Download DNA Sequencing Troubleshooting Guide

DNA Sequencing
Troubleshooting Guide
© Copyright 2011 Eurofins MWG Operon, Inc.
Table of Contents
Successful DNA Sequencing Read
1
Failed DNA Sequencing Reaction or “Dirty” Sequence
2
Double Sequence Data
3
Noisy Background
4
Stuttering after Repetitive Sequences
5
Mid-Sequence Drop-Off
5
Top-Heavy Sequences
6
Spikes
Early Peak Deterioration
6
7
Successful DNA Sequencing Read
• Peaks are well formed and separated with good quality scores. There is a small area at the beginning
of the
run before theTroubleshooting
chemistry stabilizes. Guide
DNA
Sequencing
• Signal strength is high, typically in the thousands
Sequence Appearance:
- Examples of successful plasmid and PCR sequencing results
Examples:
Failed DNA sequencing reaction or “dirty” sequence
Sequence Appearance:
- Chromatogram data looks messy or is mostly blank.
- Many ‘N’s in the sequence, if bases are called at all.
- Blast search from .seq or fasta file yields unexpected results.
- Raw data has signal intensity in the low hundreds.
1
Failed DNA Sequencing Reaction or “Dirty” Sequence
Sequence Appearance:
• Chromatogram data looks messy or is mostly blank.
• Many ‘N’s in the sequence, if bases are called at all.
• Blast search from .seq or fasta file yields unexpected results.
• Raw data has signal intensity in the low hundreds.
Examples:
Possible
Causes:
Possible Causes:
It is difficult to pinpoint a specific cause
cause for
for the
the failure
failure when
when there
there are
are no
no data
data for
for review.
review. Common causes
Common
causes are:
are:
1. DNA Template concentration is too low – Note: measuring DNA concentration by UV absorption
• DNA Template
concentration
is too low – Note:
measuring
DNA concentration
UV are
absorption
is often inaccurate
and concentration
is frequently
overestimated.
Agarosebygels
a betteris often
method
estimate quality
and quantity
of DNA samples.
inaccurate
andtoconcentration
is frequently
overestimated.
Agarose gels are a better method to estimate quality
2. quantity
Wrong primer
no primer was added to the reaction – binding between the DNA and the primer
and
of DNAorsamples.
cannot occur in either of these instances.
• Wrong
primer
or no
primer
wasincomplete
added to the
reaction
– binding
between
the
3. Low
quality
prep
– yields
removal
of protein
and/or
RNA,the
or DNA
bufferand
salts
orprimer
other cannot occur
in either
of
these
instances.
chemical contamination may be present. Any of these conditions can inhibit sequencing reaction
enzymes.
Treatment:
Treatment:
1. Check the concentration of your template by agarose gel to ensure that it falls with the ranges
• Checklisted
the concentration
your template
by agarose gel to ensure that it falls with the ranges
in the Sampleof
Submission
Guidelines.
listed
in
the
Sample
Submission
Guidelines.
2. Check the primer sequence against the template sequence to ensure that there is a proper
binding site.
• Check the primer sequence against the template sequence to ensure that there is a proper binding site.
3. Improve the quality of the template prep
a. quality
Prepare
fresh
stock solutions
for template prep and dilution just prior to submitting
• Improve the
of the
template
prep
samples
for
sequencing.
a. Prepare fresh stock solutions for template prep and dilution just prior to submitting samples for sequencing.
Prepare
plasmid
using a commercial
mini-prep kit.
b. Prepareb.plasmid
DNA
using DNA
a commercial
mini-prep kit.
c.
A
final
ethanol
precipitation
after
the
prep
may
ensure success.
a. A final ethanol precipitation after the prep may help ensurehelp
success.
2
Double Sequence Data
Double Sequence Data
Sequence Appearance:
Sequence Appearance:
- Sequencing peaks are not “clean”. Multiple peaks with the same height or of differing hei
overlapping one another.
- Raw data has adequate signal intensity, indicating that the double peaks are not due to w
and/or background noise.
Example:
• Sequencing peaks are not “clean”.
Multiple peaks with the same height or of
differing heights, overlapping one another.
• Raw data has adequate signal intensity,
indicating that the double peaks are not due
to weak signal and/or background noise.
Possible Causes:
• Clone contamination – in this case, the
beginning of the sequence is often clean and becomes ‘dirty’ as the sequence progresses past the clone insertion site.
