Illumina dual index sample sheet The software that calls bases from clusters flips out when the entire flowcell lights up for a single base. Full primer sequences can be downloaded from the product page at activemotif. Depending on the run type, a sample sheet might be required or optional. The format of this sample sheet is designed for the instrument when it is running in Diagnostic mode. IDT provides several indexing options with its Custom NGS Adapters. Jan 23, 2015 · Information on dual index sequencing workflows and requirements for the TruSeq Nano DNA HT Sample Prep kit. Pool any plexity ≥ 2 from the top or Do you have Sample Sheets for use with the NEBNext ® Multiplex Oligos for Illumina ® (96 Unique Dual Index Primer Pairs Set 4)? What should I do if the sample sheet from the NEB. LRM v3 and v4 sample sheets can be exported from the LRM interface, or templates downloaded with the available module. Sample Sheet A sample sheet (SampleSheet. Unique dual indexes allow you to increase the number of samples sequenced per run and reduce per-sample cost compared to other indexing strategies. Learn more about how unique dual indexes can help ensure high accuracy on Illumina sequencing systems. Local Run Manager The sample sheet is a comma-delimited file (SampleSheet. Oligonucleotide sequences ©2019 Illumina Inc. com. The Apr 25, 2016 · Common issues are that one or more samples were omitted from the sample sheet, errors entering the barcodes, incorrect library chemistry (e. We are writing to inform you that Illumina will soon be obsoleting IDT for Illumina DNA/RNA UD Indexes Sets A-D, Tagmentation (96 Indexes, 96 Samples) and IDT for Illumina RNA UD Indexes Sets A-D, Ligation (96 Indexes, 96 Samples). Prepared libraries are amenable to single-read, paired-end, and multiplexed sequencing on all Illumina sequencing instruments. May 9, 2023 · FAQ: Where can I find the sample sheets for the NEBNext Unique Dual Index UMI Adaptors DNA Sets 1-4? Instrument-specific sample sheets can be downloaded from the Usage Guidelines section located under the “Protocols, Manual & Usage” tab on the relevant Set’s product page, or find it in the below table: The Unique Dual Indexes kits (UDI) provide combinations of barcodes where each barcode is uniquely attributed to one sample. Settings Section The software uses the settings section of the sample sheet to specify adapter trimming, cycle, UMI, and index options. The sample sheet consists of a list of samples and their index sequences. csv) file format used by Illumina instruments, platforms, and analysis pipelines to store settings and data for sequencing and analysis. The index adapter sequences are 10 bases long. Apr 3, 2019 · Just omit index 2 from your sample sheet, and redo the demultiplexing. If needed, use one of the following options to generate a sample sheet: After sequencing, the unique combination of the two indexes is used to demultiplex the data and assign reads to the proper sample. NEBNext Unique Dual Index UMI Adaptors DNA Set 1, IDT for Illumina (TruSeq DNA UD Indexes) and Kapa Dual-Indexed Adaptors were used for the NEBNext, Illumina, and Kapa kits, respectively, following manufacturers’ recommendations. After sequencing, the unique combination of the two indexes is used to demultiplex the data and assign reads to the proper sample. The 10x sample index name aids demultiplexing but isn't needed for Illumina setup. The file includes a list of samples, their index sequences, and the sequencing workflow. bin) is a binary file in BIN format that contains index statistics for each sample and index combination per lane provided to BCL Convert in the sample sheet. csv) records information about samples, the corresponding indexes, and other information that dictates the behavior of DRAGEN. Along with best practices, using the unique dual indexing strategy will make sure that libraries sequence and demultiplex with the highest accuracy across all Illumina sequencing platforms. The following table shows index adapters (wells) that can be combined in a two-plex through eight-plex pool, while the color-coded figure illustrates each combination. Creating a custom index adapter kit can be done from within Run Planning (when creating a Configuration) or from the Resources page (select Index Adapter Kit tab). For all indexing workflows, the Index 1 Read directly follows Read 1. When used with Illumina DNA PCR-Free* and the NovaSeq 6000 System or the NovaSeq x Series,† certain index pairs consistently give higher or lower sequence yield. An IEM sample sheet has several fields specific to running on Illumina platforms, including a [Data] section where sample and index information is specified. 