Illumina Experiment Manager User Guide

Transcription

1 Illumina Experiment Manager User Guide For Research Use Only. Not for use in diagnostic procedures. What is Illumina Experiment Manager? 3 Getting Started 4 Creating a Sample Plate 7 Creating a Sample Sheet 10 Revision History 28 Technical Assistance ILLUMINA PROPRIETARY Document # v03 January 2016

2 This document and its contents are proprietary to Illumina, Inc. and its affiliates ("Illumina"), and are intended solely for the contractual use of its customer in connection with the use of the product(s) described herein and for no other purpose. This document and its contents shall not be used or distributed for any other purpose and/or otherwise communicated, disclosed, or reproduced in any way whatsoever without the prior written consent of Illumina. Illumina does not convey any license under its patent, trademark, copyright, or common-law rights nor similar rights of any third parties by this document. The instructions in this document must be strictly and explicitly followed by qualified and properly trained personnel in order to ensure the proper and safe use of the product(s) described herein. All of the contents of this document must be fully read and understood prior to using such product(s). FAILURE TO COMPLETELY READ AND EXPLICITLY FOLLOW ALL OF THE INSTRUCTIONS CONTAINED HEREIN MAY RESULT IN DAMAGE TO THE PRODUCT(S), INJURY TO PERSONS, INCLUDING TO USERS OR OTHERS, AND DAMAGE TO OTHER PROPERTY. ILLUMINA DOES NOT ASSUME ANY LIABILITY ARISING OUT OF THE IMPROPER USE OF THE PRODUCT(S) DESCRIBED HEREIN (INCLUDING PARTS THEREOF OR SOFTWARE) Illumina, Inc. All rights reserved. Illumina, 24sure, BaseSpace, BeadArray, BlueFish, BlueFuse, BlueGnome, cbot, CSPro, CytoChip, DesignStudio, Epicentre, ForenSeq, Genetic Energy, GenomeStudio, GoldenGate, HiScan, HiSeq, HiSeq X, Infinium, iscan, iselect, MiniSeq, MiSeq, MiSeqDx, MiSeq FGx, NeoPrep, NextBio, Nextera, NextSeq, Powered by Illumina, SureMDA, TruGenome, TruSeq, TruSight, Understand Your Genome, UYG, VeraCode, verifi, VeriSeq, the pumpkin orange color, and the streaming bases design are trademarks of Illumina, Inc. and/or its affiliate(s) in the U.S. and/or other countries. All other names, logos, and other trademarks are the property of their respective owners.

3 What is Illumina Experiment Manager? The Illumina Experiment Manager (IEM) software enables you to create and edit wellformed sample sheets for Illumina instruments and analysis software. Create your sample sheet before starting sample or library preparation. IEM detects and warns you when an index combination specified is suboptimal. By creating the sample sheet before sample or library preparation, you can try a different index combination without risking your samples. There are 2 steps to creating a sample sheet with the IEM for Illumina sequencing systems: } First, you create a sample plate. The sample plate stores information about the samples in each well of a 96-well (or fewer) plate, including: the type of library prep that is performed, the plate name, and sample indexes. } Second, you create a sample sheet based on the information previously defined on the sample plate. The sample sheet passes run and sample information in the correct file format (*.csv) to the instrument control and analysis software. Creating a sample sheet for the NeoPrep Library Prep System makes sure that the sample sheet passes run and sample information in the correct file format (*.csv) to the NeoPrep Control Software. This guide describes detailed general information on how to use the IEM application. For instructions specific to the sample or library preparation kit you are using, see the IEM quick reference cards. NOTE You can download the IEM quick reference cards from the Illumina website at Go to the Illumina Experiment Manager support page or the support page for your kit and select the Documentation & Literature tab. What is Illumina Experiment Manager? Illumina Experiment Manager User Guide 3

4 Getting Started Downloading the Software You can download the IEM software from the Illumina website at support.illumina.com/sequencing/sequencing_software/experiment_manager.ilmn. From the Experiment Manager Support page, select Downloads. A MyIllumina account is required. Installing the Software The IEM software can be run on any Windows platform. To start the installation, doubleselect the Setup.exe file. The installation wizard opens. Click Next to accept all default settings. Starting the Software To start the software, go to Start All Programs Illumina Illumina Experiment Manager, or select the IEM icon on your desktop. Setting your Preferences You can specify where your sample plates and sample sheets are saved by selecting Folder Settings on the main screen of the IEM software. Click Browse to navigate to the desired folders for each type of file, and then select OK. TIP To make sure that the files are accessible from the sequencing instrument in your lab, save the files in a shared network folder. Alternatively sample sheets and manifests can be copied to a USB drive on an individual basis. Downloading Manifests Illumina provides manifests for library prep kits that use the TruSeq Amplicon, Enrichment, or Targeted RNA applications. Download the manifests from the Illumina website at } For library prep kits that use the TruSeq Amplicon or Enrichment applications, go to the support page for the library prep kit that you are using, and then select Downloads. Save manifests in the IEM Manifest Repository specified in the Folder Settings. } For library prep kits that use the Targeted RNA application, log in to your MyIllumina account. Follow the links in the Custom Products tab to access the Product Files for TruSeq Targeted RNA Expression. To download a manifest, select Download next to the file name on the Product Files page. If you ordered an add-on project, the 2 manifest files created for the project must be merged using the Merge Files function on the Product Files page. Save manifests in the IEM Manifest Repository specified in the Folder Settings. } For Nextera Rapid Capture Custom Enrichment, download target and probe manifest files from the Export Manifest function in the Project Dashboard of your DesignStudio project. Target and probe manifest files can also be downloaded from the Product Files section of Custom Products in your MyIllumina account. } For TruSight Tumor 15, manifest files for library MixA and MixB are preloaded with IEM v1.11 or later. 4 Document # v03

