SOLiD™ System S O D - NDSUmcclean/plsc411/SOLiD... · SOLiD System: Development Status Dedicated...
Transcript of SOLiD™ System S O D - NDSUmcclean/plsc411/SOLiD... · SOLiD System: Development Status Dedicated...
![Page 1: SOLiD™ System S O D - NDSUmcclean/plsc411/SOLiD... · SOLiD System: Development Status Dedicated teams at Beverly and Foster City with Instruments up and running Anticipated Performance](https://reader033.fdocuments.in/reader033/viewer/2022053007/5f0b63747e708231d4304665/html5/thumbnails/1.jpg)
SOLiD™ SystemSequencing by Oligonucleotide Ligation and Detection
![Page 2: SOLiD™ System S O D - NDSUmcclean/plsc411/SOLiD... · SOLiD System: Development Status Dedicated teams at Beverly and Foster City with Instruments up and running Anticipated Performance](https://reader033.fdocuments.in/reader033/viewer/2022053007/5f0b63747e708231d4304665/html5/thumbnails/2.jpg)
2 November 2006 © 2006 Applied Biosystems
Workflow
Application specificsample
preparation
Application specificsample
preparation
Emulsion PCR &
substrate preparation
Emulsion PCR &
substrate preparation
Imaging &basecallingImaging &
basecalling
Application specific
Data analysis
Application specific
Data analysis
Sequencing chemistry
Sequencing chemistry
![Page 3: SOLiD™ System S O D - NDSUmcclean/plsc411/SOLiD... · SOLiD System: Development Status Dedicated teams at Beverly and Foster City with Instruments up and running Anticipated Performance](https://reader033.fdocuments.in/reader033/viewer/2022053007/5f0b63747e708231d4304665/html5/thumbnails/3.jpg)
3 November 2006 © 2006 Applied Biosystems
Fragment Library (directed resequencing)
Mate Pair Library (whole genome sequencing)
DNA Fragment
60-90 Bases
Create library of DNA fragments:2 methods
1µmbead
P1 Adapter P2 Adapter
1µmbead
P1 Adapter Internal Adapter
25 baseTag #1
25 baseTag #2
P2 Adapter
![Page 4: SOLiD™ System S O D - NDSUmcclean/plsc411/SOLiD... · SOLiD System: Development Status Dedicated teams at Beverly and Foster City with Instruments up and running Anticipated Performance](https://reader033.fdocuments.in/reader033/viewer/2022053007/5f0b63747e708231d4304665/html5/thumbnails/4.jpg)
4 November 2006 © 2006 Applied Biosystems
Creating a mate-paired library (i)
Randomly Fragmentsample
Complex sample
Ligate Internal adapters
Circularize
+
size select(eg:1, 2, 3, 5, 10 KB)
![Page 5: SOLiD™ System S O D - NDSUmcclean/plsc411/SOLiD... · SOLiD System: Development Status Dedicated teams at Beverly and Foster City with Instruments up and running Anticipated Performance](https://reader033.fdocuments.in/reader033/viewer/2022053007/5f0b63747e708231d4304665/html5/thumbnails/5.jpg)
5 November 2006 © 2006 Applied Biosystems
Creating a mate-paired library (ii)
Cleave EcoP15I
27bp 27bp
Ligate P1 and P2
+
Internal Adapter
25 baseTag #1
25 baseTag #2
P1 P2
![Page 6: SOLiD™ System S O D - NDSUmcclean/plsc411/SOLiD... · SOLiD System: Development Status Dedicated teams at Beverly and Foster City with Instruments up and running Anticipated Performance](https://reader033.fdocuments.in/reader033/viewer/2022053007/5f0b63747e708231d4304665/html5/thumbnails/6.jpg)
6 November 2006 © 2006 Applied Biosystems
Emulsion PCR (i)
+
Templates
Primers P1<<P2
Enzyme
P1-coupled beads
Polymerase
