1. Prior to using the Assay Design Software (ADS), all CpG sites in your genomic DNA sequence
should be converted to reflect the sequence changes that the bisulfite treatment produces. It is
important to determine whether or not the region that you will be analyzing has any known SNPs
or mutations prior to doing this sequence conversion. Any SNPs or mutations should be noted.
It is best to account for this sequence variation with a “/” between the bases that are possible at
the position (i.e. A/C). Once the sequence variation has been accounted for, the sequence
conversion can be done on your genomic DNA sequences using Microsoft Word. Paste the
unconverted sequence into Microsoft Word, and then select the edit menu at the top of the
screen. Select "find" option from the drop-down edit menu. A window will appear. In the "find
what" field, enter "CG" and select the “find all” option. Now all of the CpG sites will be
highlighted. Next, select the replace tab and then replace "CG" with "YG". Y is the IUPAC code
for a T/C variable position. All of the CG sequences will be converted to YG. Next, do another
search for “C” to find the cytosines that are not associated with CpG sites. Finally, replace all “C”
with “T”.
2. Copy and paste the converted sequence into the Assay Design Software (ADS). The ADS will
identify the best combinations of PCR primers, as well as the best sequencing primer for
whatever target region is selected. Researchers generally are interested in sequences that are
close to the transcription start sites for the methylation analysis. You will need to select a target
region that includes between 50 and 100 bp and run the software in AQ mode. Larger target
regions can also be selected, but it is usually much easier to work with relatively small target
regions, especially when designing assays for methylation analysis.
It can be challenging to select the optimal target region in methylation assays because you also
need to consider the sequences upstream and downstream of this target region for placement of
your primers. Ideally, it is best to find a target region that has very few, if any, CpG sites
upstream and downstream of it. It is not recommended to place primers in regions with a lot of
repetitive sequences. It is also best to work with small pcr amplicons when working in this
application area. Ideally the CpG amplicons should be less than 200 bp, with an absolute
maximum amplicon size of no more than 300 bps.
For information on how to select regions of interest, please refer to section 4.2.4 in the Assay
Design User’s manual. An example of an excellent Target Region is shown below:
3. The Assay Design Software uses certain “Default” settings for determining the best primers for
analyzing a particular target region. These default settings were originally designed to be used
with normal genomic DNA sequences, not CpG-rich, bisulfite-converted sequences. Because of
the nature of these bisulfite-converted sequences, the ADS settings should be adjusted to make
the settings more favorable for methylation designs. The default settings can be found by clicking
on the File menu at the top of the screen.
A. These are the recommended PCR settings for CpG Designs.
The original default settings for the ADS will not allow the PCR primers or sequencing
primers to cover any methylation targets in the template. It is always *preferable* to avoid
covering CpG sites with your primers, but in most cases, you will not be able to avoid sitting
your primers over methylation sites. This practice routinely appears in methylation papers,
and is perfectly acceptable as long as the methylation sites occur near the 5’ end of the
primer region. You should change the default settings in the Assay design software to allow
the primers to sit over methylation sites by clicking on the allow Primer over SNP box in the
default settings window. The ADS will automatically change the PCR primer base that
appears directly over the cytosine in the CpG site so that the base would be a mismatch for
the methylated and unmethylated forms in the amplification template. In other words, the
primer will not form any hydrogen bonds over the C position of the CpG dinulceotide,
regardless of the methylation state. You can also use degenerate primers as well if you
would prefer to do so.
As previously mentioned in section 2, it is preferable to work with small amplicons when
designing CpG primer sets. The optimal Amplicon length should be adjusted to be between
50 and 200 bases. The maximum amplicon length should be decreased from the factory
setting, down to 300 bp.
It is also recommended that the Max PCR Primer Length be increased from 24 to 30. This
will allow the software to identify additional primers that it may have normally been ignored.
Once the primer sets have been identified by the software, it is easy to manually edit the 5’
end of the pcr primers to make the primers smaller if the primer set has excessive Tm values.
