Module 3: Overview - Broad Institute · 3 3 4 6 84 83 83 80 2 5 5 7-229 400 500 -240 7,459 8,059...

SequencingModule 3: Overview

2

Sequencing Workflow

Sample

Preparation

Cluster

Generation Data AnalysisSequencing

3

► 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

► Paired-end runs require turnaround chemistry similar

to cluster generation

– This happens on the Genome Analyzer, with the

Paired-End Module

Sequencing

Sequencing

4

Basic Sequencing Workflow

Pre-Run Instrument Wash

Clean and Install Prism

Clean and Install Flow Cell

Apply Oil

First-Base Incorporation and Auto Calibration

Steps for completing the

sequencing run vary, depending

on the type of sequencing you are

performing.

Check Quality Metrics

Continue Sequencing Run

Post-Run Instrument Wash

5

Add 4 Fl-

NTP’s +

Polymerase

Incorporated

Fl-NTP is

imaged

Terminator and

fluorescent dye are

cleaved from the Fl-

NTP

X 36 - 100

Sequencing

6

Oil

Flow cell

Prism

Water CavityChiller

Obj.

lens

Camera

Tile

Genome Analyzer Imaging System

7

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 AF spot in x-ordinate

x

y

zMovement of objective

8


The x-position of the

AF spot moves as

the AF images are

taken at varying z-

positions of the

objective lens

► 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

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

X

Y

R

Q

10

A flow cell contains eight lanesLane 1

Lane 8

.

.

.

Column 1

Column 2

Tile

Each lane contains two columns of tiles

Each column contains 50 (GAII) or 60 tiles (GAIIX)

Each tile is imaged four times per cycle –

one image per base.

Flow Cell Images

11

100 Million Clusters

Per Flow Cell

20 Microns

100 Microns

Genome Analyzer Imaging

12

► Sequencing Control Software (SCS) software controls GA and

takes and stores images

► Real Time Analysis (RTA) software analyzes images

– 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

Sequencing Software

13

Read length 36 bp 50 bp 75 bp 100 bp

Number of

clusters130-170M clusters

Gigabases

single read /

run time

4.7 – 6.1 Gb /

2.2 days

6.5 – 8.5 Gb /

3 days

9.75 – 12.75

Gb / 4.5 days

13 – 17 Gb /

6 days

Gigabases

paired-end

reads / run time

9.4 – 12.2 Gb

/ 4.3 days

13 – 17 Gb /

6 days

19.5 - 25.5

Gb / 9 days

26 – 34 Gb /

12 days

Avg. raw

accuracy99.25% 99% 98.5% 98.0%

% reads with

no errors> 90% > 80% > 70% >60

GAIIx Performance Metrics

14

Paired-End Sequencing

► Provides long range information

► Important for many short read applications

– 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

15

Valve Flowcell8-way

pump

SBS reagents

1 2 3 4 5 6 7

VICI

8

9 21

SBS reagents

Genome Analyzer

PE module

Priming

pumpwaste

waste


Connected

to GA

16

► Sequenced strand

is denatured at the

end of the first read

► 3’-ends of template

strands and lawn

primers are

unblocked


Blocked

3’-ends

Sequenced

strand

17

► Single-stranded

template loops

over to form a

bridge by

hybridizing with a

lawn primer

► 3’-ends of lawn

primer is extended


Bridge

formation

3’ extension

18

► Single-stranded

template loops

over to form a

bridge by

hybridizing with a

lawn primer

► 3’-ends of lawn

primer is extended


Double

stranded

DNA

19

► Bridges are

linearized and the

original forward

template is cleaved

off


Double

stranded

DNA

Blocked

3’-ends

20

► Free 3’ ends of the

reverse template

and lawn primers

are blocked to

prevent unwanted

DNA priming


Blocked

3’-ends

Reverse

strand

template

21

Add 4 Fl-

NTP’s +

Polymerase

Incorporated

Fl-NTP is

imaged

Terminator and

fluorescent dye

are cleaved from

the Fl-NTP

X 36 - 100

Hybridize

read 2

sequencing

primer

Sequencing Reverse Strand

22

► 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

Y

300b

p

Short Insert Long Insert

A A

X

FragmentLigate

(circularize)

Capture, End-

Repair, Ligate

adapters, PCR

X

Y

XFragment, End repair with biotin-NTP YX

X YX Y

Short and Long Insert Paired Ends

23

► 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

► 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

Best Practices for Sequencing

SequencingBest Practices from The Broad Institute

25

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.