![Page 7: SOLiD™ System S O D - NDSUmcclean/plsc411/SOLiD... · SOLiD System: Development Status Dedicated teams at Beverly and Foster City with Instruments up and running Anticipated Performance](https://reader033.fdocuments.in/reader033/viewer/2022053007/5f0b63747e708231d4304665/html5/thumbnails/7.jpg)
7 November 2006 © 2006 Applied Biosystems
Emulsion PCR (ii)Mix PCR aqueous phase into a water-in-oil (w/o) emulsion and carry out emulsion PCR
![Page 8: SOLiD™ System S O D - NDSUmcclean/plsc411/SOLiD... · SOLiD System: Development Status Dedicated teams at Beverly and Foster City with Instruments up and running Anticipated Performance](https://reader033.fdocuments.in/reader033/viewer/2022053007/5f0b63747e708231d4304665/html5/thumbnails/8.jpg)
8 November 2006 © 2006 Applied Biosystems
Emulsion MetricsBead size: 1 μm
Reactor size: 4 μmReactor volume: 34 fL
Beads / emulsion plate (96-well): 2-4 x 109
Post Enrichment: ~500M / plate
SOLiD system: Clonal Amplification
Dressman, PNAS 2003Embleton, 1994
![Page 9: SOLiD™ System S O D - NDSUmcclean/plsc411/SOLiD... · SOLiD System: Development Status Dedicated teams at Beverly and Foster City with Instruments up and running Anticipated Performance](https://reader033.fdocuments.in/reader033/viewer/2022053007/5f0b63747e708231d4304665/html5/thumbnails/9.jpg)
9 November 2006 © 2006 Applied Biosystems
emulsion PCR Individual Bead
2) Polymerase extends from P1
1) Template Anneals to P1
3) Complementary sequence is extended off bead surface
4) Template disassociates
![Page 10: SOLiD™ System S O D - NDSUmcclean/plsc411/SOLiD... · SOLiD System: Development Status Dedicated teams at Beverly and Foster City with Instruments up and running Anticipated Performance](https://reader033.fdocuments.in/reader033/viewer/2022053007/5f0b63747e708231d4304665/html5/thumbnails/10.jpg)
10 November 2006 © 2006 Applied Biosystems
Results of emulsion PCR,after breaking emulsion
Bead contains ~20K amplified products from original single strand molecule Beads with no product
![Page 11: SOLiD™ System S O D - NDSUmcclean/plsc411/SOLiD... · SOLiD System: Development Status Dedicated teams at Beverly and Foster City with Instruments up and running Anticipated Performance](https://reader033.fdocuments.in/reader033/viewer/2022053007/5f0b63747e708231d4304665/html5/thumbnails/11.jpg)
11 November 2006 © 2006 Applied Biosystems
Enrichment
P1 P2
P2’
P2 P1
LargePolystyrenebead coated
with P2
Centrifuge in glycerol gradient
Supernatant Captured beads with templates
Pellet Beads with no template
![Page 12: SOLiD™ System S O D - NDSUmcclean/plsc411/SOLiD... · SOLiD System: Development Status Dedicated teams at Beverly and Foster City with Instruments up and running Anticipated Performance](https://reader033.fdocuments.in/reader033/viewer/2022053007/5f0b63747e708231d4304665/html5/thumbnails/12.jpg)
12 November 2006 © 2006 Applied Biosystems
P2-hybridization, Pre- and post-enrichment
Pre-enrichment (30%)
Post-enrichment (80%)
em6395
![Page 13: SOLiD™ System S O D - NDSUmcclean/plsc411/SOLiD... · SOLiD System: Development Status Dedicated teams at Beverly and Foster City with Instruments up and running Anticipated Performance](https://reader033.fdocuments.in/reader033/viewer/2022053007/5f0b63747e708231d4304665/html5/thumbnails/13.jpg)
13 November 2006 © 2006 Applied Biosystems
Bead Deposition
3’-end modification
Beads attached to glass surface in a random array
