Secvențierea ADN-ului

Secvențierea ADN-ului este o metodă

ce permite determinarea ordinii
nucleotidelor prezente în moleculele de
ADN și ARN izolate de la animale,
plante, bacterii, sau orice altă formă de
viață.
Secvențierea ADN-ul permite stabilirea
structurii/ compozitiei unei regiuni,
genei, grupuri de gene, cromozomi
întregi sau chiar genomuri.
 Istoric

• Ray Jui Wu- Cornell University

• 1970- Journal of Molecular Biology - When this terminal region is present as a single strand, as in

bacteriophage lambda, Escherichia coli DNA polymerase can be used to repair the single-stranded region

with the addition of radioactive nucleotides to the 3′-end copying the protruding 5′-terminated single

strand. The partially labeled DNA can be degraded with nucleases, the radioactive oligonucleotides

isolated, and their sequence determined.-specific-primer-extension principle- secventiat 12 nucleotide

Frederik Sanger –MRC Center Cambridge, DNA sequencing with chain-terminating

inhibitors" in 1977.

Walter Gilber si Allan Maxam, Hardvard- "DNA sequencing by chemical degradation“

Primul genom secventiat a a fost in 1977- bacteriofag φX174 in 1977

Istoric: Secvențierea Sanger

Secvențierea Sanger este o metodă de

secvențiere ADN bazată pe incorporarea
selectivă a di-deoxinucleotidelor de către
enzima ADN polimerază în timpul replicării ADN
in vitro. Metoda a fost dezvoltată de către
Frederick Sanger și colegii săi în 1977.

Metoda clasică de secvențiere Sanger ia un șablon

simplu de ADN, un primer ADN, enzima ADN
polimerază, deoxinucleozidetrifosfate normale
(dNTP) și di-deoxinucleozidetrifosfate modificate
(ddNTP), cele din urmă terminând prematur
elongarea șirului ADN. ddNTP-urilor le lipsesc grupul
hidroxil (OH) de la capătul 3', deci nu pot forma
legătura necesară dintre două nucleotide, ceea ce
împiedică polimeraza ADN să extindă ADN-ul.
Rezultatul unei
ddNTP-urile sunt etichetate radioactiv sau
secventieri Sanger
fluorescent pentru a putea fi detectate in gel sau
automat.
 Fluorescent Sanger Sequencing

Load on gel
(modern machines use
capillaries, not slab gels)
dGTP
dATP +
dTTP
dCTP Direction
of electro-
phoresis
One-tube sequencing reaction
(note: cycle sequencing with modified Taq Polymerase)
Istoric: Electroforeza capilara (CE)
Evolutia secventierii

NGS
Routine
Tests
Clinical
Diagnostic

Basic
Research
Dezvoltarea tehnicilor de secventiere

2001 2011 2016

100 mil $ 10,000 $ 1000 $

8
WGS – Whole Genome Sequence

9
Second/ Next generation sequencing technology

NGS- secventierea prin sinteza -permite secventierea intregului genom prin fragmentarea
acestuia si apoi polimerizarea acestora

Illumina (Solexa) sequencing- https://www.youtube.com/watch?v=9YxExTSwgPM

Roche 454 sequencing- https://www.youtube.com/watch?v=KzdWZ5ryBlA
Ion torrent: Proton / PGM sequencing - ttps://www.youtube.com/watch?v=DyijNS0LWBY
SOLiD sequencing- https://www.youtube.com/watch?v=nlvyF8bFDwM&t=9s

Avantaje Dezavanataje
-Acoperire ridicata (50-1000×) - Citeste fragmente mici (25-500pb);
comparativ cu Sanger 10×; - erori in citirea fragmentelor
-pret scazut repetitive;
-Volum mare de probe (0.4-300Gb) - Genereaza un volum mare de date
(2-30Gb) si necesita softuri multiple
- timp redus de analiza (3h- 8zile)
pt procesarea datelor
Tipuri de probe

Small
RNA

ChIP
DNA
ARNm

ADNg
 Illumina Sequencing Technology
Robust Reversible Terminator Chemistry Foundation
3’ 5’

DNA
(0.1-1.0 ug) A G
T
C G
A
C
T T
A
C C
G
G A
T
A A
C
T C
C
C G G
A
T
T C
Sample G
A
preparation Single molecule
Cluster growtharray T
5’

Sequencing
1 2 3 4 5 6 7 8 9
T G C T A C G A T …

Image acquisition Base calling

 Sequencing

250+ Million Clusters

Per Flow Cell

20 Microns

100 Microns
1
Library Prep

2
Cluster generation

3
Sequencing

4
Data analysis
1
Library Prep

2
Cluster generation

3
Sequencing

4
Data analysis
LIBRAIRY PREPARATION
A critical step for good sequencing

Dual Index Library shown

Same general template architecture regardless of application or

input starting material
 Sample preparation
Library prep:
Nextera DNA sample prep Illumina KIT

