CpG Tips Draft-JH
1. Generate Bisulfite Converted Sequence
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.
a. Note: Determine whether or not the region that you will be analyzing has any
known SNPs or mutations prior to doing this sequence conversion. Account for
this sequence variation with a “/” between the bases that are possible at the
position (i.e. A/C).
b. Paste the unconverted sequence into Microsoft Word
c. Select “Edit”. Select "Find" option from the drop-down edit menu.
d. 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".
e. 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).
3. From the Assay Type drop-down menu, select the “allele quantification”.
4. Select a Target Region
Highlight a 50-100bp section of sequence containing the CpG sites of interest. Right-click
and choose “Target Region”, then “Set Target Region”
a. Researchers generally are interested in sequences that are close to the
transcription start sites for the methylation analysis.
b. Start and end the Target Region with a CpG site.
c. Ideally, it is best to find a target region that has very few, if any, CpG sites
upstream and downstream of it.
d. It is not recommended to place primers in regions with a lot of repetitive
sequences.
5. Change Assay settings for optimization with Bisulfite converted sequence.
The default settings can be found by clicking on the File menu at the top of the screen.
a. Under “PCR Primer”, change the settings to:
b. Under “Sequencing Primer, change settings to:
6. Press “Play”
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.
7. Examine the Reports:
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 most important penalties in this report include the
following:
a. Biotinylated PCR Primer Hairpin Analysis:
b. Duplex Formation Analysis:
c. Mispriming Analysis:
d. Template Loop Analysis:
e. Duplex Formation Analysis (Biotinylated PCR primer):
f. Mispriming Analysis (Biotinylated PCR primer): *note the location of the
mispriming event. Mispriming outside of the amplicon can be ignored.
g. Hairpin Loop Analysis (Biotinylated PCR primer):
h. Duplex Formation Analysis (Sequencing primer):
i. Hairpin Loop Analysis (Sequencing primer):
Penalties of over 50 are considered to be significant in any of these categories.
It is important to minimize these penalties by editing 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.
8. Edit Primers if necessary: 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. If you cannot design a clean assay by editing the
primers or moving the target region, see “Tips and Tricks” below for more information on
optimizing the design.
9. Optimize your PCR
a. 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.
b. You should run a pcr gradient with at least 10 to 15 different annealing
temperatures. 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.
c. Run a gel and pick out the cycling conditions that produce strong, specific
products.
d. 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.
e. 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 48Tips and Tricks
1. 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 than 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.
2. There are ways of correcting the penalties aside from your typical primer editing
strategies.
a. 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.
b. 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.
c. 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.
d. Biotinylated PCR primer mispriming events on the amplicon can sometimes be
destabilized by creating a mismatch near the 5’ end of the primer. A mismatch of
this type will usually allow the primer to continue to anneal to the target, but
destabilize a non-specific priming site enough to reduce the penalty.
3. Location: The location that 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.
a. 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.
b. 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.
c. 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.
4. 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.
5. 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.