• PCR template may be heterozygous
due to indels present in a diploid
(or polyploid) organism.
• Two primers may have been mistakenly added
to the sequencing reaction.
Possible Causes:
1. Clone
contamination
– in this case, the beginning of the sequence is often clean a
• PCR products were not purified (or the purification was not
performed
properly).
‘dirty’ as the sequence progresses past the clone insertion site.
In this case, residual PCR primers may participate in the sequencing
reaction.
2. PCR template may be heterozygous due to indels present in a diploid (or polyploid
3. Two primers may have been mistakenly added to the sequencing reaction.
• Primer may also be binding to another area of the template
withproducts
sufficient
4. PCR
werehomology.
not purified (or the purification was not performed properly). In
residual PCR primers may participate in the sequencing reaction.
5. There may be enough homology in another area of the template that where the se
• Primer may have degraded.
primer begins extension.
Treatment:
1. If clone contamination is expected, please return to your clone resource and repla
Treatment:
onto selection media. You may want to select up to 12 clearly separated clones fo
ensure you find the actual clone of interest.
• If clone contamination is expected, please return to your2.clone
resource and replate the bacteria onto selection
Where PCR template heterozygosity is suspected, examination of the ‘dirty’ seque
media. You may want to select up to 12 clearly separated clones
for sequencing
you find
the
actualPCR primers that y
some indication
of the areato
of ensure
heterozygosity.
Newly
designed
products may allow you to discern where the troublesome area is. Alternatively, yo
clone of interest.
to redesign the sequencing primer close to the area where the problem first arises
a primer
sequence
one of the
product
• Where PCR template heterozygosity is suspected, examination
of that
thewill
‘dirty’
sequence
may
yieldspecies.
some If the PCR product is large
template into shorter pieces may also provide a strategy for discerning the true se
indication of the area of heterozygosity. Newly designed PCR
primers that yield shorter products may allow
area of interest.
you to discern where the troublesome area is. Alternatively,
you may
decide
redesign
the sequencing
primer
3. When
you suspect
thattotwo
different primers
may have been
added to the reaction
simply
repeat the
reaction.
If the working
solution
forproduct
the sequencing primer may b
close to the area where the problem first arises hoping to find
a primer
that
will sequence
one
of the
then going back to your original uncontaminated stock solution to prepare a new w
species. If the PCR product is large, subcloning the template
into shorter pieces may also provide a strategy
or resynthesize the primer.
for discerning the true sequence of the area of interest. 4. Check your PCR purification protocol and the solutions used in the clean-up react
• If you suspect that two different primers may have been added to the reaction, repeat the reaction using the
original stock primer solution, in case your working solution is the source of the contamination.
• Check your PCR purification protocol and the solutions used in the clean-up reaction.
• Check the primer sequence against the template sequence to ensure that there is a single binding site. Where
sequence is unknown, you may need to switch to a different sequencing primer to eliminate the problem.
• Order new batch of sequencing primers.
3
Noisy Background
Sequence Appearance
DNA Sequencing Traces with Noisy Background
• Background noise and odd peaks are present underneath the main sequence peaks
•Sequence
Raw dataAppearance:
has signal intensity in the low hundreds.
- Background noise and odd peaks are present underneath the main sequence peaks
Example:
Possible
Causes
Possible Causes:
1. Clone contamination – in this case, the beginning of the sequence is often clean and becomes
• Clone contamination – in this case, the beginning of the sequence is often clean and becomes ‘dirty’ as the
‘dirty’ as the sequence progresses past the clone insertion site.
sequence progresses past the clone insertion site.
2. PCR template may be heterozygous due to indels present in a diploid (or polyploid) organism.
3. template
Two primers
have been mistakenly
added
to theinsequencing
• PCR
may may
be heterozygous
due to indels
present
a diploid (orreaction.
polyploid) organism.
4. PCR products were not purified (or the purification was not performed properly). In this case,
• Two primers
may
have
been may
mistakenly
added
to the
sequencing
reaction.
residual
PCR
primers
participate
in the
sequencing
reaction.
5. There may be enough homology in another area of the template that where the sequencing
• PCR products were not purified (or the purification was not performed properly). In this case,
primer begins extension.
Treatment:
residual PCR primers may participate in the sequencing reaction.
