Sequencing
Module 3: Overview
Sequencing Workflow
Sample
Preparation
2
Cluster
Generation
Sequencing
Data Analysis
Sequencing
►
The goal of this step is to capture images of the
sequenced DNA to allow the sequence to be
determined
– Given clonal clusters, incorporate fluorescent
nucleotides and take images
►
This can be repeated for many cycles
– Incorporate fluorescent nucleotide
– Image tiles
– Cleave terminator
Sequencing
►
Paired-end runs require turnaround chemistry similar
to cluster generation
– This happens on the Genome Analyzer, with the
Paired-End Module
3
Basic Sequencing Workflow
Pre-Run Instrument Wash
Clean and Install Prism
Clean and Install Flow Cell
Apply Oil
First-Base Incorporation and Auto Calibration
Check Quality Metrics
Continue Sequencing Run
Post-Run Instrument Wash
4
Steps for completing the
sequencing run vary, depending
on the type of sequencing you are
performing.
Sequencing
Add 4 FlNTP’s +
Polymerase
Incorporated
Fl-NTP is
imaged
X 36 - 100
5
Terminator and
fluorescent dye are
cleaved from the FlNTP
Genome Analyzer Imaging System
Tile
Camera
Obj.
lens
Flow cell
Prism
Chiller
6
Oil
Water Cavity
Autofocus Laser System
►
Independent laser system used to calibrate the Z-height
adjustments needed during the run.
►
First imaging action performed at start of recipe.
►
A series of 30 images are taken, used to create a standard curve.
Movement of objective
z
y
Movement of AF spot in x-ordinate
x
7
Autofocus Laser System
The x-position of the
AF spot moves as
the AF images are
taken at varying zpositions of the
objective lens
8
►
Each image taken at increments of 1000nm in the Z direction
►
When starting a run the results are saved in the run folder :
D:\Runs\...\calibration\AFCalResult.txt
Autofocus Laser System
►
►
►
9
Goodness of fit
►
Ideally R(z, r) ~1
►
Extraction of spots in x,y coord.
S(q) value
►
Ideally be less than 0.2
►
Standard deviation of R values
Sensitivity
►
Ideally: 350 – 400 nm/pixel
►
X crossing value of best fit line
Y
Q
R
X
Flow Cell Images
A flow cell contains eight lanes
Lane 1
.
.
.
Lane 8
Each lane contains two columns of tiles
Column 1
Column 2
Tile
Each column contains 50 (GAII) or 60 tiles (GAIIX)
Each tile is imaged four times per cycle –
one image per base.
10
Genome Analyzer Imaging
100 Million Clusters
Per Flow Cell
20 Microns
100 Microns
11
Sequencing Software
►
Sequencing Control Software (SCS) software controls GA and
takes and stores images
►
Real Time Analysis (RTA) software analyzes images
–
–
–
–
–
►
12
Identifies clusters
Determines intensities of clusters
Calls bases for each cluster
Assigns base call quality scores
Handles data transfer to Pipeline server
RTA carries out first two steps of data analysis
GAIIx Performance Metrics
Read length
36 bp
50 bp
Number of
clusters
Gigabases
single read /
run time
13
75 bp
100 bp
130-170M clusters
4.7 – 6.1 Gb / 6.5 – 8.5 Gb / 9.75 – 12.75
2.2 days
3 days
Gb / 4.5 days
13 – 17 Gb /
6 days
Gigabases
9.4 – 12.2 Gb
paired-end
/ 4.3 days
reads / run time
13 – 17 Gb /
6 days
19.5 - 25.5
Gb / 9 days
26 – 34 Gb /
12 days
Avg. raw
accuracy
99.25%
99%
98.5%
98.0%
% reads with
no errors
> 90%
> 80%
> 70%
>60
Paired-End Sequencing
►
Provides long range information
►
Important for many short read applications
–
–
–
–
–
14
Repeat sequences
Characterize copy number variants & rearrangements
De novo assembly
Di-tag sequence cDNAs, ChIP, etc.
