PN 100-5066 E3 USER GUIDE SINGuLAR™ Analysis Toolset 3.0 © Fluidigm Corporation. All rights reserved. © Fluidigm Corporation. All rights reserved. Contents ™ Chapter 1: SINGuLAR Analysis Toolset 3.0 ........................................... 11 Gene Expression .............................................................................................. 11 Variant Analysis ............................................................................................... 12 The Key Features of SINGuLAR Analysis Toolset 3.0 ..................................................... 12 For Gene Expression in SINGuLAR Analysis Toolset 3.0 ............................................ 12 For Variant Analysis in SINGuLAR Analysis Toolset 3.0 ............................................. 14 Where to Get the Information You Need .................................................................. 15 Fluidigm Related Products ............................................................................. 15 Chapter 2: Installing R and SINGuLAR Analysis Toolset 3.0 ........................ 17 Install R 3.0 for Windows .................................................................................... 17 Install R 3.0 for Mac .......................................................................................... 18 Install SINGuLAR Analysis Toolset 3.0...................................................................... 19 Updating on the Mac .................................................................................... 21 Create the Directory for SINGuLARTM Analysis Toolset 3.0 .............................................. 22 Rules and Guidelines for Naming Genes and Samples ................................................... 22 The Terminology in Your Dataset ..................................................................... 22 Naming Conventions .................................................................................... 22 Restrictions .............................................................................................. 22 Dialogs Unrelated to the R Application .................................................................... 23 Useful R Function Assistance ................................................................................ 23 Basic Concepts in R ........................................................................................... 23 Example 1: Creating an Object from a File.......................................................... 24 Example 2: Creating an Object from a Calculation ................................................ 24 Overwriting© Objects Fluidigm .................................................................................... Corporation. All rights reserved. 24 Useful Functions for Viewing Files and Objects ..................................................... 24 To get a list of the objects created in the current working session .................................... 24 To display the R actions taken for a function .............................................................. 24 To copy and paste within R .................................................................................... 25 To view the set of data frames for an exp or VC object ................................................. 25 To see a only a specific column within an list object ..................................................... 25 Get R Help ..................................................................................................... 25 SINGuLAR Analysis Toolset 3.0 User Guide 3 Contents Chapter 3: SINGuLAR Analysis Toolset 3.0 for Gene Expression ................... 27 Single-Cell Studies: Gene Expression ...................................................................... 27 Prepare and Process mRNA Seq Data ...................................................................... 28 Pre-Processing and Initial Assessment ................................................................ 29 Mapping ................................................................................................... 29 Prepare Reference Sequences ................................................................................ 29 Map RNA Seq Reads to Reference Genome or Reference Sequences ................................... 29 Calculate Expression Values of Annotated Genes or Transcripts ....................................... 30 Example of an mRNA Seq Expression File ................................................................... 30 Prepare and Process qPCR Data ............................................................................ 31 Some Guidelines for Data Preparation ............................................................... 31 Prepare Sample/Gene Group Annotation Files ........................................................... 31 Prepare the Input File ....................................................................................... 32 Workflow for Gene Expression with SINGuLAR Analysis Toolset 3.0 .................................. 33 Log2EX and Limit of Detection (LoD) ...................................................................... 34 Log2EX and LoD in mRNA Seq Experiments .......................................................... 34 Log2EX and LoD in qPCR Experiments ................................................................ 34 How to Handle Missing Data ................................................................................. 35 Example 1: Data for an LOD of 24 for qPCR ......................................................... 36 Example 2: Data for mRNA Seq data LOD =1 ........................................................ 36 Mathematical and Plotting Functions ...................................................................... 36 Principal Component Analysis (PCA) .................................................................. 36 ANOVA .................................................................................................... 40 Hierarchical Clustering (HC) Analysis ................................................................. 40 Correlation Analysis ..................................................................................... 41 Visualization of Expression Profiles ................................................................... 41 The Outlier Identification Method ......................................................................... 42 © Fluidigm Corporation. All rights reserved. The Automatic Analysis Method ............................................................................ 43 The autoAnalysis Workflow ............................................................................ 44 Perform Outlier Identification .............................................................................. 45 Perform Automatic Analysis ................................................................................. 47 A Note About Using Functions Without Automatic Analysis ....................................... 47 Run the Automatic Analysis ............................................................................ 48 Examine the Results .................................................................................... 49 Violin Plot ......................................................................................................... 49 4 SINGuLAR Analysis Toolset 3.0 User Guide Contents Hierarchical Clustering (HC) Plot ............................................................................. 50 PCA Plots .......................................................................................................... 51 The Selected Gene List Output File .................................................................. 53 Advanced Functions ..................................................................................... 54 Reading Experimental Data ............................................................................ 54 To read experimental objects ................................................................................. 54 To read mRNA Seq data while changing the LoD value to 6 ............................................. 54 Annotating Samples ..................................................................................... 54 To update a sample list (sample name plus group ID) with a file ...................................... 54 Annotating Genes ....................................................................................... 55 To update genes with group information ................................................................... 55 Setting Custom Colors and Symbols .................................................................. 55 To customize the colors and symbols for sample groups of given Exp object ........................ 55 Saving Data ............................................................................................... 55 To save the EXP object to files for future use ............................................................. 56 To save a gene or sample list to a *.txt file ................................................................ 56 To save a plot as an image file ............................................................................... 56 Working with the Analysis Output in Excel .......................................................... 56 Selecting and Trimming Genes ........................................................................ 56 To display the number of detected genes for samples above a threshold of 1 .....................