Solexa测序原理及流程
深圳华大基因研究院
Agenda
DNA样品制备 Cluster Formation
Binding to flowcell Bridge Amplification
SBS (Sequencing By Synthesize)
One Cycle One Base
P5
3’ddN
O
ttactatgccgctggtggctctagatgt aatgatacggcgaccaccgagatctaca
s(T)10
agtgtagatctcggtggtcgccgtatcatt
gaagagctcgtatgccgtcttctgcttgaaaaaaaaaa
NaIO4
DNA insert
Melt and hybridize 3’ to primers
5’
1st
PCR oligo 2
P7
round
PCR 5’
3’
3’ 5’
3’ extension
3’
3’
5’
3’ extension
Insert
3’ 5’
3’ 5’
P7
P5
5’ 3’
SBS oligo
P7 反向互补序列
5’ 3’ 5’
Pair End
DNA样品制备
Cluster formation
Cluster Formation
Bridge Amplification Cycle
DNA synthesization
P7 P5
SBS oligo
Sequencing By Synthesization
三种特殊序列
P7序列：flowcell上结合的序列，边合成边边 测序（SBS）过程模板序列的5’端。
3’ 5’
SBS oligo P7 反向互补序列
5’po4 3’ 5’po4 3’A 5’
Phosphorylation T4 polynucleaotide kinase, ATP
3’ po45’
2.Add 3’ Adenosine with
Klenow (3’exo- ) and dATP
A3’
ctaga tctagccttctcgcagcacatccctttc gatct agatcggaagagcgtcgtgtagggaaag
P7
sttttttttttcaagcagaagacggcatacgagctcttc
aaaaaaaaaagttcgtcttctgccgtatgctcgagaagg sttttttttttcaagcagaagacggcatacgagctcttcc
3’ddN
O
P7
P7
hybridization of sequencing primer
3’ddN
O
Pair End
Standard sample prep
Paired-end sample prep
P5 SBS3
5’
Insert
5’
P7
P5 PESP#2
5’
Insert
5’
PESP#1 P7
Different Cluster template
2nd round PCR
Cluster template
P7
3’ 5’
Insert
5’
5’
OH 3’
3’
P7
OH 3’diolFra bibliotek3’P5
OH 3’
NaIO4 diol
NaIO4
OH 3’
diol
P5
P7
P5
P7
Bridge Amplification
ddNTP
ddNTP
P5
P7
P5 P7
SBS (Sequencing By Synthesize)
8. ddNTP block & Hyb SBS 8
SBS READ 2
OH
7. Linearise P7 (Fpg)
Fpg
OH
6. Re-synthesis of P5-strand
(Isothermal amp)
OH OH
5. De-phosphorylate P5-PO4(PNK)
Specification comparison:
P7
O
s(T)10
aatgatacggcgaccaccgagatctacact
P5
3’ddN
O
Linearization of Clusters (cont.) denature
DNA insert
gagaaagggatgtgctgcgagaaggctaga tctagc
DNA insert
gatct agatcggaagagcgtcgtgtagggaaag
OH
OH
U
8oxo-G
8oxoG-P7 U-P5
1. Grafting
OVERVIEW OF THE METHOD
=
PO4
OH
USER OH
OH
OH
U
U
PNK
2. Cluster ampn
3. USER Linearisation
4. ddNTP () block & Hyb SBS 3
SBS READ 1
PCR oligo 1
PCR enrichment (cont.)
3’ 5’
Melt and hybridize to primers
2nd round PCR
3’ extension
3’
Insert
PCR oligo 2
3’
5’
3’
5’
SBS oligo P5
5’ 3’
Melt and hybridize to primers
P5序列：flowcell上结合的序列。 SBS引物序列(oligo)
Fragment DNA
5’ 3’
Genomic DNA
sample prep
5’
+
5’ 3’
5’
1.End repair
T4 polymerase , DNA Pol 1 (Klenow fragment)
5’ + 3’
R2 preparation SBS kit
15 reagents 4 h 50 mins
36 cycle
36 cycle
36 cycle
THE END!
THANKS!
ttactatgccgctggtggctctagatgt
s(T)10
sttttttttttcaagcagaagacggcatacgagctcttc sttttttttttcaagcagaagacggcatacgagctcttcc
s(T)10
agtgtagatctcggtggtcgccgtatcatt
Bridge Amplification
3’ extension
melt
hybridize
P5
P7
P5 P7
P7 P5
P7 P5
O
Linearization of Clusters
NaIO4
DNA insert
gagaaagggatgtgctgcgagaaggctaga tctagc ctctttccctacacgacgctcttccgatct agatcg
+
po45’
5’po4 3’T
3.Ligation of adaptors
DNA insert
3’
3’
5’ P7反向互补序列
3’
SBS oligo
5’
P7 P5 SBS oligo 5’ P7 反向互补序列
3’
PCR oligo 1
5’
5’
P5 SBS oligo
5’ 3’
5’ 3’
5’ 3’
PCR enrichment 3’
Flowcell
Single read 2 primer
2-primer PE method
Read 1
Read 2
2 (different) primer
Linearisation Blocking 1 Blocking 2
Diol
USER
Fpg
✓
✓
✓
-
✓
✓
Sequencing primer SBS3(+T) SBS3(+T) SBS8(+T)