5 reagent kits, and NovaSeq X System performs the Index 2 Read after Read 2 resynthesis. We recommend that customers transition to the new alternative replacement products listed in Table 1. With just one unique dual index plate, you can pool 96 samples together. A custom Index kit can be configured in For information on using v2 sample sheet (*. csv) into Local Run Manager. Overview The NEBNext Multiplex Oligos for Illumina (96 Unique Dual Index Primer Pairs) contains adaptors and primers that are ideally suited for multiplex sample preparation for next-generation sequencing on the Illumina platform (Illumina, Inc. Using this dual-barcode approach, Nextera XT Index Kits only require 40 unique index oligos to process up to 384 samples Following the addition of two indices to each gDNA fragment, up to 96 uniquely indexed samples can be pooled and sequenced together in a single lane on any Illumina sequencer. NEBNext® Multiplex Oligos for Illumina® (Index Primers Set 1) This product will be discontinued on December 15, 2025. This kit includes 8 i5 index primers and 12 i7 index primers for dual indexing. The Illumina Experiment Manager can also be used to create sample sheets for bcl2fastq. csv file provides the number of reads mapping to every combination of provided index and index2 values, including via mismatch tolerance. csv. How to setup a 10x Genomics sample sheet in Illumina's BaseSpace for demultiplexing AI summary: To create a Sample Sheet CSV in Illumina BaseSpace, log in, set up the run specifying instrument and demultiplexing options, select the latest BCL Convert version and library prep kit, enter Sample ID with 4 index rows per sample (for ATAC/Multiome), leave AdapterRead and BarcodeMismatches fields Method Illumina NGS systems use different i5 index read strategies, but it is simple to co-sequence single-indexed and dual-indexed libraries together. The NEBNext Adaptor has a unique hairpin loop structure that minimizes adaptor-dimer formation, while NEBNext index PCR primers enable index (barcode) incorporation during library Feb 17, 2016 · We are using bcl2fastq-v2 for nextseq runs without problems (one index runs). 4. Each kit component must pass rigorous quality control standards, and for each new lot the entire set of reagents is functionally validated Demultiplexing Visium data with Illumina's BCL Convert or bcl2fastq software requires the correct specification of the sample sheet and command line options. Using the full complement of Nextera XT indices, up to 96 libraries can be pooled together for sequencing. The parameter CreateFastqForIndexReads,0 under [BCLConvert_Settings] tells BCL Convert not to generate FASTQ files for indices. However, most Illumina sample sheet generation software handles this automatically. Sequencing kits include a paired-end flow cell. Each of these components must pass rigorous quality control standards and are lot controlled, both individually and as a set of reagents. By default, the automation is triggered by a button on the Record Details screen. NEBNext Multiplex Oligos provide adaptors and primers to enable high yield multiplex Illumina library production. The TruSeq Nano DNA LT kit provides up to 24 indices for low-throughput studies (with both Sets A and B), while the TruSeq Nano DNA HT kit includes 96 dual-index combinations for high-throughput studies. Every run in BaseSpace Sequence Hub requires an associated sample sheet to define projects and samples, assign indexes, and run workflow apps. 5: Strict by default but configurable to relaxed mode using sample sheet option: Sep 3, 2024 · Run Planning provides a list of Illumina index adapter kits used for sequencing. Generate the sample sheet for the libraries with the longest indexing scheme, then manually edit the CSV to include the other libraries as appropriate. Using this dual barcode approach, Nextera Index Kits only require 20 unique Local Mode —NextSeq 1000/2000 Integration service generates the sample sheet. Plates are desalted, not HPLC purified. New protocols reduce the number of purification, sample transfer, and pipetting steps. Prepare Sample Sheet To automatically generate FASTQ files from the run folder using BaseSpace Sequence Hub, you must create a sample sheet prior to initiating a sequencing run. Mar 25, 2025 · In this example, the NextSeq 2000 is compatible with a v2 sample sheet from Illumina’s run setup software. Question: I need to use BCL Convert to generate FASTQ files for my dual indexed gene expression libraries. Users may copy the sample sheet to a network location or copy the sample sheet from a USB drive using the "Manage Files" feature in MCS. Use Illumina Cloud Run Planning on BaseSpace Sequence Hub to generate a sample sheet specifically for your TSO 500 ctDNA run Single and Dual Indexing The number of index sequences added to samples differs for single-indexed and dual-indexed sequencing. UDIs are particularly important when using instruments with patterned flow cells, such as the NovaSeq ® system from Illumina. 