Tm values should always be kept below 73 degrees. A Tm of 73 degrees would likely require
a Ta of approximately 68 degrees for the amplification. Qiagen normally recommends a
maximum Ta of 68 in their amplification protocols. Reducing the length of a pcr primer at the
5’ end of the pcr primers will reduce the Tm of the primer, but it may also affect the template
loop penalties as well (non-biotinylated PCR primer only). Caution should be exercised when
manually editing primers to reduce high Tm values. Please refer to section 4.5.2 in the Assay
Design Software User’s manual for an explanation of how to manually edit primers.
B. These are the recommended Sequencing Primer Default Parameters for CpG Designs:
There are several other default settings that should be changed. You should allow the
sequencing primer to anneal more than three bases away from the first CpG site. You should
increase the default setting of three so that the sequencing primer can be placed up to 15
bases from the first CpG site.
It is also recommend that the sequencing primer length be increased from 20 to 25 bases.
4. Once the Default settings have been edited, and the target region has been identified, then the
play button in the Assay Design Software should be pressed. The Assay Design software will
then identify the best primer sets based on your target region selection and the Default settings
that you currently have set.
5. Once the primer sets have been identified by the software, the individual designs should be
scrutinized and one final primer set should be identified, along with several other backup
candidate designs. To determine the best design for a target region, you should double click on
the top 5 or 10 assays that appear in the primer set area, and then read through the primer set
reports. The total quality scores for a given design is not always the best indicator of a good CpG
assay design. It is always best to refer to the individual warnings within the primer set report to
determine whether a design is worth pursuing further. The most important penalties in this report
include the following:









Biotinylated PCR Primer Hairpin Analysis: Evaluates hairpin structures on the
biotinylated PCR primer.
Duplex Formation Analysis: Identifies possible annealing between the sequencing
primer and the biotinylated PCR primer.
Mispriming Analysis: Identifies alternate annealing sites for the sequencing primer on
the amplicon.
Template Loop Analysis: Identifies possible template loops on the amplicon.
Duplex Formation Analysis (Biotinylated PCR primer): Identifies possible Biotinylated
PCR primer self-annealing.
Mispriming Analysis (Biotinylated PCR primer): Identifies alternate annealing sites for
the Biotinylated PCR primer on the DNA template.
Hairpin Loop Analysis (Biotinylated PCR primer): Investigates possible hairpin
structures on the PCR primer.
Duplex Formation Analysis (Sequencing primer): Identifies possible sequencing
primer self-annealing.
Hairpin Loop Analysis (Sequencing primer): Investigates possible hairpin structures
on the Sequencing primer.
These non-specific mispriming, duplex, and loop structure events are made worse when they
become sources of background in the pyrosequencing reaction. If these non-specific structures
have stable 3’ ends, these double-stranded structures will be used as alternate priming sites by
the polymerase. It is important to understand that not all of these non-specific events are going to
be stable enough to actually cause detectable levels of background in the Pyrosequencing
reaction. The primer reports will show the non-specific, annealed double-stranded structures
which are expected to form at 28 degrees C. The base pair positions that are able to produce
hydrogen bonds with the template will be clearly presented in these figures. The most stable
non-specific annealing events will be given the highest penalties. In most cases, penalties of
over 50 are considered to be significant in any of these categories. It is important to minimize
these penalties by edit primer sets or redesigning the primers altogether. If these penalties can
be reduced into the 30s or even the 20s, the design should be relatively clean. There are also
ways of correcting the penalties aside from your typical primer editing strategies.



Biotinylated primer penalties are the easiest of these penalties to correct. By using less
than 0.2 micro molar of the pcr primers in the pcr setup and increasing the cycle numbers
up to 45 or 50, these biotin problems are easily corrected in most cases.
Implementing a slow cooling step during sample prep can also, in some cases, help to
promote specific sequencing primer annealing which can reduce sequencing primer
mispriming.
Template loops structures can sometimes be destabilized by adding or removing one or
more bases to the 5’ end of the non-biotinylated pcr primer. In some extreme cases, one
might consider adding one or more “G” bases at the 5’ end of the non-biotinylated pcr
primer on forward designs, and one or more “C” bases on 5’ non-biotinylated pcr primer
for reverse assays. These “G” or “C” bases would only be capable of annealing to
methylated sequences, so the addition of these bases should, in many cases, prevent the
template loop from having a stable 3’ end. The affect of adding these extra bases should
be carefully considered using the ADS before ordering the primers with these 5’ end
sequence modifications.
