Sequencing troubleshooting
There are a number of factors that can adversely affect the
quality and length of sequence you receive. Source BioScience
LifeSciences have compiled a list of common problems we find
after completing a sequencing job and where appropriate
the services that we can offer to increase the chances of obtaining
a satisfactory sequence from your precious template. You may also
be interested in help on preparing
sequencing templates.
Secondary Structure Resolution
Certain template characteristics can prevent the progression of
DNA polymerase along the growing strand, and the sequencing read
can stop abruptly. It is well known that conventional
sequencing chemistry is unable to progress past some secondary
structures, and templates rich in GC, GT, or G content.
dGTP BigDye Terminator Ready Reaction Cycle Sequencing Kit
can be used on difficult templates where the standard terminator
kits give data with early signal loss. Templates can be
sequenced with dGTP if the appropriate option is selected when
placing your online sequencing order.
Because of band compressions, we do not recommend using the dGTP
BigDye Terminator Kit for routine sequencing. It should be used
only if the standard terminator kits do not give good data.
Template Amplification Service
The quality of the template DNA used in automated sequencing is
of the highest importance. Templates that are too low in
concentration or that contain sequencing reaction inhibitors such
as salt, carbohydrate and or protein will all generate sequences
low in signal intensity. When signal intensity is low the analysis
software has difficulty in resolving the base peaks from background
noise. Typically this results in a shorter read length with other
random peaks being generated under the main data set. Source
BioScience LifeSciences recommends that plasmid templates are
submitted at 100ng/ul and eluted in pure water.
Source BioScience LifeSciences offer a Template Amplification
Service to enhance the quality and concentration of plasmid
templates that have previously proven to be unsuitable for
sequencing. The sequencing reads below shows analysed data with a
low signal intensity.
If you would like us to try the Template Amplification on your
templates please indicate this on your online
sequencing order.
n-1 Primer
Oligonucleotide primers can degrade to n-1 after multiple freeze
thawing or this can be due to poor purification at manufacture. An
n-1 primer will generate a second sequence lagging one base to the
left or right of the main peak. Below is an example of a sequence
generated with a mildly degraded n-1 primer. The main data is still
visible but the detection of sequence variants in this example
would be difficult. In more extreme conditions the data might prove
completely unusable. If your custom primer is n-1, why not consider
using one of our stock
primers? These primers are successfully used every day in a
large number of reactions and are thus proven to be free of n-1
variants.
Mixed Template
In certain circumstances sequencing will fail because two or
more populations of sequencing products are made in the same
reaction and are impossible to properly resolve. This may be caused
by there being mixed plasmids, or two or more template inserts
present in the cloning vector, or there being more than one primer
binding site. Multiple reads appear superimposed over each other.
To stop this happening plasmid DNA extractions (or cultures for
extraction) should always be prepared from a single bacterial
colony. Ensure that PCR products have been cleaned before receipt
so they are free from PCR primers which could also initiate
extension in a sequencing reaction.
Sequencing a mixed population mixed plasmid clones will generate
messy data from the insert site, prior to this (in the vector) the
read might look perfect.
No Data
NNNN indicates that the sequencing reaction has failed to make
any extension products that are detectable during
electrophoresis.
This problem can be caused by the primer not annealing to the
template or the template being of extremely low concentration or
extremely low quality. Primer should be designed using the
following guidelines: 18-22bp long, GC content of 50-55%, Tm of 55
to 60 degrees C.
Dye Blobs
Unincorporated dye terminators (commonly called "Dye Blobs")
appear at positions 70 to 80bp and again at approximately 100bp.
The chromatogram below shows unincorporated dye-terminators
superimposed over and partially obscuring the real peaks.
Dye blobs are caused by an imbalance of primer:BigDye:template.
We use proprietary clean-up plates to remove dye blobs but in
extreme cases of imbalance they can still remain. Source
BioScience LifeSciences have optimized primer:BigDye concentrations
for specified template concentrations (below) and so it is
important that you work to send the correct template concentration
if you find that Dye Blobs are a problem for you.
Material
|
Concentration
|
Volume (μl)
|
| Extracted plasmids |
100 ng/μl |
5 |
| PCR products |
1 ng/μl per 100bp |
5 |
| BACs, PACs |
1000 ng/μl |
25 |
| Custom sequencing primers (Tm 55 to 60°C) |
3.2pmol/μl |
5 |
Note: We require 5μl of sample and 5μl of primer per
reaction.
If dye blobs are interfering with your read then please let us
know. We will remove them for you free of charge in the first
instance and are happy to discuss optimizing your template
concentrations so that this is not a problem for you.