplet technology
    • Sequential DEXAS
    • SOLiD system: sequencing by ligation
    • PCR-based DNA sequencing technologies
      • Bridge amplification PCR system
      • COLD-PCR and sequencing
      • Digital PCR
      • Dual primer emulsion PCR
      • Emulsion PCR
      • Multiplex PCR
    • Non-PCR based sequencing
    • Nucleic acid sequence-based amplification
    • Microarray-based DNA sequencing technologies
      • Arrayit's® H25K
      • High-throughput array-based resequencing
      • Sequencing by hybridization
      • SOLiD-System based ChIP-Sequencing
      • Companies developing whole genome chips/microarrays
    • Next generation sequencing vs microarrays for gene expression profiling
    • RNA sequencing
      • Strand specific RNA sequencing
    • Exome sequencing
      • Human exome microarrays
    • Third generation sequencing
      • SOLiD4 System
      • SOLiD PI System
      • Helicos™ Genetic Analysis System
      • Molecular Combing
    • Nanotechnology-based sequencing
      • DNA sequence by use of nanoparticles
      • Denaturation mapping of DNA in nanofluidic channels
      • Nanopore sequencing
    • Convex lens-induced nanoscale templating
    • Detection of single molecules for sequencing
      • Helicos™ Genetic Analysis System
      • Molecular Combing
      • Optical Mapping
      • Nanopore-based single-molecule detection of specific DNA sequences
      • Sequencing-by-synthesis for single-molecule sequencing
      • Single molecule DNA sequencing by use of carbon nanotubes
      • Single molecule sequencing using Qdot nanocrystals
      • Single-molecule DNA sequencing in a sTOP chip nanowell
      • Single-molecule real-time sequencing
    • Single cell sequencing
      • MALBAC for single cell DNA sequencing
      • Microfluidic single-cell whole-transcriptome sequencing
      • Single cell sequencing of uncultured microbes
      • Single-sperm sequencing
      • Fluorescent in situ RNA sequencing
      • Future prospects of single cell sequencing
    • Thermosequencing
    • Whole genome sequencing for haplotyping
    • Mitochondrial exome sequencing
    • ImmunoSEQ technology
  • Future prospects of next generation sequencing
    • Devices for NGS
    • Reduction of errors in NGS
      • Artifactual mutations during the sample preparation process
      • Duplex sequencing

3. Role of Bioinformatics in Sequencing

  • Introduction
  • Growth of the sequencing database
  • Sequencing data storage
    • Cloud computing for sequencing data
      • Cloud computing facilities
  • Bioinformatics challenges of new sequencing technology
  • Construction of libraries for NGS
  • Bioinformatic tools for analysis of genomic sequencing data
    • Software for DNA sequencing
      • Software from academic and open sources
      • Commercial software for sequencing
    • ChIA-PET tool for analysis with paired-end tag sequencing
    • CLC Cancer Research Workbench
    • Compressive genomics
    • Detection of CNVs and gene duplications
    • Detection of SVs in massively parallel sequencing data
    • Differential expression analysis for sequence count data
    • Expression profiling without genome sequence information
    • Ingenuity® Variant Analysis™
    • Ion Reporter Software
    • Opal platform
    • VAAST
    • knoSYSTM100
  • Accessing DNA sequence information
  • Analysis of genomic variation by sequencing of large populations
    • Analysis of rare variants in NGS studies
  • Human gene connectome
  • Funding of research for interpretation of sequencing data
  • Future challenges for managing sequencing data

4. Comparative Analysis of Sequencing Technologies

  • General findings of the study
    • Sanger versus second generation marketed sequencers
    • Common features and differences among second generation sequencers
  • Third generation large sequencers
    • SOLiD4 versus competing large sequencers
    • Illumina's HiSeq sequencer
  • Third generation small sequencers
    • Genometrica™ desk top sequencer
    • Illumina's MiSeq sequencer
    • Roche/454 GS Junior System
    • Life Technologies' Benchtop Ion Proton™ Sequencer
    • Oxford Nanopore's MinION™ system
  • The ideal small sequencer
  • SWOT analysis of small sequencers
  • Concluding remarks on SWOT analysis