Read 1 Prep

and

Sequencing

Read 2 Prep

and

SequencingAnalysis

Cluster

Gen. &

SYBR QC

Sample

Prep.

26


Pre-Run Sequencer NaOH Wash

Linearization, Blocking, and Primer Anneal

(LBPA)

Read I Sequencing Reagent Preparation

First Base Incorporation and Calibration

Check Quality Metrics

Prism & Flow Cell Cleaning and Installation

Oil Application

Read I Start & Progression

LBPA

Read 2 Cluster Resynthesis

and Read 2 LBPARead II

Ga2x Post-Run Wash

Cluster Generation SYBR QC

Analysis

Read I

Sequencer Preparation

27


Read II Sequencing Reagent Preparation

Paired-End Module Preparation

Read II Start & Progression

First Base Incorporation

LBPA

Read 2 Cluster Resynthesis

and Read 2 LBPARead II

Ga2x Post-Run Wash

Cluster Generation SYBR QC

Analysis

Read I

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

– 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)

Tracking During Run Set-Up

Paper and LIMS tracking

29

Checking Calibration Results

► Standard recipe: Focus Calibration and Edge Find start automatically

after First Base Incorporation chemistry

Recipe change:

► Add User Waits before “Calibration” and “Edge Find”

– 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

First Base Incorporation and Calibration

30


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.

Illumina Calibration Specs

AutoFocus Z-height Calibration

31

BestZ Plot Shows Focus Quality


► 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

32

188,103 189,265 194,003 183,802

6,983 9,054 3,690 7,773

1,063 1,220 1,128 1,093

73 104 53 97

996 1,087 1,000 971

44 75 51 89

1,379 1,666 1,537 1,496

202 141 263 253

1,190 1,397 1,330 1,184

153 116 220 205

88 87 88 86

2 4 1 5

87 84 86 84

1 4 1 4

88 87 87 85

3 3 4 6

84 83 83 80

2 5 5 7

-229 400 500 -240

7,459 8,059 8,400 8,050

7,688 7,659 7,900 8,290Flow cell Tilt 8,559 9,339 11,240 12,249

Foc Pos Min -750 -2,130 -3,840 -5,340

Foc Pos Max 7,809 7,209 7,400 6,909

T Focus Metric 84 83 86 85

Standard Dev 2 2 2 3

G Focus Metric 88 87 90 89


C Focus Metric 86 85 86 87


A Focus Metric 88 87 89 89


T Intensity 1,416 1,329 1,413 1,396

Standard Dev 114 128 112 186

G Intensity 1,759 1,645 1,778 1,714

Standard Dev 151 224 178 218

C Intensity 1,094 1,008 1,059 1,082


A Intensity 1,216 1,132 1,226 1,202


# of Clusters 191,614 188,073 191,788 180,913

Standard Dev 4,872 4,830 4,749 12,726

First Cycle (1)

MetricName Lane1 Lane2 Lane3 Lane4 Lane5 Lane6 Lane7 Lane8

Machine

Run Date: ReadPrep1

Run Id:

First Base Report: Overview

Calibration and First Base Incorporation

► 1st base reports show

average metrics for each

lane, based on image

analysis of a subset of

tiles

► 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

33

188,103 189,265 194,003 183,802

6,983 9,054 3,690 7,773

1,063 1,220 1,128 1,093

73 104 53 97

996 1,087 1,000 971

44 75 51 89

1,379 1,666 1,537 1,496

202 141 263 253

1,190 1,397 1,330 1,184

153 116 220 205

88 87 88 86

2 4 1 5

87 84 86 84

1 4 1 4

88 87 87 85

3 3 4 6

84 83 83 80

2 5 5 7

-229 400 500 -240

7,459 8,059 8,400 8,050

7,688 7,659 7,900 8,290Flow cell Tilt 8,559 9,339 11,240 12,249

Foc Pos Min -750 -2,130 -3,840 -5,340

Foc Pos Max 7,809 7,209 7,400 6,909









T Intensity 1,416 1,329 1,413 1,396

Standard Dev 114 128 112 186

G Intensity 1,759 1,645 1,778 1,714

Standard Dev 151 224 178 218

C Intensity 1,094 1,008 1,059 1,082


A Intensity 1,216 1,132 1,226 1,202


# of Clusters 191,614 188,073 191,788 180,913

Standard Dev 4,872 4,830 4,749 12,726

First Cycle (1)


Machine

Run Date: ReadPrep1

Run Id:First Base Report: Focus


► Focal Quality is dependent on

library insert size and cluster

uniformity. Larger inserts will

produce larger clusters

(reducing FQ)

