The Discovery Moment:

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The Discovery Moment discovery Applied
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AB Sequencing Milestones
1988 373
1995 377
1996 310
1999 3700
2000 3100
2002 3100-Avant
2002 3730 3730xl
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The Applied Biosystems Family of Capillary
Electrophoresis Genetic Analysers
3100-Avant
3730
310
3100
3730xl
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Electrokinetic Injection Electrophoresis

• Capillary and electrode are placed into the
  sample
• Voltage is applied
• - ve charged DNA enters the capillary as it
  migrates toward the ve electrode at the other
  capillary end

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Fluorescence Excitation Detection

• DNA fragments labeled with one of up to five
  different dyes electrophorese past the laser
• Laser excites dyes causing them to emit light at
  wavelengths longer than that of the laser
• Emitted light is collected by a CCD camera
• Software converts pattern of emissions into
  coloured peaks

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Cycle Sequencing
Dideoxy chain termination (Sanger) method
Dye primer or dye terminator labelling methods
- Dye primer - fluorescently-labelled primers -
Dye terminator - fluorescently-labelled ddNTPs
Dye Terminator
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Introducing BigDye Terminator v3.1 and
v1.1New generation DNA Sequencing
chemistries for difficult templates and assured
heterozygote detection
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BigDye v3.1 and v1.1 Improvements Over v3.0 and
v1 Kits
Improvement
Result

• Greater success with difficult templates
• Longer, higher quality reads
• More accurate mixed base detection

• Enhanced Robustness
• Greater Peak height uniformity
• Optimized Signal Balance

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BigDye v3.1 and v1.1 Cycle Sequencing Kit
Characteristics

• v3.1 and v1.1 are reformulations of v3.0 and
  original v1 chemistries
• No new software or instrument recalibration
  required for use
• Optimized protocols will enhance performance
• Complementary sequencing buffer formulated
  specifically for v3.1 and v1.1 chemistries

Note Assumes instrument currently has
corresponding BigDye chemistry calibration files
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BigDye v3.1 and v1.1 Cycle Sequencing Kit
Characteristics (contd)

• v3.1 kit uses the same dyes as v3.0 kits while
  v1.1 kit uses the same dyes as v1 and v2.0 kits
• v3.1 and v1.1 kits feature a new enzyme
• v3.1 (v1.1) kit is designed to be no less
  dilutable than v3.0 (v1)kit. For most
  templates, v3.1 (v1.1) kits dilutability will
  either be equivalent to or incrementally greater
  than the v3.0 (v1) kit.

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Optimised Protocols for v3.1 and v1.1 Cycle
Sequencing Kits

• Chemistries will perform well with current
  protocols but new optimised protocols will
  enhance performance
• New Cycle Sequencing Protocol includes the
  addition of an initial 1 minute 96C denaturation
  step (rest of profile the same as for v3/v1)
• Ethanol/EDTA is recommended for cleanup
• EDTA/Sodium Acetate/Ethanol recommended for
  recovery of low molecular weight fragments

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BigDye v3.1 and v1.1 Kits Applications

• v3.1 kit for most applications
• de novo sequencing
• Resequencing
• v1.1 kit for specialty applications
• Sequencing short PCR products using rapid run
  modules
• Other applications requiring optimal basecalling
  adjacent to primer
• Customers using v3.0 kit will likely prefer v3.1
  kit, and those using v1 kit will likely prefer
  v1.1kit
• v2.0 kit customers should select appropriate
  chemistry for their application(s)

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Customer 1 data shows greater number of Phred
Q20 bases with v3.1 chemistry
Data courtesy of Agencourt
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Customer example of more Q20 bases with a
difficult template using BigDye Terminator v3.1
Chemistry
Kits
v3.0
v3.1
Data courtesy of Agencourt
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Customer 2 data shows greater number of Phred
Q20 bases with v3.1 chemistry
Data courtesy of Washington University
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Customer example of more Q20 bases with difficult
templates using BigDye Terminator v3.1 Chemistry
Kits
v3.0
v3.1
Data courtesy of Washington University
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Customer example of more Q20 bases with difficult
templates using BigDye Terminator v3.1 Chemistry
Kits
v3.0
v3.1
Data courtesy of Washington University
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Customer 3 data shows greater number of Phred
Q20 bases with v3.1 chemistry
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Long read with BigDye Terminator v3.1 run on
3100 Genetic Analyzer, 80 cm array, and POP-4
Polymer
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Long read with BigDye Terminator v3.1 on the
3730xl DNA Analyzer, 50 cm array, and Pop-7
Polymer
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Customer example of BigDye Terminator v3.1
reading through G-rich region
Kits
v3.0
v3.1
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Core lab customer example of BigDye Terminator
v3.1 Chemistry sequencing a difficult template
Kits
v3.0
v3.1
Data courtesy of DNA Sequencing Core Lab,
University of Utah
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Customer example of more Q20 bases with difficult
templates using BigDye Terminator v1.1 Chemistry
Kits
v1.0
v1.1
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Customer example of improved performance with
BigDye Terminator v1.1 Reading through region
with high G/T
Kits
v1.0
v1.1
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Customer example of improved performance with
BigDye Terminator v1.1 - Reading through Poly T
stretch
Kits
v1.0
v1.1
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Customer example of improved performance with
BigDye Terminator v1.1 Reading through C/T
repeat
Kits
v1.0
v1.1
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A Generational Improvement in Dye Terminator Peak
Pattern Facilitates Mutant Detection
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Improved peak height uniformity with customer
samples
Note 100 peak height uniformity represents
idealized situation in which all analyzed data
peaks are of equivalent height
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Better Uniformity with BigDye Terminator v3.1
yields more high quality bases/template
Kits
v3.0
v3.1
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Mixed Base Calling with v3.1 kit MicroSeq
Template
Kits
v3.0
v3.1
Samples run on 3700 instrument with POP-6 Polymer
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Customer Example of Increased Q Values Leading to
More Q20 Bases
Data Courtesy of Dr. Tim Holzer,The Whitehead
Institute/MIT Center for Genome Research
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BigDye Terminator v3.1 Sequencing Chemistry
delivers