5 Genome Repository Preferences The IEM requires a reference genome for the creation of a sample sheet, particularly for runs that are analyzed using the PCR Amplicon workflow. When the IEM is installed, a series of folders mimicking the genomes folder structure on the MiSeq are created on your computer. The folder structure that is created is a copy of the latest genome build. Download the latest genomes from MyIllumina. To save space on the computer that the IEM is installed on, these folders are blank except for a genomesize.xml file. The genomesize.xml file provides the IEM with the genome information necessary for the creation of the sample sheet. If you are using a custom reference genome not included in the default IEM installed folder structure, follow these steps: 1 Copy your custom genome to the local directory that the IEM is using. IEM recognizes the directory structure of the custom genome folder when it is the following: The path{repository}/genome_name exists. At least 1 FASTA file is required in the folder. Getting Started a b If your custom genome has a genomesize.xml file, you can delete the *.fa files in the folder. If your custom genome does NOT have a genomesize.xml file, the IEM creates 1 the first time a PCR Amplicon manifest is created. After the genomesize.xml file is created, you can delete the *.fa files in the folder. NOTE If the genome is ever edited, repeat step b. Loading a Custom Genome To load a custom genome into the Genome Repository folder, follow these steps: } Make that the genome is in a FASTA format. } Place the FASTA file in a folder labeled with the 'genome' name inside the genome folder in the genome repository. } Close and reopen the IEM. } Create a manifest file and select the new genome. } When asked if you want to convert the file to an xml file, select Yes. Creating a Custom Library Prep Kit Type WARNING Any non-illumina library prep kits are required to meet the specified parameters (eg Index Read number and Index Read length) for the chosen application. If these parameters do not match, there is a risk of an issue with the recipe when setting up the run. For example, when using the Default recipe file, there is only a single Index Read supported. Illumina is unable to provide guidance or support for library prep kits that Illumina does not make, or for custom recipes on Illumina sequencing systems. 1 Go to your Program Files folder and find the Illumina\Illumina Experiment Manager\SamplePrepKits folder. 2 Copy an existing library prep kit *.txt file and rename the file a unique name for the custom library prep kit. 3 Open the new *.txt file in a text editor. 4 Under the [Name] section, rename the entry the same unique name you named the file. Illumina Experiment Manager User Guide 5

6 5 Update the sequences accordingly. 6 Save and close *.txt file. If you have the IEM open, close it and reopen the IEM. 7 After you reopen the IEM and select Create Sample Plate, your new custom Library Prep Kit is a new option. Creating a Custom Library Prep Kit NOTE Create a library prep kit type before you create a custom Library Prep Kit. Only new library prep kit types added to the Illumina\Illumina Experiment Manager\SamplePrepKits folder can be added to existing Applications. 1 Go to your Program Files folder and find the Illumina\Illumina Experiment Manager\Applications folder. 2 Copy an existing Library Prep Kit.txt file and rename the file a unique name for the custom library prep kit. 3 Open the existing Application.txt file of where you want your custom library prep kit to display. 4 Under the [Compatible Sample Prep Kits] section, add the file name of a new custom Library Prep Kit Type you have already created. 5 Save and close.txt file. If you have the IEM open close and relaunch the IEM. 6 After you reopen the IEM, your new custom Library Prep Kit is an option in the Library Prep Kit drop-down list of the selected Application. 6 Document # v03

7 Creating a Sample Plate NOTE These instructions describe the creation of a new sample plate. You can also adapt them to edit an existing sample plate by selecting Edit Sample Plate and navigating to the sample plate you want to edit. 1 On the main screen, select Create Sample Plate. 2 On the Library Prep Kit Selection screen, select the appropriate kit, and then select Next. 3 On the Assay Parameters screen, do the following: a b c In the Unique Plate Name field, type a name for the sample plate. In the Index Reads field, select the number of indexes you run for the samples on this plate: 0, 1, or 2 (if applicable). Click Next. Creating a Sample Plate 4 On the Plate Samples screen, select the Table or the Plate tab, depending on your preference. You can enter information for the wells in your plate using either view. } Table tab displays the wells on a 96-well plate in a list, identifiable by the column and row (A01, A02... B01, B02... and so on) All information for a well is visible at a time, horizontally across the row: Sample Name, Sample ID, Index 1 (if applicable), Index 2 (if applicable), Sample Project, and Description. } Plate tab mimics the layout of a 96-well plate, with wells laid out in columns A-H and rows Only 1 type of information for a well is visible at a time, as determined by your selection in the Currently Displaying drop-down menu. 5 For each well containing sample, enter a unique sample ID. This sample ID is used to track the sample from preparation through sequencing and analysis. The ID is often a barcode, but any value is acceptable. Illumina Experiment Manager User Guide 7