0 reagent kits, MiniSeq ® with Rapid reagent kits, MiSeq ®, HiSeq ® 2000/2500 (paired-end flow cell), HiSeq 3000/4000 (single-read For unique dual index inputs, the Index_Hopping_Counts. 15 analysis software. For information, see the instrument support pages. Run/Analysis configuration is imported manually into NextSeq 1000/2000 instrument via a sample sheet to start the run. Thus the demultiplexing step will generate a fastq file for each sample using the information from the sample sheet (which associate barcodes - singe or dual - to sample names). Applications The NEBNext Multiplex Oligos for Illumina (Dual Index Primers Set 1) contains the adaptor and index primers that are ideally suited for multiplex sample preparation for next-generation sequencing on the Illumina platform. Avoid using methods that measure total nucleic acid content (eg NanoDrop or other UV absorbance methods) because common contaminants such as ssDNA, RNA, and oligos are not substrates for the Nextera DNA Library Prep assay. A sample sheet is a comma-separated value (*. } IEM—A desktop application that helps you create a sample sheet for dual-indexed sequencing on the HiSeq®, HiScanSQTM, GAIIx, MiSeq®, or NextSeq® systems. Prepare library–The protocol describes the steps to amplify the V3 and V4 region and using a limited cycle PCR, add Illumina sequencing adapters and dual‐index barcodes to the amplicon target. The default location of the sample sheet is the root output folder. May 9, 2023 · FAQ: Where can I find the sample sheets for the NEBNext Unique Dual Index UMI Adaptors DNA Sets 1-4? Instrument-specific sample sheets can be downloaded from the Usage Guidelines section located under the “Protocols, Manual & Usage” tab on the relevant Set’s product page, or find it in the below table: NEBNext LV Unique Dual Index Primers Set 2 includes 96 unique pairs of i5 and i7 index primers optimized for low-volume (LV) NEBNext workflows, including NEBNext EM-seq™ v2, E5hmC-seq™, and NEBNext Low-bias Small RNA Library Prep Kit, with Illumina ® sequencing. What settings should I use in the sample sheet CSV, and will the output from the BCL Convert work with Cell Ranger? For unique dual index inputs, the Index_Hopping_Counts. If a run is set up with single-indexed and dual-indexed samples in the same run, it is possible to demultiplex the single-indexed samples using BCL Convert, use the following steps. Oct 24, 2025 · Index guidelines for Illumina sequencing Indexes, also known as barcodes, are used to bioinformatically identify which read corresponds to which sample after sequencing. Sample sheet options and requirements depend on After sequencing, the unique combination of the two indexes is used to demultiplex the data and assign reads to the proper sample. Each of the 96 wells has unique sample indexes; when pooling, use distinct indexes per library per lane. com website is not compatible with the software version I am using? Import the appropriate definition file (*. For additional Can be combined with Dual Index Primers Set 2 for Illu-mina® (Catalog No. The Resources & References The NextSeq 1000/2000 Sequencing System pages on the Illumina Support Site provide additional system resources. Applications The NEBNext Multiplex Oligos for Illumina (Dual Index Primers Set 2) contains the adaptor and index primers that are ideally suited for multiplex sample preparation for next-generation sequencing on the Illumina platform. . g. In contrast, combinatorial dual indexing is limited to index combinations of 8 unique dual pairs. These resources include software, training, compatible products, and the following documentation. This set (Unique Dual Index UMI Adaptors RNA Set 1; NEB #7416) was designed and optimized for use in RNA sequencing workflows Illumina recommends creating the sample sheet using Illumina Experiment Manager before preparing sample libraries. Index sequences are available for various 10x library types. Nextera DNA CD Indexes (24 Indexes, 24 Samples) Nextera DNA CD Indexes (24 indexes, 24 samples) contain six Index 1 (i7) adapters and four Index 2 (i5) adapters packaged in tubes. 