ELŐADÁS KIVONAT CLASSROOM LECTURE HANDOUT. financed by the program
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1 TÁMP C-13/1/KNV projekt Az élettudományi-klinikai felsőoktatás gyakorlatorientált és hallgatóbarát korszerűsítése a vidéki képzőhelyek nemzetközi versenyképességének erősítésére program keretében finanszírozott ELŐADÁS KIVNAT CLASSRM LECTURE HANDUT financed by the program Practice-oriented, student-friendly modernization of the biomedical education for strengthening the international competitiveness of the rural Hungarian universities Dátum / Date: ÁPRILIS 20. / APRIL 20, 2016 Helyszín / Place: MTA SZBK Biofizika Tanácsterem / Lecture room of the Institute of Biophysics, BRC SZEGED, TEMESVÁRI KRT. 62. Az előadás címe / Title of the presentation: NEXT GENERATIN SEQUENCING: NEW PSSIBILITIES IN MEDICINE Előadó / Speaker: ATTILA KERESZT Biological Research Centre Address: H-6726 Szeged, Temesvári krt. 62. Mail: H-6701 Szeged, PB
2 Practice-oriented, student-friendly modernization of the biomedical education for strengthening the international competitiveness of the rural Hungarian universities TÁMP C-13/1/KNV Next generation sequencing: new possibilities in medicine Attila Kereszt Institute of Biochemistry April 20, 2016 DNA SEQUENCING DNA sequencing is the process of reading nucleotide bases in a DNA molecule GENME SEQUENCING Sequencing the whole genetic material of an organism 1953: Structure of DNA 1973: First sequence of 24 bp published (lac perator) 1977: Phage ΦX : Haemophilus influenzae 1996: Methanococcus jannaschii 1996: Saccharomyces cerevisiae 1997: Escherichia coli 1998: Caenorhabditis elegans 2000: Drosophila melanogaster 2000: Arabidopsis thaliana 2001: Homo sapiens (draft) 2002: Mus musculus 2006: Homo sapiens (complete) 2009: 1000th prokaryotic genome (complete) 1
3 THE EVLUTIN F GENME SEQUENCING Sanger (First generation) sequencing Need for library preparation in a host Labour and time - intensive, expensive Toxic regions are not represented Host genome contaminations Low throughput strand synthesis and base determination are separated need for electrophoretic step high unit cost (cost/bp) NGS (Next Generation Sequencing) No need for library preparation in a host immobilized template fragments, PCR methods labour, time and cost effective High throughput several millions-billions of sequencing /run synthesis and sequencing are not separated Long read, low coverage No competition, but complementation Short read, huge coverage (Second generation) Very longong read, large coverage (Third generation) FIRST GENERATIN DNA SEQUENCING: LIBRARY CNSTRUCTIN IN A HST FIRST GENERATIN DNA SEQUENCING: SEQUENCE ASSEMBLY STRATEGY 2
4 FIRST GENERATIN DNA SEQUENCING TECHNLGY IS BASED N THE ACTIVITY F DNA PLYMERASE FIRST GENERATIN DNA SEQUENCING TECHNLGY IS BASED N THE ACTIVITY F DNA PLYMERASE FIRST GENERATIN DNA SEQUENCING TECHNLGY IS BASED N THE ACTIVITY F DNA PLYMERASE AND THE USE F DIDEXY NUCLETIDES (SANGER) datp ddatp NH 2 NH 2 N N N N N N N N - P P H P - P P P H H H H H H H H H H H H The 3 hydroxyl has been changed to a hydrogen in ddntp s, which terminates a DNA chain because a phosphodiester bond cannot form at this 3 location 3
5 CHAIN TERMINATIN FIRST GENERATIN DNA SEQUENCING TECHNLGY: 4 reactions: each contains 4 dntps and 1 ddntp primer or one dntp radioactively labeled DNA molecules of different length are separated by gel electrophoresis A C G T FIRST GENERATIN DNA SEQUENCING TECHNLGY: 4 reactions: each contains 4 dntps and 1 ddntp Primers in the 4 reactions are labeled with different fluorescent dyes DNA molecules of different length are separated by gel electrophoresis A C G G A C T T 4