BAC-end sequencing
►
Sample multiplexing (identifier tags)
►
Increases output per flowcell
Paired-End Sequencing
Genome Analyzer
SBS reagents
1234567
Flowcell
Valve
8-way
pump
waste
8
Priming
pump
VICI
9
21
SBS reagents
15
waste
PE module
Connected
to GA
Paired-End Sequencing
Blocked
3’-ends
Sequenced
strand
►
►
16
Sequenced strand
is denatured at the
end of the first read
3’-ends of template
strands and lawn
primers are
unblocked
Paired-End Sequencing
►
Bridge
formation
►
3’ extension
17
Single-stranded
template loops
over to form a
bridge by
hybridizing with a
lawn primer
3’-ends of lawn
primer is extended
Paired-End Sequencing
►
Double
stranded
DNA
►
18
Single-stranded
template loops
over to form a
bridge by
hybridizing with a
lawn primer
3’-ends of lawn
primer is extended
Paired-End Sequencing
Blocked
3’-ends
Double
stranded
DNA
19
►
Bridges are
linearized and the
original forward
template is cleaved
off
Paired-End Sequencing
Blocked
3’-ends
Reverse
strand
template
20
►
Free 3’ ends of the
reverse template
and lawn primers
are blocked to
prevent unwanted
DNA priming
Sequencing Reverse Strand
Hybridize
read 2
sequencing
primer
Add 4 FlNTP’s +
Polymerase
Incorporated
Fl-NTP is
imaged
X 36 - 100
21
Terminator and
fluorescent dye
are cleaved from
the Fl-NTP
Short and Long Insert Paired Ends
►
The GA is the only platform that can do both
– Short Insert Paired Ends
– Long Insert Paired Ends
►
Need both for discovery of genome variation
Long Insert
Short Insert
X
A
X
300bp
Ligate
(circularize)
X
22
Y
X
Y
Fragment, End repair with biotin-NTP
A
Y
Y
Fragment
Capture, EndRepair, Ligate
adapters, PCR
X
Y
X
Best Practices for Sequencing
►
Store and prepare reagents as recommended
– 6 month shelf life
– Pool all reagents for a read (2 full kits for 76 cycles)
– Chill reagents before loading on sequencer
►
Control ambient temperature
– Ambient temperature specification is 22 +/- 3 degrees
– Temperatures over 28 (or fluctuations) can be seen in intensities
23
►
Monitor first base report and establish a threshold for intensity
►
Confirm auto-calibration and monitor autofocus laser performance
►
Maintain sufficient network throughput (10 Mbit/second file transfer)
►
Ensure that Genome Analyzer is washed regularly
Sequencing
Best Practices from The Broad Institute
Broad Sequencing Workflow
►
We usually perform Linearization/Blocking/Primer Annealing (LBPA)
as a single recipe, before sequencing, the day after Cluster
Generation and SYBR QC.
Sample
Prep.
25
Cluster
Gen. &
SYBR QC
Read 1 Prep
and
Sequencing
Read 2 Prep
and
Sequencing
Analysis
Broad Sequencing Workflow
Pre-Run Sequencer NaOH Wash
Cluster Generation SYBR QC
LBPA
Read I
Linearization, Blocking, and Primer Anneal
(LBPA)
Read I Sequencing Reagent Preparation
Sequencer Preparation
Read 2 Cluster Resynthesis
and Read
Read2IILBPA
Prism & Flow Cell Cleaning and Installation
Oil Application
Ga2x Post-Run Wash
First Base Incorporation and Calibration
Analysis
Check Quality Metrics
Read I Start & Progression
26
Broad Sequencing Workflow
Cluster Generation SYBR QC
LBPA
Read I
Read 2 Cluster Resynthesis
and Read
Read2IILBPA
Ga2x Post-Run Wash
Analysis
27
Paired-End Module Preparation
Read II Sequencing Reagent Preparation
First Base Incorporation
Read II Start & Progression
Tracking During Run Set-Up
28
►
As a beta testing site for Illumina, running various versions of the technology in testing,
limited production and full production, tracking is critical.