5. Sequencing for Research

  • Introduction
  • Applications in basic research
    • ChIA-PET technology for 3D study of the genome
    • ChIP-Seq for study of gene expression
    • Chromatin profiling by direct DNA sequencing
    • Discovery of immunoglobulin gene by pyrosequencing
    • Epigenetic modifications analyzed by next generation sequencing
    • Exome sequencing for study of human variation
    • Genome sequencing with combinatorial probe anchor ligation
    • Identifying protein-coding genes in genomic sequences
    • Mutation rate measured by direct sequencing
    • Protein-protein interactome network mapping
    • RNA sequencing
      • Applications of RNA sequencing
      • Contribution of RNA structure to gene regulation revealed by RNA-Seq
      • Molecular indexing for quantitative targeted RNA sequencing
    • Sequencing for the study of microchimerism
    • Sequencing for the study of CNVs
      • CNVnator
      • GS-FLX sequencing for simultaneous detection of mutations and CNVs
    • Sequencing the transcriptomes of stem cells
    • Sequencing and synthetic biology
      • Synthetic sequence in a bacterial cell
      • Functional synthetic proteins
  • Sequencing of human genomes
    • Whole genome sequencing
    • Whole-genome sequencing of methylome
    • Whole genome resequencing
    • Personal genome sequencing
    • Sequencing 1000 human genomes
    • 100,000 Genomes Project
    • Saudi Arabian human genome program
    • Genome of the Netherlands
    • Missing human genome sequences
    • Role of sequencing in identification of human remains
    • Sequence map of the human pan-genome
    • Sequencing of African genomes
    • Sequencing of Korean genomes
    • Sequencing mitochondrial genome
    • Sequencing of ancient genomes
      • Hominin genome
      • Neandertal genome
      • Anzick-1 genome
      • Saqqaq genome
    • Future prospects of human genome sequencing
  • Sequencing genomes of non-human primates
    • Sequencing of chimpanzee genome
    • Sequencing of macaque genome
    • Sequencing of gorilla genome
  • Sequencing genomes of other organisms
    • Species biodiversity study by the Canadian Centre for DNA Barcoding
    • Ant genome
    • Bat genome
    • Body louse genome
    • Camel genome
    • Cat genome
    • Dog genome
    • Frog genome
    • Goat genome
    • Horse genome
    • Ancient horse genome
    • House fly
    • Mouse genome
    • Tibetan antelope genome
    • Turkey genome
    • Water flea genome
    • Whale genome
  • Applications in drug discovery and development
    • Resequencing
    • RNA profiling
    • Transcriptome sequencing for mRNA Expression
    • RNA splice variants
    • Quantitative selection of aptamers through sequencing
  • Sequencing projects supported by US Government
    • NHGRI's sequencing initiatives
    • JGI's Community Sequencing Program
    • NIH funding for interpreting sequence variants in the human genome
    • NIH to fund studies of gene-environmental interactions in human diseases
    • Approved medical sequencing projects
  • 1000 Genomes Project
    • Findings of some studies of the 1000 genomes project
    • HapMap catalog as a foundation
    • Role of SOLiD™ System in 1000 Genomes Project
    • Protection of privacy of participants in 100 Genomes project
    • Concluding remarks
  • Human Variome Project
  • Academic centers conducting research on sequencing
    • Important academic collaborations
    • Large-scale genomic sequencing projects at Joint Genome Institute
    • Projects supported by NHGRI's Clinical Sequencing Exploratory Research grants
    • Manpower for sequencing
    • New York Genome Center
    • USTAR Center for Genetic Discovery