1. It provides a feature for recording parameters for the sample plate, such as sample ID, project name, dual indices, and barcode information. The following table shows strategies for pooling three to eight dual-indexed libraries prepared with these indexes. Mar 31, 2025 · Guidelines for preparing libraries with balanced index combinations for sequencing on Illumina systems. If your index adapter kit is not available, please follow the following instructions to create a custom one. However, for dual-indexing on a paired-end flow cell, the rest of the workflow differs. Typically, dual indexes are used during library preparation, with one index on Illumina Experiment Manager does not allow for different indexing lengths for sample sheet creation. Places the reads with valid index adapter sequences in the sample FASTQ file. Nextera DNA Indexes enable multiplexing of high-quality sequencing libraries generated from the Nextera XT DNA Library Preparation Kit or Illumina DNA Prep. This section has an example dual index GEX sample sheet and an example single index ATAC sample sheet that can be customized for your experiment. When a sample sheet contains one unindexed sample, all reads are placed in the sample FASTQ files (one each for Read 1 and Read 2). Use Illumina This kit includes 8 i5 index primers and 12 i7 index primers for dual indexing. Aug 13, 2024 · The sequencing process for single- and dual-indexed runs on Illumina instruments. The driver_file_generator script is invoked in an automation command line, typically configured on the step prior to the instrument run. Jan 16, 2018 · A custom product file for use with IDT for Illumina TruSeq UD Indexes and the Illumina Experiment Manager (IEM) software. Overview The NEBNext Multiplex Oligos for Illumina (Unique Dual Index UMI Adaptors DNA Set 1) contains adaptors and primers that are ideally suited for multiplex sample preparation for next-generation sequencing on the Illumina platform (Illumina, Inc. Illumina recommends using 2 μl of each DNA sample with 198 μl of the Qubit working solution for sample quantification. Use the Worklow A template for the HiSeq 2000, HiSeq 2500, MiSeq, or NovaSeq 6000 Systems. selecting NexteraXT instead of TruSeqHT) or that the barcodes (especially sometimes index 2 on dual-indexed samples) need to be reverse-complemented on the sample sheet. Or more generally, to demultiplex without using either the i7 (index 1) or i5 (index 2), use the following steps. After library preparation, all that is necessary is to specify the i5 index sequence in the sample sheet. Each kit component must pass rigorous quality control standards, and for each new lot the entire set of reagents is functionally validated together For optimal workflow flexibility, NEBNext ® Multiplex Oligos are available separately from library preparation kits, providing adaptors and primers for high-yield multiplex Illumina ® library production. 18. The default location of the sample sheet is the input folder. Always check support pages for the latest versions. The NEBNext Adaptor has a unique hairpin loop structure that minimizes adaptor-dimer formation, while NEBNext index PCR primers enable index (barcode) incorporation during library For unique dual index inputs, the Index_Hopping_Counts. Sample Sheet Preparation To ensure proper index combinations for pooled libraries with dual-indexed experiments, use the Illumina Experiment Manager (IEM) or BaseSpace before performing library prep. Use the Template File Generator tool to generate custom sample sheets for upload to Illumina instruments. Apr 27, 2023 · Single indexing refers to when the barcode is found only on the i7 index read, therefore allowing a limited amount of unique combinations or barcodes. Create a Planned Run To plan a new sequencing run, use the on-instrument or cloud run planning interface. Single and dual-indexed samples should be processed in separate instances of the cellranger mkfastq pipeline. When used as a set, these kits offer unique dual indexes (UDIs) for multiplexing up to 384 samples. You can download IEM from the Illumina website and install it on any Windows computer. All reads that do not demultiplex to the samples defined in the Data section of the sample sheet are placed in Undetermined_S0. The TruSeq DNA PCR-Free LT kit provides up to 24 indices for low-throughput studies (with both Sets A and B), while the TruSeq DNA PCR-Free HT kit includes 96 dual-index combinations for high-throughput studies. Dual-indexed sequencing includes two index reads after Read 1: the Index 1 Read and the Index 2 Read. Method Illumina NGS systems use different i5 index read strategies, but it is simple to co-sequence single-indexed and dual-indexed libraries together. IDT for Illumina TruSeq RNA UD Indexes (96 Indexes, 96 Samples) Sample Information