Manual ® Archer Analysis 4.1 User Manual CS001-02 Revision 2 August 26, 2016 For use with versions 4.1 and above View videos and additional resources for Archer products at: http://archerdx.com/support For Research Use Only. Not for use in diagnostic procedures. Table of Contents Table of Contents .................................................................................................................... 2 1 Introduction and Summary ................................................................................................ 4 1.1 Introduction ................................................................................................................ 4 2 Analyzing Samples ........................................................................................................... 4 2.1 Run an analysis using the web interface .................................................................... 4 2.1.1 Create a login account ........................................................................................ 4 2.1.2 Run an analysis ................................................................................................... 5 2.1.3 Selecting the CONTROL samples for CNV runs ............................................... 10 2.1.4 Advanced Options ............................................................................................. 12 2.1.5 Progress Bar ..................................................................................................... 13 2.1.6 Progress bar and status updates ...................................................................... 14 2.1.7 Analyzing data from multiple runs ..................................................................... 14 2.1.8 Stopping a running or queued job ..................................................................... 15 3 Understanding Results .................................................................................................... 15 3.1 Home screen ............................................................................................................ 15 3.1.1 Reviewing the analysis settings ........................................................................ 16 3.1.2 Reviewing the (error) logs ................................................................................. 16 3.1.3 Rerun or clone a previous run ........................................................................... 17 3.1.4 View overview of results for the entire job ......................................................... 17 3.2 Job detail page ......................................................................................................... 17 3.2.1 Sample Name column ....................................................................................... 18 3.2.2 Assay Result column ......................................................................................... 18 3.2.3 QC Result column ............................................................................................. 20 3.2.4 Visualize Sample Data in JBrowse .................................................................... 21 3.2.5 Detailed Summary pages .................................................................................. 22 3.2.6 Strong Evidence and Weak Evidence Criteria .................................................. 23 3.2.7 Strong Evidence and Weak Evidence Tabs ...................................................... 25 3.2.8 Read Statistics Page ......................................................................................... 29 3.2.9 Assay Targets Page .......................................................................................... 29 3.2.10 Definition of RNA, DNA and AMBIGUOUS fragments .................................... 30 3.2.11 CNV Summary results ..................................................................................... 31 3.2.12 SNP/InDel detection ........................................................................................ 33 3.2.13 Filtering of results ............................................................................................ 35 3.2.14 Visualization of the variants using JBrowse .................................................... 36 3.2.15 Customizable and printable PDF reports ........................................................ 37 3.2.16 Jobs that complete with errors ........................................................................ 41 3.2.17 Adding and managing custom content ............................................................ 41 3.2.18 Adding custom targeted mutation sets ............................................................ 43 3.2.19 Adding custom reports .................................................................................... 46 3.2.20 Changing the analysis settings ........................................................................ 47 4 Description of output files and formats ............................................................................ 48 4.1 Full results summary ................................................................................................ 52 4.1.1 CNV Results file format ..................................................................................... 61 5 Workflow Automation ...................................................................................................... 63 5.1 Set up a watched folder in the web interface ........................................................... 63 5.2 Setup workflow automation definition in the web interface ...................................... 65 5.2.1 Analysis settings for the executed workflows .................................................... 65 2 | P a g e User Manual, Archer Analysis 4.1 CS001-02 Rev 2 5.2.2 Removing a watched directory .......................................................................... 66 5.2.3 Removing target region files that are used in watched directory workflows ...... 66 5.2.4 Develop procedure/script for the movement of FASTQ files ............................. 66 5.2.5 How the systems knows when to execute a workflow ....................................... 67 5.2.6 Example for job containing a single sample ...................................................... 67 5.2.7 Example for job containing multiple samples Contents of the watch folder ...... 67 5.2.8 Fate of the files in the watch directory ............................................................... 