1. If clone contamination is expected, please return to your clone resource and replate the bacteria
• Primer may also be binding to another area of the template with sufficient homology.
onto selection media. You may want to select up to 12 clearly separated clones for sequencing to
ensure you find the actual clone of interest.
Treatment
2. Where PCR template heterozygosity is suspected, examination of the ‘dirty’ sequence may yield
some indication of the area of heterozygosity. Newly designed PCR primers that yield shorter
• If clone
contamination
is expected,
please
return
your clone resource
and replate the
onto selection
products
may allow
you to discern
where
thetotroublesome
area is. Alternatively,
youbacteria
may decide
media.toYou
may want
to select upprimer
to 12 clearly
clones the
for problem
sequencing
ensure
you find
the actual
redesign
the sequencing
close toseparated
the area where
firsttoarises
hoping
to find
clone aofprimer
interest.
that will sequence one of the product species. If the PCR product is large, subcloning the
template into shorter pieces may also provide a strategy for discerning the true sequence of the
• Where PCR template heterozygosity is suspected, examination of the ‘dirty’ sequence may yield some
area of interest.
indication of the area of heterozygosity. Newly designed PCR primers that yield shorter products may allow
3. When you suspect that two different primers may have been added to the reaction, you can
you to discern where the troublesome area is. Alternatively, you may decide to redesign the sequencing primer
simply repeat the reaction. If the working solution for the sequencing primer may be contaminated
close then
to thegoing
area back
where
problem
arises hopingstock
to find
a primer
will sequence
one of
the product
to the
your
originalfirst
uncontaminated
solution
to that
prepare
a new working
solution
species.
If
the
PCR
product
is
large,
subcloning
the
template
into
shorter
pieces
may
also
provide
a strategy for
or resynthesize the primer.
discerning
truePCR
sequence
of theprotocol
area of and
interest.
4. Checkthe
your
purification
the solutions used in the clean-up reaction.
thethat
primer
sequenceprimers
againstmay
the have
template
ensure
thatrepeat
there isthe
a single
• If 5.
youCheck
suspect
two different
beensequence
added toto
the
reaction,
reaction using the
binding
site.
Where
sequence
is
unknown,
you
may
need
to
switch
to
a
different
sequencing
original stock primer solution, in case your working solution is the source of the contamination.
primer to eliminate the problem.
• Check your PCR purification protocol and the solutions used in the clean-up reaction.
• Check the primer sequence against the template sequence to ensure that there is a single binding site. Where
sequence is unknown, you may need to switch to a different sequencing primer to eliminate the problem.
4
Stuttering after Repetitive Sequences
Stuttering Appearance
after Mononucleotide Stretches
Sequence
•Sequence
Sequencing
data quality is poor after mononucleotide strectches or tandem repeats.
Appearance:
- Sequencing after
data quality
is poor after stretches
of 7 or more nucleotides of the same base
Stuttering
Mononucleotide
Stretches
Example:
Sequence Appearance:
- Sequencing data quality is poor after stretches of 7 or more nucleotides of the same base
Possible
Causes
Possible Causes:
slippage
during
DNA
synthesis.
This
is aisrecognized
limitation
of the
Sanger
method.
•Polymerase
Polymerase
slippage
during
DNA
synthesis.
This
a recognized
limitation
of the
Sanger
method.
Treatment:
•Options
Polymerase
frameshift
error during
PCRare
amplification.
go around
the stuttering
artifact
PossibletoCauses:
1.
To
sequence
from
the
reverse
direction
Polymerase slippage during DNA synthesis. This is a recognized limitation of the Sanger method.
2. Use a poly-mononucleotide primer with a degenerate base (wobble) at the 3’ end.
Treatment:
Treatment:
Options to go around the stuttering artifact are
• Sequence
from the reverse direction
1. To sequence from the reverse direction
2. aUse
a poly-mononucleotide
a degenerate
(wobble)
3’ end.
• Use
poly-mononucleotide
primerprimer
with awith
degenerate
base base
(wobble)
at theat3’the
end.
Mid sequence stop or drop-off
Mid-Sequence
Stop or Drop-Off
Sequence Appearance:
- The sequence
DNA sequence
suddenly
stops or peak intensity drops off substantially. NOTE: Many popular
Mid
stop
or drop-off
Sequence
Appearance:
cloning vectors
have palindromes flanking their linker and may show the drop-off effect when sequenced.
This is a limitation
of the Sanger method, but it can many times be overcome with PowerRead
•Sequence
The DNAAppearance:
sequence suddenly stops or peak intensity drops off substantially.
technology.