Before preparing libraries, create a sample sheet to record the index adapter combinations and other information about your samples. For a dual index run, requires only 1 index (index1 or index2) to have a collision in order to error out based on a barcode collision. Start a Manual Run Use the following instructions to initiate a manual run. It is possible to configure different index 1 and 2 lengths in Local Run Manager or Manual Run Mode. Then, the sample plate information can be imported to the sample sheet using the Illumina Experiment Manager. Mar 15, 2024 · Explore barcode series for sample multiplexing in NGS, using multiplex sequencing with indexes of varying lengths to improve accuracy and efficiency. For additional information on available indices and formats for multiplexing, please refer to the NEBNext Multiplex Oligos Selection Chart. 53221) to multiplex up to 96 samples. Documentation, software downloads, and other support resources for Illumina products The Illumina MiSeqDx Integration Package allows for automatic generation of a sample sheet to be used with the MiSeqDx instrument software. When preparing libraries for multiplexing, we recommend using unique dual indexes. Identical indexes within index1 or index2 (combinatorial dual indexes) still supported. If you are sequencing on a NovaSeq ® 6000 with v1. Maximize the effectiveness of your Illumina system, train new employees, or learn the latest techniques and best practices. Index pairs that underperform lead to This allows concurrent execution of BCL conversion with DRAGEN analysis. NEBNext ® Multiplex Oligos provide adaptors and primers to enable high yield multiplex Illumina ® library production. Sample Sheet Requirements The DRAGEN TruSight Oncology 500 ctDNA Local Analysis Software requires a sample sheet for each analysis. This page will walk you through generating Space Ranger-compatible FASTQs with BCL Convert and bcl2fastq. Illumina Experiment Manager v1. 53222) to multiplex up to 96 samples. Before starting the run, make sure the sample sheet is accessible to the instrument. The Nextera XT DNA Library Preparation Kit offers a fast and easy workflow for preparing, quantifying, and normalizing libraries for sequencing small genomes, PCR amplicons, plasmids, microbial genomes, concatenated amplicons, and double-stranded cDNA from a low amount of input DNA (1 ng), making this kit amenable for use with precious samples available in limited quantity. csv) files with Illumina instruments, platforms, and analysis pipelines, refer to Illumina Connected Software. BaseSpace Run Planning has been updated to include the new Illumina DNA/RNA UD Indexes and can produce Sample Sheets to export for use with BCL Convert software. The pipeline will select the appropriate mode depending on the sample indexes used, and enable index-hopping filtering automatically for dual-indexed flow cells. One unique dual index plate enables 96 samples to be pooled together. Alternatively, you can import a sample sheet v2 file to create a planned run. The new replacement Illumina UD Index kits have some new UD Note that sample sheets from Illumina Experiment Manager are not compatible with LRM v3 or v4. The multiplexed sample FASTQ files are assigned to projects and samples based on a user-generated sample sheet, and stored in corresponding project and sample directories (see also Generating the Sample Sheet on page 13). Confirm the sample sheet version applicable to your sequencer to ensure the appropriate barcode sequences and sample sheets are supplied. Does anyone know which is the samplesheet format for illumina nextseq dual index run? I have been experimenting with different columns but does not work ,the manual lists 2 columns index, index2, but I have not seen any tutorial to be used my csv data section is: [Data] This section shows an example sample sheet for libraries created with the Dual Index Plate TT, Set A or Dual Index Plate TN, Set A. The Two-plex through eight-plex pooling strategies are only recommended for Illumina UD Indexes Set A and Set B. Aug 14, 2013 · Guidance for setting up sample sheets for sequencing on the MiSeq. To specify any CSV file in any location, use the command --sample-sheet. Using this dual-barcode approach, Nextera XT Index Kits only require 40 unique index oligos to process up to 384 samples Note: At this point you may either import a prefilled Sample Sheet OR manually enter the information into the LRM. This first set of UDI primer pairs (Set 1 of 5) includes 96 pre-mixed unique pairs of i5 and i7 Sample Sheet Information for NovaSeq v 1. If importing a Sample Sheet, Select Import Sample Sheet and navigate to the