Quality Measures for 24sure Microarrays

Transcription

1 Quality Measures for 24sure Microarrays How to evaluate 24sure quality in BlueFuse Multi software. Introduction Data quality is one of the most important aspects of any microarray experiment. This technical note outlines methods for users to check the quality of their microarray data and assess laboratory procedures, before proceeding with downstream analyses. Checking data quality enables users to assess whether the genomic changes seen in a sample are real or artefacts. All 24sure microarrays come with BlueFuse Multi software. Quality control (QC) metrics are displayed in the report for each experiment. The composite image for each array can also be viewed to check for any major artefacts (Subarray QC shortcut). In addition, a confidence call is given to the imbalance calling of the chromosomes. It is also possible to view QC metrics across a database to assess data quality over time (detailed in the BlueFuse Multi Reference Manual). This document should be used as a guide only. It remains the responsibility of the laboratory personnel to make the final decision on the usability of array data. For any further assistance, contact Illumina technical support at techsupport@illumina.com. Overview of QC Metrics QC Measures In BlueFuse Multi, the report displays QC measures that are relevant to the selected experiment. These measures should be consulted together with the Subarray QC shortcut and the confidence values for calling when reviewing results. SD Autosome/Robust is a measure of the dispersion of log 2 ratio of all clones on the array. A small value indicates that the log 2 ratios of the autosome clones are tightly clustered around zero, which is their expected value. It is calculated on the normalized but unsmoothed data. SD autosome is calculated using all autosomal clones on the array; SD robust is calculated using the middle 66% of the data, allowing the removal of outliers. The SD robust value is used by the software for region calling to determine gain and loss thresholds. An SD value that is too low suggests a problem with washing or hybridization, resulting in lots of non-specific signals left on the array. % Included Clones percentage of all clones that were not excluded due to inconsistency between clone replicates on an array. This measure is used for bacterial artificial chromosome (BAC) arrays (CytoChip Focus Constitutional and 24sure). If the level is low, it may indicate a major artefact on the array. There is no exclusion on oligo arrays; therefore, this value is always 100%. Mean Spot Amplitude Ch1/Ch2 the mean fluorescent signal intensities for the two channels; channel 1 = sample (typically Cy3) and channel 2 = reference (typically Cy5). This metric is variable due to the differences between available scanners. The mean spot amplitude metric can give an indication of how well the DNA has labeled with fluorescent dyes. More importantly, extremely high values can indicate over-scanning of the microarray image or poor washing so that there is lots of non-specific signal left on the array. The balance between channels can be assessed but the Cy5 signal tends to give a higher intensity than Cy3. Major differences in the channels may indicate a labelling or a scanner problem. SBR Ch1/Ch2 the overall signal-to-background ratio for each fluorescent channel. This value is calculated by dividing the background-corrected fluorescent signal intensity from the DNA clones by the raw background signal level. SBR indicates how clearly the spots can be detected above the background. The SBR metric gives an indication of how well the labeling and washing steps have been performed. DLR Raw/Fused derivative log ratio (DLR) measures the spread of the difference in log 2 ratios between all the pairs of adjacent clones along the genome. For a simple profile, this statistic will be very similar to autosome standard deviation. The presence of many step changes in a complex profile will cause the autosome standard deviation to become inflated, whereas the DLR metric is much less sensitive to this problem. DLR fused is the DLR calculated on the normalized but unsmoothed data. 24sure QC Guidance For 24sure experiments, it is difficult to offer definitive QC measure values for results, because there are so many factors that can affect the Fused Chart result and the QC parameters. Factors such as the biopsy sample type (polar body [PB], blastomere, trophectoderm), the amount of amplification during the SurePlex protocol, and the length of the labeling or hybridization steps, all impact the final result. 24sure analysis is designed to assess whole-genome aneuploidy so high-resolution results are not necessary. Often, even with a poor experiment, results can still be interpreted. Factors that increase noise include: Fewer cells results obtained with polar bodies and blastomere biopsies with only one cell can be more noisy than a trophectoderm biopsy with three to five cells. Shorter labeling less labeled product being formed and available for hybridization can lead to noisier data. Shorter hybridization hybridization kinetics mean that longer hybridization times result in more product specifically bound to the target. Incorrect wash temperature if the wash temperature is too low, it will cause a lot of non-specific binding to the array and will lead to noisy data. Temperatures that are too high can remove too much real signal.

