DNA Sequencing Setup and Troubleshooting Reviewing

DNA Sequencing Setup and Troubleshooting Reviewing
DNA Sequencing Setup and Troubleshooting
Lara Cullen, PhD
Scientific Applications Specialist
Australia and New Zealand
Reviewing Sequencing Data
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Review the Electropherogram
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Review the Raw Data (Signal intensity, data start points, baseline,
length of read, artefacts)
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Review the EPT Plot
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Review Data Analysis settings (correct basecaller, mobility file?)
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Is there any pattern to the problem? (eg: specific capillary?, specific
primer?)
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Good Quality Sequencing Data
Electropherogram
Lara Cullen, PhD
Scientific Applications Specialist
Australia and New Zealand
Peaks should be evenly spaced, not too broad and no tailing
Read length (QV =20) should be ~600bp with POP6 on 50cm Array
Baseline “noise” under the main peaks should be minimal
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Quality Values are an
indicator of the chance of
an incorrect basecall in
your sequencing data
QV 10 = 1 in 10 chance
QV 20 = 1 in 100 chance
QV 30 = 1 in 1000 chance
QV 40 = 1 in 10000 chance
Quality Values of 20 or
higher will give blue bars
when the default settings
are used in Sequencing
Analysis
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Good Quality Sequencing Data
Raw Data
Aim for signal strength in the range of a few hundred to a couple of thousand.
There is no official spec for Signal to Noise Ratio, but samples that have ratios
>100 are generally considered to be good.
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Good Quality Sequencing Data
EPT Data
Check the current and voltage both have the normal profile, anything unusual
could indicate a reagent or instrument problem
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Sequencing Troubleshooting:
Defining the Problem
● Poor data can occur for many reasons
• Instrument Problem?
• Array Problem?
• Polymer Problem?
• Sequencing Primer Problem?
• Template DNA Quality Problem?
• Sequencing Primer Problem?
• Data Analysis Problem?
Controls are an essential part of defining the problem
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BigDye® Terminator v3.1 Sequencing Standard
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Lyophilized sequencing
reactions that require only
resuspension and
denaturation before use
●
Validate the instrument
performance and rule out
problems with common
reagents and consumables
such as polymer, array,
buffer, plasticware and
septa
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Sequencing Standard showing early loss of resolution
Tailing peaks in all 4 colors or early occurrence of broad peaks (LOR) can
indicate the array is starting to wear out and needs to be replaced or that the
polymer needs to be replaced.
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pGEM®-3Zf(+) control DNA + M13 forward primer
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High quality plasmid DNA
and primer that can be used
in a control reaction with
BigDye Terminator ready
reaction mix
●
Run and clean up the pGEM
reaction in the same way as
you do your samples
●
Validates the Chemistry
(BDT kit), thermal cycling
conditions, thermal cycler
and the sequencing reaction
clean-up
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pGEM control reaction showing trailing peaks on C’s only
Tailing in the C peaks alone may indicate a problem with the BDT ready reaction
mix. BDT exposed to light during storage or excessive freeze thaw cycles may
show this problem. Samples loaded in water instead of Hi-Di formamide may also
be more prone to this problem
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Sequencing Reaction Setup Example
Reaction Component
Volume/reaction
BDT Ready Reaction Premix (2.5x)
BigDye Sequencing Buffer (5x)
Primer — 3.2 pmol/ul
Template DNA (10ng/ul)
Water
Final Volume (1X)
1.0 µl
3.5 µl
1.0 µl
1.0 µl
13.5 µl
20 µl
Sequencing Reactions contain only BDT Reaction premix, buffer and
your template DNA and primer.
If sequencing standard and pGEM controls give good quality data the
problem may lie with the template DNA preparation method, template
DNA quantity or the primer quality.
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Additional controls for DNA and Primer quality
● Are some samples giving good quality data and others
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●
●
●
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bad with the same primer?
Are you sequencing the same template DNA with more
than one primer (eg: forward and reverse) and finding one
works much better than the other?
Have you recently switched DNA preparation methods?
Do you quantitate your DNA before sequencing?
Do you have a sample that you know works well that you
can use as a lab specific control?
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Primer Design
● Use Generic Sequencing primers if possible (eg: M13
Forward and M13 Reverse or T3 and T7) when
sequencing plasmids
● When designing primers for Sequencing keep in mind the
following:
– Primers should be at least 18bp and avoid runs of identical
nucleotides to ensure they are specific to the intended target and
hybridise well
– Avoid primers that have the potential to form dimers or have
secondary structure
– GC content should be between 30-80% (50% optimal)
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Primer Design
● In most cases PCR primers with Tm of about 60°C will
work as sequencing primers
● If you are having problems, primers with Tm of about
55°C may work better than higher or lower Tm since
reaction conditions are as follows:
96°C for 1 min
1 cycle
96°C for 10 sec
50°C for 5 sec
60°C for 4 min
25 cycles
4°C Hold
Dilute fresh primers from stocks regularly as they
aren’t as stable when stored at low concentrations
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Template DNA Quality
● When Sequencing PCR products ensure the PCR is
specific
– High Tm primers (60 degrees or more)
– One specific band when run on a gel
– PCR clean up kits or ExoSAP-IT® should work well if the PCR
product is specific
● Commercial plasmid miniprep kits generally give DNA of
sufficiently high quality for sequencing
– Try not to overload the columns
– Some spin column based kits may leave residual resin that can
interfere with injection and cause failed samples. Centrifuge then
take from the top of the sample for sequencing
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Template DNA Quantity
Too much DNA or too little
will reduce the length of
read and the quality of
base calls
The suggested template
DNA quantities should be
used as a guide however
you may need to optimise
your own quantities in
some cases
Quantitate your DNA by gel
electrophoresis or UV
absorbance
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Running controls helps in focusing troubleshooting
efforts and reduces time taken to determine the cause
of the problem
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Common Sequencing Problems
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Sample Prep Issues
● No Injection/sample
● Overloading
– Too much template
– Spectral pull up
● Multiple Sequencing Products
● Poor Sequencing Primer quality
● Dye Blobs
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No Injection/sample
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No Injection/sample
● Failed Reaction
– Component left out of reaction
– Wrong primer used
– Enzyme not working
– Thermal cycler problem
– Not enough DNA
– Inhibitor in DNA
● Labelled product lost during cleanup
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Low Signal
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Low Signal
● Sequencing reaction failed
● Not enough primer/tempate/Big Dye Terminator
● Partial loss of product during cleanup
● Difficult template sequence (Adding 5% DMSO or 1M
Betaine to the reaction may help in some cases)
● Salts in sample interfering with electrokinetic injection
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Sample Overloading
● Can refer to too much template being present and/or a
very robust reaction resulting in a very strong signal.
