SOLiD Technology. library preparation & Sequencing Chemistry (sequencing by ligation!) Imaging and analysis. Application specific sample preparation

Átírás

1 SOLiD Technology Application specific sample preparation Application specific data analysis library preparation & emulsion PCR Sequencing Chemistry (sequencing by ligation!) Imaging and analysis

2 SOLiD V4System Summary: Read length: bases Generated tags: 1000 Million tags/run Throughput: 50 GBase/slide/run (10 000x coverage on 5 MB bact. genome) Highest accuracy (no homopolymer issue, two-base encoding)

3 ~50 Gbase sequence/slide/run x coverage of 5Mb bacterium 4000 x coverage of 12Mb yeast ~400x coverage of 128Mb Arabidopsis

4 Available Library Protocols Three Methods Available for Different Applications Fragment Library Bead Forward Adapter DNA Fragment Reverse Adapter Whole Transcriptome (RNA-Seq) Targeted Resequencing SAGE or 5 SAGE (DGEP) ChIP SNP Discovery Mate Paired Library Bead Forward Adapter DNA Fragment Tag #1 Internal Adapter DNA Fragment Tag #2 Reverse Adapter Whole Genome Sequencing SNP Discovery DNA Methylation Small RNA Library Bead Forward Adapter mirna Reverse Adapter mirna Discovery and/or Profiling Digital Gene Expression (DGEP)

5 SOLiD Mate-Paired Library Workflow Whole Genomes Sheared DNA Size Selection Internal Adaptor Ligation Internal Adapter Internal Adapter

6 SOLiD Mate-Paired Library Workflow Protocol A Circularization Protocol B EcoP15I cut 25bp 25bp Pixi dust Adaptor Ligation MP Library LMP Library P1 Adapter P2 Adapter P1 Adapter P2 Adapter 25bp 25bp 50-75bp 50-75bp Emulsion PCR

7 SOLiD Chemistry

8 epcr: Oil Phase Premix automatization

9 SOLiD Chemistry

10 SOLiD Chemistry

11 1 st Base Properties of the Probes Cleavage site is between 5th and 6th base ligation site, cleavage site and dye are spatially separated X X n n n z z z Blue - probe Fluorescent dye interrogates bases on 1 st + 2 nd position Probes are octamers N=degenerate bases, Z=universal bases 1024 probes, 256 probes per color A C G T 2 nd Base A C G T

12 SOLiD 4-color ligation Ligation reaction universal seq primer p5 ligase Y-probe 5 X Xn n n z z z G-probe 5 X X n n n z z z B-probe 5 X X n n n z z z R-probe 5 X X n n n z z z 5 P1 Primer Template Sequence

13 SOLiD 4-color ligation Ligation reaction ligase Y-probe 5 X Xn n n z z z G-probe 5 X X n n n z z z universal seq primer ligase p5 x x 5 P1 Primer Template Sequence B-probe 5 X X n n n z z z R-probe 5 X X n n n z z z

14 SOLiD 4-color ligation Visualization universal seq primer x x 5 P1 Primer Template Sequence Y 1-2

15 SOLiD 4-color ligation Cleavage universal seq primer x x 5 P1 Primer Template Sequence Y p5 1-2

16 SOLiD 4-color ligation Ligation (2 nd cycle) ligase Y-probe 5 X Xn n n z z z G-probe 5 X X n n n z z z universal seq primer x x ligase 5 Adapter Oligo Sequence Template Sequence Y x x B-probe 5 X X n n n z z z R-probe 5 X X n n n z z z 1-2

17 SOLiD 4-color ligation Visualization (2 nd cycle) universal seq primer X X 5 Adapter Oligo Sequence Template Sequence Y x x R

18 SOLiD 4-color ligation Cleavage (2 nd cycle) universal seq primer X X 5 Adapter Oligo Sequence Template Sequence Y x x R p

19 SOLiD 4-color ligation universal seq primer X X X X X X X X X X 5 Adapter Oligo Sequence Template Sequence Y R R B G

20 SOLiD 4-color ligation Reset 5 Adapter Oligo Sequence Template Sequence

21 SOLiD 4-color ligation (1 st cycle after reset) universal seq primer n-1 p5 ligase Y-probe 5 X Xn n n z z z G-probe 5 X X n n n z z z ligase universal seq primer n-1 p5 x x 5 Adapter Oligo Sequence Template Sequence B-probe 5 X X n n n z z z R-probe 5 X X n n n z z z

22 SOLiD 4-color ligation (1 st cycle after reset) universal seq primer n-1 x x 5 Adapter Oligo Sequence Template Sequence R 0-1

23 SOLiD 4-color ligation (2 nd Round) universal seq primer n-1 X X X X X X X X X X 5 Adapter Oligo Sequence Template Sequence R R R B G

24 Sequential rounds of sequencing Multiple cycles per round 5 Adapter Oligo Sequence Template Sequence universal seq primer reset universal seq primer n reset reset reset universal seq primer n+3 universal seq primer n+2 universal seq primer n+1 spacer spacer spacer

25 SOLiD Chemistry

26 1 st Base 2 Base Pair Encoding Using 4 Dyes 2 nd Base A C G T A Red-probe A T n n n z z z 5 C G Blue-probe T T n n n z z z 5 T

27 1 st Base Advantages of 2 base pair encoding Each base is interrogated twice, by two independent probes A C G G T C G T C G T G T G C G T A C G G T C G T C G T G T G C G T 2 nd Base A A C G T C G T

28 1 st Base 2 base pair encoding reference alignment in color space A C G G T C G T C G T G T G C G T A C G G T C G C C G T G T G C G T To be acceptable, the SNP must be encoded by two color changes A reference expected observed 2 nd Base A C G T C G T

29 1 st Base Advantages of 2 base pair encoding Miscall A C G G T C G T C G T G T G C G T reference expected A C G G T C G C T A C A C A T A C observed Single color change, represents sequencing error. A 2 nd Base A C G T C G T

30 5 additional bases on the P2 adapter Multiplexing

31 SOLiD System Barcodes Sequencing read (35 bases) BC read (5 bases) P1* Target DNA P2* 20 barcode available with SOLiD 3 (96 barcodes in R&D) Fragment libraries only (not currently available for mate-paired libraries)

32 Multiplex Analysis Libraries epcr Enrichment Deposition

33 Alkalmazások: -de novo szekvenálás (ismeretlen genomú faj szekvenálása, majd nukleotidsorrendjének (genomjának) meghatározása), -újraszekvenálás (ismert referenciagenomú fajok szekvenálása, pl. baktériumok filogenetikai rendszerezése, szekvenciavariációk és SNP-k azonosítása), -összehasonlító-, meta-, populációgenomika -gén-kifejeződés (RNA-Seq; Digital Gene Expression Profiling, DGEP), -mikrorns-kifejeződés, -azonosítás, -kromatin immunprecipitációt követő szekvenálás (ChIP-Seq; transzkripciós faktorok kötőhelyeinek azonosítása; aktív vagy passzív kromatin feltérképezése), -DNS metilációs analízis (Meth-Seq)

34 Gyakorlati példák: -genetikai betegségekre való hajlam kimutatása, -terápiás lehetőségek felfedezése (személyre szabott gyógyászat), -fertőzések diagnosztikája, prognosztikája, -kórokozók azonosítása (strain-to-reference), -két egyed közötti rokonsági fok meghatározása (pl. apasági vizsgálatok, filogenetika) -személyazonosítás (pl. bűnüldözés), -oltóanyag fejlesztés, -nemesítés, -génváltozatok összehasonlítása.