►
Important points to track from run set-up through post-run analysis:
– Flow Cell ID
– Libraries contained per lane
Paper and LIMS tracking
– Cycle count
– Indexed chemistry
– Recipe version
(Stored off-rig, selected at run startup using integrated recipe selector)
– Reagents/FC version
(Selected at SCS software start up, tracked in LIMS)
– Lot numbers of reagents
(LIMS and paper tracking sheets)
– Software version
(Selected at SCS software start up, tracked in LIMS)
Checking Calibration Results
First Base Incorporation and Calibration
►
Standard recipe: Focus Calibration and Edge Find start automatically
after First Base Incorporation chemistry
Recipe change:
►
Add User Waits before “Calibration” and “Edge Find”
–
–
–
–
–
29
Allows for manual course focusing and “four corners check” for oil
Allows user to monitor Focus Calibration and Edge Find
Allows user to assess calibration reports
Allows user intervention (necessary if auto calibration fails)
Convenient point in recipe to restart if necessary
Checking Calibration Results
AutoFocus Z-height Calibration
Illumina Calibration Specs
Spec
Read 1
R(z, r)
Above 0.99
S(q)
Below 0.25
MxBrt
1700-2200
Sensitivity
350-400
FQ*
Above 65
*See BestZ Plot.
If Max Bright is out of spec, it requires a
service call to adjust the autofocus laser.
Repeating issue may signify dying AF laser.
30
Checking Calibration Results
BestZ Plot Shows Focus Quality
►
►
31
FQ is also reported in 1st Base
Report (for each base)
Identifies tile’s highest Focus
Quality value
►
If recalibration is necessary (e.g.
lane too sparse, too dense), switch
to a different lane/tile for each
attempt to reduce tile bleaching
►
FQ values are dependent on
cluster uniformity and density
►
Typical: Lane 4, Column 1, Tile 30
Machine
Run Date: ReadPrep1
Run Id:
First Base Report: Overview
First Cycle (1)
Calibration and First Base Incorporation
►
1st base reports show
average metrics for each
lane, based on image
analysis of a subset of
tiles
MetricName
# of Clusters
►
32
Generally the images are
taken using a different
exposure than standard
cycle imaging
Values are similar to what
will be seen during the
run, but not the same
Lane7
Lane8
191,788 180,913
Standard Dev
6,983
9,054
3,690
7,773
4,872
4,830
4,749
12,726
A Intensity
1,063
1,220
1,128
1,093
1,216
1,132
1,226
1,202
73
104
53
97
69
34
51
142
996
1,087
1,000
971
1,094
1,008
1,059
1,082
44
75
51
89
27
52
50
165
1,379
1,666
1,537
1,496
1,759
1,645
1,778
1,714
202
141
263
253
151
224
178
218
1,190
1,397
1,330
1,184
1,416
1,329
1,413
1,396
Standard Dev
153
116
220
205
114
128
112
186
A Focus Metric
88
87
88
86
88
87
89
89
Standard Dev
2
4
1
5
1
2
1
1
C Focus Metric
87
84
86
84
86
85
86
87
Standard Dev
1
4
1
4
1
2
1
2
G Focus Metric
88
87
87
85
88
87
90
89
Standard Dev
3
3
4
6
2
3
1
2
T Focus Metric
84
83
83
80
84
83
86
85
Standard Dev
Standard Dev
C Intensity
Standard Dev
G Intensity
Standard Dev
►
Lane1 Lane2 Lane3 Lane4
Lane5 Lane6
188,103 189,265 194,003 183,802 191,614 188,073
T Intensity
2
5
5
7
2
2
2
3
Foc Pos Min
-229
400
500
-240
-750
-2,130
-3,840
-5,340
Foc Pos Max
7,459
8,059
8,400