![Page 14: SOLiD™ System S O D - NDSUmcclean/plsc411/SOLiD... · SOLiD System: Development Status Dedicated teams at Beverly and Foster City with Instruments up and running Anticipated Performance](https://reader033.fdocuments.in/reader033/viewer/2022053007/5f0b63747e708231d4304665/html5/thumbnails/14.jpg)
14 November 2006 © 2006 Applied Biosystems
Properties of the ProbesSpatial separation among dye, ligation & cleavage sites
3’
n n n T C z z z
Fluorescent dye
Cleavage site,
3’ Ligation site
1,024 Octamer Probes (45)4 Dyes, 4 dinucleotides, 256 probes per dyeN= degenerate bases Z= Universal bases
![Page 15: SOLiD™ System S O D - NDSUmcclean/plsc411/SOLiD... · SOLiD System: Development Status Dedicated teams at Beverly and Foster City with Instruments up and running Anticipated Performance](https://reader033.fdocuments.in/reader033/viewer/2022053007/5f0b63747e708231d4304665/html5/thumbnails/15.jpg)
15 November 2006 © 2006 Applied Biosystems
SOLiD Chemistry System 4-color ligationLigation reaction
ligase3’ p5’
universal seq primer
Template Sequence 5’ 3’P1 Adapter
1µmbead
5’
3’ 5’
3’ 5’
3’ 5’
n n n T A z z z
n n n T C z z z n n n A T z z z
n n n G G z z z
1µmbead
universal seq primer p5’
![Page 16: SOLiD™ System S O D - NDSUmcclean/plsc411/SOLiD... · SOLiD System: Development Status Dedicated teams at Beverly and Foster City with Instruments up and running Anticipated Performance](https://reader033.fdocuments.in/reader033/viewer/2022053007/5f0b63747e708231d4304665/html5/thumbnails/16.jpg)
16 November 2006 © 2006 Applied Biosystems
ligase
SOLiD Chemistry System 4-color ligation Ligation reaction
ligase3’ p5’
universal seq primer
Template Sequence 5’ 3’P1 Adapter
1µmbead
universal seq primer
1µmbead
p5’T AT A
5’
3’ 5’
3’ 5’
3’ 5’
n n n T A z z z
n n n T C z z z n n n A T z z z
n n n G G z z z
![Page 17: SOLiD™ System S O D - NDSUmcclean/plsc411/SOLiD... · SOLiD System: Development Status Dedicated teams at Beverly and Foster City with Instruments up and running Anticipated Performance](https://reader033.fdocuments.in/reader033/viewer/2022053007/5f0b63747e708231d4304665/html5/thumbnails/17.jpg)
17 November 2006 © 2006 Applied Biosystems
SOLiD Chemistry System 4-color ligation De-Phosphorylation
Template Sequence 5’ 3’P1 Adapter
1µmbead
universal seq primer
1µmbead
Template Sequence 5’ 3’P1 Primer
1µmbead
universal seq primer
1µmbead
p5’
T AT A
![Page 18: SOLiD™ System S O D - NDSUmcclean/plsc411/SOLiD... · SOLiD System: Development Status Dedicated teams at Beverly and Foster City with Instruments up and running Anticipated Performance](https://reader033.fdocuments.in/reader033/viewer/2022053007/5f0b63747e708231d4304665/html5/thumbnails/18.jpg)
18 November 2006 © 2006 Applied Biosystems
SOLiD Chemistry System 4-color ligation Visualization
4,5 Template Sequence 5’ 3’P1 Adapter
1µmbead
universal seq primer
1µmbead
T AT A
![Page 19: SOLiD™ System S O D - NDSUmcclean/plsc411/SOLiD... · SOLiD System: Development Status Dedicated teams at Beverly and Foster City with Instruments up and running Anticipated Performance](https://reader033.fdocuments.in/reader033/viewer/2022053007/5f0b63747e708231d4304665/html5/thumbnails/19.jpg)
19 November 2006 © 2006 Applied Biosystems
T AT A
SOLiD Chemistry System 4-color ligationCleavage
Template Sequence 5’ 3’P1 Adapter
1µmbead
universal seq primer
1µmbead
p5’
4,5