8 TIP Use the IEM Fill feature to populate any type of data for the wells in your sample sheet quickly. The Fill feature can copy data as-is from cell to cell or can automatically increase numerical data by 1 to create sequential data. The IEM determines which method is appropriate based on what type of data are in the cell or cells being copied. To generate sequential data, enter sequential data in 2 adjacent wells, then highlight the other cells you want to populate. Right-select the highlighted area and select Fill Down or Fill Right. The highlighted cells are numbered in sequential order. To copy data, populate 1 cell, then highlight, right-select, and fill the other cells you want to populate with the same information. Content can be pasted into the Sample ID column directly from an excel table. 6 If you selected 1 or 2 index reads on the Assay Parameters screen, specify what index adapter you use for each Index Read. Use combinations that result in at least 1 A or C base (red) and at least 1 G or T base (green) at every cycle. After entering a sample ID and index adapter, the gray shading is removed from the well, indicating that there is enough information for the well to populate a sample sheet. } Illumina provides a recommended default index layout. Click Apply Default Index Layout to autopopulate the indexes for all index reads for 96 wells. After the index adapters are populated, you can edit them if you want to try a different combination of adapters. } If you want to use your modified index layout in the future, select Save As Default Index Layout. The next time you create a sample plate, select Apply Default Index Layout to use your version. } You can always restore the Illumina layout as the default later by selecting Restore Illumina Default Index Layout. 7 If you want to capture more detailed information about the plate, enter a sample name, project, description. 8 [Optional] Click the Plate Graphic tab to view the sample ID and index adapter in each well. 8 Document # v03

9 If you want to copy an image of your sample plate for use in a presentation or paper, go the Plate Graphics tab and select Copy to Clipboard. You can paste the image into any graphics-enabled program, such as Paint, Microsoft PowerPoint, Microsoft Word, and Adobe Photoshop. [Optional] If you want to print an image of your sample plate, go the Plate Graphics tab, and select Print. 9 Click Finish and save the sample plate file in the desired folder. WARNING You can use any file naming convention that makes sense for your organization, but the file must contain the default *.plt file extension. If you use a different file extension, the IEM does not recognize the sample plate when it comes time to generate a sample sheet. For the default file extension specific to the sample or library preparation kit you are using, see the IEM appropriate quick reference card for your kit. Creating a Sample Plate Illumina Experiment Manager User Guide 9

10 Creating a Sample Sheet A sample sheet can be generated for the following types of instruments: } MiSeq } HiSeq } HiScanSQ } Genome Analyzer } NextSeq in standalone mode with the bcl2fastq2 software package } NeoPrep The sample sheet can be used to set up secondary analysis. For a HiSeq run, the loaded samples are specified for each lane. The analysis workflows available in IEM vary by sequencing system. } For analysis of MiSeq sequencing data, see MiSeq-Compatible Sample Sheets on page 10. } For analysis of HiSeq, HiScanSQ, or Genome Analyzer sequencing data, see HiSeq-, HiScanSQ-, or Genome Analyzer-Compatible Sample Sheets on page 19. } For analysis of NextSeq sequencing data, see NextSeq-Compatible Sample Sheets on page 23. The sample sheet can be used to support a NeoPrep library prep run. See NeoPrep- Compatible Sample Sheets on page 26. MiSeq-Compatible Sample Sheets This section provides instructions for creating a sample sheet for the analysis of MiSeq sequencing data. MiSeq Applications and Library Prep Kits To process a sequencing run fully on the MiSeq, MiSeq Reporter requires that an analysis application is specified in the sample sheet. 10 Document # v03

11 The following categories and their applications are supported: Category Application Description Targeted Resequencing TruSight Tumor 15 TruSeq Amplicon PCR Amplicon Designed explicitly for the TruSight Tumor 15 assay, this workflow performs alignment and somatic variant calling for FFPE samples. Two libraries per sample are required to perform analysis. The manifest files for these libraries come preloaded in IEM and MiSeq Reporter. Sequencing of samples prepared using TruSeq Amplicon chemistry kits, including TruSight Myeloid. A manifest file from DesignStudio or the TruSight Myeloid support page is required for alignment. Reads are aligned against one or more manifest files specified in the sample sheet. Sequencing of PCR amplicon samples prepared using the Nextera XT Library Prep kit. The PCR amplicons are generated from primers targeting particular genome positions (up to approximately 384 loci from up to 96 samples). Reads are aligned against the specified reference genome. A usergenerated manifest file (specified in the sample sheet) gives the targeted regions; variant calling is performed only within these regions, and coverage statistics are reported for the target regions. Creating a Sample Sheet Metagenomics 16S rrna Enrichment Clone Checking Amplicon - DS TruSight Tumor 26 Sequencing of genetic material from uncultured samples. No genomic reference is required for a Metagenomics workflow. Reads are classified using a database of 16S rrna data included with the MiSeq Reporter software. Sequencing of DNA obtained from Nextera (hybrid capture-based) enrichment. Reads are aligned against the specified reference genome. An Illumina manifest file (specified in the sample sheet) gives the targeted regions; variant calling is performed only within these regions, and coverage statistics are reported for the target regions. Verification of clone production by sequencing typically done with custom primers. A pseudogene (in FASTA format) representing the clone of interest is required for mapping and alignment. Designed explicitly for the TruSight Tumor 26 assay, the Amplicon - DS workflow is suited for detection of somatic mutations in formalin-fixed paraffinembedded (FFPE) samples. This workflow independently processes variants from the forward and reverse strands of the sample material, and then algorithmically reconciles the calls. Illumina Experiment Manager User Guide 11

