Coffalyser 2.4 troubleshooting on capillary data v2. COMMON PROBLEMS WITH .... of signal sloping which cannot be corrected by data analysis techniques.
Coffalyser 2.4 troubleshooting on capillary data v2
COMMON PROBLEMS WITH MLPA This page discusses the most common problems observed when performing separation of MLPA products on a capillary electrophoresis device. These are the problems which should be resolved either during the experimental procedure of MLPA or by adjusting your fragment separation procedure. These problems cannot be resolved using smart analysis techniques (except for a normal amount of size to signal drop). Please note that many problems may be related.
Discussed Topics Problem 1: Too much Signal Sloping Problem 2: Unequal signal intensities of size marker Problem 3: Spectral pull up / pull down Problem 4: Spiky peaks Problem 5 : Low MLPA probe peak intensities Problem 6: Raised baseline Problem 7: High background signals / Shoulder peaks Problem 8: Split Peaks Problem 9: Incomplete denaturation Problem 10: Primer dimer formation Problem 11: Truncated peaks Problem 12: Irregular current patterns
Coffalyser 2.4 troubleshooting on capillary data v2
Problem 1: Too much Signal Sloping Recognized by: Compare the signal intensity of the smaller fragment signals with those of the larger fragments of both the size marker and the MLPA probe signals. Size to signal drop in your size marker should be almost non-existent (Figure 1), size to signal drop in the MLPA probe signals is expected but signal decrease shouldn’t exceed a 60-70% (shortest MLPA fragments to longest MLPA fragments).
Figure 1 Normal separation pattern of a GS-500 rox marker.
Possible causes: This effect may be enhanced by sample contaminants or evaporation during the hybridization reaction. Signal sloping may further be influenced by injection bias of the capillary system and diffusion of the MLPA products within the capillaries. In addition, the peak heights will be more affected by diffusion of the MLPA products than peak areas, but are influenced less by the presence of shoulder peaks than peak areas. Recommendations: Signal sloping visible in the size marker (figure 2): Size standards contain fragments of known sizes in equal concentrations. The overall signal intensity of these fragments should therefore be equal after separation and quantification assuming that the capillary system doesn’t introduce size to signal sloping. When sloping of the size marker is spotted, the capillary system thus introduces structural variation, which under optimal conditions is not expected. This effect will also be present in the fragment you want to analyze making normalization of these fragments even more difficult A number of factors may play a role in this, most importantly being:
Coffalyser 2.4 troubleshooting on capillary data v2 •
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Old gel (capillaries): old gel creates more resistance resulting in wider and lower peaks for the longer fragments. A gel pump operated by a stepper motor may be used to deliver fresh gel to one or more capillaries through one or more manifolds and valves. By controlling the valves, fresh gel delivered by the pump will replace the old gel in the capillaries (automated gel replacement system). Make sure that the correct number of injection steps are set for your gel type, capillary type and length (standard run modules are usually good). If you don’t use your device for longer times, store your gel at 4 degrees Celsius. Too high injection voltage: very high injection voltages (2.5 kv>) causes a similar effect as old gel, an injection bias causes larger fragments to be injected slower causing the peaks of the larger fragments to be wider. A injection voltage of 2.0 Kv usually provides good results and can be used as a starting point to further adjust your device from here (if needed). Low quality formamide: The quality of the formamide affects the sensitivity. Low quality formamide not only requires higher sample concentrations but may also cause a size to signal drop. It is therefore required to use hi-di formamide (ABI). Regular formamide can be made more pure with an ion exchange resin.
