next generation sequencing: new possibilities in medicine · ngs (next generation sequencing) no...

Next generation sequencing:new possibilities in medicine

Attila KeresztInstitute of Biochemistry

12th April, 2017

„Practice-oriented, student-friendly modernization of the biomedical education for strengthening the international competitiveness of the rural Hungarian universities”TÁMOP-4.1.1.C-13/1/KONV-2014-0001

DNA SEQUENCING

DNA sequencing is the process of reading nucleotide bases in a DNA molecule

GENOME SEQUENCINGSequencing the whole genetic material of an organism

1953: Structure of DNA1973: First sequence of 24 bp published (lac Operator)

1977: Phage ΦX-1741995: Haemophilus influenzae

1996: Methanococcus jannaschii1996: Saccharomyces cerevisiae

1997: Escherichia coli1998: Caenorhabditis elegans

2000: Drosophila melanogaster2000: Arabidopsis thaliana2001: Homo sapiens (draft)

2002: Mus musculus2006: Homo sapiens (complete)

2009: 1000th prokaryotic genome (complete)

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)

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

Sanger (First generation) sequencing

NGS (Next Generation Sequencing)

No competition, but complementation

Long read, low coverage

Short read, huge coverage(Second generation)

THE EVOLUTION OF GENOME SEQUENCING

Very longong read, large coverage(Third generation)

FIRST GENERATION DNA SEQUENCING: LIBRARY CONSTRUCTION IN A HOST

FIRST GENERATION DNA SEQUENCING:SEQUENCE ASSEMBLY STRATEGY

FIRST GENERATION DNA SEQUENCING TECHNOLOGYIS BASED ON THE ACTIVITY OF DNA POLYMERASE

FIRST GENERATION DNA SEQUENCING TECHNOLOGYIS BASED ON THE ACTIVITY OF DNA POLYMERASE

AND THE USE OF DIDEOXY NUCLEOTIDES (SANGER)

N

NN

N

NH2

O

HOH

HH

HH

OPH

O-

O

POP-O

O

O-

O

O-

N

NN

N

NH2

O

HH

HH

HH

OPO

O-

O

POP-O

O

O-

O

O-

dATP ddATP

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

CHAIN TERMINATION

A

C

G

T

FIRST GENERATION DNA SEQUENCING TECHNOLOGY:4 reactions: each contains 4 dNTPs and 1 ddNTPprimer or one dNTP radioactively labeledDNA molecules of different length are separated by gel electrophoresis

FIRST GENERATION DNA SEQUENCING TECHNOLOGY:4 reactions: each contains 4 dNTPs and 1 ddNTPPrimers in the 4 reactions are labeled with different fluorescent dyesDNA molecules of different length are separated by gel electrophoresis

A

C

G

T

A CG T

FIRST GENERATION DNA SEQUENCING TECHNOLOGY:1 reaction: contains 4 dNTPs and 4 ddNTPsthat are labeled with 4 different fluorescent dyesDNA molecules of different length are separated by gel/capillary electrophoresis

C Y C L E S E Q U E N C I N GC Y C L E S E Q U E N C I N G

SECOND GENERATION DNA SEQUENCING: LIBRARY CONSTRUCTION WITHOUT A HOST

ISOLATE DNA/RNA TO BE SEQUENCEDISOLATE DNA/RNA TO BE SEQUENCED

FRAGMENTATION OF DNALIGATION OF ADAPTORS, PRIMERS (BARCODE)

SIZE SELECTION

EMULSION PCR SOLID-PHASE PCR

PYRO-SEQUENCING

454Roche

SEMICONDUCTORSEQUENCING

IonTorrentLife Technologies

SEQUENCINGBY LIGATION

SOLiDLife Technologies

REVERSIBLETERMINATORSEQUENCINGIllumina Solexa

sequencingin picowells

sequencingon solid surface

sequencingby synthesisby ligation

LIBRARY PREPARATIONLIBRARY PREPARATION

CLONAL AMPLIFICATION OF THE LIBRARYCLONAL AMPLIFICATION OF THE LIBRARY

LIBRARY PREPARATIONISOLATE DNA/RNA RANDOM FRAGMENTATION

END REPAIRADAPTORSPRIMERS

LIGATION

SIZESELECTION

CLONALAMPLIFICATION

CLONAL AMPLIFICATION: EMULSION PCR

CLONAL AMPLIFICATION: SOLID-PHASE PCRcluster generation for Illumina sequencing

originaltemplate

originaltemplate

newstrand

newstrand

CLONAL AMPLIFICATION: SOLID-PHASE PCRcluster generation for Illumina sequencing

4 nucleotides flow sequentially

CYCLE SEQUENCING35-1000cycles

A

step1

add A

detect A

washaway A

C

step2

add C

detect C

washaway C

G

step3

add G

detect G

washaway G

T

step4

add T

detect T

washaway T

PYROSEQUENCING (ROCHE 454)

C

T

A

G

4 nu

cleo

tides

flow

seq

uent

ially

hν

PYROSEQUENCING (ROCHE 454)SIGNAL DETECTION (PYROGRAM)

Parameters Roche GS Junior Roche GS FLXRead length 700 nt 700 ‐1000 ntReads per run 100 000 1 000 000Throughput 70 Mbp 700 Mbp

SEMICONDUCTOR SEQUENCING(LifeTechnologies IonTorrent)

No camera, just a pH sensor in each well

SEMICONDUCTOR SEQUENCING

SEMICONDUCTOR SEQUENCINGION TORRENT

ProtonIon 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 ntReads per run 400‐550 thousand 2‐3 million 4‐5.5 million 60‐80 millionThroughput 30‐50/60‐100 Mbp 300‐600/600‐1 000 Mbp 0.6‐1/1.2‐2 Gbp up to 10 Gbp

