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Massively Parallel Signature Sequencing (MPSS)

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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  1. Massively Parallel Signature Sequencing (MPSS)

  2. 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

  3. Micro-Bead Monolayer

  4. 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

  5. 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

  6. Track the positions of microbeads and capture the fluorescence intensity for each decoder (G, A, T,C) at each base position

  7. NNNN NNNN 4321 NNNN NNNN NNNN 4321 NNNN NNNN NNNN 4321 NNNN

  8. (75,179) G (188,358) G 1 (75,179) (188,358)

  9. (75,179) G A (188,358) G A 1 2 (75,179) (188,358)

  10. (75,179) G A T (188,358) G A T 1 2 3 (75,179) (188,358)

  11. G A T C (75,179) G A T C (188,358) 1 2 3 4 (75,179) (188,358)

  12. ^ 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

  13. G A T C (75,179) G A T C T (188,358) G 1 2 3 4 5 (75,179) (188,358)

  14. 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)

  15. 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

  16. 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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