Figure 2 Signal sloping in size marker (in red)
Signal sloping of MLPA probe fragments (Figure 3a-d): Caused by a decreasing efficiency of amplification of the larger MLPA probes, a systematic probe length effect or size to signal drop can be seen in all MLPA runs (electropherograms). This effect may be enhanced by sample contaminants or evaporation during the hybridization reaction. •
Normal MLPA probe signal sloping (figure 3b): a normal amount of signal sloping of the MLPA probes is always expected, however because this effect
Coffalyser 2.4 troubleshooting on capillary data v2
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is present in both reference and sample runs, normalization removes this artifact. Normal MLPA signal sloping can be recognized when signal intensity the largest MLPA fragments is approximately 1/3 of the signal intensity of the shortest MLPA fragment (or higher). The sloping effect should furthermore be equal between reference and sample runs, if this is not the case normalization methods without signal sloping are compromised and results may not be reliable. It should be noted that correction of signal sloping by analysis techniques is permitted, as long as the longest fragments do not get into the error range of the capillary device (3x the intensity of the baseline, ABI (>300 units), CEQ (5000>)). Sample contaminants (figures 3a, 3b): sample contaminations may further increase the size to signal sloping effect by inhibiting the polymerase during the PCR reaction. This will usually result in sloping differences between reference and sample runs, which if not correct for, causes problems when using classical normalization methods. Analysis of a limited amount of size to signal drop (Coffalyser): For every run, prior to normalization in the Coffalyser, the amount of signal sloping is be approached by a function where the pre-normalized probe signals are regressed linearly on the probe lengths. These probe signals may include signals of all probes (population / tumor analysis method) or reference probes (control probes method) only. Depending on the samples and MLPA mix used a choice should be made. To minimize the effect of large values, the user may choose to first convert all values to a log scale. Please be noted that too much sloping cannot be completely corrected and that these samples should be repeated after cleaning up the DNA or diluting the samples. More information on correction of sloping correction can be found in the Coffalyser manual and in the MLPA results interpretation section. Sample contaminants 2 (figure 3c, 3d): too much signal sloping often causes the larger fragments to become almost undetectable by the capillary device. This may be recognized when the signal intensities decrease in a more or less exponential (polynomial) fashion. Correction for this kind of artifact proves to be really difficult, it is therefore recommended to clean the DNA and repeat the MLPA reaction. A simple ethanol precipitation usually works for these purposes. Alternatively you can try to dilute your samples deionized or distilled water if concentrations allow this.
Coffalyser 2.4 troubleshooting on capillary data v2
Figures 3a-e Top to bottom, figures A and B illustrate signal sloping differences between pure and paraffin DNA. Figures C and D show an amount of signal sloping which cannot be corrected by data analysis techniques.
Coffalyser 2.4 troubleshooting on capillary data v2
Problem 2: Unequal signal intensities of size marker Recognized by (figure 4): High salt concentrations in the injection mixture may cause re-annealing of DNA fragments which can be recognized in the separation pattern of the size marker by unequal signal intensities or unexpected signal loss of size marker fragments. Possible causes: High salt concentrations in the injection mixture are usually caused by adding too much PCR product in the mixture. Recommendations: Do not use higher PCR product volumes than about 1/10 of the total volume of the injection mixture. Higher PCR product volumes decrease the signal intensities rather than increasing them and also cause other problems as noted before. The runs that failed due to high salt concentration should be rerun with lower PCR product concentrations after checking the capillary system for any flaws (gel, waste, tubing, cleaning tray, laser, etc). This can be done best by diluting your products in distilled or deionized water. Alternatively the product can also be cleaned using a column purification method.
Figure 4 High salt concentrations cause re-annealing.
Coffalyser 2.4 troubleshooting on capillary data v2
Problem 3: Spectral pull up / pull down Recognized by (figure 6a, 5b): Spectral pull up or pull down can be recognized by consistent patterns in the colors that are pulled up or pulled down for example, green always below blue. Possible causes: This can be attributed to an improper spectral/matrix being applied to the sample. The function of the spectral is to subtract any overlap between the dyes. Optimal emission spectra have minimal spectral overlap of different dye colors and therefore need minimal dye subtraction (figure 5). The dyes that were used (MLPA probe dye, and the size marker dye) should match the filter set used on your capillary device (also see capillary separation protocol). Always make sure to set the right filter-set! If the pull up and pull down does not exhibit a consistent pattern then it may be sample related. Other causing may be replaced or realigned optics or usage of a new polymer type. Recommendations: Make sure that you have used the proper dye set / filter set matching your chemistry. If the settings were correct you need to rerun the spectral calibration. Spectral calibration is always needed when: the optics are changed, a different polymer type is used or new dyes or being used on the system.
Figure 5 Emission spectra of a five dye set on an ABI machine.