6. Applications of Sequencing in Healthcare

  • Introduction
  • Applications of sequencing in molecular diagnostics
    • Clinical exome sequencing
      • ACE Clinical Exome test
    • Diagnosis and screening of genetic disorders
      • Carrier screening using high-throughput NGS
      • CNV sequencing for diagnosis of chromosomal disorders
      • Role of WGS in screening of newborns
    • Guidelines for use of sequencing for diagnosis
    • NGS for diagnosis of CNS infections
    • NGS for detection of solid organ transplant rejection
    • NGS for forensic diagnosis
    • NGS-based HLA typing
      • High-throughput HLA genotyping with deep sequencing
      • Approaches to NGS-based HLA typing
      • Applications of NGS-based HLA typing
      • Tandem repeat variability for detection of genetic factors in diseases
    • Companies developing sequence-based molecular diagnostics
  • Applications of sequencing in oncology
    • A project to assess sequencing technologies for tumor DNA
    • A universal NGS-based oncology test system
    • Amplicon sequencing in cancer
    • Cancer Genome Atlas
    • Catalog of cancer genes
    • Detection of cancer biomarkers
      • Sequencing mitochondrial DNA to identify cancer biomarkers
      • Biomarkers for personalizing cancer treatment
    • Digital proteomics for cancer profiling
    • Epigenome profiling
    • Exosome sequencing
    • Gaining insights into mutational processes
    • High throughput sequencing for anticancer drug discovery
    • Multiple-gene sequencing panel to assess risk of hereditary cancer
    • Multiplexed cancer gene mutation analysis
    • NGS for enhancing verification rate of chromosomal structural rearrangements
    • NGS-based molecular profiling of cancer in FFPE specimens
    • Paired-end sequencing
    • Pathology tissue-ChIP
    • Quality control of NGS in oncology
    • RNA-Seq to study cancer transcriptome
    • Sequencing cancer cell lines
    • Sequencing for studying chromothripsis in cancer
    • Sequencing of complex human cancer genomes
    • Sequencing for identification of FGFR gene fusions in cancer
    • Sequencing single cells to study evolution of cancer
    • Sequencing circulating tumor cell genomes
    • Sequencing for assessing resistance to anticancer therapy
    • Sequencing of brain tumors
      • Sequencing for genetic alterations in gliomas
      • Sequencing for genetic alterations in medulloblastoma
    • Sequencing of breast cancer
      • BRCA mutations
      • Circulating nucleic acids as biomarkers of cancer
      • Deep sequencing of miRNA for signatures of invasiveness
      • NGS reveals heterogeneity of breast cancer
      • Sequencing of breast cancer metastases
      • Triple negative breast cancer
      • Whole genome sequencing in breast cancer
    • Sequencing of gastric cancer
    • Sequencing of colorectal cancer
    • Sequencing of head and neck cancer
      • NGS for detection of HPV sequences in carcinoma of oropharynx
    • Sequencing of hematological malignancies
      • Myelodysplastic syndromes
      • Acute myeloid leukemia
      • Acute promyelocytic leukemia
      • Chronic myelomonocytic leukemia
      • Hairy-cell leukemia
      • Sequencing in chronic neutrophilic leukemia and atypical CML
    • Sequencing in hepatocellular carcinoma
    • Sequencing of melanoma
    • Sequencing of ovarian cancer
    • Sequencing of prostate cancer
      • Identification of mutations in prostate cancer by exome sequencing
      • Role of sequencing in liquid biopsy for prostate cancer patients
    • Future prospects and challenges of NGS applications in oncology
    • Actionable Genome Consortium to guide use of NGS in clinical oncology
  • Sequencing in genetic disorders
    • Approaches to sequencing in genetic disorders
      • DNA sequencing for prenatal disorders
      • High-throughput sequencing in Undiagnosed Disease Program at NIH
      • Sequencing of maternal plasma for detection of fetal aneuploidy
      • Sequencing for study of transposons
      • Sequencing genomes of the newborn to screen for genetic disorders
      • Study of rare variants in pinpointing disease-causing genes
      • Whole genome sequencing for diagnosis of genetic disorders
      • Whole exome sequencing for diagnosis of genetic disorders
      • Whole genome sequencing of a human fetus from maternal plasma
    • Genetic disorders investigated by sequencing
      • Bartter syndrome
      • CHARGE syndrome
      • DiGeorge syndrome
      • Discovery of the gene for Miller syndrome
      • Discovery of the gene for Kabuki syndrome
      • Familial combined hypolipidemia
      • Familial thoracic aortic aneurysm