![Page 20: SOLiD™ System S O D - NDSUmcclean/plsc411/SOLiD... · SOLiD System: Development Status Dedicated teams at Beverly and Foster City with Instruments up and running Anticipated Performance](https://reader033.fdocuments.in/reader033/viewer/2022053007/5f0b63747e708231d4304665/html5/thumbnails/20.jpg)
20 November 2006 © 2006 Applied Biosystems
ligase
SOLiD Chemistry System 4-color ligation Ligation (2nd cycle)
ligase
Template Sequence 5’ 3’P1 Adapter
1µmbead
universal seq primer
1µmbead
p5’
5’
3’ 5’ 3’ 5’
n n n T A z z z
G G
n n n T C z z z n n n A T z z z
3’ 5’n n n G G z z z
4,5
T AT A
![Page 21: SOLiD™ System S O D - NDSUmcclean/plsc411/SOLiD... · SOLiD System: Development Status Dedicated teams at Beverly and Foster City with Instruments up and running Anticipated Performance](https://reader033.fdocuments.in/reader033/viewer/2022053007/5f0b63747e708231d4304665/html5/thumbnails/21.jpg)
21 November 2006 © 2006 Applied Biosystems
SOLiD Chemistry System 4-color ligation Visualization (2nd cycle)
Template Sequence 5’ 3’P1 Adapter
1µmbead
universal seq primer
1µmbead
G G
9,104,5
T AT A
![Page 22: SOLiD™ System S O D - NDSUmcclean/plsc411/SOLiD... · SOLiD System: Development Status Dedicated teams at Beverly and Foster City with Instruments up and running Anticipated Performance](https://reader033.fdocuments.in/reader033/viewer/2022053007/5f0b63747e708231d4304665/html5/thumbnails/22.jpg)
22 November 2006 © 2006 Applied Biosystems
SOLiD Chemistry System 4-color ligation Cleavage (2nd cycle)
Template Sequence 5’ 3’P1 Adapter
1µmbead
universal seq primer
1µmbead
G GT AT A
9,104,5
p5’
![Page 23: SOLiD™ System S O D - NDSUmcclean/plsc411/SOLiD... · SOLiD System: Development Status Dedicated teams at Beverly and Foster City with Instruments up and running Anticipated Performance](https://reader033.fdocuments.in/reader033/viewer/2022053007/5f0b63747e708231d4304665/html5/thumbnails/23.jpg)
23 November 2006 © 2006 Applied Biosystems
C CT TG AG A
19,2014,15
SOLiD Chemistry System 4-color ligationinterrogates every 5th base
Template Sequence 5’ 3’P1 Adapter
1µmbead
universal seq primer
1µmbead
G GT AT A
9,104,5 24,25
![Page 24: SOLiD™ System S O D - NDSUmcclean/plsc411/SOLiD... · SOLiD System: Development Status Dedicated teams at Beverly and Foster City with Instruments up and running Anticipated Performance](https://reader033.fdocuments.in/reader033/viewer/2022053007/5f0b63747e708231d4304665/html5/thumbnails/24.jpg)
24 November 2006 © 2006 Applied Biosystems
SOLiD Chemistry System 4-color ligation Reset
Template Sequence 5’ 3’P1 Adapter
1µmbead
1µmbead
![Page 25: SOLiD™ System S O D - NDSUmcclean/plsc411/SOLiD... · SOLiD System: Development Status Dedicated teams at Beverly and Foster City with Instruments up and running Anticipated Performance](https://reader033.fdocuments.in/reader033/viewer/2022053007/5f0b63747e708231d4304665/html5/thumbnails/25.jpg)
25 November 2006 © 2006 Applied Biosystems
ligase
SOLiD Chemistry System 4-color ligation (1st cycle after reset)
ligase3’ p5’
universal seq primer n-1
Template Sequence 5’ 3’P1 Adapter
1µmbead
1µmbead
universal seq primer n-1 p5’
5’
3’ 5’
3’ 5’
3’ 5’
n n n T A z z z
n n n T C z z z n n n A T z z z
n n n G G z z z
A T
![Page 26: SOLiD™ System S O D - NDSUmcclean/plsc411/SOLiD... · SOLiD System: Development Status Dedicated teams at Beverly and Foster City with Instruments up and running Anticipated Performance](https://reader033.fdocuments.in/reader033/viewer/2022053007/5f0b63747e708231d4304665/html5/thumbnails/26.jpg)