2 Table 1: Confidence Calls Chromosome X Y Imbalances G Confidence The first part of this technical note outlines the QC parameters expected for the reference subarrays that are hybridized with every batch of 24sure experiments. Illumina has compiled basic QC criteria settings that can be used as a guide to assessing 24sure reference subarray results. It is important to check the reference subarray QC metrics because, if they have failed in a batch, it is likely that the sample subarrays have also failed. The examples shown are from sexmatched references. The second part of this technical note shows examples of good quality and bad quality 24sure sample subarray results with their corresponding QC metrics, in addition to an example of good quality array data of a chaotic sample. Detailed Result Confidence The Results section of the report in BlueFuse Multi contains a table called Detailed Result. This table shows the imbalances in the genome, together with a value that relates to the confidence given to the calling. An example is shown in Table 1. The values of the confidence call can range from 0.5 to 1; chromosomes with confidence values nearer 1 have a higher confidence in their calling. The confidence relates to chromosomes with no change, as well those that are gained or lost. In BlueFuse Multi, the 24sure V3 (single-channel) algorithm uses absolute threshold values to determine whether a chromosome is gained or lost. As part of this calculation, all the data points on a chromosome are combined to calculate a median log 2 ratio value. If a chromosome has a median log 2 ratio value of or greater, it will be called as a gain; if a chromosome has a median log 2 ratio value of or less, it will be called as a loss. A chromosome with median log 2 ratio between and will be called as a no change. have a confidence of 1 as definitely no change. If the ratio is closer to , the confidence will drop so that when it reaches it will have a confidence 0.5; i.e., a 50% chance it could be gained or normal. Once the ratio starts to move even higher, e.g., to 0.25, then the chromosome will be called as a gain. The higher the ratio the more confidence that it is a gain and the higher the confidence value will be. Do not perform calling of chromosomes using the confidence values alone. Always check the fused chart to confirm results. 24sure Reference Subarray QC Metrics Table 2 lists basic QC criteria settings that can be used as a guide to assess 24sure reference subarray results. Table 2: Coverage Details SD Autosome/Robust Good Too low < 0.02 Too high > 0.17 % Included Clones Good > 95% Mean Spot Amplitude (signal)* Good 700 3,000 Too low < 500 Too high > 3,000 SBR Good 3 12 Too low < 3 Too high > 15 DLR Raw/Fused Good < 0.2 *Scanner-dependent OK < 0.22 Fail > 0.24 The confidence given to each call relates to how close the median log 2 ratio value for a given chromosome is to the ideal value. For example, if the chromosome has a median log 2 ratio value of 0, then it will

3 Figure 1: Fused Chart for Good Reference Subarray A Good Quality 24sure Reference Subarray Result Figure 1 shows a fused chart plot from BlueFuse Multi and is an example of a result from a good experiment. The plot is totally flat (sexmatched design) and data points are tight to the log 2 ratio zero axis. Figure 2: Composite Image for Good Reference Subarray Figure 2 is the scan image of this 24sure reference subarray. It shows a clean image, and an even dark background with no wash-related artefacts or other flaws. Table 3 shows the QC metrics for the good reference subarray. Table 3: QC Metrics for Good Reference Subarray SD Autosome/Robust 0.02/0.02 SD autosome is acceptable % Included Clones Less than 2% of clones excluded; data should be very interpretable Mean Spot Amplitude Ch1/Ch / Signal intensities are good; array does not appear over-scanned SBR Ch1/Ch2 5.22/9.84 Good SBR values DLR Raw/Fused 0.06/0.04 Excellent fused DLR value; any value below 0.15 is a very good result

4 Figure 3: Fused Chart for Poor Reference Subarray A Poor Quality 24sure Reference Subarray Result Figure 3 shows a fused chart plot from BlueFuse Multi and is an example of a result from a poor or failed experiment. it is not very easy to judge the quality of this hybridization from the fused chart; the profile is perhaps not as tight as it should be. Figure 4: Composite Image for Poor Reference Subarray Figure 4 is the scanned image of this 24sure reference subarray. It is evident that the slide has not been washed effectively. There is low signal and high uneven background. It is possible that the array dried out during hybridization. The image is also quite red. Table 4 shows the QC metrics for the poor reference subarray. Table 4: QC Metrics for Poor Reference Subarray SD Autosome/Robust 0.06/0.06 SD autosome is still in the very good range % Included Clones % clones excluded leading to poor data Mean Spot Amplitude Ch1/Ch / Very low Cy3 (Ch1) signal SBR Ch1/Ch2 0.78/2.50 Poor SBR values due to low signal and high background DLR Raw/Fused 0.16/0.12 DLR is acceptable, so it is unlikely to be a DNA issue