12 Category Application Description Small Genome Sequencing RNA Sequencing Resequencing Plasmids Assembly Targeted RNA Small RNA RNA-Seq Sequencing of a small genome, such as E. coli. Reads are aligned against the reference and variants are reported. Sequencing of entire plasmid DNA. A reference plasmid genome is required for mapping and alignment. Output files are *.bam and *.vcf. Assembly of small (< 20 Mb) genomes from reads without the use of a genomic reference. If a genomic reference is included, a dot-plot is generated with regarding the reference. Sequencing of TruSeq Targeted RNA Expression libraries. A target reference is needed as well as a manifest file from DesignStudio. Reads are aligned against one or more manifest files specified in the sample sheet. This workflow is not used for TruSight RNA Pan- Cancer libraries. Sequencing of cdna following reverse transcription of small RNA. Annotation backed by mirbase and Rfam. FASTQ files and stats files are available for subsequent downstream analysis. Sequencing of cdna following reverse transcription and library preparation, including TruSight RNA Pan-Cancer libraries. FASTQ files are generated and can be used with third-party software for subsequent analysis. Other TruSight HLA Sequencing of Long Range PCR HLA gene amplicons prepared using Nextera XT library prep technology. Library QC FASTQ Only ChIP-Seq Fast resequencing of a reference genome to QC the DNA library and generate per-sample statistics. Generation of demultiplexed FASTQ files from any type of library. Sequencing of TruSeq ChIP libraries. FASTQ files are generated and can be used with third-party software for subsequent analysis. The following tables list the applications of each category and the associated analysis workflow written to the sample sheet. For supported library prep kits for each application, see the Illumina Experiment Manager support page. Targeted Resequencing Application in IEM Analysis Workflow in Sample Sheet TruSight Tumor 15 TruSight Tumor Document # v03

13 Application in IEM TruSeq Amplicon PCR Amplicon Metagenomics 16S rrna Enrichment Clone Checking Amplicon - DS TruSight Tumor 26 Small Genome Sequencing Analysis Workflow in Sample Sheet Custom Amplicon PCR Amplicon Metagenomics Enrichment GenerateFASTQ Amplicon - DS Creating a Sample Sheet Application in IEM Resequencing Plasmids Assembly Analysis Workflow in Sample Sheet Resequencing GenerateFASTQ Assembly RNA Sequencing Application in IEM Targeted RNA Small RNA RNA-Seq Analysis Workflow in Sample Sheet Targeted RNA SmallRNA GenerateFASTQ Other Workflows Application in IEM TruSight HLA LibraryQC FASTQ Only ChIP-Seq Analysis Workflow in Sample Sheet GenerateFASTQ LibraryQC GenerateFASTQ GenerateFASTQ Illumina Experiment Manager User Guide 13

14 Creating a MiSeq Sample Sheet NOTE These instructions describe the creation of a new sample sheet. You can also adapt them to edit an existing sample sheet by selecting Edit Sample Sheet and navigating to the sample sheet you want to edit. 1 On the main screen, select Create Sample Sheet. 2 On the Instrument Selection screen, select MiSeq, and then select Next. 3 On the MiSeq Application Selection screen, select the desired category and application and for your kit, and then select Next. Reference MiSeq Applications and Library Prep Kits on page On the Workflow Parameters screen, do the following: a In the Reagent Cartridge Barcode field, enter the barcode number of the MiSeq reagent cartridge. The barcode number is on the cartridge label. b From the Library Prep Kit drop-down menu, select the appropriate option for the kit you are using. c In the Index Reads field, select the number of indexes you run for the samples on this plate: 0, 1, or 2 (if applicable). The same number of indexes you selected when you created the sample plate must be selected here. The number of Index Cycles is selected automatically based on the selection you make in the Index Reads field. d Type an experiment name, investigator name, and description. e Select the date from the calendar. f Select Paired End or Single Read sequencing run, depending on the options available. g Select the number of cycles for each read in your sequencing run, plus 1. h Depending on which application you selected in step 3, check or uncheck the appropriate checkboxes or specify the following settings for your sequencing run: i Click Next. Workflow- Specific Setting BWA-backtrack Custom Primer for Read 1 Custom Primer for Index Custom Primer for Read 2 Description Available for Enrichment, Library QC, PCR Amplicon, and Small Genome Resequencing. Allows selection of the previous version of the BWA aligner (v0.6.1). This setting only applies to MiSeq Reporter v2.6 (and later) workflows, which use the newer BWA aligner (BWA-MEM, v0.7.9a). Available for all applications except TruSeq Amplicon. Select this checkbox if you used a custom Read 1 primer and not an Illumina Read 1 primer. Available for all applications except TruSeq Amplicon. Select this checkbox if you used a custom Index primer and not an Illumina Index primer. Available for all applications except TruSeq Amplicon. Select this checkbox if you used a custom Read 2 primer and not an Illumina Read 2 primer. 14 Document # v03