The location that these different non-specific annealing events occur on the amplicon should not
be ignored. The locations of these non-specific annealing events can be found in the Primer
Report, the Sequence Tab, and the Assay Overview Area (see section 2.4).



All non-specific events that don’t occur on the pcr amplicon can be safely ignored, along
with any non-specific events that involve the non-biotinylated pcr primer.
Non-specific annealing events that occur just downstream of your sequencing primers
can often block the “specific” polymerase activity that you are trying to measure.
Mispriming events and loops that occur further downstream of the sequencing primer are
usually more favorable.
The further that these mispriming events occur from your sequencing primer annealing
site, the more information you’ll get before the sequence pattern starts to deviate from the
expected sequence pattern. Even if these downstream mispriming events block the
specific polymerase activity, you might still be able to get valuable information from the
first portion of the sequence run if the mispriming occurs far enough away from the
sequencing primer.
CpG designs are very rarely perfect, so careful consideration is generally required to determine
the “lesser of two (or more) evils” when designing a methylation assay. If the primer design
results are poor with one target region, it is best to try manually editing the primers to improve the
designs. For example, if you have a bad sequencing primer, but good pcr primers, the
sequencing primer should be the focus of this editing. Please refer to section 4.5.2 in the Assay
Design Software User’s manual for an explanation of how to manually edit primers. If your design
efforts fail for a particular target region, then try another target region and repeat your primer
design efforts on the new region.
In order to improve your success rate while keeping your assay development costs down, it is
recommended that you order the most favorable primer set, along with a few back up sequencing
primers and non-biotinylated primers. If you require assistance in determining what should be
done with a particular design, Biotage Technical Support can assist with these types of questions.
6. It is also recommend that forward assays be used whenever possible to avoid dealing with “A”
dispensations in the CpG site dispensations. Reverse methylation assays involve dispensing G/A
instead of C/T. In pyrosequencing, we use chemically modified Adenosine, resulting in "A peaks"
that a slightly larger that the rest of the nucleotide peaks. The bottom line is that “reverse”
methylation assays are less quantitative than forward methylation assays due to these "A
peak" artifacts. Our Pyromark and CpG software packages both have an "A peak" correction
factor, but it is always better to avoid “A” dispensations in variable positions whenever possible. If
the ADS scores are equal when comparing two similar assay design candidates, the forward
assay is almost always a better choice than a reverse assay. If the ADS scores and penalties are
favorable for the reverse assays and unfavorable for the forward assays, ignore the direction of
the design and go with the design that will give you cleanest results.
7. Once the assays have been designed, it is important to optimize the PCR assay to ensure
that your PCR products amplify properly. You should run a pcr gradient with at least 10 to 15
different annealing temperatures. Ideally the middle temperature in this gradient should be 5
degrees less than the predicted Tm of your pcr primers. For each temperature, it is best to run as
many as three different MgCl2 concentrations: 1.5 mM, 3.0 mM, and 4.5 mM. More information
on PCR optimization can be found in Biotage’s PCR Optimization document.
8. Next, you should run a gel and pick out the cycling conditions that produce strong, specific
products. Then, you should run these products through the pyrosequencing reaction on some
cell lines as well as some samples. Pick the set of cycling conditions that give you the best
pyrograms with the most accurate methylation measurements. It is recommended that assay
designs and cycling conditions be tested on samples with known methylation percentages to test
for bias. There was an excellent publication that discussed the importance of this bias testing in
CpG methylation testing.
Optimizing annealing temperature overcomes bias in bisulfite PCR methylation analysis
Lanlan Shen, Yi Guo, Xinli Chen, Saira Ahmed, and Jean-Pierre J. Issa. BioTechniques.
January 2007. Volume 42, Number 1: pp 48-58
http://www.biotechniques.com/default.asp?page=aop&subsection=article_display&display=full&id
=112312