EmbryoCellect. RHS Scanning and Analysis Instructions. for. Genepix Pro Software

Transcription

1 EmbryoCellect RHS Scanning and Analysis Instructions for Genepix Pro Software EmbryoCellect Genepix Pro Scanning and Analysis Technical Data Sheet Version 1.0 October

2 Copyright Reproductive Health Science Limited West Thebarton Road Thebarton, South Australia, 5031 EmbryoCellect Genepix Pro Scanning and Analysis Technical Data Sheet Version 1.0 October 2015 Reproductive Health Science Limited (RHS) makes no representations and gives no warranties of any kind in relation to the contents of this document and all warranties, conditions and other terms implied by statute or common law are, to the fullest extent permitted by law, hereby excluded. In particular, RHS assumes no responsibility for any errors or omissions that may appear in this document and makes no commitment to update or keep current the information contained in this document. RHS retains the right to make changes to this document (including any specifications contained herein) at any time without notice. This document is confidential. No part of this document may be modified, copied, reproduced, republished, published, transmitted or distributed in any form or by any means without the prior written consent of RHS. The contents of this document are to be used solely for the purpose for which they are provided by RHS and for no other purpose. All content, text, graphics and all other materials contained in this document are owned by RHS, and all proprietary and intellectual property (names, logos and trademarks) wherever arising in relation to this document, rest in RHS (or its licensors) and all such rights are reserved. EmbryoCellect is a Research Use Only product and is not to be used for diagnostic procedures EmbryoCellect is for Research Use Only and should not be used in diagnostic procedures. You are responsible for ensuring that you accurately follow the protocols provided in this Genepix Pro Scanning and Analysis Technical Data Sheet (TDS) and analysing and interpreting the results you obtain. RHS does not guarantee any results obtained. EmbryoCellect Genepix Pro Scanning and Analysis Technical Data Sheet Version 1.0 October

3 Contents 1. Genepix Pro Software Compatibility General Instructions Slide format Scanner requirements Prescan Scanning Scan parameters Saving TIFF images Scanning Instructions Check the settings Perform and save a Preview Scan of the slide Scan each array individually Array quality control Quantifying the Data Analysing the Data using the RHS Macro Final Result Quality Control Macro Troubleshooting EmbryoCellect Genepix Pro Scanning and Analysis Technical Data Sheet Version 1.0 October

4 1. Genepix Pro Software Compatibility The instructions described in this technical data sheet for the analysis of the EmbryoCellect TM array using the Genepix Pro software (Molecular Devices, USA) are compatible with version 6.1 and should be compatible with other versions. These instructions require the use of the Genepix Pro.gal files (Genepix_A, Genepix_B, Genepix_C, Genepix_D) and Macro 6.2, which are available from the RHS dropbox or the RHS website. The RHS analysis macro only works when the Excel decimal separator is a period (.) not a comma (,). While every effort has been made to ensure the protocol steps are compatible across all versions of Genepix Pro software, there may be a version where the user may experience a run error during the analyses of array data. If an error is encountered, please contact RHS immediately by ing support@rhsc.com.au and we will provide you with assistance. 2. General Instructions 2.1 Slide format Each EmbryoCellect TM slide contains four microarrays with identical probes in each (Figure 1). A B C D Figure 1: Microarray slide template for sample loading displaying the locations of individual arrays Four sample batching is possible, making EmbryoCellect TM ideal for processing small numbers of samples. Slides cannot be reused so the recommended minimum number of samples to be hybridised is four. If all four microarrays are not hybridised, the unused microarrays will not be able to be utilised in subsequent hybridisations. 2.2 Scanner requirements A dual channel scanner equipped with a green and red laser (532nm and 635nm wavelength, respectively) is required to scan EmbryoCellect TM slides. 2.3 Prescan A prescan allows low-resolution visualisation of the entire slide surface, showing the array locations (see Figure 2). Most scanners will also read the slide barcode. When the prescan has completed, the scan area can be adjusted independently for each microarray for the higher resolution analysis scan. Figure 2: Example of a prescan image illustrating individual array locations EmbryoCellect Genepix Pro Scanning and Analysis Technical Data Sheet Version 1.0 October