ParametersPersonal Genome Machine (PGM)

Illumina SolexaREVERSIBLE TERMINATOR SEQUENCING

Library amplification on solid surface

ILLUMINA SEQUENCINGreversible terminator sequencing

fluorescently labeled3′-blocked reversible terminators

ILLUMINA SEQUENCINGreversible terminator sequencing

3′-blockedreversible

terminators

incorporationof a singlenucleotide

detectionof the

fluorescence

cleavage offluorophore,

blocker

ILLUMINA SEQUENCINGdata processing

ILLUMINA SEQUENCINGsysytems with different throughputs

Parameters MiSeq NextSeq500 HiSeq2500 HiSeq4000 HiSeq XRead length up to 2x300 nt up to 2x150 nt up to 2x125 nt up to 2x150 nt up to 2x150 nt

Clusters per run 22‐25 million 400 million 4 billion 4.3‐5 billion 5.3‐6 billionThroughput 13‐15 Gbp 100‐120 Gbp 0.9‐1 Tbp 1.3‐1.5 Tbp 1.6‐1.8 Tbp

SEQUENCING BY LIGATIONLifeTechnologies (SOLiD)

ACCAGTTG


A CC AG TT G


A TC GG CT A


Parameters SOLiD5500xlRead length 1x75 or 2x50Reads per run 3.2 billionThroughput 160‐320 Gbp

THIRD GENERATION DNA SEQUENCING: SINGLE MOLECULE SEQUENCING

Pacific Biosciences has developedSingle Molecule Real Time (SMRT™)

DNA sequencing technology

Oxford 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.

3rd GENERATION SEQUENCINGPacific 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 polymeraseattached to te bottom


4 dNTPs labeled with different, phospholinked fluorophores


backgroundsignal fromfluorescentdNTPs fromabove the

detection zone

DNA poly-merase bindsfluorescentdNTP thatresults inlight burst

DNA poly-merase

cleaves offphospholinked

linkedfluorophore

DNA poly-merase bindsfluorescentdNTP thatresults inlight burst

DNA poly-merase

cleaves offphospholinked

linkedfluorophore


Simple workflow: library preparation then sequencing (no amplification)

DNA ISOLATION

DNA FRAGMENTATION

END REPAIR, HAIRPIN LIGATION


Key advantages of SMRTbell templates:structurally lineartopologically circularstructural homogeneity of templatesprovides sequences of both forwardand reverse strands in the same trace

3rd GENERATION SEQUENCINGOxford Nanopore


Nanopore sensingIonic current flows through the pore

Introduce analyte of interest into the poreIdentify target analyte by the characteristic disruption of the electrical current


adding hairpin to one fragment end allows sequencing of both

strands

NEXT GENERATION SEQUENCINGAPPLICATIONSdiscovery phase

I. GENOME SEQUENCING:1. de novo genome sequencing

no previous sequence information availableusually combining long and short reads

2. genome re-sequencingsequencing the whole genome and compare it to a reference sequence

3. targeted re-sequencingsequence 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.

targeted re-sequencingSureSelect target enrichment (Agilent)

an example


fragmented DNA labeledprobes (baits)

unbound fraction(discarded)

hybridization

target capture

target recovery

sequencing


II. TRANSCRIPTOME SEQUENCING:

RNA sequencingsequencing the RNA pools of cells, tissues, organs

micro/small RNA sequencingsequencing the small RNA pools of cells, tissues, organs sequence

Deep-SAGE/CAGE sequencingSAGE: 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 profilingsequencing of ribosome-protected mRNA fragments

investigation of the expressed genomegenome-wide or targeted comparison of gene expression profiles

between different cells, tissues, organs, conditions...


III. DNA-PROTEIN INTERACTIONS:

1. Chromatin-immunoprecipitation sequencing (Chip-Seq)after DNA fragmentation and protein-DNA crosslinking protein-bound fragments are

isolated with the help of specific antibodieshiston modifications (acetylation, methylation, phosphorilation, ubiquitination)

DNA binding proteins/transcription factors

2. MNase sequencingMicrococcal nuclease (MNase) digests naked DNA

nucleosome-associated DNA is protected, enriched and sequenced

3. ATAC sequencingassay for transposase-accessible chromatin using sequencing (ATAC-seq)

captures open chromatin sitesbased on direct in vitro transposition of sequencing adaptors into native chromatin


ATAC-SEQ

NEXT GENERATION SEQUENCING APPLICATIONS: in clinical practice

INHERITED DISEASESOver 6000 monogenic inherited diseases

CFTR: Cystic fibrosis transmembrane conductance regulatorhigh occurance of carriers

FDA approved NGS detection method for 139 clinically relevant CFTR variants

TueSight One 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) typingHLAs 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 ...)


IN VITRO FERTILIZATION AND NONINVASIVE PRENATAL DIAGNOSTICSproblem 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 pregnancyThe 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 DNAThrough 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


CANCER GENOMICScancer: disease of the genome

certain mutations predispose to cancermutations affect the progression of the disease and the prognosis of the patient

there are targeted therapies with >200 drugscertain mutant oncogene proteins are targeted by given drugs

certain mutations cause the inefficacy of given drugs

Cancer panelsto detect germline mutationsto detect somatic mutations

to detect mutations associated with certain cancer types(for example, colon, lung, myeloid, ...)

Thank you for your attention!

This work is supported by the European Union, co-financed by the European Social Fund, within the framework of " Practice-

oriented, student-friendly modernization of the biomedical education for strengthening the international

competitiveness of the rural Hungarian universities " TÁMOP-4.1.1.C-13/1/KONV-2014-0001 project.

next generation sequencing: new possibilities in medicine · ngs (next generation sequencing) no...

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