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CNG NGH C TRNH T GEN TH H MI

(NEXT-GENERATION SEQUENCING)

TS. Dng Quc Chnh

Sinh hc Phn t, Tin-Sinh hc

TNG QUAN V C TRNH T GEN

Mc ch:

Xc nh trnh t sp xp cc nucleoCde ca h gen
Gii hn cng ngh:

Cc h thng hin ti ch c c cc on gen ngn <1kb

Gii php:
Phn mnh ADN thnh cc on ngn;
c trnh t;
S dng phn mm my ^nh lp gip thnh h gen hon
chnh

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LCH S PHT TRIN CNG NGH C TRNH T GEN

M. Stra(on et al. Nature 458 (2009)

PHT TRIN CC H THNG NGS

Roche 454/FLX (2004)

Illumina Genome analyzer (2006), HiSeq, MiSeq, Nextseq

Applied Biosystem SOLiD (2007)
Helicos Heliscope (2008)
Pacic Bioscience SMRT (2010)

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CHI PH THC HIN GII TRNH T GEN

2001: Human Genome Project
2.7G$, 11 years

Chi ph (Log10)

10

2007: 454
1M$, 3 months

8
6

2008: ABI SOLiD

60K$, 2 weeks

2001: Celera
100M$, 3 years

2009: Illumina,
Helicos
40-50K$

2005

2000

2010: 5K$,
a few days?

2012: 100$,
<24 hrs?
2010

H THNG NGS PH BIN TI VIT NAM

VIT NAM

Illumina MiSeq, Nextseq

Life technologies: Ion Torrent

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QUY TRNH CHUN B TH VIN ADN

D Koboldt et al. Briengs in Bioinforma>cs (2010)

QUY TRNH CHUN B TH VIN ADN

D Koboldt et al. Briengs in Bioinforma>cs (2010)

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PHNG PHP KHUCH I ADN

Emulsion PCR




Ligaon

Extension/Enrichment

100-200 triu bead c gn ln phin knh hoc cc h trn khay PTP

(PicoTiterPlate).

PHNG PHP KHUCH I ADN

Solid Phase amplicaon

Gn adaptor

Khuch i v to cluster

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PHNG PHP C TRNH T GEN

Solid Phase amplicaon

C TRNH T ADN SI N
Single molecule sequencing

Cng ngh c trnh t (thng mi)
mi nht hin nay.
c trnh t trn si n ADN
c trnh t thng qua phn ng ko
di chui

Nanowell

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ROCHE 454/FLX

Roche/454 GS FLX

ROCHE 454/FLX
Chuyn th vin ADN vo cc ging c trnh t














Sau khuch i, th vin ADN c chuyn vo cc ging c trnh t.
1 bead/ging

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ROCHE 454/FLX
B sung vi ht gn enzyme














Sulphurylase, luciferase enzyme coated beads
Trnh t Nu c trong qu trnh ko di chui, bng CCD camera.

ILLUMINA

Illumina
Genome
Analyzer

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ILLUMINA
c trnh t trn h thng Illumina.

ILLUMINA
c trnh t trn h thng Illumina.

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ILLUMINA
c trnh t trn h thng Illumina.

Pht hin Nu bng n hiu hunh quang, chnh xc cao.

Hiu sut cao

APPLIED BIOSYSTEM - SOLiD

SOLiD
Applied Biosystems

10

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APPLIED BIOSYSTEM - SOLiD

c trnh t trn h thng SOLiD

Vi ht gn ln phin knh theo lin kt cng ho tr v lin kt gDNA-ch gn

(target linker)

APPLIED BIOSYSTEM - SOLiD

c trnh t theo phng php gn mi

11

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APPLIED BIOSYSTEM - SOLiD

Round 1

Round 2

Round

(staggered by 1bp)

1
2

APPLIED BIOSYSTEM - SOLiD

c trnh t mi Nu lp li 5 ln vi mi primer (n, n-1, n-4).

Kt qu: Cc Nu u c c lp li vi kh nng pht sinh li thp.

12

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PACIFIC BIOSCIENCE

Pacic Bioscience
PacBio RS II

PACIFIC BIOSCIENCE

Polymerase c nh vo y ging v mi phn t ADN c c trnh t

ti ch theo thi gian thc (real-Cme)
Khng cn khuch i ADN bng PCR -> gim thiu sai st

13

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PACIFIC BIOSCIENCE

NGS vs SANGER

Capillary
454
Illumina
SOLiD

Read length
(bases)
1000
450
75 (max 600)
50

Runme
(days per gigabase)
500
2
0.5
0.5

Cost
($ per 1000 bases)
$0.10
$0.02
$0.001
$0.001

14

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KHUCH I GEN CH

Sequence capture enrichment (SCE)

Agilent: eArray SureSelect capture
Illumina: Target enrichment
Roche: NimbleGen

NHNG YU T QUAN TRNG

Chun b th vin.
Processing of sequence reads
Kiu c trnh t (vd. mate pairs)
Lp ghp d liu
Phn ch bin i di truyn

15

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MT S VN TN TI CA NGS

Sai s cao (so vi Sanger sequencing)

<0.1 - 10% ph thuc mi h thng
Sai s h thng (systemac bias)
on c ngn (max 600 bases)
D liu thu c rt ln
Qun l d liu
Phn ch d liu

CI THIN CHT LNG C TRNH T VI NGS

Tnh ton bao ph (coverage) da trn kt qu lp ghp.

Ung th: yu cu bao ph cao
Loi tr cc kt qu c trng lp (duplicate reads).
Pht sinh do khuch i ADN.
S dng cc kt qu c cht lng cao
S dng cc on lp trong h gen.
QC30

16

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CNG NGH MI NANOPORE SEQUENCING

Si DNA inserted in a nanopore

in lipid membrane
speed control provided by a
phi29 DNA polymerase
TranslocaCon via an electrical
eld and polymerase DNA
sequence via changes in the
ionic current

CNG NGH MI NANOPORE SEQUENCING

17

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NG DNG NGS TRONG NGHIN CU/LM SNG

Xc nh cc bin i di truyn

Ton b h gen

Exome

Nhm gen

Transcriptomes (vd. RNASeq)

Chip-seq
Epigenomes (methylaCon)
ng dng khc!

XC NH BT THNG DI TRUYN BNG NGS

t bin im
Chn/xo Nu (indels)
Bt thng s copy (CNVs)
Bt thng cu trc (SV)
Bn trong NST
Chn/xo on ln
Lp on
o on
Trao i cho NST
i xng
Bt i xng

18

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XC NH BT THNG DI TRUYN BNG NGS

P Sohrab et al. Nature, 461 (2010)

XC NH BT THNG DI TRUYN BNG NGS

Xc nh nhanh cc t bin
im
Phn ^ch cc yu t gy ung
th
Xc nh t bin gy bnh

Normal

Tumor

19

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XC NH BT THNG DI TRUYN BNG NGS

Silent

= synonymous

SubsCtuCon = nonsynonymous
Nonsense

= premature stop

hp://commons.wikimedia.org

XC NH BT THNG DI TRUYN BNG NGS

20

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MT S H THNG NGS TI VIT NAM

C quan/ T chc

H thng gii trnh t

Vin Nghin cu h gen

Ion torrent, NextSeq

Vin Huyt hc Truyn mu Trung

ng

MiSeq

Vin Khoa hc Hnh s

MiSeq

Vin Ph sn H Ni

MiSeq

Vin Cng ngh sinh hc

MiSeq, HiSeq

21