5 2.4 Scanning Some microarray scanners have automatic scanning settings which adjust the power of the laser and/or gain of the photomultiplier tube (PMT). The effects of these settings can be viewed on a histogram (see Figure 3). If required, these settings can usually be changed manually and should be adjusted until the red and green lines on the histogram overlap as much as possible (see Figure 4). Figure 3: Example of Histogram before scanning parameter optimisation. The high peak close to the Y axis represents the background signal. The secondary smaller peaks represent the microarray spot signal. The green line represents results from the green laser (532 wavelength test) and the red line represents results from the red laser (635 wavelength - reference). This image shows poor overlap between the green and red signals. Figure 4: Example of Histogram with ideal scan parameters. The red and green lines for the test and reference signal are overlapping. 2.5 Scan parameters In addition to the laser and PMT settings, other scanner software parameters should be adjusted for ideal EmbryoCellect TM slide scanning. Spot signals depend on several parameters, from sample DNA quality to hybridization and washing process success. Scan parameters should be adapted to each slide or array in order to fit within the following image quality control parameters; Pixel Saturation: EmbryoCellect TM slides must be scanned with minimal saturation because saturated pixels are excluded from the image. GenePix Pro s Auto-PMT option allows you to set the percentage of saturated pixels allowed. Set the option Saturation tolerance (%) to 1e-4 Pixel size: Set the Pixel Size (µm) to 10µm EmbryoCellect Genepix Pro Scanning and Analysis Technical Data Sheet Version 1.0 October

6 2.6 Saving TIFF images Images should be saved with the following settings as a TIFF file containing both colour images (this is usually the default scanner setting). RHS recommends that files are saved using the slide barcode plus the array suffix as a minimum. E.g. RHS001234_A. Figure 5: Save settings 3. Scanning Instructions Switch on Scanner Start GenePix Pro Software Allow scanner to warm up for ~15 minutes before use Insert Slide Ensure that the slide will be scanned from the top to the bottom array by placing the slide in the correct orientation (the barcode is at the bottom of the slide) EmbryoCellect Genepix Pro Scanning and Analysis Technical Data Sheet Version 1.0 October

7 3.1 Check the settings Open the options box Open the Workflow tab: Check the box: Search for Barcode after each Data Scan or Image load. Check the box: Register Images after a Data Scan. Open the Analysis tab: In the Ratio Formulation select 532/635. Open the Alignment tab: Change value for Minimum Diameter % to 50. Change value for Maximum Diameter % to 200. Change value for Composite Pixel Intensity (CPI) threshold to include a pixel in a feature during alignment to 500. Open the File save tab: In the Image and Results File Naming section, check the Prefix-Barcode box. Select OK EmbryoCellect Genepix Pro Scanning and Analysis Technical Data Sheet Version 1.0 October

8 3.2 Perform and save a Preview Scan of the slide Open the Hardware Settings dialog box: Set PMT levels at 600 for both 635 and 532. Leave the Power % setting at 100% Set the desired Pixel size to 10µm Leave the Lines to Average setting at 1. Leave the desired Focus Position setting at 0. Press Preview Scan Save the Preview Scan Select File/save Select Export Images Save the preview scan in a desired location as a JPEG 3.3 Scan each array individually Define the region of interest: Select the Scan Area tool Press F10 to bring up the Scan Area options (alternatively right click with the mouse and select Scan Area Options). Array Left Top Width Height A 7000 B C D Enter the following coordinates to select the scan areas for each of the RHS arrays, starting with array A. Press OK EmbryoCellect Genepix Pro Scanning and Analysis Technical Data Sheet Version 1.0 October

