ncounter PlexSet Data Analysis Guidelines NanoString Technologies, Inc. 530 airview Ave North Seattle, Washington 98109 USA Telephone: 206.378.6266 888.358.6266 E-mail: info@nanostring.com Molecules That Count MAN-10044-01 March 2017
OR RESEARCH USE ONLY. Not for diagnostic purposes. Intellectual Property Rights ncounter PlexSet reagents and its contents are the property of NanoString Technologies, Inc. ( NanoString ), and are intended for the use of NanoString customers solely in connection with their operation of the ncounter Analysis System. The ncounter Analysis System (including both its software and hardware components) and this User Manual and any other documentation provided to you by NanoString in connection therewith are subject to patents, copyright, trade secret rights, and other intellectual property rights owned by or licensed to NanoString. No part of the software or hardware may be reproduced, transmitted, transcribed, stored in a retrieval system, or translated into other languages without the prior written consent of NanoString. or a list of applicable patents, see www.nanostring.com/company/patents. Limited License Subject to the terms and conditions of sale of the ncounter Analysis System, NanoString grants you a limited, non-exclusive, non-transferable, non-sublicensable, research use only license to use this proprietary nsolver software with the ncounter Analysis System only in accordance with this manual, the manual for the ncounter Analysis System, and other written instructions provided by NanoString. Except as expressly set forth in the terms and conditions, no right or license, whether express, implied, or statutory, is granted by NanoString under any intellectual property right owned by or licensed to NanoString by virtue of the supply of this software or the proprietary ncounter Analysis System. Without limiting the foregoing, no right or license, whether express, implied, or statutory, is granted by NanoString to use the nsolver Analysis Software or ncounter Analysis System with any third-party product not supplied or licensed to you by NanoString, or recommended for use by NanoString in a manual or other written instruction provided by NanoString. Trademarks NanoString Technologies, NanoString, the NanoString logo, ncounter, nsolver, PlexSet and Molecules That Count are trademarks or registered trademarks of NanoString Technologies, Inc., in the United States and/or other countries. All other trademarks and/or service marks not owned by NanoString that appear in this document are the property of their respective owners. Copyright 2017 NanoString Technologies, Inc. All rights reserved. Molecules That Count 2
Introduction ncounter PlexSet Data Analysis Guidelines This guide provides an overview of PlexSet data analysis using nsolver 4.0 analysis software. NOTE: PlexSet data is not supported in nsolver versions 3.0 and below. nsolver allows you to easily import, process, and analyze your data. Due to the unique nature of the PlexSet assay, the nsolver workflow is slightly different from that of other assays. The PlexSet assay uses a 96- well plate for hybridization and allows you to pool all 8 samples in a column (A through H) into a single cartridge lane for analysis on the Digital Analyzer. The barcodes used in the PlexSets (A through H) are all unique, allowing the nsolver software to assign the data back to individual samples/wells during the analysis. Molecules That Count 3
Import To open your data folder and unzip the data files, right click and select Extract All. Note: If you do not have a Zip/Unzip application on your system, download a freeware program (e.g., 7-Zip). Open nsolver 4.0 TM and select Import RCC iles. Do not use Import RL for this Alpha software release. Navigate to your unzipped data folder, select the files, and click Next. QC Choose your QC parameters. If these are hidden, select the double-arrow icon at the right of the screen to reveal parameters. The Imaging QC is a measure of the percentage of each lane that was successfully scanned. The Binding Density QC is a measure of barcode density within each sample lane. The Positive Control Linearity QC and the Positive Control Limit of Detection QC parameters are not used in the PlexSet assay; these QCs are intended for assays containing 6 Positive controls covering a range of concentrations. Instead, each PlexSet reagent contains a single unique Positive control and single unique Negative control (one for each row A H). You may change the QC parameters, but this is not usually recommended or necessary. Select Import. Your RCC data files will now be stored under the corresponding RL CodeSet ile on the Raw Data tab. Select the CodeSet name to view all RCC files in a table format. The main raw data table columns are labeled as Set A, Set B, etc. through Set H. Rows are labeled with RCC file names, which correspond to the different cartridge lanes 1-12. The default sample names are the standard well coordinates of a 96-well plate. Custom sample names can also be pasted or typed into the cells of this table. Scroll to check for QC flags. Select samples and click the Table button to review the individual counts of each sample; column headers are sorted by well identity (A1, B1, etc). Examine the data to ensure that counts of POS/NEG controls and Housekeeping/Endogenous genes meet expectations, especially for samples with QC flags. Molecules That Count 4