• Improved Heterozygote Detection
• Sequencing through otherwise difficult templates
  where other chemistries fail
• Higher quality data

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Dilution of BDT v1.1 v3.1

• Keep to 20 ul rxn volume
• Use diln buffer (included)
• Run a control
• Sequence in both directions if possible

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Analysis and Interpretation of Data .ab1 Sample
File Format
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Printouts contain lots of useful info

• Electropherogram
• Basecalls
• Signal Strength
• Analysis Settings
• Base Spacing
• Heterozygotes (if required)
• Quality Values (NEW)

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The electropherogram printout
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ABI Prism data files are industry standard

• Sequence files are imported by products from
  these vendors
• SequencherTM (Genecodes)
• BioEdit (Dept Micro at North Carolina)
• Phred/Phrap/Consed (Uni Washington)
• ABIview (Stanford)
• STADEN (Medical Research Council UK)
• EMBOSS (EMBnet)
• Trace Viewer Plus (Arizona Res. Labs.)
• Chromas (Griffith Uni)

• SeqMan IITM (Lasergene/DNASTAR)
• CGG (Sanger)
• SeqMergeTM (Accelrys)
• VectorNTITM (Informax)
• JellyfishTM (BioWIRE)
• DNASISTM (MiraiBio/Hitachi)
• RIDOM (Uni. Wurzburg)
• Sequencing AnalysisTM
• EditViewTM
• SeqScapeTM
• MatchToolsTM
• ViroSeqTM
• MicroSeqTM

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Electropherogram view
Data view icons
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Electropherogram View (NEW)
Quality Values
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Annotation / information view
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Sequence (text) view
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Raw data view
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EPT view
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A successful sequencing Reaction!
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Too much template
Try to optimise Primer/Template ratio for better
balance.
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Top Heavy Data (with short read length)
Cause - excess template - poor
primertemplate ratio
Solution - reduce template - adjust primer
template ratio

• average signal intensity should be lt 4000 rfu
• (between 150 1500 ideal)

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Too Much Template
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Broad Peaks
Cause - excessive amount of sample injected into
capillary
Solution - reduce concentration of sample
(dilute) and re-inject - reduce time of EKI
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How much template? - New for v3.1
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Primers

• Purity
• Mismatch
• Second priming site
• No hybridisation site
• Secondary Structure
• Primer Dimer
• Slippage

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Primers

• gt18 bases long to ensure good hybridisation (also
  minimises chance of secondary hybridisation site
  on the target DNA).
• Avoid runs of an identical nucleotide, especially
  gt 4 Gs
• Keep the G-C content in the range 3080.
• Keep Tm above 45 C to produce better results
• For primers with a G-C content lt 50, it may be
  necessary to extend the primer sequence beyond 18
  bases to keep the Tmgt45C.
• Avoid primers that have secondary structure or
  that form primer dimers

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Primer Tips for cleaner sequences

• Try not to sequence with the amplification primer
  set!
• consider
• nested primers
• or
• tailing the amp primers

M13for
M13rev
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Calculating Primer Tm
For DNA 10-40 bases
Tm (?H/(?S So - Rxln(C/4))) -273.15
16.6xlogK
?H nearest neighbor enthalpy ?S nearest
neighbor entropy So initiation entropy C
oligo concentration K monovalent salt conc. R
molar gas constant (1.987 cal/oC-mol)
NAR 186409 (1990)
Included in 9700 thermal cycler keypad!
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16 mer, 37C anneal
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16 mer, 50C anneal, Low Signal
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30 mer, 37C anneal, Good Data
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Primer Dimer
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No Binding Site
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Failed Reaction

