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 One Cycle One Base
SBS (Sequencing By Synthesize)
Pair End
DNA样品制备
Cluster formation
Cluster Formation
Bridge Amplification Cycle
DNA synthesization
P7
SBS oligo
P5
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。 P5序列：flowcell上结合的序列。 SBS引物序列(oligo)
Genomic DNA
sample prep
Fragment DNA
5’ 3’ 5’ + 5’ 3’ 5’ 5’ + 3’ 3’ 5’
1.End repair
T4 polymerase , DNA Pol 1 (Klenow fragment)
SBS oligo
P7 反向互补序列
Phosphorylation T4 polynucleaotide kinase, ATP
5’po4 3’ 3’ po45’
2.Add 3’ Adenosine with
Klenow (3’exo- ) and dATP
5’po4 3’A A3’ po45’ 3’ 5’po4 3’T 5’ P7反向互补序列 3’
+
3.Ligation of adaptors
5’ DNA insert
SBS oligo
5’
3’
P7 P5 SBS oligo
5’
PCR enrichment
3’
P7 反向互补序列
3’
5’
PCR oligo 1
5’ 5’ 3’
Melt and hybridize to primers
1st round PCR
PCR oligo 2
5’ 3’ 3’ 5’
P7
P5 SBS oligo
5’ 3’ 3’
3’ extension
3’
5’
3’ extension
Insert
3’ 5’ 3’ 5’
5’ 3’ 5’ 3’
P7
P5 SBS oligo P7 反向互补序列
5’ 3’
PCR enrichment (cont.)
3’ 5’
Melt and hybridize to primers
2nd round PCR
3’
Insert
3’ extension
5’ 3’ 5’ 3’
PCR oligo 2
5’ 3’
5’
PCR oligo 1
SBS oligo P5
5’ 3’
Melt and hybridize to primers
2nd round PCR
3’ 5’
P7
Cluster template
Insert
5’
5’
OH
3’ 3’
OH
3’
OH
3’
OH
3’
diol
3’
NaIO4
P5
diol
P7
NaIO4
diol
P7
P7
P5
P5
Bridge Amplification
ddNTP P5 P7
ddNTP P5 P7
SBS (Sequencing By Synthesize)
Bridge Amplification
3’ extension
melt
hybridize
P5
P7
P5 P7
P7 P5
P7 P5
3’ddN
O
s(T)10
ttactatgccgctggtggctctagatgt aatgatacggcgaccaccgagatctaca
P5
Linearization of Clusters
NaIO4
caagcagaagacggcatacgagctcttc stttttttttt
gagaaagggatgtgctgcgagaaggctaga ctctttccctacacgacgctcttccgatct
aaaaaaaaaagttcgtcttctgccgtatgctcgagaagg caagcagaagacggcatacgagctcttcc tttttttttt s
O
aatgatacggcgaccaccgagatctacact
s(T)10
agtgtagatctcggtggtcgccgtatcatt
gaagagctcgtatgccgtcttctgcttgaaaaaaaaaa
O DNA insert
tctagc agatcg
P7 NaIO4
…… 此处隐藏421字 ……
5’
Paired-end sample prep
5’ 5’
P5 SBS3
P5 PESP#2
Insert