15 Workflow- Specific Setting Use Somatic Variant Caller Indel Realignment GATK Flag PCR Duplicates Description Available in the TruSeq Amplicon, PCR Amplicon, Enrichment, and Resequencing applications. Select this checkbox if you are using the somatic variant caller. Somatic variant caller is an Illumina variant calling algorithm specifically for TruSeq Amplicon - Cancer Panel or TruSight Myeloid to detect low frequency mutations (even below 5%) in a mixed cell population. For more information, see the Somatic Variant Caller Tech Note, which you can download from the Illumina website at Available in the Resequencing and Enrichment applications. Select this checkbox to perform a local realignment of reads around indels such that number of mismatches is minimized. Available in the Resequencing, PCR Amplicon, Enrichment, and Library QC applications. Select this checkbox to flag apparent PCR duplicates in the BAM files and not used for variant calling. PCR duplicates are defined as 2 clusters from a paired-end run where both clusters have the exact same alignment positions for each read. Creating a Sample Sheet Variant Quality Filter Use Adapter Trimming Use Adapter Trimming Read 2 Run Picard HsMetrics Available in the TruSeq Amplicon, PCR Amplicon, Resequencing, and Enrichment applications. Cutoff parameter. The default setting is 30. For more information, see the MiSeq Sample Sheet Quick Reference Guide, which you can download from the Illumina website at Available for all applications except TruSeq Amplicon. This setting is recommended when sequencing libraries prepared with Nextera, Nextera XT, Nextera Rapid Capture Enrichment, or TruSight Enrichment kits. It is possible that some clusters can sequence beyond the sample DNA and read bases from the adapter. Select this checkbox to cause MiSeq Reporter to mask the adapter sequence, improving accuracy and speed during secondary analysis. The default adapter sequence is the adapter present in all Nextera libraries. If a different adapter sequence is used, edit the sequence that is displayed in the final sample sheet. For TruSight HLA, do not change the default. Available in the Plasmids, Assembly, RNA-Seq, Library QC, FASTQ Only, and ChIP-Seq applications. Select this checkbox to set the AdapterRead2 setting in the sample sheet to trim a different adapter sequence in read2. Available in the Enrichment application. Select this checkbox to perform Picard hybrid selection (HS) analysis of the *.bam file. Illumina Experiment Manager User Guide 15

16 Workflow- Specific Setting Reverse Complement K-mer size Genome Export to gvcf Genus-Level Classification Description Available in the Resequencing, Library QC, FASTQ Only, and Assembly applications. Select this checkbox to convert Nextera Mate Pair libraries from a mate pair to a paired-end orientation as required by BWA and Velvet. Available in the Assembly application. Set the k-mer sized used for assembly. The range is Larger k-mer sizes require more memory and can dramatically affect the stability and performance of the analysis. K-mer optimization is suggested for optimal assemblies. Available in the Small RNA application. Select from the Genome drop-down menu Provide the relative or absolute path to the following reference sequence folders. The typical settings for human runs are in parentheses: Contaminants (HumanRNAContaminants) RNA (HumanRNA) mirna (HumanRNAMature) Available in the PCR Amplicon, TruSeq Amplicon, and Enrichment applications. Select the checkbox to enable gvcf files to output to the run folder. Available in the Metagenomics application. Select the checkbox to enable genus-level classification, which overrides the species-level taxonomic classification default. } Click Next. 5 On the Sample Selection screen, select Select Plate and navigate to a sample plate you created previously. TIP Uncheck Maximize to view the sample plate and sample sheet portion of the screen. NOTE If you have not yet created a sample plate, you can do so now by selecting New Plate. For more information, see Creating a Sample Plate on page 7. 6 Click Select All to include all wells in this sequencing run or highlight the wells you want to include in this sequencing run. 7 Click Add Selected Samples. 8 [Optional] Click Add Blank Row to add rows and manually enter the sample information. 16 Document # v03

17 TIP Use the IEM Fill feature to populate any type of data for the wells in your sample sheet quickly. The Fill feature can copy data as-is from cell to cell or can automatically increase numerical data by 1 to create sequential data. The IEM determines which method is appropriate based on what type of data are in the cell or cells being copied. To generate sequential data, enter sequential data in 2 adjacent wells, then highlight the other cells you want to populate. Right-select the highlighted area and select Fill Down or Fill Right. The highlighted cells are numbered in sequential order. To copy data, populate one cell, then highlight, right-select, and fill the other cells you want to populate with the same information. Content can be pasted into the Sample ID column directly from an excel table. 9 [Optional]Highlight one or more fields, and then select Remove Selected Rows to remove the entire rows that contain those fields. 10 Type a sample name, sample project, and description for each sample. TIP Check Maximize to view only the sample sheet portion of the screen. 11 For the following applications that use a genomic reference, select where the FASTA reference files are saved from the Genome Folder drop-down menu. Creating a Sample Sheet Category Targeted Resequencing Application TruSeq Amplicon PCR Amplicon Enrichment Amplicon - DS TruSight Tumor 26 Small Genome Sequencing Resequencing Assembly Other LibraryQC 12 Do one of the following: } If you are using the PCR Amplicon application, proceed to Creating a Manifest File on page 18. } For all other applications, proceed to Finish Creating a MiSeq Sample Sheet on page 19. Illumina Experiment Manager User Guide 17

18 Creating a Manifest File The manifest file is required for analysis using the PCR Amplicon workflow. The manifest contains information about each sample that focuses analysis results only to user-defined regions of interest. There can be multiple manifests per sample sheet. Manifest creation requires input of: } Reference genome used for alignment. } Chromosome coordinates (start, stop) of each amplicon/roi. } Length of each PCR primer used for generation of the PCR amplicon. 1 Select 1 of the following from the Nextera Manifest drop-down menu: } Select New to create a manifest. } Select Edit to choose an existing manifest. 2 If you are creating a new manifest file, the Create New Amplicon Manifest screen opens. 3 Select a single reference genome for each new manifest from the Genomes dropdown menu. The genome must be located in your IEM genome repository directory. Each sample sheet you are creating must have the same reference genome for all manifests associated with it. TIP Make sure that the reference genome matches the reference genome used to design the PCR amplicon primers. NOTE Make the IEM genome repository directory the same as your MiSeq Reporter Genomes location, where the actual reference genome is located. 4 Add a blank row for each ROI and name the Amplicon. 18 Document # v03