SampleSheet. This document provides the nucleotide sequences that comprise Illumina oligonucleotides used in Illumina sequencing technologies. cellranger-arc mkfastq supports listing either index set names or the oligo sequences. --no-lane-splitting true —Output all lanes of a flow cell to the same FASTQ files consecutively. Apr 19, 2022 · Learn when to use combinatorial dual indexing (CDI) or unique dual indexing (UDI) for each particular application and sample type. Cell Ranger's mkfastq supports single-indexed and dual-indexed flow cells. This workflow requires a reverse complement of the Index 2 (i5) primer sequence compared to the primer sequence used on other Illumina platforms. Alternative products are the range of NEBNext® Multiplex Oligos for Illumina® (96 Unique Dual Index Primer Pairs Sets 1-5) (NEB #E6440, E6442, E6444, E6446, E6448). Also see more on NextSeq 1000/2000 sample sheets in this blog from Illumina. The additional reads allow the post-run analysis software to demultiplex with increased accuracy. TruSeq DNA PCR-Free kits are an eficient solution for preparing and indexing sample libraries. csv) that stores the information needed to set up and analyze a sequencing experiment. When preparing libraries for multiplexing, Illumina recommends using unique dual indexing (UDI) whenever possible for the most accurate demultiplexing. bin) is a binary file in BIN format that contains index statistics for each sample and index combination per lane provided to BCL Convert Analysis app in the sample sheet. We offer support webinars, online courses, expert video tips, and instructor-led trainings. Use the following sample sheet templates with IDT for Illumina Nextera DNA UD Indexes. For optimal workflow flexibility, NEBNext ® Multiplex Oligos are available separately from library preparation kits, providing adaptors and primers for high-yield multiplex Illumina ® library production. 0 Reagent Kits, MiSeq, and HiSeq 2000/2500) Headers for the data section should be [Data] or [data] for sample sheet v1 and [BCLConvert_Data] for sample sheet v2. The unique hairpin loop structure of the NEBNext Adaptor minimizes adaptor-dimer formation, and NEBNext index PCR primers enable index incorporation during library amplification. This is particularly frequent with the NovaSeq6000, and so the use of Unique Dual-Indexing (UDI) is Product Description IDT for Illumina DNA/RNA UD Indexes are extension-based unique dual (UD) indexes with unrelated adapters for both index reads. Sample Information Before preparing libraries, create a sample sheet to record the index adapter combinations and other information about your samples. Aug 14, 2024 · FAQ: Where can I find the sample sheets for these Multiplex Oligos? Instrument-specific sample sheets can be downloaded from the Usage Guidelines section located under the “Protocols, Manual & Usage” tab. In addition to unique dual indexes, another strategy for dual index sequencing is to use combinatorial dual indexes. Detailed Steps for Run The samples are identified by index sequences that were attached to the template during sample prep. The Unique Dual Index Kits contain unique, dual-indexed PCR primers for amplification of Illumina®-compatible NGS libraries. DRAGEN uses columns in the Data section to sort samples and index adapters. With a dual-indexed library, the sequencing run includes two additional reads. Different types of sequencing runs use different index adapters. csv) records information about samples, the corresponding indexes, and other information that dictates the behavior of the software. Mixed index lengths are not supported on the NextSeq 500/550. Sample sheet options and requirements depend on the instrument. A phenomenon known as index hopping can lead to misattribution of some reads to the wrong sample. This is a great tool to identify mistakes during index sequencing . This process is also referred to as index masking (the index that is not used is masked). ). IDT for Illumina PCR Index Sets 1-4 (384 IDX) Four 96-sample unique dual index plates used with Illumina library prep for multiplexing up to 384 samples. One mismatch would also result in barcode collision. Illumina UD Indexes Set C and Set D are not designed for low-plexity pooling. Sep 6, 2024 · Index performance affects sample coverage Illumina DNA/RNA Unique Dual (UD) Indexes, tagmentation are available in four sets of 96 index pairs (Sets A, B, C, and D) for 384 total index pairs. fastq per Can be combined with Dual Index Primers Set 1 for Illumi-na® (Catalog No. When demultiplexing, the final sample sheet provided to the FASTQ generation software needs to have the i5 index in the correct