Coffalyser digitalMLPA performs multiple quality checks on digitalMLPA data and reports the results in its output. Only samples that pass quality requirements are suitable for result interpretation. If a quality warning is shown, troubleshooting may be necessary.
This article provides an overview of the quality checks in Coffalyser digitalMLPA, including common causes and possible solutions for issues related to each check. Related plausible root causes that may be reported by Coffalyser digitalMLPA are also listed – for more information on plausible root causes, see the Coffalyser digitalMLPA User Manual.
Sample information |
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| Description | Explanation |
|---|---|
| Sex |
Sample sex detection is only based on the presence or absence of X- and Y-chromosome control probes. These control probes can be influenced by SNPs or copy number loss of the targeted region. For example, Y chromosome loss is a common phenomenon in older males. Y-chromosome specific probes may also be influenced by sample contamination of a male sample with female DNA, or vice versa. Furthermore, some females possess part of the Y chromosome which is translocated to another chromosome (e.g. Morel et al. 2002). Sex-chromosome aneuploidies, such as Turner syndrome (45,X0), Klinefelter syndrome (47,XXY) or Trisomy X (47,XXX), are not detected by these control probes.
Related plausible root cause: sex detection. |
Sequence run |
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| Description | Explanation |
| MTRQ & MDRQ |
Median total reads (MTRQ) and median distinct reads (MDRQ) are measures for the read depth and number of independent ligation events, respectively.
Related plausible root cause: probe read depth too low. |
| Unrecognised reads |
Ensure the correct Product Sheet is defined in the Coffalyser Definition File and sufficient DNA is used as input for each reaction (see digitalMLPA NXtec Protocol and/or probemix-specific product description). Check the read quality of the NGS run as reads with many errors cannot reliably be assigned to probes, which increases the number of unrecognised reads. To check read quality, we recommend using Illumina's BaseSpace analysis environment or Illumina's Sequence Analysis Viewer software. Related plausible root causes:
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| Sequence quality |
Check the Q30 score of the NGS run. When sequencing quality issues arise, consult Illumina. To check read quality, we recommend using Illumina's BaseSpace analysis environment or Illumina's Sequence Analysis Viewer software. Ensure adapter trimming is disabled. If adapter trimming was enabled, please regenerate the FASTQ file from the original sequencer files with adapter trimming switched off. Related plausible root causes: too many sequence errors & variable read length. |
| Sample uniformity |
Ensure that equal amounts of all reactions have been pooled when preparing the NGS library prior to the sequencing run (see the digitalMLPA NXtec Protocol for details). Related plausible root cause: unbalanced sample read depths. |
Identification |
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| Description | Explanation |
| SNP id code |
The SNP id code is shown in a red and bold font when sample DNA is considered contaminated with DNA from another individual. See DNA contamination below for more information. |
| Probemix lot |
Ensure the correct Product Sheet is defined for your experiment in the Coffalyser Definition File, that a single probemix lot has been used for each sample, and that there is no contamination (e.g. from another probemix lot). If your Product Sheet is not present in the configuration, please redownload Coffalyser digitalMLPA. Related plausible root causes: possible incorrect sheet & probemix lot detection. |
| Barcode lot |
If the error known as incompatible or warning not known as compatible are present, you may have used a barcode lot incompatible with your probemix lot. Consult the probemix-specific product description to check which barcode plate lots are compatible. In case the error too many is present, two or more barcode lots were mixed in one reaction. Always use a single barcode solution per reaction. Related plausible root cause: barcode lot detection. |
Reaction |
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| Description | Explanation |
| DNA quantity |
Ensure sufficient DNA is used as input for each reaction (see digitalMLPA NXtec Protocol and/or probemix-specific product description). Related plausible root cause: DNA quantity. |
| DNA contamination |
Possible causes and solutions include:
Related plausible root cause: DNA contamination. |
| DNA denaturation 1, 2 |
A warning/error in a single or a few samples: dilute the DNA sample if the DNA concentration is sufficient, or perform an extra DNA purification step. A warning/error in all samples: ensure the thermocycler used is properly calibrated and the thermocycler program is correct. A warning in all samples can also be due to e.g. the use of a suboptimal DNA extraction method for all samples, or all samples having suffered from evaporation (resulting in high salt concentrations). Related plausible root cause: DNA denaturation. |