19 5 Choose the appropriate chromosome and input the amplicon coordinates (start and end-including primers). Include the primer lengths in the amplicon start and end. 6 Specify the length of each primer (upstream and downstream). The specific length allows variants called in these regions to be flagged during analysis. TIP All content needed for a new manifest file can be copied from an Excel table into IEM. Make sure that the columns contain the appropriate contents after pasting in from the Excel table. The upstream and downstream probe lengths use the PCR primers to generate the amplicon. 7 Name your manifest file to save it into the Manifest Repository. NOTE Copy the new manifest file into the MCS Manifest Repository location on the MiSeq instrument before starting your run. Creating a Sample Sheet Finish Creating a MiSeq Sample Sheet 1 If you are using the Enrichment application, enter the name of the Illumina Manifest file for your assay or control. Leave off the.txt file extension part of the file name. TIP If you do not see a manifest file for your library prep kit in the drop-down menu, go to the Manifest Repository folder for IEM. Copy an Illumina manifest file there. See Downloading Manifests on page 4. 2 Click Finish and save the sample sheet file in the desired folder. 3 When prompted, select Yes if you want to review the sample sheet in Microsoft Excel or No to exit the sample sheet wizard without reviewing the sample sheet. HiSeq-, HiScanSQ-, or Genome Analyzer-Compatible Sample Sheets This section provides instructions for creating a sample sheet for the analysis of HiSeq, HiScanSQ, or Genome Analyzer sequencing data. HiSeq, HiScanSQ, and Genome Analyzer Applications and Library Prep Kits To process a sequencing run fully on HiSeq, HiScanSQ, or Genome Analyzer, an analysis application is specified in the sample sheet. Illumina Experiment Manager User Guide 19

20 The following categories and their applications are supported: Application Human Genome Resequencing HiSeq FASTQ Only HiSeq Enrichment HiSeq CASAVA Description Sequencing of a human genome. Reads are aligned against the hg19 reference and variants are reported. Generation of demultiplexed FASTQ files from any type of library. Sequencing of DNA obtained from Nextera (hybrid capture-based) enrichment. Reads are aligned against the specified reference genome. An Illumina manifest file (specified in the sample sheet) gives the targeted regions; variant calling is performed only within these regions, and coverage statistics are reported for the target regions. Sequencing of human and other genomes available from Illumina igenomes. Outputs include aligned reads and variant calls. For more information on igenomes, see support.illumina.com/sequencing/sequencing_ software/igenome.ilmn. The following table lists the applications and the associated analysis workflow written to the sample sheet. For supported library prep kits for each application, see the Illumina Experiment Manager support page. Application in IEM Human Genome Resequencing HiSeq FASTQ Only HiSeq Enrichment HiSeq CASAVA Analysis Workflow in Sample Sheet Resequencing GenerateFASTQ Enrichment Not applicable Creating a HiSeq-, HiScanSQ-, or Genome Analyzer Sample Sheet These instructions describe the creation of a new sample sheet. 1 On the main screen, select Create Sample Sheet. 2 On the Instrument Selection screen, select HiSeq, HiScanSQ, GA, and then select Next. 3 On second the Instrument Selection screen, select HiSeq 3000, 4000, HiSeq 1500, 2500, HiSeq 1000, 2000, HiScanSQ, or Genome Analyzer, and then select Next. 4 On the HiSeq Application Selection screen, select the desired application and for your kit, and then select Next. Reference HiSeq, HiScanSQ, and Genome Analyzer Applications and Library Prep Kits on page On the Workflow Parameters screen, enter the reagent kit barcode from the label of either box 1 or box 2 of the SBS kit. 6 From the Library Prep Kit drop-down menu, select the appropriate option for the kit you are using. 20 Document # v03

21 NOTE For the appropriate option for your kit, reference the appropriate IEM quick reference card for your kit. 7 In the Index Reads field, select the number of indexes for your library type: 0, 1, or 2. The same number of indexes you selected when you created the sample plate must be selected here. The number of Index Cycles is selected automatically based on the selection you make in the Index Reads field. 8 If creating a sample sheet for Human Genome Resequencing, HiSeq FASTQ Only, or HiSeq Enrichment, do the following: a b c d Type an experiment name, investigator name, and description. Select the date from the calendar. Select a Paired End or Single Read sequencing run, depending on the options available. Select the number of cycles for each read in your sequencing run, plus 1. If you are performing a paired-end sequencing run, perform the same number of cycles in both reads. Creating a Sample Sheet 9 If creating a sample sheet for HiSeq CASAVA, do the following: a b c In the Flow Cell ID field, enter the barcode number of the flow cell. In the Operator field, enter the name of the person running the sequencing instrument. In the Recipe field, enter the name of the recipe to be used for the run of the sequencing instrument. 10 Depending on which application you selected in step 4, check or uncheck the appropriate checkboxes or specify the following settings for your sequencing run: Workflow- Specific Setting Custom Primer for Read 1 Custom Primer for Index Custom Primer for Read 2 Indel Realignment GATK Flag PCR Duplicates Description Available for the HiSeq FASTQ Only application. Select this checkbox if you used a custom Read 1 primer and not an Illumina Read 1 primer. Available for the HiSeq FASTQ Only application. Select this checkbox if you used a custom Index primer and not an Illumina Index primer. Available for the HiSeq FASTQ Only application. Select this checkbox if you used a custom Read 2 primer and not an Illumina Read 2 primer. Available for the HiSeq Enrichment application. Select this checkbox to perform a local realignment of reads around indels such that number of mismatches is minimized. Available for the Human Genome Resequencing and HiSeq Enrichment applications. Select this checkbox to flag apparent PCR duplicates in the BAM files and not used for variant calling. PCR duplicates are defined as 2 clusters from a paired-end run where both clusters have the exact same alignment positions for each read. Illumina Experiment Manager User Guide 21