No separated fragmentation
required

Tag with mix enzymes

Incorporate indexes and

primers
RECOMMANDATIONS –
Sample preparation

Prep kits on Illumina sequencers

– TruSeq DNA ans SMALL RNA sample preparation

– TruSeq Custom Amplicon
– TruSeq Amplicon Cancer Panel
– Nextera DNA
– Nextera XT DNA Sample Prep

Quantification methods recommanded

 Qubit

To obtain a good cluster density and high performance of sequencing,

an accurate quantification of library quality is required
1
Library Prep

2
Cluster generation

3
Sequencing

4
Data analysis
CLUSTER GENERATION

Cluster generation in flowcell

Glass slide with lines and

canals

Each ligne coated of

complementary oligos to
library adapters
 Forward
Reverse
strand
strand

200,000 clusters per tile 62.5 million reads ear line 100 bp reads -> 12.5 Gb per line
1
Library Prep

2
Cluster generation

3
Sequencing

4
Data analysis
 SEQUENCING BY SYNTHESIS

3’
T
DNA (1 ng – 1 µg)

C
Sample Cluster
Single molecule
growth array
preparation T
(3 million – 3 billion)
n=250

5’ Base calling
Data analysis
1
Library Prep

2
Cluster generation

3
Sequencing

4
Data analysis
PRIMARY DATA ANALYSIS WORKFLOW

Image analysis,
Initiation of run cluster template
folders generation and
matrix calculation

Intensity extraction
Basecalling
and correction

Quality Scoring Filtering

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SOFTWARES – DATA ANALYSIS

• Images
MiSeq Control
• Base Calling
Software (MCS)
• Q score

Sequence
Analysis Viewer • Data Run Monitoring
(SAV)

MiSeq Reporter
Software (MSR) • Alignment
• Data Assembling
BaseSpace

ANNOTATION & variant filters

 NOVASEQ - 2017
HiSeq X Ten
iSeq 100 - 2018
Seul système NGS sur le
marché

HiSeq 2500
Hautement efficace,
performant

NextSeq 550
Intégré tout en un

MiSeq Dx
NextSeq 500
Seul système NGS
Rapide, efficace
certifié CE-IVD sur le
abordable
marché

MiSeq
Puissant, rapide
évolutif
MiSeq FGx
MiniSeq
Compatibilité
Puissant, rapide
CODIS, forensic
• Specifications:
– Integrated system: Workflow easy to use
– Throughput: 1,5-15 Gb
– Able to sequence all clinical exome
– 150.000 time performant than CE system
– Covers large range of applications

N.B: Double role identify fragments size plus sequencing

Applicatii in Biologia Medicala

Epidemiologie

PGS/PGD
Imunogenetica
NIPT

Aplicatii ale secventierii

Identificarea
Diagnostic umana

Oncologie
552 genes

101 genes
FORENSIC SCIENCES (Forensic panel)

NGS Data compatibility with mondial existed database

MICROBIOLOGIE
 Aplicatiile NGS in studiie de taxonomie

Tulpini individuale Comunitati

Determini %GC Identificare si cuantificare
Hibridizarea in silico Evidentiere MO
Structura gene necultivabile
ribozomale Descoperirea unor noi
MLSA si MLST specii
SNP genotyping

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Third generation sequencing technology

PacBio –SMRT- https://www.youtube.com/watch?v=v8p4ph2MAvI

Nanopore-https://www.youtube.com/watch?v=E9-Rm5AoZGw
Quantapore - citirea optica (cost mai mic si viteza mai mare de transmisie a datelor)
Stratos - https://www.youtube.com/watch?v=ADzIXItP4hE

• Avantaje • Dezavanataje
- Citeste framente lungi (50-150kb) • acuratete scazuta Nanopore
- Timp redus de analiza (3-4h) • Cost ridicat- PacBio
- Usor de manipulat
- Pret si dimensiune scazuta (nanopore)
- Detecteaza modificarile epigenetice
- asamblarea de novo este posibila
 Metode de secventiere Third generation
sequencing
Second generation Framente lungi (50-
sequencing/NGS 150kb)
Fragmente 50-300pb Timp redus de
analiza (3-4h)
Volum mare de date
(0.4-600Gb) Evidentiaza
secventele repetitive
Sanger Timp redus de analiza
(3h- 12 zile) Face asamblare de
Secventiere prin
novo
sinteza Acoperire 30-1000x
Pret 1000 €/genom
Fragmente scurte Pret 100 €/genom bacterian
300-900pb bacterian
PacBio, Nanopore,
Acoperire 10x Illumina, IonTorrent, Quantapore, Stratos
Pret 15€ /400pb Roche 454, SOLiD.

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