68 5.2.9 File collisions ..................................................................................................... 69 6 Troubleshooting .............................................................................................................. 70 6.1 Job finished with job status “COMPLETED_ERROR” ............................................. 70 6.2 Job is stuck with job status “NEW” ........................................................................... 71 6.3 Job is stuck with job status “HOLD” ......................................................................... 71 7 Managing Users And Groups .......................................................................................... 72 7.1 The admin user ........................................................................................................ 72 7.2 Users ........................................................................................................................ 72 7.2.1 Adding users ..................................................................................................... 73 7.2.2 Editing users ...................................................................................................... 74 7.3 Groups ..................................................................................................................... 76 7.3.1 Adding groups ................................................................................................... 76 7.3.2 Editing groups ................................................................................................... 78 8 Appendix ......................................................................................................................... 79 8.1 Settings Menu – Analysis Settings ........................................................................... 79 8.2 Detailed Summary - Strong and Weak evidence tabs ............................................. 81 8.3 Detailed Summary – Read Statistics tab .................................................................. 82 8.3.1 Molecular Barcode Statistics ............................................................................. 82 8.3.2 Read Statistics .................................................................................................. 82 8.3.3 DNA/RNA Statistics ........................................................................................... 83 8.3.4 DNA/RNA Fragment Lengths ............................................................................ 84 8.4 Detailed Summary – Assay Targets tab ................................................................... 85 8.4.1 Unique Molecular Bins sub-tab ......................................................................... 85 8.4.2 Unique Start Sites sub-tab ................................................................................ 85 8.4.3 Raw Alignment sub-tab ..................................................................................... 85 8.4.4 DNA sub-tab ...................................................................................................... 85 8.4.5 RNA sub-tab ...................................................................................................... 85 8.4.6 Ambiguous sub-tab ........................................................................................... 86 8.4.7 Total sub-tab ..................................................................................................... 86 8.4.8 Details Summary – Variant Summary tab ......................................................... 86 3 | P a g e User Manual, Archer Analysis 4.1 CS001-02 Rev 2 1 Introduction and Summary 1.1 Introduction This document describes the installation process and running procedure for the Archer Analysis Software. The Analysis software is available as a fully contained VM (Virtual Machine), which provides a web-based interface to the analysis software. Instructions for downloading and installing the VM are contained in a separate Installation Guide. 2 Analyzing Samples This section describes the execution of an analysis on the Archer Analysis Software. 2.1 Run an analysis using the web interface The Archer Analysis Software can be accessed through most modern Web Browsers, with the exception of Internet Explorer® version 7 (Windows® XP) which has been shown to have some problems. Google Chrome™ is the recommended web browser. Refer to the Archer Analysis Virtual Machine Installation Guide for instructions on how to discover the IP address of the Archer Analysis software. Start a web browser application and enter the IP address for the virtual machine in the address box. The login screen for the Archer Analysis Software will appear as shown below: Figure 1. Archer login page If the web page does not appear, check that the virtual machine is running (see the Archer Analysis Virtual Machine Installation Guide for instructions on how to install the virtual machine). 2.1.1 Create a login account The Archer Analysis Software is a fully contained and secure environment that allows users to run their analyses under their own account. Create a login account by selecting the “Create Account” link on the bottom left corner of the screen. The account creation webpage will appear as shown below: Figure 2. Archer Analysis account creation page Use your email address as your login and create a new password. Alternatively, your site administrator can create a login for you with your e-mail address. See section 7.2.1 for more details. At this time email cannot be sent from the VM. Enter a Password Retrieval Question and the appropriate answer. Later, if you lose your password, you will be asked the Password Retrieval Question to reset your password. Read the End User License Agreement (EULA) and check the box to indicate you have read and agree to the EULA, then click the “Create Account” button to create your account. This will automatically log you in. After successful login, the home screen will be displayed as shown below: Figure 3. Archer home screen 2.1.2 Run an analysis To start a new analysis, click the “Perform Analysis” button on the top right side of the screen. The Archer run analysis page will then appear: 5 | P a g e User Manual, Archer Analysis 4.1 CS001-02 Rev 2 Figure 4. Archer run analysis page 2.1.2.1 Name the analysis Give the analysis a name in the “Name of Analysis” box. The name is not required to be unique. 