Kỹ thuật New generation sequencing (NGS)

Lecture 3: New generation sequencing (NGS):  Technology National Institute of Infectious Disease January 16, 2017 Scope of investigation covered by epidemiology Identifying…  disease occurrence and distribution in time and place  reservoir of infectious agents  modes and pattern of disease transmission  setting of disease transmission  pathogen-related biologic factors that influence transmission  host-related (demographic, behavioral, clinical, genetic) factors that influence transmission  environmental factors (socioeconomic, anthropologic, ecologic) that influence transmission  etiologic role of a microbe for a newly-recognized disease or a disease previously not recognized to be associated with an infectious agent Scope of investigations covered by next generation molecular epidemiology Identifying …  risk factors that could not be identified by conventional or early-generation molecular biology laboratory methods  new or hidden transmission pathways  direction of transmission of an infectious agent  endogenous reactivation vs exogenous reinfection  ecological niche from which clonal pathogenic strains are selected and disseminate  pathogen microbial population structures associated with a syndrome  host commensal microbial population structures that determine noncommunicable disease outcomes Types of sequencing applications  Targeted sequencing  Whole genome sequencing  Metagenomics (deep sequencing) bgiamericas.com  RNAseq WWW.clcbio.com www.sanger.ac.uk Technology of NGS • Sequencing methods—microbiology/infectious disease applications • Types of sequencing • Deep sequencing (metagenomics) • Whole genome sequencing • RNA‐seq • Chemistry • Sequencing‐by‐synthesis • Sequencing‐by‐ligation • Sequencing platforms (instruments used to generate sequence data) • Bioinformatics (Computational biology) • Algorithms used to analyze sequence data • Statistics for computational biology High‐throughput (next generation) sequencing • General features: • Sequencing done in parallel  • Designed to increase speed and reduce  cost • Makes possible whole genome  sequencing and deep sequencing of a  large number of microorganisms High‐throughput sequencing (HTS)—general features • Sequencing‐by‐synthesis • Bridge PCR • Emulsion PCR: water‐in‐oil • Single‐molecule real‐time(SMRT) • Sequencing‐by‐ligation Chemistry: based on chemical  reaction of hybridization or  ligation or nucleotide assembly • Differences in preparation of samples for HTS • PCR‐ vs single‐molecule‐based methods • Differences in nucleotide incorporation signal detection NGS platforms • 454 (Roche) • Illumina (Solexa) • SOLiD (Applied Biosystems) • Ion Torrent PGM and Proton (Thermo Fisher) • PacBio (Pacific Biosciences) • MinION (Oxford Nanopore) • GeneReader (Qiagen) Sequencing—basic concepts • Sanger sequencing (dye‐terminator  Primer sequencing) 5’ Template DNA ddTTP ddGTP ddATP ddCTP Uses ddNTPS (terminator) labeled  with fluorescent dyes 454, Roche: Ultra‐high‐throughput parallel sequencing:  pyrosequencing using beads adapter Emulsion  PCR Pyrosequencing PicoTiter Plate http://scott.sherrillmix.com/blog/tag/pyrosequencing/ Pyrosequencing www.Medscape.com 1. Incorporation of dNTP releases PPi 2. ATP sulfurylase converts PPi into ATP in  the presence of adenosine 5´ phosphosulfate.  3. ATP supplies energy for luciferase to  convert luciferin to oxiluciferin to emit light 4. Light is detected by camera genomesequence.blogspot.com Apyrase continuously degrades  unincorporated ATP and nucleotides Bridge amplification,  reversible dye termination  (Illumina) Davetang Illumina: GA, HiSeq, MiSeq, NextSeq500 • Most widely used, due to its cost and adaptability to different tasks • Uses flow cells with attached oligos complementary to adapter  sequence • Uses reversible dye terminators • Advantages • Longest reads increasing (~300 bp with MiSeq; paired‐end reads) • Very high throughput: 150 million clusters/flow cell (~1000 copies/cluster) • can produce 4 billion reads/run (~125 bp/read)—useful for “shot‐gun”  sequencing • Low cost (~$0.50/Mb) • Disadvantages • Short reads compared to 454—makes assembly more difficult • Slower than 454 (27 h for MiSeq; 11 days for HiSeq) Ion Torrent Sequencers (Life Technologies):  PGM, Ion Proton • Sequence‐by‐synthesis • Starts with emulsion PCR  • Nucleotide incorporation detected by microchip sensitive to change in pH due to  release of proton • Advantages • Affordable (~$0.63/Mb) • Fast • Ion Proton produces ~50 million reads/run (~200 bp/read); ~400/read by PGM • Disadvantages • • • • Not as high output as 454. SOLiD, or Illumina platforms Paired‐end reads not reliable Errors with homopolymers Errors with AT‐rich sequences PacBio sequencer • Based on single molecule sequencing approach • Does not use any amplification step • High error rate of earlier‐generation machines corrected by  incorporating circular consensus sequencing (CCS) • Sequencing done on a chip containing zero‐mode  waveguide (ZMW) detectors • DNA polymerase attached to ZMW detectors • Dye‐labeled nucleotide incorporation is detected in real  time PacBio Sequencer • Advantages • Long reads: average read ~10,000 bp/read—makes  assembly and alignment easier • Multiplexing done by “in‐line” barcoding  • Paired‐end reads not necessary due to CCS • Disadvantages • Cost • Low number of reads/run (~47,000/run) MinION (Oxford Nanopore DNA strand Technologies, UK) • Advantages: Protein nanopore • Small size (100 grams) • Derives power from USB port on a portable computerElectrically resis polymer memb • Long reads: ~75,000 bases/run • Disadvantages: • Error‐prone Current generated by ions passing through the nanopore https://nanoporetech.com/science‐technology/how‐it‐works Infectious disease epidemiological problems addressed by molecular biology techniques (2016)               Tracking strains across time and geography Distinguishing endemic from epidemic disease occurrence Stratification of data to refine study designs Distinguishing pathovars vs commensal flora or saprophytes Identifying new modes of transmission Studying microorganisms associated with healthcare or institutional infections Surveillance and monitoring response to intervention Characterizing population distribution and determinants of distribution of parasites Identifying genetic basis for disease transmission Validating microdiversity genotyping methods applied to epidemiology Virus quasispecies population structure analysis Identifying direction and chain of transmission Identifying hidden social networks and transmission links Analyzing microbiomes to study non-infectious disease epidemiology NGS ... Sequencing? ?methods—microbiology/infectious disease applications • Types of? ?sequencing • Deep? ?sequencing? ?(metagenomics) • Whole genome? ?sequencing • RNA‐seq • Chemistry • Sequencing? ??by‐synthesis • Sequencing? ??by‐ligation • Sequencing? ?platforms (instruments used to generate sequence data)... Types of sequencing applications  Targeted sequencing  Whole genome sequencing  Metagenomics (deep sequencing) bgiamericas.com  RNAseq WWW.clcbio.com www.sanger.ac.uk Technology of NGS • Sequencing? ?methods—microbiology/infectious disease applications... High‐throughput (next? ?generation) ? ?sequencing • General features: • Sequencing? ?done in parallel  • Designed to increase speed and reduce  cost • Makes possible whole genome  sequencing? ?and deep? ?sequencing? ?of a