- The
DNAMany
sequence
suddenly
or peak
drops
off substantially.
: Mshow
any popular
NOTE:
popular
cloningstops
vectors
have intensity
palindromes
flanking
their linkerNOTE
and may
the drop-off
cloning
have palindromes
theiroflinker
and may
show but,
the drop-off
effect
when
effectvectors
when sequenced.
This is flanking
a limitation
the Sanger
method
many times
it can
besequenced.
overcome with This
is a limitation
of the Sanger
method,
but it canMWG
manyOperon.
times be overcome with PowerRead
Power
Read chemistry,
available
from Eurofins
technology.
Example:
Possible Causes:
1. Secondary structures in the DNA template (e.g. hairpin loops, palindromes)
2. GC or GT rich regions
Possible
Causes
3. Sample
is an siRNA construct
Possible
Causes:
Treatment:
1. Secondary
structures
the DNA
template
(e.g. hairpin
palindromes)
• Secondary
structures
in theinDNA
template
(e.g. hairpin
loops,loops,
palindromes)
Power
yields excellent results with these template types.
2. Read
GC ortechnology
GT rich regions
• GC
GT richisregions
3. orSample
an siRNA construct
Treatment:
• Sample is an siRNA construct
Power Read technology yields excellent results with these template types.
Treatment
• Power Read technology yields excellent results with these template types.
5
Top-Heavy Sequences
Top-heavy
sequences
Sequence Appearance
Sequence
Appearance:
• Sequencing
signal gradually drops off
- Sequencing signal gradually drops off
Example:
Top-heavy sequences
Sequence Appearance:
- Sequencing signal gradually drops off
Possible Causes
Possible
• ExcessCauses:
DNA template or primer.
1. Excess DNA template or primer.
• GC
GTorrich
(from (from
bisulfide
treated
DNA,DNA,
for example)
2. orGC
GTtemplate
rich template
bisulfide
treated
for example)
Treatment:
• Long stretch of short tandem repeats.
Carefully quantify your DNA template and primer prior to sequencing.
Treatment
•Possible
Carefully
quantify your DNA template and primer prior to sequencing.
Causes:
Spikes
1. Excess DNA template or primer.
• Sequence from the reverse direction
2. GC or GT rich template (from bisulfide treated DNA, for example)
Sequence
Appearance:
Treatment:
• Use a poly-mononucleotide
primer with a degenerate base (wobble) at the 3’ end.
-Carefully
Sharp, high-intensity,
peaks
that randomly
in the sequence
quantify your multicolored
DNA template
and primer
prior to appear
sequencing.
Spikes
Sequence
Appearance
Spikes
•Sequence
Sharp, high-intensity,
Appearance:multicolored peaks that randomly appear in the sequence
- Sharp, high-intensity, multicolored peaks that randomly appear in the sequence
Example:
Possible Causes:
The cause of spikes is not completely understood. One possible explanation is that an impurity or micro
bubble passes through the camera view of the sequencing instrument, scattering light.
Treatment:
The surrounding sequence should still be correct and accurate if of good quality, please request your
sample to be rerun if necessary.
Possible
Causes
Possible Causes:
cause
of spikes
is not
completely
understood.
One
possible
explanation
is that
an an
impurity
or micro
•The
The
cause
of spikes
is not
completely
understood.
One
possible
explanation
is that
impurity
or micro bubble bubble passes through the camera view of the sequencing instrument, scattering light.
passes through the camera view of the sequencing instrument, scattering light.
Treatment:
The surrounding sequence should still be correct and accurate if of good quality, please request your
Treatment
sample to be rerun if necessary.
• The surrounding sequence should still be correct and accurate. Please request your sample to be rerun,
if necessary.
6
Early Peak Deterioration
Early peakAppearance
deterioration
Sequence
Appearance:
•Sequence
Peaks become
broad or oddly shaped very early on in the sequence.
- Peaks become broad or oddly shaped very early on in the sequence.
Example:
Possible
Causes
Possible Causes:
Salt or other impurities carried over from DNA isolation protocols, especially “home made” protocols.
•Treatment:
Salt or other impurities carried over from DNA isolation protocols, especially “home-made” protocols.
Use a commercial desalting kit to further purify the DNA template before sequencing.
Treatment
• Use a commercial desalting kit to further purify the DNA template before sequencing.
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7