Sequencing Troubleshooting:
The Genomics Core Technology Unit is happy to consult with any of its
customers having difficulty with DNA sequencing. Please don’t hesitate to
contact us with any questions you may have.
Cycle Sequencing Reactions
PCR Product
100-200bp
200-500bp
500-1000bp
1000-2000bp
>2000bp
Recommended Quantity
1-3ng
3-10ng
5-20ng
10-40ng
40-100ng
Below is the Master Mix that we recommend using the ABI cycle sequencing
kit:
X1
Big Dye Reaction Buffer X5
2.0µl
ddH2O
6.28µl
Sequencing Primer (10µM in TE 0.1M)
0.16µl
Big Dye Reaction v3.1
0.16µl
8.6µl
1. Pipette 8.6µl of Master Mix into the required number of wells in the
sequencing plate
2. Add 1.4µl of PCR product/plasmid.
3. Include a NTC as a control for each set of sequence reactions.
4. If the sequence is GC rich, add 0.2 µl Big Dye reaction v 3.1
5. Perform cycle sequencing on PCR machine as follows:
96ºC for 20 secs
96ºC for 10 secs
50ºC for 5 secs
60ºC for 4 minutes
25 cycles
4ºC soak until required.
Samples need to be cleaned-up either prior to bringing over for electrophoresis
or Clean-up requested on form.
Sequencing plasmids
Select a single colony and use 400 ng of template in the sequencing reaction. It
is good practice to select a single colony and re-streak on a fresh agar plate and
then the following day select a single colony again for sequencing. This should
ensure that a single pure template is sequenced. Below is an example of a
plasmid sequence with reads up to approximately 700 – 800 bp.
Sequencing PCR product
Optimise the PCR reaction to ensure there is only one product and check on
agarose gel. Clean-up product and use 10-20 ng for sequencing reaction. Below
is an example of a sequence profile for a PCR product.
The main sequencing problems fall into 2 categories:
1 Reaction problems
A. Blank Samples
Sequencing strands are not produced leading to a lack of callable peaks. Two or
three large ddNTP peaks (commonly referred to as dye-blobs) are the only
observable peaks present. Typical result in processed data is a sequence of
“NNNN”. On chromatogram view you will see data with undefined peaks close to
the baseline. Signal strengths below 200 are considered blank. See examples
below:
Possible causes:
1.
An insufficient amount of template/primer -The most common reason
for a reaction failure is insufficient amount of template due to overestimation of template concentration. It is very important that template
concentration is accurately measured. Dirty template preps will often
give inaccurate high Abs. 260nm readings leading you to think you have
more DNA than you really do.
Solution: Increase amount of template in reaction.
2.
Incorrect primer used or no primer annealing site in the template.
Even a one base mismatch between primer and template can result in a
blank sample. The best way to test this possibility is to perform a regular
PCR reaction using the sequencing primer and a second primer. Use 1 ng
template and 100 ng each primer for 25 cycles. If you don’t see a strong
band of your correct product, you know your primer(s) aren’t annealing.
Solution: Primers may need to be re-designed. Primers should be designed
using the following guidelines: 18-22 bp long, 50-55% GC content and Tm of 5560oC.
3.
Primers have degraded through freeze-thaw cycles
Solution: Make up new primer dilutions.
4.
The presence of PCR inhibitors such as alcohols and EDTA-
Solution: Samples containing PCR inhibitors will have to be re-purified using a
high quality commercial kit.
Our facility carefully monitors our BigDye and cycling conditions by using controls
on every full sequencing plate.
B. Weak Signal
Causes: The reasons for low signal strength are nearly identical to those of a
reaction failure, but to a lesser degree. As with blank samples, the most
common reason is an insufficient amount of template.
Solution: All actions mentioned in the reaction failure section can be used to
overcome low signal strength.
C. Truncated sequence – ski slope effect
Causes: This is usually caused by too much template or by impurities inhibiting
the sequencing reaction.
Solution: Reduce concentration of template in sequencing reaction. If this fails
re-purify template.
D. Multiple peaks throughout
Causes:
1.
Multiple primer sites.
Solution: Design different primers.
2.
Carry-over of residual primers from PCR stage.
Solution: Ensure PCR sample is well cleaned–up so that PCR primers have
been removed.
3.
Poor purification during primer synthesis – full length primer is mixed
with primer missing one base giving a shadow sequence one base behind
the real sequence (n-1).
Solution: order new sequencing primer, preferably HPLC purified.
4.