● In the Raw Data, if the signal exceeds the values below,
the Analysis Software may not analyze the data properly:
– 3100 and 3130/3130xl Genetic Analyzers: >8000 rfu
– 3730/3730xl Genetic Analyzers: >32,000 rfu
Note: RFU values for overloading are based on software analysis values.
Sample overloading and miscalling may occur with rfu values much lower than
listed. Pull-up (peaks under peaks) in the Sequencing data may occur with very
strong/overloaded signal.
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Overloading – Too Much Template
If samples contain too much DNA, the labelled ddNTPs can be
incorporated early on, creating a “top heavy” reaction where
the data looks strong in the front and then gets weaker as the
run progresses, resulting in shorter reads. Careful quantitation
of the DNA template can usually avoid this.
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Ratio of template, primer dye terminator is incorrect, check reaction
components and ensure correct amounts are being used
Template may be degraded
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Overloading – Spectral Pull up
Raw data showing overloaded data on an Applied Biosystems 3100 Genetic Analyzer.
In the areas where the signal is very strong, you can start to see pull down (negative
peaks) in the baseline on multi-capillary instruments.
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Overloading – Spectral Pull up
Raw Data – Peaks are off scale on the Applied Biosystems 3730 Instrument.
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Overloading – Pull Up Peaks
Peaks appearing under peaks in a discernable pattern. Similar
pattern can also occur if the wrong spectral/matrix is used or if the
spectral needs to be re-done due to changes in the optics.
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Sample Prep Issues
Multiple Products
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Multiple Products: Heterozygous insertion or Deletion
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Sample Prep Issues - Multiple Products
Multiple products can be caused by:
• Non-specific binding of the primer to the template
during PCR or Cycle Sequencing
• Multiple clones or colonies present during sample
prep or PCR products
• Heterozygous insertions or deletions (HIM)
• Contamination from water/environment
• Re-using Septa
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Poor Sequencing Primer quality
N+1 effect due to poor primer manufacturing
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Dye Blobs – Problem with sequencing clean-up
Electropherogram showing Dye Blobs
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Raw Data showing Dye Blobs
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General Issues - Hardware
● Hazes – Red, Blue, Green
● Dust
● Bubbles
● Electrophoresis Problem
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Hazes – Red, Blue, Green
Hazes can appear in almost any color and are usually the result of some contaminant
getting into the system.
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Hazes – Red, Blue, Green
Potential Reasons:
● Improper/little maintenance
● Contaminant in the water used to clean the system
● Use of solvents or cleaners to clean instrument
components.
● Residual Carbon or Ozone from an Arcing event.
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Dust causing Spikes in the Data
Raw Data view. The figure on the left is a zoomed in view of the
spike in the data seen in the zoomed out view on the right. Note that
all 4 colors are present in a thin spike.
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Dust causing Spikes in the Data
Analyzed data view of a dust spike crossing the read region.
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Bubbles
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Bubbles
●
Check for leaks on the system.
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Polymer should be allowed to equilibrate and degas for 30 – 60
minutes prior to placing on the instrument.
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Run the Bubble Remove Wizard on the Applied Biosystems
3100/3130 and 3730 series to remove bubbles.
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Make sure all fittings are tight.
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Make sure the Ferrule Tip area is clear of bubbles or microbubbles.
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If bubbles persist in spite of all fittings being tight please call AB
Technical Support for assistance
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Electrophoresis Problem
Electropherogram
Raw Data
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Electrophoresis Problem
EPT Data
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Analysis Troubleshooting
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Dye Sets/Virtual Filter Sets
Dye set/Filter set needs to be chosen before the run,
normally samples need to be re-run if the wrong dye set
is chosen
Sequencing Dye Sets
E: BigDye® Terminator v.1.1
Z: BigDye® Terminator v.3.1
Fragment Analysis Dye Sets
D: 6-FAM™, NED™, HEX™, ROX™
D: 6-FAM™, NED™, VIC®, ROX™
™
™
™
™
F: 5-FAM , JOE , NED , ROX
™
®
™
G5: 6-FAM , VIC , NED , PET®, LIZ®
E5: dR110, dR6G, dTAMRA™, dROX™, LIZ®
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Analysis Problem – Wrong Dye Set/Primer File
The sample above was Analysed with the Dye Set/Primer file for BDTv1.1
but the chemistry used to set up the reactions was BDTv3.1
Overlapping bases and poor quality values result
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Analysis Problem – Wrong Dye Set/Primer File
This is the same sample re-analysed with the Dye Set/Primer file for BDTv3.1
Note the improvement in the spacing and quality values
This is the only circumstance in which you can change the dye set after the run
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For any questions you have or assistance you
need contact AB Technical Support:
[email protected]
1800 636 327 (Aus) / 0800 636 327 (NZ)
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