      • Hereditary blindness
      • Noonan syndrome
      • Proteus syndrome
      • Syndrome of hypogonadotropic hypogonadism, ataxia, and dementia
      • Syndrome of polyarthritis nodosa vasculopathy
      • X-linked disorder due to N-terminal acetyltransferase deficiency
  • Sequencing for study of the human immune system
    • Sequencing for investigating drug-virome interactions in organ transplants
  • Sequencing in neurological and psychiatric disorders
    • Sequencing in Alzheimer disease
    • Sequencing in Parkinson disease
    • Sequencing in Huntington's disease
    • Sequencing in Wilson's disease
    • Sequencing in ataxias
    • Sequencing in epilepsy
    • Sequencing for mutations in familial amyotrophic lateral sclerosis
    • Sequencing of whole genome in Charcot-Marie-Tooth disease
    • Sequence-based detection of a new variant of Lambert-Eaton syndrome
    • Sequencing in muscular dystrophy
    • Sequencing in acute brain injury due to hemorrhage
    • Sequencing for mutations associated with autism spectrum disorders
    • Sequencing for diagnosis of intellectual disability
    • Sequencing in neurodevelopmental disorders
      • NGS for identifying mutations in RNA gene
      • Sequencing in attention-deficit/hyperactivity disorder
    • Sequencing in schizophrenia and bipolar disorder
    • Sequencing in drug addiction
  • Sequencing in cardiovascular disorders
    • Inherited cardiomyopathies
    • Exome sequencing and mutations associated with risk of coronary heart disease
  • Sequencing genomes of microbes
    • DNA sequencing for study of bacterial epidemics
      • Genome sequencing of H. influenzae to identify population structure
      • Role of sequencing in cholera epidemics
      • Role of sequencing in epidemic of Shiga toxin-producing E. coli
      • Sequencing study of Salmonella emergence in Sub-Saharan Africa
      • Sequencing of ancient specimes from past epidemics
    • Sequencing for tracking hospital acquired infections
      • Sequencing for investigation of MRSA outbreaks
      • Role of sequencing in tracking a hospital infection of K. pneumoniae
      • Role of whole genome sequencing in identification of C. difficile
    • Human Microbiome Project
    • Role of sequencing in the management of bacterial infections
      • Pyrosequencing of microbial flora in leg ulcers
      • Sequencing for study of antibiotic resistance in bacteria
      • Sequencing for predicting the virulence of MRSA
    • Sequencing the human gut microbiome for new immunomodulatory molecules
      • Sequencing of gut microbes in obesity
    • Sequencing for mapping genomic variation in Mycobacterium ulcerans
    • Sequencing for mapping genetic interactions in bacteria
    • Sequencing of DNA from single cells of bacteria
    • Sequencing of the fungal genomes
    • Sequencing for detection of drug resistance in Plasmodium falciparum
    • Sequencing of human salivary microbiome
    • Sequencing in the management of HIV/AIDS
      • Long read sequencing for personalizing HIV therapy
      • NGS for studying neuroAIDS
      • Sequencing plus immunological analyses to study HIV evolution
      • Surveillance of drug resistance in HIV-infected individuals
    • Sequencing in the management of Ebola virus infection
    • Sequencing in the management of HBV
    • Sequencing in the management of HCV
    • Sequencing genome of Lassa fever virus
    • Sequencing genome of a rhabdovirus associated with acute hemorrhagic fever
    • Surveillance of H1N1 influenza A virus using resequencing arrays
  • Population targeted sequencing studies
  • Sequencing in aging research
  • Next generation sequencing and pharmaceutical industry
    • Next generation sequencing and drug design and discovery
    • Next generation sequencing and drug safety
    • Next generation sequencing for antibacterial therapeutic discovery
    • Applications of human transcriptome array in clinical trials
  • Role of sequencing in personalized medicine
    • Whole genome sequencing and personalized medicine
    • Whole exome sequencing and personalized disease risk
    • Personal Genome Project
    • Role of sequencing in personalized cancer management
    • Standardization of sequencing for personalized medicine
    • Future of sequencing and personalized medicine
  • Current status and future prospects of clinical applications of NGS
    • Needs of the clinical market for NGS
    • Sequencers for the clinical market
    • Challenges for clinical applications of NGS
    • ACMG clinical laboratory standards for NGS
    • Future trends in clinical sequencing
    • Rare Diseases Genomes Project
    • Ethical aspects of sequencing