5’ Primer Degradation -The primer consists of a mix of regular length
primer and primer degraded at the 5’ end. When a primer of length n loses
a single nucleotide at the 5’ end, it produces an n-1 strand. A loss of two
nucleotides at the 5’ end would be an n-2 strand, and so on. 3’ primer
degradation does not produce similar results since missing nucleotides will
be replaced during elongation.
Solution: use a fresh aliquot of primer.
5.
Mixed template – more than one PCR product or more than a single
colony picked for plasmid prep.
Solution: run PCR product out on gel to check for single band or re-streak
colony on a new agar plate and select a single colony for plasmid prep.
E.. Multiple Signals In Sections
Causes: Multiple (hetero) alleles are being amplified in the sequencing reaction.
The location of these regions are useful for identifying the specific reason.
1.
A single nucleotide polymorphism (SNP)- Appearance of two
superimposed peaks in a given position with a corresponding reduction in
their heights compared to neighboring peaks.
2.
INDELS will have multiple signals after the point where they occur.
Solution:– sequence complimentary strand to confirm.
3.
Multiple cloning sites
Multiple
cloning site
Solution: A plasmid with multiple signals starting at the multiple cloning site will
need to be re-picked, with care being taken not to pick more than one colony. It is
best to re-streak colony and then pick off a single colony from new plate for
plasmid preparation.
4.
Multiple PCR templates
Solution: Optimise PCR conditions to amplify one product or carry out gel
extraction.
F. Multiple peaks downstream of homopolymer
Cause: Enzyme slippage occurs giving varying lengths of the same sequence
after this region (n-1, n-2, n-3 etc).
Solution: sequence the complimentary strand.
G.
Short Read
Sequence starts well but signal stops abruptly.
Causes:
1.
GC or AT rich templates.
Solution: add more Big Dye
2.
Secondary structure (GC and AT rich templates can cause the DNA to
loop and form hairpins. The formation of hairpin then blocks polymerase
proceeding through the region.).
Solutions:
1. Add (5-10%) DMSO to sequencing reaction to help relax structure and
denature at 98oC.
2. Add 4X mix of Betaine solution below to sequencing master mix:
90 µl 10X PCRx (Invitrogen 11495-017)
160 µl 5M Betaine (Sigma-Aldrich B0300)
this gives a final reaction concentration of 0.9X PCRx and 0.80M Betaine.
3. Linearize template or select different primer. Since linear DNA denatures more
efficiently than super-coiled DNA, digesting template outside region of interest
sometimes helps. Alternatively, selecting a different primer even though it may
anneal very near original primer can result in improved sequence due to
differences in hybridization efficiency.
4. Sub-clone the insert into smaller fragments (<200 bp). Sub-cloning insert into
smaller fragments minimizes the amount of high GC template and reduces
opportunity for stable secondary structures to form.
G. Artifacts
Cause: Large peaks obscuring the real sequence e.g. Dye blobs caused by
unincorporated BigDye and are typically seen at 70bp and 120bp. They are
usually seen in failed or weak sequences. Real sequence can still be read
underneath these blobs.
Solution: they can be eliminated by adding more DNA template or less BigDye
to the sequencing reaction.
2. Electrophoresis Problems
Internal controls are included on each of our plates to carefully monitor Clean-up
and electrophoresis processes.
A.
Delayed Separation
The signal appears much later than expected. Peaks are wide and not uniform.
Cause: This occurs because the sample contains an excess of negatively
charged ions that compete with sequencing products for migration through
capillary. The excess ions are almost always salt or excess DNA.
Solution: Repeating electrophoresis usually resolves problem. Some samples
may require a slight dilution before repeating cycle sequencing or a decreased
injection time.
B.
Unusual Electropherogram
The peak shape can start off sharp, but become unusual. This can last for a few
nucleotides or for rest of the run.
Cause:
1.
Moisture or a bubble has found its way into the capillary. The
moisture/bubble changes the polymer’s consistency, leading to
inconsistent separation.
Solution: Repeating electrophoresis usually resolves problem.
2.
A contaminant in the sample.
Solution: Clean up template DNA
3.
Degradation of polymer or array
Solution: Inform us if loss of resolution continues.
C.
Dye-Spikes
A high-intensity fluorescent spike is observed. The spike usually consists of all
four colors and is in a random location (not at the usual dye-blob locations.).
Cause:
A bubble is inside the capillary, near or at the site of the laser. The bubble is
reflecting the fluorescence of the neighboring tags, making it appear as if multiple
tags are at the same location.
Solution: Running the sample through the electrophoresis a second time usually
fixes the problem.