22 Workflow- Specific Setting Variant Quality Filter Use Adapter Trimming Use Adapter Trimming Read 2 Run Picard HsMetrics Reverse Complement Flow Cell ID Operator Recipe Description Available for the HiSeq Enrichment application. Cutoff parameter. The default setting is 30. Available for the Human Genome Resequencing, HiSeq FASTQ Only, and HiSeq Enrichment applications. This setting is recommended when sequencing libraries prepared with Nextera, or Nextera XT kits. It is possible that some clusters can sequence beyond the sample DNA and read bases from the adapter. Selecting this checkbox causes the analysis software to mask the adapter sequence, improving accuracy and speed during secondary analysis. The default adapter sequence is the adapter present in all Nextera libraries. If a different adapter sequence is used, edit the sequence that is displayed in the final sample sheet. Available for the Human Genome Resequencing and HiSeq FASTQ Only applications. Select this checkbox to set the AdapterRead2 setting in the sample sheet to trim a different adapter sequence in read2. Available in the HiSeq Enrichment application. Select this checkbox to perform Picard hybrid selection (Hs) analysis of the *.bam file. Available for the HiSeq FASTQ Only application. Select this checkbox to convert Nextera Mate Pair libraries from a mate pair to a paired-end orientation as required by BWA and Velvet. Available for the HiSeq CASAVA Only application. Enter the barcode number of the flow cell. Available for the HiSeq CASAVA Only application. Enter the name of the person running the sequencing instrument. Available for the HiSeq CASAVA Only application. Enter the name of the recipe to be used for the run on the sequencing instrument. 11 Click Next. 12 On the Sample Selection screen, select Select Plate and navigate to the sample plate you created previously. TIP Uncheck Maximize to view the sample plate and sample sheet portion of the screen. NOTE If you have not yet created a sample plate, you can do so now by selecting New Plate. For more information, see Creating a Sample Plate on page For each lane you are using in the flow cell, do the following: a b Click the lane number: 1 through 8 on the sample sheet area of the screen. Click Select All to include all wells in this sequencing run or highlight the wells you want to include in this sequencing run. 22 Document # v03

23 c d e Click Add Selected Samples. [Optional]Click Add Blank Row to add rows and manually enter the sample information. [Optional]Highlight one or more fields, and then select Remove Selected Rows to remove the entire rows that contain those fields. TIP Use the IEM Fill feature to populate any type of data for the wells in your sample sheet quickly. The Fill feature can copy data as-is from cell to cell or can automatically increase numerical data by 1 to create sequential data. The IEM determines which method is appropriate based on what type of data are in the cell or cells being copied. To generate sequential data, enter sequential data in 2 adjacent wells, then highlight the other cells you want to populate. Right-select the highlighted area and select Fill Down or Fill Right. The highlighted cells are numbered in sequential order. Creating a Sample Sheet To copy data, populate one cell, then highlight, right-select, and fill the other cells you want to populate with the same information. Content can be pasted into the Sample ID column directly from an excel table. 14 Type a sample name, sample reference, sample project, and description for each sample in each lane. TIP Check Maximize to view only the sample sheet portion of the screen. 15 When the wells and samples for every lane in the flow cell have been defined, select Finish and save the sample sheet file in the desired folder. 16 When prompted, select Yes if you want to review the sample sheet in Microsoft Excel or No to exit the sample sheet wizard without reviewing the sample sheet. NextSeq-Compatible Sample Sheets This section provides instructions for creating a sample sheet for the analysis of NextSeq sequencing data. NOTE The NextSeq sample sheets generated by IEM are for use when operating the NextSeq in standalone mode, with the bcl2fastq2 software package. NextSeq Applications and Library Prep Kits To process a sequencing run on NextSeq, an analysis application is specified in the sample sheet. The following categories and their applications are supported: Application NextSeq FASTQ Only Description Generation of demultiplexed FASTQ files from any type of library. Illumina Experiment Manager User Guide 23

24 The following table lists the applications and the associated analysis workflow written to the sample sheet. For supported library prep kits for each application, see the Illumina Experiment Manager support page. Application in IEM NextSeq FASTQ Only Analysis Workflow in Sample Sheet GenerateFASTQ Creating a NextSeq Sample Sheet NOTE These instructions describe the creation of a new sample sheet. You can also adapt them to edit an existing sample sheet by selecting Edit Sample Sheet and navigating to the sample sheet you want to edit. 1 On the main screen, select Create Sample Sheet. 2 On the Instrument Selection screen, select NextSeq, and then select Next. 3 On the NextSeq Application Selection screen, select the desired application and for your kit, and then select Next. Reference NextSeq Applications and Library Prep Kits on page On the Workflow Parameters screen, enter the reagent kit ID from the label of either box 1 or box 2 of the SBS kit. 5 In the Reagent Kit Barcode field, enter the reagent kit ID from the label of either box 1 or box 2 of the SBS kit. 6 From the Library Prep Kit drop-down menu, select the appropriate option for the kit you are using. NOTE For the appropriate option for your kit, reference the appropriate IEM quick reference card for your kit. 7 In the Index Reads field, select the number of indexes you run for the samples on this plate: 0, 1, or 2 (if applicable). The same number of indexes you selected when you created the sample plate must be selected here. The number of Index Cycles is selected automatically based on the selection you make in the Index Reads field. 8 Type an experiment name, investigator name, and description. 9 Select the date from the calendar. 10 Select a Paired End or Single Read sequencing run, depending on the options available. 11 Select the number of cycles for each read in your sequencing run, plus 1. If you are performing a paired-end sequencing run, perform the same number of cycles in both reads. 12 Check or uncheck the appropriate checkboxes or specify the following settings for your sequencing run: 24 Document # v03