5 Figure 5: Fused Chart for Good Sample Subarray A Good Quality 24sure Sample Subarray Result Figure 5 shows the single-channel view from BlueFuse Multi and is an example of a result from a good experiment. The plot is totally flat against the female reference (sex-matched design) with good separation against the male reference, indicating a female embryo. Data points are tight to the log 2 ratio zero axis. For this type of result, there is really no need to rely on QC metrics. Figure 6: Composite Image for Good Sample Subarray Figure 6 is the corresponding scan image of the good 24sure sample subarray. It shows a clean image, and even dark background with no wash-related artefacts or other flaws. Table 4 shows the QC metrics for the good sample subarray. Table 5: QC Metrics for Good Sample Subarray SD Autosome (Male/Female/Combined) 0.04/0.03/0.03 SD autosome is in the very good range % Included Clones (Male/Female/Combined) 94.80/96.08/97.01 Only 3 5% of clones excluded; data should be easy to interpret Mean Spot Amplitude Ch1/Ch Signal is a little high but with the other QC metrics it is not a concern SBR 9.22 Very good SBR value DLR Raw/Fused (Male) 0.07/0.06 Excellent fused DLR value; any value below 0.15 is a very good result DLR Raw/Fused (Female) 0.06/0.05 Excellent fused DLR value; any value below 0.15 is a very good result DLR Fused (Combined) 0.05 DLR is very good

6 Figure 7: Fused Chart for Poor Sample Subarray A Poor Quality 24sure Sample Subarray Result Figure 7 is an example of a result from a bad or failed experiment. It is easy to see the quality of this array is poor, as the data are very noisy. Figure 8: Composite Image for Poor Sample Subarray Figure 8 is a scan image of a poor 24sure sample subarray. It is evident that the slide has not been washed effectively. There is high uneven background. The image also has wash artefacts, further confirming that it was not washed properly. Table 6 shows the QC metrics for the poor sample subarray. Table 6: QC Metrics for Poor Sample Subarray SD Autosome (Male/Female/Combined) 0.40/0.40/0.39 Very bad robust SD score; any value above 0.17 is a bad result and almost impossible to call accurately % Included Clones (Male/Female/Combined) 74.95/74.05/83.83 Between 20 25% clones excluded, leading to poor data Mean Spot Amplitude Ch1/Ch Signal is on the low side SBR 0.77 Poor SBR values due to low signal and high background DLR Raw/Fused (Male) 0.18/0.20 DLR is acceptable, so it is unlikely to be a DNA issue DLR Raw/Fused (Female) 0.18/0.20 DLR is acceptable DLR Raw/Fused (Combined) 0.19 DLR is acceptable

7 Figure 9: Fused Chart for Chaotic Sample Subarray A Chaotic 24sure Sample Subarray Result Figure 9 shows an example of a chaotic profile from a good quality experiment. At first glance, the data look noisy: in fact, the data quality is good, but it is a complex/chaotic sample. The complexity of the profile is reflected in the high SD autosome metric, but the quality of the data is good as shown by the low DLR values. Figure 10: Composite Image for Chaotic Sample Subarray Figure 10 is the corresponding scan image from the 24sure sample subarray of a chaotic sample. The subarray is clear, with a good signal and the background is not too high. Table 7 shows the QC metrics for the chaotic profile. Table 7: QC Metrics for Chaotic Sample Subarray SD Autosome (Male/Female/Combined) 0.45/0.46/0.45 SD autosome is too high due to complex profile % Included Clones (Male/Female/Combined) 94.51/95.23/97.72 Only around 5% of clones excluded; data should be very interpretable Mean Spot Amplitude Ch1/Ch Signal intensity is good SBR 4.37 SBR value is in the good range DLR Raw/Fused (Male) 0.05/0.06 Excellent fused DLR value; any value below 0.15 is a very good result DLR Raw/Fused (Female) 0.05/0.06 Excellent fused DLR value; any value below 0.15 is a very good result DLR Raw/Fused (Combined) 0.05 DLR is very good

8 Illumina toll-free (U.S.) tel FOR RESEARCH USE ONLY 2014 Illumina, Inc. All rights reserved. Illumina, IlluminaDx, BlueFuse, 24sure, the pumpkin orange color, and the Genetic Energy streaming bases design are trademarks or registered trademarks of Illumina, Inc. All other brands and names contained herein are the property of their respective owners. Pub. No Current as of 12 August 2015