Coffalyser 2.4 troubleshooting on capillary data v2
Figure 6a/b Spectral pull-up / pull-down. Figure a shows little pull up (blue to green), while figure b shows extreme bleeding through (blue to green to yellow to red).
Coffalyser 2.4 troubleshooting on capillary data v2
Problem 4: Spiky peaks Recognized by (Figure 7): Spiky peaks are artifacts that are seen from time to time in electropherograms. It is a narrow peak that appears in all four colors. It usually does not interfere with data interpretation, because size calling software usually corrects for these peaks. However when these peaks are not ignored during size calling, there may be a shift in the estimated probe lengths. When such a peak aligns with a MLPA probe peak it may furthermore contribute to the measures intensity of this peak. Possible causes: Spiky peaks are thought to be caused by air bubbles in the sample plate or array, also leakages in the system may allow particulates to enter the system. Recommendations: Inspect the system and sample tray for air bubbles. Most capillary systems have flush modules under the direct control option / wizard option. Run this module, if problem persist, inspect the polymer lines for any source of bubbles or particles and correct these leaks. If urea is used on the same device, beware of urea crystal formation on the pump block or connection pieces. Small leaks will cause formation of urea / gel crystals on the spot (figure 8a). Also if needed clean the area around the capillaries without touching the capillaries (figure 8b)! If needed replace the inline filter and replace the polymer with a fresh bottle. After replacing the polymer regenerate the array.
Figure 7 Spiky peak, is a narrow peak that appears in all four (five) colors
Coffalyser 2.4 troubleshooting on capillary data v2
Figure 8a Pump block should be well cleaned to avoid problems with urea crystal formation.
Figure 8b Keep the area around the capillaries clean (do not touch the capillaries!).
Coffalyser 2.4 troubleshooting on capillary data v2
Problem 5 : Low MLPA probe peak intensities Recognized by (Figure 9): Overall low signal intensities may result in too much noise which interferes with your MLPA product signals. Peak intensities should always be evaluated on the raw electropherograms. While too low signals will result in unreliable results, too high signals may result in size calling errors or oversaturation of the capillary system. In general it is recommended to have a signal intensity which is approximately between the 10-50% of the maximum signal intensity. Samples and references having more or less equal signal intensities are furthermore easier to compare and provide more accurate results. Possible causes / Recommendations: Low signal intensities may have a number of causes which are partially discussed in the electropherogram troubleshooting scheme. First always inspect the DQ fragments (see MLPA_DQ_DDQ_fragments protocol) which will be high when the input of sample DNA was low at the MLPA reaction. If these fragments were high, repeat the MLPA increasing your amount of template. When the signal intensities are low and the current (EPV) pattern is straight (figure 10) but no other dyes are seen, rerun the products using a fresh batch of formamide (degraded formamide may also cause low signal intensities), make sure to mix your injection mixture well. Also run a sample in a mixture with a volume of 20 ul (if good signals appear, recalibrate the autosampler). Also consult the protocol 2.1_Settings_sequencers_v1.doc, on how to adjust your injection time / injection voltage to increase the signal intensities. When these actions do not help, check your samples for reaction inhibitors and repeat your samples after dilution in deionized/distilled water or after cleaning up (ethanol precipitation may help). When the signal intensities are low and the current is also crashing (figure 11), your injection mixture (MLPA products) probably contain an injection inhibitor (salt, metal ions, protein). High salt concentrations may also cause re-annealing of DNA. Signal intensities may become better by decreasing the amount of MLPA product you are putting in the injection mixture. This can be done best by diluting your products in distilled or deionized water. Alternatively the product can also be cleaned using a column purification method. Also make sure that he pre-denaturation step is performed (gradual declining of the current may also be caused by super coiled DNA), if not rerun the MLPA products making sure to perform the preheating step, and if necessary adjust this step. Try one minute longer for three times, if the current is still dropping and raw signal drop rapidly then adjust the heating settings to 5 min on 86 degrees Celsius.
Coffalyser 2.4 troubleshooting on capillary data v2
Figure 9 Emission spectra of a four dye set on an ABI machine giving low probe peak signals.
Figure 10 Normal current pattern
Coffalyser 2.4 troubleshooting on capillary data v2
Figure 11 Gradually crashing current. Gradual declining of the current is usually caused by super coiled DNA, caused by complex DNA or no preheat treatment.