orientation for that platform. This article discusses the Illumina sequencer integration packages that generate a sample sheet for use with bcl2fastq v2. Dual indexed libraries contain barcodes at both the i7 and i5 index reads, allowing for many more unique combinations and therefore greater sample multiplexing. Available in two adapter kits, the Nextera XT Index Kit v2 and Nextera DNA CD Indexes suit a range of throughput next-generation sequencing (NGS) applications. These short index sequences are attached to each sample during the library preparation, serving as a unique identifier for that specific sample. A dual-indexed run on the iSeq 100, MiniSeq with standard reagent kits, NextSeq Systems, NovaSeq 6000 with v1. The index metrics out file (IndexMetricsOut. The Unique Dual Indexes are 8-nt long i5 and i7 dual index sequences, corresponding to the IDT for Illumina-TruSeq® DNA and RNA UD Indexes - UDI0001–UDI0096 according to the Illumina Adapter Sequences Document (1000000002694 v10). For additional NEBNext ® Multiplex Oligos for Illumina® are an essential piece of the NEBNext suite of library preparation products, available in several different configurations for use with NEBNext products or other standard Illumina-compatible library preparation protocols. To Barcode Collison example 2: The two index sequences are distinct by 2 base pair. The metrics provide visibility into any index-hopping behavior that have occurred. These sequences are provided for the sole purpose of understanding and publishing the results of your sequencing experiments. tsv) or sample sheet (*. --bcl-sampleproject-subdirectories true —Output FASTQ files to subdirectories based on sample sheet Sample_Project column. The control software performs Read 1, any index reads, and then Read 2 based on the parameters provided for the run in the sample sheet or during run setup. Using this dual-barcode approach, Nextera XT Index Kits only require 40 unique index oligos to process up to 384 samples AI summary: Dual index plates contain unique i5 and i7 oligos per well; single index plates have 4 unique i7 oligos per well for base balance. Dual-index sequencing on a paired-end flow cell follows 1 of 2 workflows, depending on the system: All the tools you need for demultiplexing after sequencing Unique Dual Indexed samples. The IDT for Illumina UD Indexes include IDT for Illumina Nextera DNA UD Indexes, IDT for Illumina DNA/RNA UD Indexes, and IDT for Illumina PCR UD Indexes. Simple and Cost-Effective Solution Whether processing samples for RNA-Seq, genomic sequencing, or exome enrichment, the TruSeq kits provide significantly improved library preparation over previously used methods. The samples with both index and index2 values present in the sample sheet are present in the index hopping file. IDT for Illumina UD Indexes The following tables depict plate layouts and pooling strategies for IDT for Illumina UD Indexes, which are designed for dual indexing. TruSeq Nano DNA Sample Preparation Kits offer a low-input solution for preparing and indexing sample libraries. 1 was updated with the index kits. The NEBNext Adaptor has a unique hairpin loop structure that minimizes adaptor-dimer formation, while NEBNext index PCR primers enable index (barcode) incorporation during library Illumina recommends using unique dual indexes (UDIs) as a method to mitigate errors introduced by index-hopping. After sequencing, the unique combination of the two indices is used to demultiplex the data and assign reads to the proper sample in the pool. Download the template depending on your experiment: bulk template, 10X template or CRISPR template Fill out the template for this experiment, following the instructions Unique Dual Indexed UMI Adaptors DNA Set 2 for improved DNA-seq sample identification specificity or PCR-free workflows. This requirement for index diversity is compatible with any Illumina index selection for single-plex indexing except i7 [H/N]705, which uses the sequence GGACTCCT. The new universal, methylated adaptor design incorporates an index sequence at the initial With the TruSeq DNA Library Prep kits, researchers can take on virtually any DNA sequencing application, including large-scale whole-genome resequencing, targeted resequencing, de novo sequencing, metagenomics, and low-throughput methylation studies. The primers enable incorporation of unique index pairs during library amplification and have been designed to minimize index For optimal workflow flexibility, NEBNext ® Multiplex Oligos are available separately from library preparation kits, providing adaptors and primers for high-yield multiplex Illumina ® library production.