26 November 2006 © 2006 Applied Biosystems
SOLiD Chemistry System 4-color ligation (1st cycle after reset)
Template Sequence 5’ 3’P1 Adapter
1µmbead
universal seq primer n-1 A T
1µmbead
3,4
![Page 27: SOLiD™ System S O D - NDSUmcclean/plsc411/SOLiD... · SOLiD System: Development Status Dedicated teams at Beverly and Foster City with Instruments up and running Anticipated Performance](https://reader033.fdocuments.in/reader033/viewer/2022053007/5f0b63747e708231d4304665/html5/thumbnails/27.jpg)
27 November 2006 © 2006 Applied Biosystems
SOLiD Chemistry System 4-color ligation (2nd Round)
5’ 3’P1 Adapter
1µmbead
universal seq primer n-1
1µmbead
A T A GT T G A C G
3,4 8,9 13,14 18,19 23,24
![Page 28: SOLiD™ System S O D - NDSUmcclean/plsc411/SOLiD... · SOLiD System: Development Status Dedicated teams at Beverly and Foster City with Instruments up and running Anticipated Performance](https://reader033.fdocuments.in/reader033/viewer/2022053007/5f0b63747e708231d4304665/html5/thumbnails/28.jpg)
28 November 2006 © 2006 Applied Biosystems
Sequential rounds of sequencingMultiple cycles per round
3’universal seq primer
4,5 9,10 14,15 19,20 24,25
Template Sequence 5’ 3’P1 Adapter
1µmbead
1µmbead
3’universal seq primer n-1 reset
3’universal seq primer n-2reset
3’universal seq primer n-3reset
3’universal seq primer n-4reset
3,4 8,9 13,14 18,29 23,24
2,3 7,8 12,13 17,18 22,23
1,2 6,7 12,13 16,17 21,22
0,1 5,6 11,12 15,16 20,21
![Page 29: SOLiD™ System S O D - NDSUmcclean/plsc411/SOLiD... · SOLiD System: Development Status Dedicated teams at Beverly and Foster City with Instruments up and running Anticipated Performance](https://reader033.fdocuments.in/reader033/viewer/2022053007/5f0b63747e708231d4304665/html5/thumbnails/29.jpg)
29 November 2006 © 2006 Applied Biosystems
1µmbead
P1 Adapter Internal Adapter
25 baseTag #1
25 baseTag #2
P2 Adapter
Paired End two sequences generatedSequential rounds of sequencingMultiple cycles per round
![Page 30: SOLiD™ System S O D - NDSUmcclean/plsc411/SOLiD... · SOLiD System: Development Status Dedicated teams at Beverly and Foster City with Instruments up and running Anticipated Performance](https://reader033.fdocuments.in/reader033/viewer/2022053007/5f0b63747e708231d4304665/html5/thumbnails/30.jpg)
30 November 2006 © 2006 Applied Biosystems
2 base encoding● Double base interrogation eases the discrimination
between system errors and true polymorphism
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
A C G TA
C
G
T
5th Base
4thB
ase
![Page 31: SOLiD™ System S O D - NDSUmcclean/plsc411/SOLiD... · SOLiD System: Development Status Dedicated teams at Beverly and Foster City with Instruments up and running Anticipated Performance](https://reader033.fdocuments.in/reader033/viewer/2022053007/5f0b63747e708231d4304665/html5/thumbnails/31.jpg)
31 November 2006 © 2006 Applied Biosystems
Benefits of SOLiD Chemistry System● Terminating Chemistry
– No issues with homopolymers● Capping step eliminates dephasing
– Control increase in noise with subsequent cycles ● Primer reset
– removes accumulated out-of-phase ligations– longer reads (Signal recovers)
● Ligation– high fidelity– high read quality
● Double interrogation of each base– unique to ligation method– increases base calling accuracy– SNP results in a two-color change