8,050
7,809
7,209
7,400
6,909
Flow cell Tilt
7,688
7,659
7,900
8,290
8,559
9,339
11,240
12,249
Machine
Run Date: ReadPrep1
First Base Report: Focus
Run Id:
First Cycle (1)
Calibration and First Base Incorporation
►
Focal Quality is dependent on
library insert size and cluster
uniformity. Larger inserts will
produce larger clusters
(reducing FQ)
MetricName
# of Clusters
6,983
9,054
3,690
7,773
4,872
4,830
4,749
12,726
A Intensity
1,063
1,220
1,128
1,093
1,216
1,132
1,226
1,202
73
104
53
97
69
34
51
142
996
1,087
1,000
971
1,094
1,008
1,059
1,082
44
75
51
89
27
52
50
165
1,379
1,666
1,537
1,496
1,759
1,645
1,778
1,714
202
141
263
253
151
224
178
218
1,190
1,397
1,330
1,184
1,416
1,329
1,413
1,396
Standard Dev
153
116
220
205
114
128
112
186
A Focus Metric
88
87
88
86
88
87
89
89
Standard Dev
2
4
1
5
1
2
1
1
C Focus Metric
87
84
86
84
86
85
86
87
Standard Dev
1
4
1
4
1
2
1
2
G Focus Metric
88
87
87
85
88
87
90
89
Standard Dev
3
3
4
6
2
3
1
2
T Focus Metric
84
83
83
80
84
83
86
85
Standard Dev
2
5
5
7
2
2
2
3
Foc Pos Min
-229
400
500
-240
-750
-2,130
-3,840
-5,340
Foc Pos Max
7,459
8,059
8,400
8,050
7,809
7,209
7,400
6,909
Flow cell Tilt
7,688
7,659
7,900
8,290
8,559
9,339
11,240
12,249
Standard Dev
C Intensity
Standard Dev
Standard Dev
T Intensity
►
33
Focus Quality Metric > 65
Lane7
Lane8
191,788 180,913
Standard Dev
G Intensity
Read 1 Specs:
Lane1 Lane2 Lane3 Lane4
Lane5 Lane6
188,103 189,265 194,003 183,802 191,614 188,073
Machine
Run Date: ReadPrep1
Run Id:
First Base Report: # of Clusters
First Cycle (1)
Calibration and First Base Incorporation
MetricName
►
First Base Report does not
always find the same number
of clusters as are found
during run (which uses two
cycles to locate clusters), but
the range should be similar
# of Clusters
6,983
9,054
3,690
7,773
4,872
4,830
4,749
12,726
A Intensity
1,063
1,220
1,128
1,093
1,216
1,132
1,226
1,202
73
104
53
97
69
34
51
142
996
1,087
1,000
971
1,094
1,008
1,059
1,082
44
75
51
89
27
52
50
165
1,379
1,666
1,537
1,496
1,759
1,645
1,778
1,714
202
141
263
253
151
224
178
218
1,190
1,397
1,330
1,184
1,416
1,329
1,413
1,396
Standard Dev
153
116
220
205
114
128
112
186
A Focus Metric
88
87
88
86
88
87
89
89
# Clusters: 160-220K
– SCS v2.5, Pipeline v1.5
Standard Dev
2
4
1
5
1
2
1
1
C Focus Metric
87
84
86
84
86
85
86
87
Standard Dev
1
4
1
4
1
2
1
2
# Clusters: 265-320K
– SCS v2.6, Pipeline v1.6
G Focus Metric
88
87
87
85
88
87
90
89
Standard Dev
3
3
4
6
2
3
1
2
T Focus Metric
84
83
83
80
84
83
86
85
Standard Dev
Standard Dev
C Intensity
Standard Dev
G Intensity
T Intensity
Read 1 Specs:
►
This FC: 188K @ 2.5/1.5
34
Lane7
Lane8
191,788 180,913
Standard Dev
Standard Dev
►
Lane1 Lane2 Lane3 Lane4
Lane5 Lane6
188,103 189,265 194,003 183,802 191,614 188,073
2
5
5
7
2
2
2
3
Foc Pos Min
-229
400
500
-240
-750
-2,130
-3,840
-5,340
Foc Pos Max
7,459
8,059
8,400
8,050
7,809
7,209
7,400
6,909
Flow cell Tilt
7,688
7,659
7,900
8,290
8,559
9,339
11,240
12,249
Machine
Run Date: ReadPrep1
First Base Report: T Intensity
Run Id:
Calibration and First Base Incorporation
►
►
Used as QC metric. Desire
intensities as high as possible.