25 Workflow- Specific Setting Custom Primer for Read 1 Custom Primer for Index Custom Primer for Read 2 Use Adapter Trimming Description Available for the NextSeq FASTQ Only application. Select this checkbox if you used a custom Read 1 primer and not an Illumina Read 1 primer. Available for the NextSeq FASTQ Only application. Select this checkbox if you used a custom Index primer and not an Illumina Index primer. Available for the NextSeq FASTQ Only application. Select this checkbox if you used a custom Read 2 primer and not an Illumina Read 2 primer. Available for the NextSeq FASTQ Only application. This setting is recommended when sequencing libraries prepared with Nextera, or Nextera XT kits. It is possible that some clusters can sequence beyond the sample DNA and read bases from the adapter. Selecting this checkbox causes the analysis software to mask the adapter sequence, improving accuracy and speed during secondary analysis. The default adapter sequence is the adapter present in all Nextera libraries. If a different adapter sequence is used, edit the sequence that is displayed in the final sample sheet. Creating a Sample Sheet Use Adapter Trimming Read 2 Available for the NextSeq FASTQ Only applications. Select this checkbox to set the AdapterRead2 setting in the sample sheet to trim a different adapter sequence in read2. 13 Click Next. 14 On the Sample Selection screen, select Select Plate and navigate to the sample plate you created previously. TIP Uncheck Maximize to view the sample plate and sample sheet portion of the screen. NOTE If you have not yet created a sample plate, you can do so now by selecting New Plate. For more information, see Creating a Sample Plate on page Click Select All to include all wells in this sequencing run or highlight the wells you want to include in this sequencing run. 16 Click Add Selected Samples. 17 [Optional]Click Add Blank Row to add rows and manually enter the sample information. TIP Use the IEM Fill feature to populate any type of data for the wells in your sample sheet quickly. The Fill feature can copy data as-is from cell to cell or can automatically increase numerical data by 1 to create sequential data. The IEM determines which method is appropriate based on what type of data are in the cell or cells being copied. Illumina Experiment Manager User Guide 25

26 To generate sequential data, enter sequential data in 2 adjacent wells, then highlight the other cells you want to populate. Right-select the highlighted area and select Fill Down or Fill Right. The highlighted cells are numbered in sequential order. To copy data, populate one cell, then highlight, right-select, and fill the other cells you want to populate with the same information. Content can be pasted into the Sample ID column directly from an excel table. 18 [Optional] Highlight one or more fields, and then select Remove Selected Rows to remove the entire rows that contain those fields. 19 Type a sample name, sample reference, sample project, and description for each sample. TIP Check Maximize to view only the sample sheet portion of the screen. 20 When the wells and samples have been defined, select Finish and save the sample sheet file in the desired folder. 21 When prompted, select Yes if you want to review the sample sheet in Microsoft Excel or No to exit the sample sheet wizard without reviewing the sample sheet. NeoPrep-Compatible Sample Sheets This section provides instructions for creating a sample sheet for a NeoPrep Library Prep System run. Creating a NeoPrep Sample Sheet NOTE These instructions describe the creation of a new sample sheet. You can also adapt them to edit an existing sample sheet by selecting Edit Sample Sheet and navigating to the sample sheet you want to edit. On the IEM main screen, select Create Sample Sheet. 1 On the Instrument Selection screen, select NeoPrep and then select Next. 2 On the Workflow Parameters screen, do the following: 3 From the Library Prep Kit drop-down menu, select the appropriate option for the kit that you are using. NOTE For the appropriate option for your kit, reference the IEM NeoPrep quick reference card. 4 Type a run name, operator name, and notes. 5 Select the date. 6 Select the number of samples. The default is 16. This number is used to define the default wells and indexes in the Sample Selection screen. 7 For TruSeq DNA, select a 350, 550, or Mixed insert size. The default is Select Next. 26 Document # v03

27 9 On the Sample Selection screen: 10 Type a sample name for each sample. The default well, index adapter, and index sequence are displayed for each sample. The default index adapters correspond to the default adapter layout in the kit reagent plate. For information on the default and optional index adapters, see the library prep guide for the kit that you are using. 11 To edit the defaults, do the following: a b Select the Well cell and select a well number from the drop-down menu. Select the Index1 (i7) cell and select an index from the drop-down menu. The index sequence is automatically displayed in the i7 Sequence column. 12 For TruSeq DNA, the selected insert size is displayed. a b To change the insert size, select the Insert Size cell and select a size from the drop-down menu. If Mixed Insert Size was selected in step 2, select the Insert Size cell and select a size from the drop-down menu for each sample. Creating a Sample Sheet 13 [Optional] Select Add Blank Row to add rows and manually enter the sample information for each sample. a b c Type a sample name. Select the Well cell and select a well number from the drop-down menu. Select the Index1 (i7) cell and select an index from the drop-down menu. The index sequence is automatically displayed in the i7 Sequence column. 14 Select Finish and save the sample sheet file. 15 When prompted, select Yes if you want to review the sample sheet in Microsoft Excel or No to exit the sample sheet wizard without reviewing the sample sheet. Illumina Experiment Manager User Guide 27