Solexa测序原理及流程_--华大基因

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

ctaga gatct

P7

3’ddN

DNA insert

tctagccttctcgcagcacatccctttc agatcggaagagcgtcgtgtagggaaag

P5

O

Linearization of Clusters (cont.)

denature

DNA insert DNA insert

caagcagaagacggcatacgagctcttc stttttttttt

P7

caagcagaagacggcatacgagctcttcc

gagaaagggatgtgctgcgagaaggctaga

ttactatgccgctggtggctctagatgt

tctagc

gatct

agatcggaagagcgtcgtgtagggaaag

agtgtagatctcggtggtcgccgtatcatt

O

s(T)10

stttttttttt

3’ddN

P7

3’ddN

O

s(T)10

hybridization of sequencing primer

Pair End

Standard sample prep

5’

Paired-end sample prep

5’ 5’

P5 SBS3

P5 PESP#2

Insert

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