| DNA depurination 1, 2 |
Ensure DNA samples contain 5–10 mM Tris-HCl buffer with a pH of 8.0–8.5. More information about depurination. Related plausible root cause: DNA depurination. |
| DNA fragmentation 1, 2 |
A warning/error indicates that the sample DNA is heavily fragmented. Collect a new DNA sample. Related plausible root cause: DNA fragmentation. |
| DNA digestion 1, 2 |
For digitalMLPA experiments (no methylation analysis):
Related plausible root cause: DNA quantity. |
| Hybridisation temperature 1, 2 |
Ensure the thermocycler is properly calibrated, and the amount of salt present in the DNA sample is not too high (see DNA denaturation above). In case a warning for hybridisation completeness is also present, this may be an indication of evaporation. Ensure tube caps are properly closed before starting the overnight hybridisation. Test for evaporation by incubating 9 µl H2O overnight at 60°C; in the morning >5 µl should remain. To reduce evaporation:
Related plausible root causes:
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| Hybridisation completeness 1, 2 |
A low value in all reactions can be due to a short hybridisation time at 60°C, e.g. due to power interruption, or can be due to an incorrect hybridisation temperature. A low value in a single reaction in combination with a low value of the hybridisation temperature control probes, can be due to pipetting less than 3 µl hybridisation master mix to the reaction. An increased value for both types of hybridisation control probes can be due to evaporation (see hybridisation temperature above). Related plausible root causes:
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| Ligase start temperature 1, 2 |
Ensure the samples are in the thermocycler at 48°C when the ligase master mix is added and use a multichannel pipet (if available). Related plausible root cause: ligation temperature. |
| Ligase activity 1, 2 |
Warning/error in a single sample: perform an extra DNA purification or dilute the DNA sample to reduce contaminants. Warning/error in all samples: gently mix the ligase master mix at room temperature (by pipetting up and down) before use. Do not vortex. Do not preheat the ligase master mix and use a multichannel pipet (if available). Related plausible root cause: ligation activity. |
| Ligase inactivation 1, 2 |
Ensure the thermocycler is properly calibrated and the thermocycler program is correct. Related plausible root cause: ligation inactivation. |
| Polymerase activity 1, 2 |
Warning/error in a single sample: perform an extra DNA purification or dilute the DNA sample to reduce contaminants. Warning/error in all samples: gently mix the polymerase master mix at room temperature (by pipetting up and down) before use. Do not vortex and use a multichannel pipet (if available). Related plausible root cause: polymerase activity. |
Data analysis |
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| Description | Explanation |
| Reference probe quality |
Ensure the tissue type used is compatible with the probemix (see probemix-specific product description). Related plausible root cause: reference probe quality. |
| Reference sample quality |
Ensure all samples used in an experiment are from a similar source and treated similarly. Dedicated reference samples should not have any copy number aberration in the targets tested. Related plausible root causes:
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| Reference sample quantity |
Ensure the reference population contains sufficient suitable samples (see probemix-specific product description for details). Related plausible root causes:
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| No-DNA control(s) |
The no-DNA control(s) included in this experiment did not meet quality criteria. More information. Ensure all DNA samples are correctly defined in the Coffalyser Definition File, and that no samples with DNA have been set as no-DNA. Related plausible root causes: no-DNA control(s) & DNA quantity (for no-DNA sample). |
- Warnings or errors for this quality check can be triggered at the intra and/or inter level. The intra check focuses on the quality of each individual sample. For control probes/fragments, this is based on comparing intra ratios to what is expected based on data generated at MRC Holland. The inter check focuses on the comparison of the sample to the reference population. A warning or error is triggered in case the tested condition differs between the sample and the reference population. Ensure all samples used in one experiment are from a similar source and are treated similarly.
- This quality check is based on a set of control probes. A warning or error is only triggered if half or more of the control probes in the set are outside of expected boundaries.