7. Markets for Sequencers

  • Introduction
  • Methods used for estimation of sequencer markets
  • Currently marketed sequencers
  • Academic and research markets for sequencing
  • Factors affecting future development of sequencing markets
    • Future needs and support of research
    • Bioinformatics in relation to sequencing
    • Reducing the cost of human genome sequencing
      • US Government-supported research on sequencing
      • Contribution of American Recovery and Reinvestment Act
      • Genome X Prize Foundation
      • Innovations to reduce cost of whole genome sequencing
      • Commercial aspects of low cost genome sequencing
    • Genome sequencing suitable for personalized medicine
      • The global sequencing markets
      • Sequencing markets according to applications
      • Sequencing markets according to therapeutic areas
  • Marketing potential for sequencers
  • Market trends for NGS
  • Challenges to developing market for sequencers
  • Recommendations

8. Companies Involved in Sequencing

  • Introduction
  • Major players in sequencing
  • Profiles of companies involved in sequencing
  • Collaborations

9. References

Tables

  • Table 1-1: Historical landmarks in DNA sequencing
  • Table 1-2: Genetic variations in the human genome
  • Table 2-1: ChIP detection platforms for sequencing
  • Table 2-2: Companies developing whole genome chips/microarrays
  • Table 2-3: Systems for single molecule sequencing
  • Table 3-1: Software programs for sequencing from open sources
  • Table 3-2: Companies providing DNA sequencing software
  • Table 4-1: Comparison of a generation I and generation II sequencers
  • Table 4-2: Similarities and differences between second generation sequencers
  • Table 4-3: SWOT of ABI 310
  • Table 4-4: SWOT of IBS sequencing
  • Table 4-5: SWOT of NABsys' Hybridization-Assisted Nanopore Sequencing
  • Table 4-6: SWOT of 4300 DNA Analysis System Li-Cor
  • Table 4-7: SWOT of Genometrica
  • Table 4-8: SWOT of Polonator
  • Table 4-9: SWOT of GS FLEX Junior
  • Table 4-10: SWOT of Oxford Nanopore's MinION™ system
  • Table 4-11: SWOT of Ion Torrent™ Personal Genome Machine
  • Table 4-12: SWOT of Pacific BioSciences' single-molecule real-time sequencing
  • Table 4-13: SWOT of Illumina's MiSeqDx
  • Table 4-14: SWOT of Illumina's NextSeq 500
  • Table 4-15: SWOT of QIAGEN's GeneReader™ sequencer
  • Table 4-16: SWOT of Bio-Rad (formerly GnuBio's) droplet-based sequencing system
  • Table 5-1: Number of genes in organisms with fully sequenced genomes
  • Table 5-2: Approved medical sequencing projects
  • Table 5-3: Academic centers conducting research on DNA sequencing
  • Table 5-4: Distribution of scientific manpower for sequencing
  • Table 6-1: Companies involved in application of sequencing in molecular diagnostics
  • Table 7-1: Marketed next generation sequencers
  • Table 7-2: De novo sequencing vs resequencing markets
  • Table 7-3: Global markets for sequencing services according to geographical regions
  • Table 7-4: Global markets for sequencing services according to applications
  • Table 7-5: Sequencing markets according to therapeutic areas
  • Table 7-6: Global markets for sequencers from 2013 to 2023
  • Table 8-1: Companies developing sequencing technologies and instruments
  • Table 8-2: Companies that provide sequencing services
  • Table 8-3: Companies that provide bioinformatics support for sequencing
  • Table 8-4: Major players in sequencing
  • Table 8-5: Selected collaborations for DNA sequencing

Figures

  • Figure 2-1: DNA sequencing process
  • Figure 2-2: Components of next generation sequencing
  • Figure 2-3: Comparison of traditional sequencing and next generation sequencing
  • Figure 2-4: Watson-Crick base pairing
  • Figure 2-5: Genome Sequencer FLX system (Roche/454)
  • Figure 2-6: Workflow of Genome Sequenser FLX system
  • Figure 2-7: Sequencing by ligation
  • Figure 2-8: Construction of SOLiD fragment library using DNA enrichment by ChIP
  • Figure 2-9: Nanopore-based sequence-specific detection of DNA
  • Figure 2-10: Single molecule, realtime DNA sequencing
  • Figure 2-11: A scheme of thermosequencing platform
  • Figure 2-12: Duplex sequencing
  • Figure 3-1: Basic workflow of NGS libraries
  • Figure 6-1: Comparison of conventional and high-throughput NGS workflows
  • Figure 6-2: Procedure for sequencing of CTC exome
  • Figure 6-3: Role of sequencing in the development of personalized medicine
  • Figure 7-1: Cost of sequencing per genome
  • Figure 7-2: Global markets for sequencing services according to applications
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