Coffalyser 2.4 troubleshooting on capillary data v2
Problem 6: Raised baseline Recognized by (Figure 12a): The baseline is more or less the minimal signal intensity which is measured after matrix subtraction when the laser is on. Most capillary devices have an option to monitor the baseline, in other cases the height of the baseline can be read from the signal intensity which is present at the start of any run. The baseline should be as low as possible; a higher baseline will always result in less sensitivity and more noise. Higher baselines (or bad matrix) may furthermore cause the color to fill up of a certain dye between peaks. This effect may furthermore cause peak detection problems during the size calling procedure. Possible causes: High baselines may be caused by several reasons. In a normal situation the laser goes through the samples in the capillaries, where after the light is dispersed and falls on a grate, the grating splits the different wavelengths which are subsequent measured on a matrix. The presence of a fluorescent label will increase the intensity of a certain wavelength which will be correlated to a certain dye. Since all colors (in the filter sets) are detected at all times by the CCD camera a matrix is used to determine the amount of spectral overlap and correlates for it (figure 14). The values that remain are the detected amount of fluorescence present in the capillaries. A poor matrix can thus also cause the baseline to be elevated, and may furthermore also cause negative measurement of certain dyes. Baselines may furthermore become higher by the natural background which can be caused by any particulates which change the original laser signal. A common problem is the capillary window to be dirty. Little spikes in the baseline indicate that the buffer is old and need to be refreshed (figure 13). Recommendations: First of all, make sure to clean the capillary window, using ethanol or methanol, avoid touching the window (blow clean) or wipe the window in one direction only! When strange results are obtained make sure you are using the right filter set, and check if the matrix is good enough (figure 6, 14).
Coffalyser 2.4 troubleshooting on capillary data v2
Figure 12 a/b High background signal and color fill up in a particular color by high
Figure 13 Cleaning the detection window.
Figure 14 Example matrix for pop4 on an ABI-310
Coffalyser 2.4 troubleshooting on capillary data v2
Problem 7: High background signals / Shoulder peaks Recognized by (Figure 15): A high background can be recognized by a lot of non-specific signals between the actual MLPA probe signals. Shoulder peaks are furthermore often seen, which are the small peaks 1 basepair before and/or after each MLPA probe specific peak. Possible causes / Recommendations: Backgrounds signals that are not originating from MLPA product should be ignored and are often caused by contamination of the capillary system or one of the MLPA ingredients. The cause of these problems can be investigated by performing a blanco MLPA reaction and/or by running only formamide (w or w/o marker) on the capillary system. The buffer and polymer furthermore affect the background fluorescence- affecting the matrix. N-1 / N+1 peaks (shoulder peaks) are often the cause of bad primer quality. Bad primer quality can be the results of often freeze-thawing of the primers or by a bad batch of primers (not enough purification, synthesis problems at manufacturer). Fluorescent labels furthermore know to deteriorate in formamide, thus minimize the time the MLPA products are in the formamide before capillary separation. Another related problem are low polymer concentrations, this will decrease the peak resolution thereby increasing background. Always run your capillary device with the standard run module recommendations for the capillary fill volumes designed for your specific capillary type and gel type.
Coffalyser 2.4 troubleshooting on capillary data v2
Figure 15 High background / shoulder peak in probe dye.
Coffalyser 2.4 troubleshooting on capillary data v2
Problem 8: Split Peaks Recognized by (Figure 16): Split peaks are peaks that look like too very close products but may in fact originate from a single product that has been altered or has a different structure. Possible causes / Recommendations: When split peaks are seen in most probes, the DNA may have been degraded. This may be resolved by using a mineral oil of molecular grade, hi-di formamide and then rerun the same MLPA products. Single probe split peaks may be due to bad probe designs targeting sequences harboring repeats resulting in enzyme slippage, investigate the sequence of these probes for repeats. Other reasons to split peaks may be high salt concentration in the injection mixture causing probes to form alternative secondary structures. Never use more than 10% volume MLPA product to the final injection mixture volume. Rather use less product with a longer injection time to increase probe signals (more about this can be found in the capillary settings protocol).
