Massively Parallel Signature Sequencing (MPSS)
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Massively Parallel Signature Sequencing (MPSS). Attachment of cDNA to microbeads via unique tags. AAAAAAAAA. mRNA from biological samples. x. AAAAAAAAA TTTTTTTTTT. cDNA. Unique tags (16.7M). (20 base). Unique complementary Anti-tag. PCR. attached.

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Massively Parallel Signature Sequencing (MPSS)

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Massively Parallel Signature Sequencing (MPSS)


Attachment of cDNA to microbeads via unique tags

AAAAAAAAA

mRNA from biological samples

x

AAAAAAAAA

TTTTTTTTTT

cDNA

Unique tags (16.7M)

(20 base)

Unique complementary Anti-tag

PCR

attached

Result: cDNA derived from each mRNA molecule a single microbead


Micro-Bead Monolayer


Parallel signature sequencing

NNNN

NNNN

4321

NNNN

Determine the overhang -NNNN with sequencing cycles

NNNN

NNNN

8765

NNNN

Signature: NNNN…NNNN, known sequences of 20 bases


Determine the overhang -NNNN with sequencing cycles

e.g. N at position 1

Fluorescence-labeled decoder for nucleotide

Fluorescence-labeled decoder for nucleotide

Fluorescence-labeled decoder for nucleotide

Fluorescence-labeled decoder for nucleotide

G

A

T

C

Call for base at position 1

G


Track the positions of microbeads

and

capture the fluorescence intensity for each decoder (G, A, T,C)

at each base position


NNNN

NNNN

4321

NNNN

NNNN

NNNN

4321

NNNN

NNNN

NNNN

4321

NNNN


(75,179)

G

(188,358)

G

1

(75,179)

(188,358)


(75,179)

G

A

(188,358)

G

A

1

2

(75,179)

(188,358)


(75,179)

G

A

T

(188,358)

G

A

T

1

2

3

(75,179)

(188,358)


G

A

T

C

(75,179)

G

A

T

C

(188,358)

1

2

3

4

(75,179)

(188,358)


^

NNNN

CODE

RS

NNNN

^

4 3 2 1

9

Digest with Type IIs enzyme to expose next 4 bases

13

NNNN

8 7 6 5

Repeat Cycle

Stepping in Four Bases


G

A

T

C

(75,179)

G

A

T

C

T

(188,358)

G

1

2

3

4

5

(75,179)

(188,358)


G

A

T

C

HUMFTE1A

G

A

T

C

T

T

A

C

C

C

G

T

G

A

C

A

A

A

A

T

HSRNPA1

G

T

G

A

C

G

C

T

G

A

A

T

A

A

A

T

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

(75,179)

(188,358)


Sequencing a Specific Bead Address

G A T C C C A G T G A A T C C T G G G T


Signature

# of Beads

1

GATCTGCATTCGGTAACAGG

1

2

GATCTTTGTCAACCAAAACA

2

3

GATCCCTGTGAAGACCCAGG

113

4

GATCTTTCCTCCTGGTTACT

16

5

GATCCAGTCAAGGAGAAAGA

3

6

GATCTGATAGTCCTGAAAAC

22

7

GATCATGGTGATGTACGGGG

1

8

GATCCAGTGCTATTCTGTCA

38

9

GATCAATCCCTCAAGATTTT

312

.

.

23,685

GATCACCATCCCAGTCACCT

2,588

856,567


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