Creating Studies & Experiments Within nsolver, a Study is an organizational folder used to store experiments, and an Experiment is a collection of samples that have been normalized to allow comparisons between samples or groups of samples. Any studies and experiments you create will be visible on the Experiments tab. Click the New Study button to create a study, then the New Experiment button to create an experiment under that study. ollow the prompts to select the samples to include in your experiment. Creating an Experiment steps you through creating annotations, background options, and normalization, just as in the standard workflow. Typically, maintain the default settings: Annotations should be assigned for experiments in which fold-change estimates and their statistical significance will be studied, or to simply provide additional sample information that could be useful during downstream analyses. Background noise in data can be filtered out using subtraction or thresholding. The settings recommended for your analyte type(s) will appear by default. They may be: o No background calculation (option clicked off or greyed out). o Background thresholding, which uses a user-defined threshold count value; all raw counts below this value will be adjusted to this value. o Background subtraction, which can be calculated by using a blank lane (if loaded) count, by assigning a defined value, or by taking the mean/geometric mean/median/max of the Negative Control counts. Positive Normalization should typically be set to the geomean of the Top 3 POS counts. o Note that this normalization method is unique to the PlexSet assay. The single Positive controls for each well are pooled down the columns (for 8 Positive controls total in each lane). These are used to normalize for instrument-based differences in assay processing across lanes. Only 3 of these positive controls are necessary for accurate normalization, so the default setting is to only include the 3 positive controls with the highest values (Top 3). CodeSet Content Normalization can be selected if you have included housekeeping genes among your targets. Housekeeping genes can be defined in the RL or can be customized at the time of analysis. Move desired housekeepers from the CodeSet Content window to the Normalization Codes window using the arrows. If you have not included housekeeping or reference genes among your targets, you may skip CodeSet Content Normalization, although this is not recommended. After normalization, you have the option to designate your reference lane in the CodeSet Calibration window: Molecules That Count 5
or calibration, a reference sample should have been loaded in all eight wells of one column of the 96-well hybridization plate. This lane(s) should be selected for CodeSet Calibration. If this calibration sample was run previously on the same lot of PlexSet reagents with the same target probes, the.rcc file for this calibration lane can also be imported into the experiment and used for calibration. Select the Sample Reference Normalization checkbox to activate the options in the window. Select the lanes in which you loaded your reference sample(s) by selecting the appropriate ile Name in the Subcode Samples window (on the left). Use the arrows to move the desired lanes to the Selected Samples window (on the right). Set the Use as Reference dropdown menu at the bottom of the screen to define the set (A through H) that all other sets will be calibrated to. The default is Set A. Select Next. You will be prompted to establish baseline data for ratios, then asked to assign ratio data names per the standard workflow. Select Next, then inish. You will be alerted to any low couts. Select Continue. Molecules That Count 6
old Changes (Ratios) can be calculated by specifying the sample(s) that represent the baseline of your experiment. All pairwise ratios compares all groups to one another, while Partitioning by allows you to choose a group as the reference (if previously defined in the Annotations step). Using user selected reference samples allows you to choose any combination of samples. Select Next, confirm the ratios you would like to calculate, and select inish. Data Tables & Export Your experiment will now be under your study on the Experiments tab. Expanding the navigation tree will reveal the following categories: The Raw Data table contains unprocessed data for all samples in this experiment. The Normalized Data table contains the processed data for all samples. Samples with unusually low counts for POS controls or Housekeeping genes may receive Normalization flags, which can be seen by scrolling to the far right in the central window. Open the normalized data and examine the normalized counts. Paying particular attention to any flagged samples, review the data to ensure that counts of POS/NEG controls and Housekeeping/Endogenous genes meet expectations. The Grouped Data table contains the geometric mean of expression levels for all samples from each group (as defined by the sample annotations). The Ratio Data table contains the fold-change results, as well as any statistical inferences surrounding those estimates. You may need to view hidden columns of data by right-clicking (or command-clicking) on the column header and choosing Select columns to choose additional fields. You may also use the table options icon to view all columns. The Analysis Data table contains any analysis you have run. Select the desired data table, highlight samples of interest in the main window, and use the Table button to examine your data or the Export button to export results. When your experiment is highlighted, the Report button can be used to create an experimental summary report that details QC and normalization. The Table Options Icon allows you to hide and move columns of data. Molecules That Count 7
Analysis Select Analysis to see the graphing options for the highlighted data table. Select the desired plot, then select Next. Select the samples, then the probes to include in your analysis. If creating a heat plot, you may be asked to set Clustering Parameters. Once your data is plotted, you can fine-tune the settings. ile allows you to save and print the plot image. Advanced Analysis is not currently available for PlexSet assays. Violin Plot Box Plot Heat Map Scatter Plot The options to the left of the Scatter Plot allow you to select the sample(s) for the plot, as well as the color designations of the data points. or additional customization (such as the axis and legend settings), select Settings. Histogram The table to the left of the Violin, Box, and Histogram plots allows you to select the probe(s) for the plot. or additional customization (such as the axis settings), select Settings. Molecules That Count 8