6 FIRST GENERATIN DNA SEQUENCING TECHNLGY: 1 reaction: contains 4 dntps and 4 ddntps that are labeled with 4 different fluorescent dyes DNA molecules of different length are separated by gel/capillary electrophoresis SECND GENERATIN DNA SEQUENCING: LIBRARY CNSTRUCTIN WITHUT A HST ISLATE DNA/RNA T BE SEQUENCED LIBRARY PREPARATIN FRAGMENTATIN F DNA LIGATIN F ADAPTRS, PRIMERS (BARCDE) SIZE SELECTIN PYR- SEQUENCING 454 Roche CLNAL AMPLIFICATIN F THE LIBRARY EMULSIN PCR SLID-PHASE PCR C Y C L E sequencing by synthesis by ligation SEMICNDUCTR SEQUENCING IonTorrent Life Technologies sequencing in picowells S E Q U E N C I N G SEQUENCING BY LIGATIN SLiD Life Technologies sequencing on solid surface REVERSIBLE TERMINATR SEQUENCING Illumina Solexa LIBRARY PREPARATIN ISLATE DNA/RNA RANDM FRAGMENTATIN END REPAIR ADAPTRS PRIMERS LIGATIN SIZE SELECTIN CLNAL AMPLIFICATIN 5
7 CLNAL AMPLIFICATIN: EMULSIN PCR CLNAL AMPLIFICATIN: SLID-PHASE PCR cluster generation for Illumina sequencing original template original template new strand new strand CLNAL AMPLIFICATIN: SLID-PHASE PCR cluster generation for Illumina sequencing 6
8 step1 CYCLE SEQUENCING cycles step2 step3 step4 add A add C add G add T A C G T detect A detect C detect G detect T wash away A wash away C wash away G wash away T 4 nucleotides flow sequentially PYRSEQUENCING (RCHE 454) 4 nucleotides flow sequentially C T A G hν PYRSEQUENCING (RCHE 454) SIGNAL DETECTIN (PYRGRAM) Parameters Roche GS Junior Roche GS FLX Read length 700 nt nt Reads per run Throughput 70 Mbp 700 Mbp 7
9 SEMICNDUCTR SEQUENCING (LifeTechnologies IonTorrent) No camera, just a ph sensor in each well SEMICNDUCTR SEQUENCING SEMICNDUCTR SEQUENCING 8
10 SEMICNDUCTR SEQUENCING SEMICNDUCTR SEQUENCING SEMICNDUCTR SEQUENCING IN TRRENT Parameters Personal Genome Machine (PGM) Proton Ion 314 Chip Ion 316 Chip Ion 318 Chip PI Chip Read length 200 nt/400 nt 200 nt/400 nt 200 nt/400 nt 200 nt Reads per run thousand 2 3 million million million Throughput 30 50/ Mbp / Mbp 0.6 1/1.2 2 Gbp up to 10 Gbp 9
11 Illumina Solexa REVERSIBLE TERMINATR SEQUENCING Library amplification on solid surface ILLUMINA SEQUENCING reversible terminator sequencing fluorescently labeled 3 -blocked reversible terminators ILLUMINA SEQUENCING reversible terminator sequencing 3 -blocked reversible terminators incorporation of a single nucleotide detection of the fluorescence cleavage of fluorophore, blocker 10
12 ILLUMINA SEQUENCING data processing ILLUMINA SEQUENCING sysytems with different throughputs Parameters MiSeq NextSeq500 HiSeq2500 HiSeq4000 HiSeq X Read length up to 2x300 nt up to 2x150 nt up to 2x125 nt up to 2x150 nt up to 2x150 nt Clusters per run million 400 million 4 billion billion billion Throughput Gbp Gbp Tbp Tbp Tbp SEQUENCING BY LIGATIN LifeTechnologies (SLiD) AC CA GT TG 11
13 SEQUENCING BY LIGATIN LifeTechnologies (SLiD) SEQUENCING BY LIGATIN LifeTechnologies (SLiD) SEQUENCING BY LIGATIN LifeTechnologies (SLiD) 12
14 SEQUENCING BY LIGATIN LifeTechnologies (SLiD) A C C A G T T G SEQUENCING BY LIGATIN LifeTechnologies (SLiD) SEQUENCING BY LIGATIN LifeTechnologies (SLiD) A T C G G C T A 13
15 SEQUENCING BY LIGATIN LifeTechnologies (SLiD) SEQUENCING BY LIGATIN LifeTechnologies (SLiD) SEQUENCING BY LIGATIN LifeTechnologies (SLiD) 14
16 SEQUENCING BY LIGATIN LifeTechnologies (SLiD) SEQUENCING BY LIGATIN LifeTechnologies (SLiD) Parameters Read length Reads per run Throughput SLiD5500xl 1x75 or 2x billion Gbp THIRD GENERATIN DNA SEQUENCING: SINGLE MLECULE SEQUENCING Pacific Biosciences has developed Single Molecule Real Time (SMRT ) DNA sequencing technology xford Nanopores developed 'Strand sequencing' which is a technique that passes intact DNA polymers through a protein nanopore, sequencing in real time as the DNA translocates the pore. 15