9 Load the Array Layout (.gal file) Click the File button Select the Load Array List command. (Shortcut: Alt + Y) Select the corresponding.gal file for the array you are analysing. E.g. Use the.gal file Genepix_A when scanning and analysing the A array. Use AUTO PMT to set the PMT Gain automatically Click the Auto-PMT button in the Hardware Settings box. Set the Saturation tolerance (%) to 1e-4. Click the Start button. Once finished, press Apply button. Perform a Data Scan Set the resolution for the data scan to 10µm in the Pixel Size field in the Hardware Settings box Press Data Scan EmbryoCellect Genepix Pro Scanning and Analysis Technical Data Sheet Version 1.0 October

10 Check for pixel saturation If there are any saturated spots; decrease PMT by 20 for both 635 and 532 then repeat the data scan. This image shows saturation in the 532 laser for some spots. The microarray should be rescanned with the PMT lowered by 20 for both lasers until no saturation is present. Align Template Line up the grid to overlap with the array by pressing F8 or click on the Gridding button and select the Find Array, Find Blocks, Align Features option. Ensure that GenePix Pro has correctly identified the position of the array and the blocks. If necessary due to incorrect spot positioning, incorrect flag, presence of debris or a scratch; If the array or the blocks are not correctly identified, move the grid manually into place with the mouse while in Block Mode. Move, resize, include or exclude spots by selecting Feature Mode Click once on a spot to select the spot + Use Ctrl + arrow keys to resize a grid spot Use the arrow keys to move a grid spot EmbryoCellect Genepix Pro Scanning and Analysis Technical Data Sheet Version 1.0 October

11 Grid spots should be adjusted to avoid dust or debris If necessary; right click on a selected spot and select Flag as Not Found or Flag as Bad to flag a spot and exclude it from the dataset Right click on selected spot and select Clear flags except Absent or Clear flags to include a spot that was previously flagged as not found EmbryoCellect Genepix Pro Scanning and Analysis Technical Data Sheet Version 1.0 October

12 If a halo effect occurs you may need to resize the spots to the inner ring of strong hybridisation otherwise it may be excluded from the dataset by the RHS macro. Save Image Select file, save images (Shortcut: Ctrl + S) In the Filename field, the slide name should already be filled in. Add the position of the array behind the barcode number (e.g. _A; _B; _C or _D Choose the correct folder for saving your image Click the Save button Proceed to steps 3.4 and 4 below before scanning other arrays on the same slide or another slide. EmbryoCellect Genepix Pro Scanning and Analysis Technical Data Sheet Version 1.0 October

13 3.4 Array quality control Each array has four replicate blocks. Spots are printed in duplicate in each of the four blocks to make a total of 8 spots for each chromosome (for example in the image to the right each spot marked with a * is the same chromosome). Verify that each of the replicates are consistent. * * * * * * * * In this example there was a bubble which caused uneven hybridisation of the spots in the middle of the array. You can see two of the replicates encompassed in this bubble hybridised differently. These spots should be flagged as bad and excluded from the data analysis. In this example some spots in the bottom left quadrant have been scratched and they are no longer consistent between the 8 spot replicates. These spots should be flagged as bad and excluded from the data analysis. EmbryoCellect Genepix Pro Scanning and Analysis Technical Data Sheet Version 1.0 October

14 Verify that there is consistent hybridisation across the whole array An array which appears like the above is considered less reliable and the sample should be re-hybridised Verify that there is consistent hybridisation across each spot 4. Quantifying the Data Press the Analyze button Arrays that have spots that have dark areas of hybridisation within them, as in the image above, are considered less reliable and the sample should be rehybridised EmbryoCellect Genepix Pro Scanning and Analysis Technical Data Sheet Version 1.0 October

15 When the analysis is finished, it will tab to the Results tab located at the top. The raw data is presented. Click the Save Results as... button (Shortcut: Alt + U) to save the results as a.gpr file. Save this in the same location as the scanned images Use the following save parameters: Repeat from 3.3 Scan each array individually with the remaining arrays Repeat from 3.2 Perform and save a Preview Scan of the slide to scan additional slides EmbryoCellect Genepix Pro Scanning and Analysis Technical Data Sheet Version 1.0 October