Library dependent (high GC,
monotemplates, libraries
containing synthetic fillers, etc.
alter T intensity)
MetricName
# of Clusters
Lane7
Lane8
191,788 180,913
6,983
9,054
3,690
7,773
4,872
4,830
4,749
12,726
A Intensity
1,063
1,220
1,128
1,093
1,216
1,132
1,226
1,202
73
104
53
97
69
34
51
142
996
1,087
1,000
971
1,094
1,008
1,059
1,082
44
75
51
89
27
52
50
165
1,379
1,666
1,537
1,496
1,759
1,645
1,778
1,714
202
141
263
253
151
224
178
218
1,190
1,397
1,330
1,184
1,416
1,329
1,413
1,396
153
116
220
205
114
128
112
186
88
87
88
86
88
87
89
89
Standard Dev
C Intensity
Standard Dev
G Intensity
T Intensity
Standard Dev
A Focus Metric
Standard Dev
2
4
1
5
1
2
1
1
87
84
86
84
86
85
86
87
1
4
1
4
1
2
1
2
88
87
87
85
88
87
90
89
3
3
4
6
2
3
1
2
84
83
83
80
84
83
86
85
2
5
5
7
2
2
2
3
Foc Pos Min
-229
400
500
-240
-750
-2,130
-3,840
-5,340
Foc Pos Max
7,459
8,059
8,400
8,050
7,809
7,209
7,400
6,909
Flow cell Tilt
7,688
7,659
7,900
8,290
8,559
9,339
11,240
12,249
C Focus Metric
#
Cycles
Illumina
Specs
Broad
Specs
36
100
400
Standard Dev
76
300
600
T Focus Metric
101
525
1000
126
525
1000
Standard Dev
G Focus Metric
35
Lane1 Lane2 Lane3 Lane4
Lane5
Lane6
188,103 189,265 194,003 183,802 191,614 188,073
Standard Dev
Standard Dev
Read 1 Specs: Starting
Intensity
First Cycle (1)
Standard Dev
Machine
Run Date:
Read 2 First Base Report: % Regeneration
Read 2 First Base Incorporation
►
% Regeneration is calculated
using average T intensity
across all lanes from R1 & R2
MetricName
# of Clusters
Standard Dev
A Intensity
Standard Dev
(Read1 T int) / (Read2 T int) x 100
C Intensity
Standard Dev
G Intensity
Read 2 Guidelines:
►
►
Standard Dev
T Intensity
Run Id:
First Cycle (1)
Lane1
Lane2 Lane3 Lane4 Lane5
Lane6 Lane7 Lane8
188,698 189,035 193,445 188,839 191,374 189,536 191,547 188,875
7,001
5,634
5,911
3,070
2,831
3,905
3,740
3,580
729
820
770
815
848
795
793
896
33
43
50
27
46
57
57
157
699
762
697
739
761
714
709
833
31
24
36
24
35
42
46
184
910
1,111
1,069
1,177
1,204
1,139
1,134
1,263
45
158
173
159
204
213
209
240
809
970
934
984
1,028
956
965
1,084
T Regeneration: > 50%*
Standard Dev
28
122
133
129
151
160
165
200
A Focus Metric
86
86
87
86
86
86
86
86
* Lower than 50% is fine if
Read 1 had particularly high T
intensities
Standard Dev
2
2
2
1
1
1
1
1
84
84
84
83
83
83
84
84
1
1
1
1
1
1
1
1
84
85
86
86
86
85
86
86
2
3
3
2
2
2
2
2
80
80
82
81
81
80
81
83
2
2
4
3
3
3
4
3
9,160
8,820
8,140
7,020
5,960
4,010
2,000
130
15,790
15,869
15,769
14,740
13,900
13,169
12,819
12,090
6,630
7,049
7,629
7,720
7,940
9,159
10,819
11,960
C Focus Metric
Standard Dev
G Focus Metric
Standard Dev
T Focus Metric
This FC: 75.67%
(7765/10655)x100%
Standard Dev
Foc Pos Min
Foc Pos Max
Flow cell Tilt
36
ReadPrep2
Fail Modes
Tracking Fail Modes
►
For each failed run, a Fail Report is
created to track relevant information
►
Assist in troubleshooting future runs
►
Identify trends, bin issues (e.g. by date)
Flowcell Fail Report
(Double click on boxes to check or uncheck)
Run Name/ FC: 42KF5AAXX
Date of Failure: 9/30/09
Sequencer/CS: XAP
Reported by: SG
Reviewed by: SC
Read 1
Read 2
Single Read
Other
Description of Failure Mode:
CS issue – describe
Reagent top off issue
Low Intensity at 1st Base = 614.13
Poor Re-synthesis for Read 2
Machine Crash due to Lack of Memory
Known Mechanical Issue:
Analysis issue:
Other Issue (please include as much detail as possible):
Action Taken:
Run Cancelled
Please update Squid for all run cancellations
Run Failed Analysis:
Please update Squid for all run failures
Unresolved : If unresolved, please list the people aware of the issue:
Other:
Please include the following FC details:
Comments/Description: Run had low t int. to begin with, cancelled b/c got worse as run
continued, would lead to a bad 126cycle run. Rehyb failed causing peeled matrix.
