Signal Generation and Imaging
PicoTiterPlate
Wells
Photons
Detected
by Camera
1600K field of
addressable wells
Reagent Flow
Sequencing
By Synthesis
Photons
Generated
• Spectral Instruments Series 800 Camera with Fairchild Imaging LM485 CCD (4096x4096,
15 μm pixels), directly bonded to a 1:1 imaging fiber bundle; cooled to -25 °C
• PTP in direct contact with imaging fiber bundle (no alignment or focusing issues); NA ~ 0.75
• Full-frame imaging mode; read-out during wash (dark portion of flow cycle)
Proprietary
Image Processing
Raw data series of
images
T
C
G
A
T
Data converted
into flowgrams”
Proprietary
Signal-Processing & Basecalling
Image Data → Signal processing Pipeline → Flowgrams → Quality Filtering
→ HQ Reads → Basecalling → HQ Bases → Mapping & Assembly
4-mer
T
A Flow
C Order
G
3-mer
TTCTGCGAA
2-mer
1-mer
Key sequence = TCAG for well identification and calibration
Proprietary
Mapping in Flowgram Space
Reference Chromosome Flowgram
Fragment Flowgrams (RN)

…,1, 3, 1, 0, 0, 2, 2, 0, 0, 1, 2, 3, 0, 1, 0…
…, 1.00, 3.14, 0.15, 0.20, 0.21, 1.84, 1.95,…
Re-sequencing and find variants to the
reference genome
Proprietary
De Novo Assembly in Flowgram Space
Overlap to form contigs
Fragment Flowgrams (RN)

Draft sequences of new genomes (species that
have not been sequenced before)
Proprietary
Potential Sources of Error
Optical Cross-Talk
• Light penetrates to adjacent fibers; leads to signal contamination
Non-DNA Well
DNA Well
False signal
True
signal
Chemical Cross-Talk
• PPi / ATP produced in DNA well appears in down-stream non-DNA wells due to
convection / diffusion; leads to signal contamination
Flow
PPi
Non-DNA
Well
False
signal
DNA Well
True
signal
Proprietary
Signal-Processing Solution
Original
Filtered
(51x51) kernel
2D intensity
contour
Pixel
Pixel
Filtering removes signal cross-contamination
Proprietary
Typical GS20 Run Results




42 Flow Cycles
~ 30MB per run
~ 300,000 reads
~ 100 bp per read
# of reads
15000
10000
5000
0
41
51
61
0
1
2
71
81
91
101
111
121
131
60%
 > 50% wells error-free
 ~ 1 % individual read
error
% of reads
50%
40%
30%
20%
10%
0%
3
4
5
6
7
8
9
# of errors per read
Proprietary
10+*
Consensus Accuracy > 99.99+% when
10x over-sampling
Example
Ref / assembled genome:
2
-GTGCGCGCGCGGGACTAATCCCGGTTCGCGCGTCGGGCATGACACGCAAC-
10 reads aligned to this position
Read #:
FlowSignal
Read 1:
2.52
Read 2:
1.95
Read 3
2.11
Read 4:
1.53
Read 5:
1.32
Read 6:
2.14
Read 7:
2.06
Read 8:
1.85
Read 9:
2.21
Read 10:
2.17
Consensus (mean): 1.99
Proprietary
De Novo Assembly Results
 4 runs of GS20 (E. Coli 4,639,675 bp)
 Each data point represents 1/2 GS20 run
100.00%
1800
# of contigs
1600
1400
# Contigs
90.00%
% Coverage
80.00%
1200
70.00%
1000
800
60.00%
600
50.00%
400
200
0
% coverage
2000
40.00%
1 run
4.5
2 runs
9
13
16.5
3 runs
20
23
4 runs
26.5
30.00%
30
Oversampling
Proprietary
Genome Sequencer FLX
Recently Launched Next Generation Sequencing Platform
in Q1, 2007 in collaboration with Roche Diagnostics





At least 400K (200K) reads of avg. 250 bp (100 bp)
8 hours (5 hours) run time
Single read avg accuracy >99.5% (99%) over 200 (100) bases
Consensus read accuracy > 99.99%
Avg yield from single run ~ 100 Mb (20-30 Mb)
(GS20 performance)





Improved fluidics for faster reagent delivery
Firmware control of reagent delivery & camera timing
On-board reagent dilution
Optimized biochemistry
Improved algorithms with corrections for
– Crosstalk (for higher densities)
– Signal droop & Phasing
 Numerical filter for improved rejection of low quality reads
Proprietary
Fluidics Modification – Air Plug Insertion
Camera cover
G
A
C
Debubbler
Air
mM
air plug in tube
PTP Inlet Nuc Conc (non-dim)
PTP Inlet Conc (non-dim)
21 s Injection: Ver
1.0 (New M
Concentration
Profile
Pre-dilution
withReagent
& w/o air
bubble Mode
11
Standard
0.80.8
Air
Bubble
Insertion
0.60.6
t
air bubbles removed
at debubbler
0.40.4
0.2
0.2
0
0
0
0
20
20
40
40
Time (s)
Time (s)
Proprietary
60
60
80
Whole Genome Sequencing Results from GS FLX
E. Coli (50% GC)
C. jejuni (35%GC)
T. thermophilus (71% GC)
Proprietary
Observed Individual Read Accuracy
Cumulative Read Error
4.0%
3.5%
E. coli run #1
09_29A
E. coli run #2
09_29B
09_14
+ 09_18A
E. coli run
#3
09_18B+09_25
E. coli run #4
Thermophilus
T. thermophilus
C
C.jejuni
jejuni
Reported in
Nature 2005
3.0%
2.5%
2.0%
GS20
Q2 2006
1.5%
1.0%
0.5%
0.0%
0
50
100
150
200
250
Base Position
 All blind-filtered reads (no reference genome required)
Proprietary
Newbler™ Assembly Results from GS FLX
C. jejuni
T. thermophilus
E. coli
E. coli
(GS20)
Genome Size:
1,641,481
2,127,575
4,639,675
4,639,675
Number of Runs:
½
½
1
3
Assembly Contigs:
25
56
105
140
Assembly Cover:
98.31%
98.15%
97.61%
97.46%
Overall Accuracy:
99.996%
99.991%
99.998%
99.998%
Avg. Contig Size:
64.6 kb
39.8 kb
43.3 kb
32.4 kb
N50 Contig Size:
116 kb
82.1 kb
105.5 kb
67.2 kb
Largest Contig:
481 kb
383.0 kb
204.7 kb
164 kb
Proprietary