16 5. Analysing the Data using the RHS Macro Ensure macros are enabled Select enable macros when prompted or change settings in Excel Options Trust Centre Trust Centre Settings Macro Settings Open the RHS Mapix Analysis Macro The macro will open in the background of your raw data or will appear as an empty excel file Open the.gpr file with Microsoft Office Excel With the raw data file and the macro open Press Ctrl + Shift + E + + EmbryoCellect Genepix Pro Scanning and Analysis Technical Data Sheet Version 1.0 October

17 6. Final Result Quality Control The Analysis tab displays the resultant Karyotype and Array SD Check the Array SD - Results where SD > 0.10 are less reliable Check the number of spots analysed for each chromosome - Results where less than 4 spots are analysed per chromosome are less reliable If a small number of spots were analysed (eg 4 or less), check the reason for exclusion. Common reasons for exclusion include: - Flagged by Mapix as bad - Flagged by user as bad - Flagged by Macro as bad This can be due to saturation, the spot being too small or too big, the spot not being uniform (eg having a halo (which is an area of strong hybridisation in the middle of the spot surrounded by an area of weaker hybridisation), donuts (which is an area of weak hybridisation in the middle of a spot surrounded by strong hybridisation) or poor hybridisation. It is recommended that the sample is rehybridised. Contact support@rhsc.com.au for additional information. EmbryoCellect Genepix Pro Scanning and Analysis Technical Data Sheet Version 1.0 October

18 The ArrayData Graph tab displays the raw signal overlap between test and reference for each chromosome. The 2 coloured lines corresponding to the raw signal of both lasers should be as close to overlapping as possible. Where the test and reference lines do not overlap, this suggests: - A difference in the labelling efficiency between test and reference samples - Poor scanning parameters Results where the lines do not overlap are less reliable Good overlap X (Inv) Poor overlap X (Inv) Ensure there is sufficient fluorescent signal by checking the signal intensity on the Y axis. RHS generally sees intensities in the range of 30,000-50,000 units Weak signals may be due to: - Poor scanning parameters i.e. not enough laser power or the PMT detection gain is too low, or; - Samples being poorly labelled. This can be seen in the spectrophotometer readings and on the agarose gel of the labelled products - Loss of labelled sample during purification and/or precipitation Results with low intensities are less reliable Normal signal intensities X (Inv) Weak signal intensities X (Inv) EmbryoCellect Genepix Pro Scanning and Analysis Technical Data Sheet Version 1.0 October

19 X (Inv) Yp Negative Mean of normalized ratios X (Inv) Y Negative Mean of normalized ratios The Array Graph tab displays the mean normalized ratio for each chromosome; along with the standard deviation across each of the 8 spots analysed. Check the error bars on the Array Graph. Large error bars indicate: - inconsistent hybridisation across the array replicates This may be caused by debris, smearing of the microarray or bubbles, all of which are evident on the scanned image (jpeg file). Results with large error bars are less reliable than array results with smaller standard deviations per chromosome (see example) Large error bars Chromosome Small error bars Chromosome EmbryoCellect Genepix Pro Scanning and Analysis Technical Data Sheet Version 1.0 October

20 7. Macro Troubleshooting The RHS analysis macro only works when the Excel decimal separator is a period (.) not a comma (,). You can change these settings in Excel Options under the advanced tab. Please contact support@rhsc.com.au for technical assistance. EmbryoCellect Genepix Pro Scanning and Analysis Technical Data Sheet Version 1.0 October

21 Reproductive Health Science Ltd (ABN West Thebarton Road THEBARTON SA AUSTRALIA 5031 E: Ph: ASX:RHS EmbryoCellect Genepix Pro Scanning and Analysis Technical Data Sheet Version 1.0 October