Libraries and loading concentrations: Solexa-14942, 41, 40, 39, 38, 37, 36, 35.
WR ID: 20066
38
Tracking Fail Modes
►
Most common on-sequencer fail modes in the past 6 months:
– Oil propagation
– Low intensities at 1st base of read I and II
– RTA processing issues
– Mechanical/hardware problems
Fail Mode
39
July –December 09
Pareto: Fail modes
Major fail modes change over time
July –December 09
40
Nov 09-Jan 10
Fail Mode: Oil
Worse in later cycles; Oil expands when heated, wicks onto FC surface, then spreads
41
►
Portions of, or the entire tile is out of focus
►
Identify oil issues as early as possible, as FC can be cleaned and run resumed
►
Images, and RTA FQ charts are key tools in identification
Fail Mode: Tile Out of Focus
►
A distorted focus dot usually precedes a tile that is out of focus
►
Possible cause: Oil or reagent (leaking from manifold) on surface of flow cell
►
Possible cause: Problem with focus laser
►
Possible cause: RTA cannot process the tile due to extreme density
42
Fail Mode: Oil on Side of Prism
►
Oil on the optical side of the prism
►
Optical pathway is compromised
reducing transmission of laser light
through the prism and into the FC.
Not here
Oil goes here
Not here
43
Fail Mode: Oil
Identify/Resolve/Prevent (Best Practices)
►
Identify
– RTA charts
– Images
►
Resolve
– At run prep: remove assembly (FC & Prism) and clean and reinstall
– During run: stop run, clean FC, prism, & peltier with methanol, restart run
►
Prevent
– Use conservative, standardized oiling method (to be presented in lab module)
– Clean peltier block frequently
44
Fail Mode: RTA Processing “Thread Death”
►
►
►
45
RTA has 3 processing threads that
are responsible for tile processing.
If a thread dies, a “checkerboard” tile
pattern will show on the charts.
Depending on the number of threads
that died, the corresponding number
of tiles will be skipped.
Fail Mode: RTA Processing “Thread Death”
Identify/Resolve/Prevent (Best Practices)
►
Identify
– RTA charts, tile status
►
Resolve
– If identified during Read 1, stopping and restarting RTA using the restart file in the
run folder will usually restart the processing threads that died.
– If identified during Read 2 after RTA has completed processing Read 1 with the
same phenotype, RTA will not be able to reprocess the lost tiles.
►
Prevent
– Monitor Read 1 closely after starting, looking for the “checkerboard” tile processing
46
Fail Mode: Poor Linearization
Read 1 & 2 Start & Progression
►
Can occur at either read 1 or 2
►
Characterized by platelet- or donutshaped clusters
►
Low flow of LMX on cluster station
or PEM is an indicator
47
What we think is happening
Good Linearization
(block)
Cluster in bridge form
Normal Linearization
Efficient Primer
incorporation
Nucleotides incorporated
only on primers
Poor Linearization
(block)
Not all bridges
get linearized
48
Primer only
incorporated
on linearized
strands
Only edge of
cluster fluoresces
Fail Mode: Poor Linearization
Identify/Resolve/Prevent (Best Practices)
►
Identify
– Insufficient flow on the CS or PEM is the primary indicator
 Volume checks at LBPA, especially of critical reagent LMX
– Inefficient LMX reagent
 Images on sequencer
►
Resolve
– Strip primer (denature with NaOH) and repeat LBPA
►
Prevent
–
–
–
–
49
Mark reagent level on each tube
Avoid freeze-thaw of reagents
Avoid storage temperature fluctuations
Record Lot #s
Fail Mode: Poor Blocking
►
Characterized by few visible
clusters and a very bright
background
►
Can be subtle (slight increase in
background noise)
►
Unblocked OH groups at the ends
of linearized strands can also
incorporate fluorophores, resulting
in increased background noise
50
What we think is happening
Good Blocking
Cluster in bridge form
Excess P7 primers
on FC have –OH
group
Normal Blocking
All excess primers
blocked
Normal Primer
incorporation
Nucleotides incorporated
only on primers
Poor Blocking
Excess P7 primers on
FC have –OH group,
51
…but not all of
them get
blocked.