17 3rd GENERATIN SEQUENCING Pacific Biosciences 3rd GENERATIN SEQUENCING Pacific Biosciences SMRT Cell: Contains arrays of thousands of zero-mode waveguides (ZMWs). A ZMW is a cylindrical hole, tens of nanometers in diameter, fabricated using semiconductor manufacturing technologies using 100 nm metal film deposited on a transparent silicon dioxide substrate. Each ZMW becomes a nanophotonic visualization chamber blocking light from penetrating past just a few nanometers due to the phenomenon of waveguide cutoff well known in microwave engineering. This provides a detection volume of just ~100 zeptoliters (10-21 liters). Limit of detection zone ZMW with 1 DNA polymerase attached to te bottom 3rd GENERATIN SEQUENCING Pacific Biosciences 4 dntps labeled with different, phospholinked fluorophores 16
18 3rd GENERATIN SEQUENCING Pacific Biosciences background signal from fluorescent dntps from above the detection zone DNA polymerase binds fluorescent dntp that results in light burst DNA polymerase cleaves off phospholinked linked fluorophore DNA polymerase binds fluorescent dntp that results in light burst DNA polymerase cleaves off phospholinked linked fluorophore 3rd GENERATIN SEQUENCING Pacific Biosciences Simple workflow: library preparation then sequencing (no amplification) DNA ISLATIN DNA FRAGMENTATIN END REPAIR, HAIRPIN LIGATIN 3rd GENERATIN SEQUENCING Pacific Biosciences Key advantages of SMRTbell templates: structurally linear topologically circular structural homogeneity of templates provides sequences of both forward and reverse strands in the same trace 17
19 3rd GENERATIN SEQUENCING xford Nanopore 3rd GENERATIN SEQUENCING xford Nanopore Nanopore sensing Ionic current flows through the pore Introduce analyte of interest into the pore Identify target analyte by the characteristic disruption of the electrical current 3rd GENERATIN SEQUENCING xford Nanopore adding hairpin to one fragment end allows sequencing of both strands 18
20 3rd GENERATIN SEQUENCING xford Nanopore 3rd GENERATIN SEQUENCING xford Nanopore 3rd GENERATIN SEQUENCING xford Nanopore 19
21 3rd GENERATIN SEQUENCING xford Nanopore NEXT GENERATIN SEQUENCING APPLICATINS discovery phase I. GENME SEQUENCING: 1. de novo genome sequencing no previous sequence information available usually combining long and short reads 2. genome re-sequencing sequencing the whole genome and compare it to a reference sequence 3. targeted re-sequencing sequence selected parts of the genome and compare it to a reference sequence hybridization and PCR based methods 4. determination of DNA modifications (epigenetics) bisulphite sequencing (identification of 5-methyl-cytosine sites) PacBio sequencing: NT modifications are recognized based on polymerase kinetics identification of mutations, structural variants: 2., 3. NEXT GENERATIN SEQUENCING APPLICATINS discovery phase targeted re-sequencing SureSelect target enrichment (Agilent) an example fragmented DNA labeled probes (baits) target recovery sequencing target capture hybridization unbound fraction (discarded) 20
22 NEXT GENERATIN SEQUENCING APPLICATINS discovery phase II. TRANSCRIPTME SEQUENCING: RNA sequencing sequencing the RNA pools of cells, tissues, organs micro/small RNA sequencing sequencing the small RNA pools of cells, tissues, organs sequence Deep-SAGE/CAGE sequencing SAGE: Serial Analysis of Gene Expression, CAGE: cap analysis gene expression sequencing tags from the 3 or the 5 ends from mrna pools of cells, tissues, organs Ribosome profiling sequencing of ribosome-protected mrna fragments investigation of the expressed genome genome-wide or targeted comparison of gene expression profiles between different cells, tissues, organs, conditions... NEXT GENERATIN SEQUENCING