2.1.2.2 Select FASTQ files for analysis Provide the demultiplexed FASTQ files for your sample(s) in the “FASTQ Files” field by selecting the “Choose Files” button. This will open a file selection dialog box to allow you to select the FASTQ files as shown below. NOTE: All files must be from a single folder. Figure 5. Archer select FASTQ file dialog box Select one or more files and click “Open”. The number of files and the total size in MB or GB of the combined set of files is listed below the section and this can be used to predict the time it will take to upload the files to the server. 6 | P a g e User Manual, Archer Analysis 4.1 CS001-02 Rev 2 NOTE: The FASTQ files can be either uncompressed (and have the extension “.fastq” or “.fq”) or compressed (using the GZIP algorithm, and have the extension “.gz”). The ZIP compression file format is currently not supported. 2.1.2.3 Choose an Analysis Template If you have a non-default set of analysis settings that you would like to routinely run, you may save them as an Analysis Template by selecting "New" from the "Analysis Templates" pull-down list, and providing a name for it in the dialog box as shown below. Figure 6. New Template Name dialog box for naming a new Analysis Template Subsequently you can load it when setting up future analyses by selecting it from the "Analysis Templates" pull-down list. 2.1.2.4 Select the Assay Type There are a number of different Archer assays that can be analyzed, based on the origin of the nucleic acids; i.e RNA vs DNA assay. For each assay, multiple analyses can be run at the same time but RNA analyses and DNA analyses cannot be run simultaneously. RNA Analyses RNA Fusion detection will detect gene fusion events by annotating the de-novo assembled mRNA reads with BLAST. Archer specific filtering logic is used to reduce misalignments and false positives. SNP and Indel analysis can also be selected to include the detection of small variations with respect to the human hg19 reference. Select the required analyses by clicking the checkbox. DNA Analyses For DNA samples, small variation, DNA anomaly, and CNV (Copy Number Variation), analyses are available. You can also now monitor DNA target coverage. Select the required analyses by clicking the checkbox. The DNA small variation workflow detects SNPs (single nucleotide polymorphisms) and Indels (insertions and deletions) in your data. This can be utilized in addition to other workflows. The software will produce a variant summary with customizable filters that can be used to filter variants of interest. These filters can be adjusted in the interactive table and saved for future analyses. The user may select which attributes of the variants to show using basic logic gates provided in the software. When satisfied with the results, the data may be exported into a PDF report or downloaded as a TSV (tab separated values) or PDF file. 7 | P a g e User Manual, Archer Analysis 4.1 CS001-02 Rev 2 Figure 7. Archer Analysis Variant Summary The DNA anomaly analysis is a new workflow starting in Archer Analysis 4.0, and features a streamlined and powerful new variant grid (Figure 7 above). This is similar to our RNA Fusion pipeline in that DNA reads are de-novo assembled and annotated. This allows us to detect when exons, introns or entire chromosomal regions are skipped or rearranged. For example, FLT3 internal tandem duplications (ITDs) and entire losses of genes can be detected. CNV detection can be selected in addition to other assay types if the user has a kit that is designed for CNV detection. This assay uses one or more control samples, if present, to compare expression levels in normal versus diseased samples. If no controls are present, all samples are normalized and used as a baseline. Selection of the proper normal sample(s) is described in 2.1.3. The analysis software uses this information to produce both a readout and a visualization of expression levels, along with a p value to convey confidence in the calls. 8 | P a g e User Manual, Archer Analysis 4.1 CS001-02 Rev 2 Figure 8. Archer Analysis CNV report 2.1.2.5 Platform The Archer Analysis Software supports the Illumina MiSeq®/NextSeq®/HiSeq® platform as well as the ThermoFisher Scientific Ion PGM™/Proton™ platforms. Select the appropriate platform as well as the library type (single read vs paired-end reads for the Illumina MiSeq/NextSeq/HiSeq platform or “demultiplexed” for the Ion Torrent platform) from the dropdown menu. Figure 9. Platform selection options 2.1.2.6 Target region Select the appropriate target region corresponding to the Archer kit/panel. 9 | P a g e User Manual, Archer Analysis 4.1 CS001-02 Rev 2 Figure 10. Target region selection options To add a different target region for a custom Archer kit, section 3.2.17, which also includes further information about adding custom content. 2.1.2.7 Targeted Mutation (Optional) When selecting the RNA or DNA SNPs/InDels assay type, it is possible to instruct the analysis software to only report on the mutations in a Targeted Mutation file (in VCF format). When a targeted mutation file is selected, and "Include Non-Targeted Variants" is set to "Off", the Variant Summary page will show the results for the targeted mutations only and not report on any other variants/mutations that may be present in the sample. Figure 11. Targeted Mutation file options When a targeted mutation file is selected, and "Include Non-Targeted Variants" is set to "On", the Variant Summary page will show the results for the targeted mutations in addition to any other variants/mutations that may be present in the sample. NOTE: Only variants within 1kb downstream of any GSP2 will be detected. Select the desired targeted mutation file from the dropdown menu or leave at “(optional)” if no targeted mutation file is desired. See section 3.2.18 for instructions on how to add/manage the set of targeted mutations files available in the VM. To start the analysis, select the “Submit Analysis” button. 2.1.3 Selecting the CONTROL samples for CNV runs If the DNA Copy Number Variation assay type was selected, a dialog box will appear that will allow the selection of the CONTROL or NORMAL sample. CNV analyses will be far more sensitive if a matched CONTROL sample is available for each CASE (tumor) sample although it is not required to be a matched control sample. It is even possible to have no control sample whatsoever. 10 | P a g e User Manual, Archer Analysis 4.1 CS001-02 Rev 2