Normal Primer
incorporation
Incorporate onto
cluster AND
unblocked primers
Fail Mode: Poor Blocking
Identify/Resolve/Prevent (Best Practices)
►
Identify
– Insufficient flow on the CS or PEM is the primary indicator
 Volume checks at LBPA, especially of critical reagent BMX
– Inefficient BMX reagent
 Images on sequencer
►
Resolve
– Strip primer (denature with NaOH) and repeat LBPA
►
Prevent
– Mark reagent level on each tube
– Avoid freeze-thaw of reagents
– Avoid storage temperature fluctuations
It’s common to see a combination of inefficient Linearization & Blocking on one FC
52
Fail Mode: Poor Primer Annealing
53
►
Inefficient primer annealing is
characterized by a lack of cluster
intensity. Clusters are visible but the
intensity is significantly below the
minimum threshold.
►
In this case, the Primer Annealing
step can be repeated (a “rescue
rehyb”)
“Rescue Rehyb”
►
If 1st Base Report shows low T intensity, the Primer Annealing step can
be repeated (a “rescue rehyb”)
►
Rescue Rehyb:
– Denature primer by flowing NaOH
– Wash
– Repeat primer annealing
►
Enables us to save both Read 1 & Read 2
►
Success rate:
– 50% over all attempts
– 100% over all instances identified certainly as poor primer incorporation
54
►
Can be performed without removing flow cell from sequencer, using the
Paired-End Module, but this requires disconnection of tubing
►
Illumina is developing Rehyb recipe and kits for CBot
“Rescue Rehyb”
MetricName
# of Clusters
Standard Dev
Initial Primer
Hyb:
1st Base Report
Avg T = 311
Lane3
115,076
Lane4
121,981
Lane5
127,178
Lane6
127,496
Lane7
140,946
Lane8
141,146
8,056
5,478
5,779
8,154
4,457
13,454
14,493
51
72
314
146
105
119
271
437
Standard Dev
12
34
93
91
58
48
135
94
C Intensity
56
75
296
140
124
139
320
523
Standard Dev
12
33
76
80
67
52
154
104
209
307
1,201
630
456
508
1,136
1,743
Standard Dev
63
163
369
392
263
213
555
350
T Intensity
93
130
465
253
216
229
529
768
(1)151
62First Cycle
134
122
82
233
166
G Intensity
MetricName
26
Lane1
131,723
Lane2
132,009
Lane3
131,675
Lane4
131,873
Lane5
131,471
Lane6
133,575
Lane7
138,406
Lane8
139,278
1,431
651
660
971
2,696
3,646
8,123
8,602
469
505
497
500
507
498
491
467
17
16
29
21
19
21
32
36
427
458
447
446
579
556
560
547
19
12
22
17
25
45
26
32
2,466
2,662
2,642
2,672
2,734
2,691
2,652
2,447
Standard Dev
131
112
219
167
140
146
187
277
T Intensity
974
1,045
1,029
1,027
1,287
1,244
1,308
1,279
49
46
69
53
113
137
118
109
Standard Dev
A Intensity
Standard Dev
C Intensity
Standard Dev
G Intensity
Standard Dev
55
Lane2
112,584
4,751
# of Clusters
Avg T = 1149
Lane1
110,611
A Intensity
Standard Dev
After Rehyb:
st
1 Base Report
First Cycle (1)
“Rescue Rehyb”
Initial Primer
Hyb:
Avg T = 311
56
After Rescue
Rehyb:
Avg T = 1149
Fail Mode: Poor Primer Annealing
Identify/Resolve/Prevent (Best Practices)
►
Identify
– Low 1st base intensity
►
Resolve
– Perform “Rescue Rehyb”
►
Prevent
– Monitor reagent consumption and flow
– Mix primer tube thoroughly after thawing
57
Fail Mode: Uneven Primer Lawn
Possible defects in the substrate used for primer seeding process in flow cell manufacturing
Tracking when and where (cycle/tile/run) these occur can distinguish between defective substrate and
problems with the laser /optics assembly (signified by reproduced pattern in subsequent runs)
58
Fail Mode: Switched Reagent
►
Incorporation Mix where Scan Mix
should be
►
Unattached fluorescent nucleotides
were not flushed away with Scan Mix
►
Labeling bottles and color matching
labels to avoid switching reagent
bottles
59
Fail Mode: Mode Scrambler Failure
►
The mode scrambler is a device that
spreads the laser illumination
uniformly over the tile.