Read 1 Specs:

► Focus Quality Metric > 65

34

First Base Report: # of Clusters


► 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

Read 1 Specs:

► # Clusters: 160-220K

– SCS v2.5, Pipeline v1.5

► # Clusters: 265-320K

– SCS v2.6, Pipeline v1.6

This FC: 188K @ 2.5/1.5

188,103 189,265 194,003 183,802

6,983 9,054 3,690 7,773

1,063 1,220 1,128 1,093

73 104 53 97

996 1,087 1,000 971

44 75 51 89

1,379 1,666 1,537 1,496

202 141 263 253

1,190 1,397 1,330 1,184

153 116 220 205

88 87 88 86

2 4 1 5

87 84 86 84

1 4 1 4

88 87 87 85

3 3 4 6

84 83 83 80

2 5 5 7

-229 400 500 -240

7,459 8,059 8,400 8,050

7,688 7,659 7,900 8,290Flow cell Tilt 8,559 9,339 11,240 12,249

Foc Pos Min -750 -2,130 -3,840 -5,340

Foc Pos Max 7,809 7,209 7,400 6,909









T Intensity 1,416 1,329 1,413 1,396

Standard Dev 114 128 112 186

G Intensity 1,759 1,645 1,778 1,714

Standard Dev 151 224 178 218

C Intensity 1,094 1,008 1,059 1,082


A Intensity 1,216 1,132 1,226 1,202


# of Clusters 191,614 188,073 191,788 180,913

Standard Dev 4,872 4,830 4,749 12,726

First Cycle (1)


Machine

Run Date: ReadPrep1

Run Id:

35

First Base Report: T Intensity

188,103 189,265 194,003 183,802

6,983 9,054 3,690 7,773

1,063 1,220 1,128 1,093

73 104 53 97

996 1,087 1,000 971

44 75 51 89

1,379 1,666 1,537 1,496

202 141 263 253

1,190 1,397 1,330 1,184

153 116 220 205

88 87 88 86

2 4 1 5

87 84 86 84

1 4 1 4

88 87 87 85

3 3 4 6

84 83 83 80

2 5 5 7

-229 400 500 -240

7,459 8,059 8,400 8,050

7,688 7,659 7,900 8,290Flow cell Tilt 8,559 9,339 11,240 12,249

Foc Pos Min -750 -2,130 -3,840 -5,340

Foc Pos Max 7,809 7,209 7,400 6,909









T Intensity 1,416 1,329 1,413 1,396

Standard Dev 114 128 112 186

G Intensity 1,759 1,645 1,778 1,714

Standard Dev 151 224 178 218

C Intensity 1,094 1,008 1,059 1,082


A Intensity 1,216 1,132 1,226 1,202


# of Clusters 191,614 188,073 191,788 180,913

Standard Dev 4,872 4,830 4,749 12,726

First Cycle (1)


Machine

Run Date: ReadPrep1

Run Id:

► Used as QC metric. Desire

intensities as high as possible.

► Library dependent (high GC,

monotemplates, libraries

containing synthetic fillers, etc.

alter T intensity)

Read 1 Specs: Starting

Intensity


#

Cycles

Illumina

Specs

Broad

Specs

36 100 400

76 300 600

101 525 1000

126 525 1000

36

Read 2 First Base Report: % Regeneration

188,698 189,035 193,445 188,839

7,001 5,634 5,911 3,070

729 820 770 815

33 43 50 27

699 762 697 739

31 24 36 24

910 1,111 1,069 1,177

45 158 173 159

809 970 934 984

28 122 133 129

86 86 87 86

2 2 2 1

84 84 84 83

1 1 1 1

84 85 86 86

2 3 3 2

80 80 82 81

2 2 4 3

9,160 8,820 8,140 7,020

15,790 15,869 15,769 14,740

6,630 7,049 7,629 7,720Flow cell Tilt 7,940 9,159 10,819 11,960

Foc Pos Min 5,960 4,010 2,000 130

Foc Pos Max 13,900 13,169 12,819 12,090









T Intensity 1,028 956 965 1,084

Standard Dev 151 160 165 200

G Intensity 1,204 1,139 1,134 1,263

Standard Dev 204 213 209 240

C Intensity 761 714 709 833


A Intensity 848 795 793 896


# of Clusters 191,374 189,536 191,547 188,875

Standard Dev 2,831 3,905 3,740 3,580

First Cycle (1)


Machine

Run Date: ReadPrep2

Run Id:

► % Regeneration is calculated

using average T intensity

across all lanes from R1 & R2

(Read1 T int) / (Read2 T int) x 100

Read 2 Guidelines:

► T Regeneration: > 50%*

► * Lower than 50% is fine if

Read 1 had particularly high T

intensities

This FC: 75.67%

(7765/10655)x100%

Read 2 First Base Incorporation

Fail Modes

38

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

39

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 July –December 09

40

Pareto: Fail modes

Nov 09-Jan 10July –December 09

Major fail modes change over time

41

Fail Mode: Oil

Worse in later cycles; Oil expands when heated, wicks onto FC surface, then spreads

► 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

42

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

43

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

44

Fail Mode: OilIdentify/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

45

Fail Mode: RTA Processing “Thread Death”

► 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.

46

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

47

Fail Mode: Poor Linearization

► Can occur at either read 1 or 2

► Characterized by platelet- or donut-

shaped clusters

► Low flow of LMX on cluster station

or PEM is an indicator

Read 1 & 2 Start & Progression

48

(block)

Not all bridges

get linearized

Only edge of

cluster fluoresces

(block)

Good Linearization

Poor Linearization

What we think is happening

Cluster in bridge form

Primer only

incorporated

on linearized

strands

Normal Linearization Efficient Primer

incorporation Nucleotides incorporated

only on primers

49

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

– Mark reagent level on each tube

– Avoid freeze-thaw of reagents

– Avoid storage temperature fluctuations

– Record Lot #s

50

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

51

…but not all of

them get

blocked.

Incorporate onto

cluster AND

unblocked primers

Poor Blocking

Good Blocking

Excess P7 primers on

FC have –OH group,

What we think is happening

Excess P7 primers

on FC have –OH

group

Cluster in bridge form Normal Blocking

All excess primers

blocked

Nucleotides incorporated

only on primers

Normal Primer

incorporation

Normal Primer

incorporation

52

Fail Mode: Poor BlockingIdentify/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

53

Fail Mode: Poor Primer Annealing

► 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”)

54

“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

► 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

55

110,611 112,584 115,076 121,981

4,751 8,056 5,478 5,779

51 72 314 146

12 34 93 91

56 75 296 140

12 33 76 80

209 307 1,201 630

63 163 369 392

93 130 465 253

26 62 134 151

T Intensity 216 229 529 768


G Intensity 456 508 1,136 1,743

Standard Dev 263 213 555 350

C Intensity 124 139 320 523


A Intensity 105 119 271 437


# of Clusters 127,178 127,496 140,946 141,146

Standard Dev 8,154 4,457 13,454 14,493

First Cycle (1)


131,723 132,009 131,675 131,873

1,431 651 660 971

469 505 497 500

17 16 29 21

427 458 447 446

19 12 22 17

2,466 2,662 2,642 2,672

131 112 219 167

974 1,045 1,029 1,027

49 46 69 53

T Intensity 1,287 1,244 1,308 1,279

Standard Dev 113 137 118 109

G Intensity 2,734 2,691 2,652 2,447

Standard Dev 140 146 187 277

C Intensity 579 556 560 547


A Intensity 507 498 491 467


# of Clusters 131,471 133,575 138,406 139,278

Standard Dev 2,696 3,646 8,123 8,602

First Cycle (1)


Avg T = 311

Avg T = 1149

“Rescue Rehyb”

After Rehyb:

1st Base Report

Initial Primer

Hyb:

1st Base Report

56

“Rescue Rehyb”

After Rescue

Rehyb:

Avg T = 1149

Initial Primer

Hyb:

Avg T = 311

57

Fail Mode: Poor Primer AnnealingIdentify/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

58

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)

59

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

60

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

61

Fail Mode: Footprint

► 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

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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

Best Practices

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Best PracticesReagent 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

– 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

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Best PracticesStage/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)

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Best PracticesRun 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

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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

► 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

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Images: Unique Phenotypes

Frosted objective Dried substrate

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► Crop circles?

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► “Ocelot”

– Jen Fostel, Hayward

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► Amoebas? (SYBR QC image)

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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

– Washes

– Flow rates

– Footprints

– Max brightness

– Focus laser

► Best practices

Module 3: Overview - Broad Institute · 3 3 4 6 84 83 83 80 2 5 5 7-229 400 500 -240 7,459 8,059...

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Transcript of Module 3: Overview - Broad Institute · 3 3 4 6 84 83 83 80 2 5 5 7-229 400 500 -240 7,459 8,059...