Coffalyser 2.4 troubleshooting on capillary data v2
Figure 16 Split peaks.
Coffalyser 2.4 troubleshooting on capillary data v2
Problem 9: Incomplete denaturation Recognized by (figure 17a-d): Incomplete denaturation can be recognized by unexplainable loss of probe signal (thus not homozygous deletions) and by the decrease in signal of the D-fragments (88, 96 bp, see MLPA_DQ_DDQ_fragments protocol) relative to the ligation dependent fragment (92 bp). Possible causes: Incomplete denaturation is often due to contaminants in the samples (salt, metal ions etc) which increases the denaturing temperature of the template. Other possible causes are problems with the thermocycler or too much evaporation during the overnight hybridization (also causes high salt). Recommendations: Check samples and cleanup if necessary. When your DNA concentration are quite high you may also choose to dilute your samples many times, thereby also diluting the concentration of the contaminants. Check initial denaturation step, increase time/temperature if necessary. Check cycle denaturing temperature and time, adjust if necessary. You can also add a tube containing 8 ul of water in the thermocycler during overnight hybridization. Next morning measure the volume in this tube, there should be at least 4 ul left. When more is evaporated, check your heated lid, or choose to use alternative tubes (brand).
Coffalyser 2.4 troubleshooting on capillary data v2
Figure 17a .Incomplete denaturation due to low DNA denaturation temperature.
Figure 17b .Incomplete denaturation due to high salt concentration in sample.
Coffalyser 2.4 troubleshooting on capillary data v2
Figure 17c .Incomplete denaturation due to high salt (incorrect dilution reagent).
Figure 17d .Incomplete denaturation due to metal ions in sample.
Coffalyser 2.4 troubleshooting on capillary data v2
Problem 10: Primer dimer formation Recognized by (figure 18a/b): The primer peak can be recognized by the largest peak which occurs right after the start around 50 bp, containing the major amount of unused primer. Closely after that a smaller peak may appear, which contain the primer-dimers. Possible causes: Primer dimers may form during the first PCR step unusually when the PCR is not HOT started or when creating the polymerase mix. Large quantities of primer dimers may be formed which negatively affect your PCR reaction by decreasing the amount of available primer. Recommendations: The following guidelines help to minimize primer dimer formation. Always create the polymerase buffer mix on ice. Add the polymerase enzyme as last and mix by pipetting up and down. Pre-heat your ligation mixture with PCR buffer at 60-72 degrees in the thermocycler and start the PCR reaction a.s.a.p. after adding the polymerase mix.
Figure 18a/b .Large amount of primer or primer/dimmers in electropherogram
Coffalyser 2.4 troubleshooting on capillary data v2
Problem 11: Truncated peaks Recognized by: Truncated peaks are made by the software when the signal intensities are so high than the peak has to be cut off in order to still show it on the available scale. Possible causes: Truncated peaks are only formed when there is a case of sincere overloading. Overloading the system doesn’t only affect the peak quantification but also raises the base line; gives higher ratio’s and disturbs the product flow in the capillaries. Recommendations: Runs that have too signals and show truncated peaks should be rerun after refreshing the gel in the capillaries using either less product in the injection mixture, a lower injection voltage and/or injection time.
Coffalyser 2.4 troubleshooting on capillary data v2
Problem 12: Irregular current patterns Recognized by (Figure 19/20): The current during capillary electrophoresis is measured on each device and can be used for troubleshooting in the raw data section. Normal current pattern start at 0 and then rise to a certain value where after it remains constant (figure 10). Possible causes: Irregular crashes (patterns) in the current pattern are cause by elements in the system impeding the capillary flow. This may be salt, protein, too much DNA, ethanol or super coiled DNA. Recommendations: High salt concentration in the injection mixture disturb the electrokinetic injection. If you signals are low while you are using much product, try to use less product instead and if needed adjust injection voltage/time to increase signal intensities. Check the formamide used and check the denaturation step and if necessary adjust. Sudden drops in the current line are caused by air bubbles in the manifold. Purge the manifold and re-run your MLPA products.
Figure 19 Uneven pattern in current line.
Coffalyser 2.4 troubleshooting on capillary data v2
Figure 20 Sudden current crash
Figure 21 Amount of sample injected according to Butler