►
Identify
– Mottled image surface
►
Resolve
– Service engineer
intervention required
– Run can continue but
expect higher error rates,
phasing and prephasing
values, low % PF
►
Prevent
– Replace mode scrambler
60
Fail Mode: Footprint
►
►
61
A “footprint” is caused by a misaligned beam shaper is dark band along any side of
the tile (it is often confused with oil edges)
User corrective measure will be demonstrated in the lab section
Fail Mode: Torn/Delaminating Substrate
►
Substrate peeling from the FC
surface
►
Sometimes occurs after
multiple rescue Rehyb
attempts
►
Sometimes occurs when
Cluster Station or Sequencer
Peltier block fails
62
Best Practices
Best Practices
Reagent Handling
►
Use 175ml Falcon & 150ml Corning bottles to eliminate top-offs in runs >100b
►
Label and color-match bottles to lines
►
Perform NaOH wash before every run start and before prolonged down-time
►
Measure and track flow volumes: dead volumes and waste volumes
►
Mark levels of reagent volumes when loading on instrument
►
Filter Incorporation mix (IMX) to reduce “super clusters”
– Filter buffer & nucleotides before adding enzyme (enzyme won’t filter)
►
Control reagent temperatures and thawing environment
–
–
–
–
–
64
Thaw each tube of reagent upright in room temp water
Immediately transfer to ice, once thawed
Change gloves after handling cleavage components
Thaw and store separately from other components
Cleavage components must not contact other reagents
Best Practices
Stage/FC/Prism Assembly
►
Clean stage with methanol before every flow cell is loaded
– Use water on ports to avoid degradation of manifold gaskets
►
Clean peltier with methanol
►
Use lens paper to clean optical surfaces such as prism and flow cell
►
Clean flow cell with water, then methanol
– Avoid ports to prevent DNA degradation
►
Check for proper seal between flow cell and manifold by flowing buffer
– A steady line of bubbles indicates improper seating
►
Oiling
–
–
–
–
–
Oil flow cell slowly from the left side to avoid dripping oil onto optical edge of prism
Ensure oil flows under the entire flowcell
Put a 10ul tip on top of 200ul tip to help guide oil under flow cell
Drag a clean tip along the right edge of flow cell can help to seal oil underneath
Check images in four flow cell corners for adequate oil
(Will be covered in the lab module)
65
Best Practices
Run Monitoring
►
RTA
(covered in Working with Data Module)
– Identify freezes and processing
errors
– Ensure sufficient intensity in all
channels
– Ensure focus quality
– Identify reagent leaks and oil issues
– Identify sudden changes
►
Images
– Ensure focus
– Identify “super clusters,” especially
near end of reads
– Identify reagent leaks and oil issues
66
Key Points
►
Careful run monitoring and tracking are keys to successful run
►
Recognizing fail mode phenotypes allows early identification of problems,
sometimes before they become detrimental to a run
►
►
67
The technology is not “set and forget”; It requires user activity throughout
the process
Each user/group must determine an appropriate set of rules and
expectations concerning tracking, QC, metrics, fail points, etc. based on
downstream effect on data quality
Image Gallery:
Unique Phenotypes
Images: Unique Phenotypes
Frosted objective
69
Dried substrate
Images: Unique Phenotypes
►
70
Crop circles?
Images: Unique Phenotypes
►
“Ocelot”
– Jen Fostel, Hayward
71
Images: Unique Phenotypes
►
72
Amoebas? (SYBR QC image)
What We’ll See in the Lab
►
Loading the GA
– Cleaning flow cell & prism
– Loading & oiling flow cell
– Quality Checks during flow cell loading
►
GA maintenance & troubleshooting
–
–
–
–
–
►
73
Washes
Flow rates
Footprints
Max brightness
Focus laser
Best practices