APPLICATINS discovery phase III. DNA-PRTEIN INTERACTINS: 1. Chromatin-immunoprecipitation sequencing (Chip-Seq) after DNA fragmentation and protein-dna crosslinking protein-bound fragments are isolated with the help of specific antibodies histon modifications (acetylation, methylation, phosphorilation, ubiquitination) DNA binding proteins/transcription factors 2. MNase sequencing Micrococcal nuclease (MNase) digests naked DNA nucleosome-associated DNA is protected, enriched and sequenced 3. ATAC sequencing assay for transposase-accessible chromatin using sequencing (ATAC-seq) captures open chromatin sites based on direct in vitro transposition of sequencing adaptors into native chromatin NEXT GENERATIN SEQUENCING APPLICATINS discovery phase ATAC-SEQ 21
23 NEXT GENERATIN SEQUENCING APPLICATINS: in clinical practice INHERITED DISEASES ver 6000 monogenic inherited diseases CFTR: Cystic fibrosis transmembrane conductance regulator high occurance of carriers FDA approved NGS detection method for 139 clinically relevant CFTR variants TueSight ne Sequencing panel (not for diagnostic purposes but it can help...) 4813 clinically relevant genes associated to a clinical phenotype Ion AmpliSeq Inherited Disease Panel (not for diagnostic purposes but it can help...) Broad survey of significant genetic disease genes with extensive 300+ gene panel Human leukocyte antigen (HLA) typing HLAs play important role in the distinction of self and non-self cells (infectious dieseases, graft ejection during transplantation, autoimmunity) difficult to typedue to the high levels of sequence homology but NGS provides accurate, unambiguous, phase-resolvedhla typing in a single assay Many more to come (autism, cardiomyopathy, sudden cardiac arrest...) NEXT GENERATIN SEQUENCING APPLICATINS: in clinical practice IN VITR FERTILIZATIN AND NNINVASIVE PRENATAL DIAGNSTICS problem of aneuploidy (abnormal number of a chromosome) Preimplantation Genetic Screening (PGS) for In Vitro Fertilization (IVF) Chromosome aneuploidy (abnormal number of chromosomes) is a major cause of in vitro fertilization (IVF) failure, pregnancy loss, and, in rare cases, abnormal pregnancy The VeriSeq PGS Kit uses NGS on the Illumina MiSeq System to screen all 24 chromosomes for aneuploidy in a single assay. The assay can be used on a single cell or a few cells from an embryo. Non-invasive prenatal testing (NIPT) During the early stages of pregnancy, fetal cfdna represents approximately 3% of the genomic content found within maternal plasma DNA Through the power of NGS, this fetal DNA can be analyzed to identify potential chromosomal aberrations and the gender of the fetus (XX, XY) (T21 Down syndrome, T18 Edwards syndromet13 Patau syndrome, Monosomy X). sequencing 28 million tags (1x25 bp) per sample NEXT GENERATIN SEQUENCING APPLICATINS: in clinical practice CANCER GENMICS cancer: disease of the genome certain mutations predispose to cancer mutations affect the progression of the disease and the prognosis of the patient there are targeted therapies with >200 drugs certain mutant oncogene proteins are targeted by given drugs certain mutations cause the inefficacy of given drugs Cancer panels to detect germline mutations to detect somatic mutations to detect mutations associated with certain cancer types (for example, colon, lung, myeloid,...) 22
24 Thank you for your attention! This work is supported by the European Union, co-financed by the European Social Fund, within the framework of " Practiceoriented, student-friendly modernization of the biomedical education for strengthening the international competitiveness of the rural Hungarian universities " TÁMP C-13/1/KNV project. 23
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