![Page 32: SOLiD™ System S O D - NDSUmcclean/plsc411/SOLiD... · SOLiD System: Development Status Dedicated teams at Beverly and Foster City with Instruments up and running Anticipated Performance](https://reader033.fdocuments.in/reader033/viewer/2022053007/5f0b63747e708231d4304665/html5/thumbnails/32.jpg)
32 November 2006 © 2006 Applied Biosystems
10-box Linux Cluster15 Tb data storage
Dual Flow CellReagent
handling
SOLiD System Instrumentation High speed automated bead imaging
![Page 33: SOLiD™ System S O D - NDSUmcclean/plsc411/SOLiD... · SOLiD System: Development Status Dedicated teams at Beverly and Foster City with Instruments up and running Anticipated Performance](https://reader033.fdocuments.in/reader033/viewer/2022053007/5f0b63747e708231d4304665/html5/thumbnails/33.jpg)
33 November 2006 © 2006 Applied Biosystems
4-Color Overlay
1/6th
Full Array Small Array
1800 panels 300 panels~20,000 beads/panel
SOLiD System Arrays: 4 Mega pixel Camera
![Page 34: SOLiD™ System S O D - NDSUmcclean/plsc411/SOLiD... · SOLiD System: Development Status Dedicated teams at Beverly and Foster City with Instruments up and running Anticipated Performance](https://reader033.fdocuments.in/reader033/viewer/2022053007/5f0b63747e708231d4304665/html5/thumbnails/34.jpg)
34 November 2006 © 2006 Applied Biosystems
Improved feature finding
Beads called per panel: 22,693 49,980
![Page 35: SOLiD™ System S O D - NDSUmcclean/plsc411/SOLiD... · SOLiD System: Development Status Dedicated teams at Beverly and Foster City with Instruments up and running Anticipated Performance](https://reader033.fdocuments.in/reader033/viewer/2022053007/5f0b63747e708231d4304665/html5/thumbnails/35.jpg)
35 November 2006 © 2006 Applied Biosystems
Cycle 1 Cycle 2 Cycle 3 Cycle 4 Cycle 5(base 4) (base 9) (base 14) (base 19) (base 24)
Round 2 after Reset
Reset prevents excessive noise accumulation
Cycle 1 Cycle 2 Cycle 3 Cycle 4 Cycle 5(base 5) (base 10) (base 15) (base 20) (base 25)
Round 1
![Page 36: SOLiD™ System S O D - NDSUmcclean/plsc411/SOLiD... · SOLiD System: Development Status Dedicated teams at Beverly and Foster City with Instruments up and running Anticipated Performance](https://reader033.fdocuments.in/reader033/viewer/2022053007/5f0b63747e708231d4304665/html5/thumbnails/36.jpg)
36 November 2006 © 2006 Applied Biosystems
SOLiD System: Development Status● Dedicated teams at Beverly and Foster City with Instruments up and running ● Anticipated Performance
– Read length: 25x2 mate pair – Raw data output rate: anticipate 100-500 Mb per day– Raw accuracy: ≥98%– Consensus accuracy: >99.99% (@ 20x coverage)– Quality metrics: Compatible with KB™ Basecaller Software– Cost per run: Competitive– Libraries: Mate pair or Fragment
○ as required by application– Application Support
○ Whole genome resequencing○ Directed (Medical) resequencing○ Gene expression○ Tag Counting
![Page 37: SOLiD™ System S O D - NDSUmcclean/plsc411/SOLiD... · SOLiD System: Development Status Dedicated teams at Beverly and Foster City with Instruments up and running Anticipated Performance](https://reader033.fdocuments.in/reader033/viewer/2022053007/5f0b63747e708231d4304665/html5/thumbnails/37.jpg)
37 November 2006 © 2006 Applied Biosystems
● For Research Use Only. Not for use in diagnostic procedures.
● Applera, Applied Biosystems, AB (Design) are registered trademarks and KB and SOLiD are trademarks of Applera Corporation or its subsidiaries in the US and/or certain other countries.