• check average signal intensity (fail is lt 50
  rfu)
• check EPT view in Sequencing Analysis software
  (diagnostic of run)
• check control pGem/M13 primer reaction
• - if successful, thermal cycler, sequencing and
  clean-up OK
• most likely template or primer problem
• - if control reaction unsuccessful
• may be thermal cycler, instrument or clean-up
  step
• (ie. lost pellet)
• - check primer and template

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Purification of Extension Products methods of
choice
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Purification of Big Dye Terminator Extension
Products
Precipitation vs Spin columns
Inexpensive, quick incomplete removal of ddNTPs
More costly, time consuming unless used in plate
format, Gold Std for efficient removal of ddNTPs
EtOHEDTA vs EthanolEDTANaOAc (Cleanest) vs (Mos
t Efficient)
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BDT v 3.1 Ethanol / EDTA Precipitation

• use this method for the cleanest
• sequencing reactions very effective at
• removal of unincorporated dye terminators
• - may result in loss of smaller mol wt fragments

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BDT v 3.1 Ethanol / EDTA / NaOAc Precipitation

• use this method when good signal
• from base 1 (3 of primer) is required
• (not ideal if dye blobs/carryover
• dye terminators is a problem)

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Precipitation Methods to Remove Residual Dye
Terminators
BDT v 3.1 final ethanol Conc should be between
67-71
Old chemistries (prior to v3.1 and v1.1)
Ethanol - recommended final concentration 60
3 ( 70 ethanol
wash) Isopropanol - recommended final
concentration 60 5 ( 75
isopropanol wash)
Isopropanol Ethanol
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Dye Blobs
T (210 bp)
G (90 bp)
G (240 bp)
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Dye Blobs at large sizing
Cause - poor ethanol precipitation procedures
- poor spin column purification procedure
Solution - remove ethanol completely after first
spin - load sample in centre of column bed
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G-Breakdown (G-blobs)
Cause - chemical degradation of Big Dye G label
(co-migrate with sequencing fragments)
- due to formamide-EDTA degradation
Solution - minimise exposure of samples to air
- use HiDi formamide
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General Precipitation Tips!

• absolute ethanol absorbs water from atmosphere,
  gradually decreasing its
• concentration open a new bottle regularly
• - ensure ethanol is removed completely from first
  precipitation step
• - always include a wash step after initial
  precipitation
• - leave products for at least 15 min, but not gt
  24 h
• - be efficient after first spin
• dont leave precipitated extension
  products in ethanol to resuspend
• - avoid drying samples in a Speedvac, air dry at
  room temp
• - protect dyes from light cover samples with
  foil

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Electrokinetic Injection

• Load Bar is Electrode
• Current is Applied
• - Charged DNA Enters the Capillary
• DNA Migrates Toward Detector

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Factors Affecting Injection

• Small molecular weight ions compete with
  sequencing products
• Template and primer quality and quantity
• Instrument conditions
• Applied voltage (run module)
• Injection time (run module)

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Contamination Problems

• Proteins
• Can be injected and adhere to the walls
• Detergents
• Triton-X and SDS will cut short array life
• Salts Unlabelled Nucleic Acids
• Prevent samples from injection

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Salts even affect your gel

• Excess RNA can act like excess salt
• Negative salt ions are preferentially injected
• Leads to shortened read length

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Therefore for CE..

• Sample Prep more important
• Salts inhibit injection
• Proteins clog capillaries
• Contaminates more easily seen
• Not as much sample used
• Lesser chance of operator error

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Spectral calibration files (matrix file)
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Spectral Calibration Files
- a file required by Sequencing Analysis
software for the correction of spectral overlap
between dyes in each dye set
When should a spectral calibration be
performed? - evidence of incomplete spectral
compensation (pull-up) - when using a new dye
set (Eg. v2.0 vs v3.1 chemistry) - whenever
polymer type (POP4 vs POP6) is changed
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Poor matrix file (operator should run new stds)
Incorrect matrix file (let operator know what
chemistry you have used)
Pull-up peaks (too strong dilute)
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More Troubleshooting
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Insertion/Deletion Heterozygote
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Sample Contamination
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Homopolymer T regions
- data is noisy after homopolymer regions due to
slippage of Taq polymerase during sequencing
reactions - sequence region using an anchored
primer 24 T bases followed by 2 bases of
sequence (G,A or C) at 3-end of primer
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Capillary Bubbles
Cause - bubbles in polymer, or bubbles generated
in the capillary Solution - replace
polymer - re-inject sample (repeat)
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www.appliedbiosystems.com
Support Hotline [phone] (NZ) Support
Email abozsupport_at_appliedbiosystems.com
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Data.
Decision.
Discovery.
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Dye Primer Chemistry
Primer dimers - seq. complementarity
between primers (3 ends) use Dye
Terminator Chemistry
A
Stop peak - old template prep. re-isolate DNA
B
C
False stop - secondary structure use Dye
Terminator Chemistry
Primer design - avoid gt 2 GCs in last 5 bases at
3 end