Questions & Answers

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General

  • What are the dimensions of the NextSeq system?

    For instrument specifications, see the Site Prep/Lab Environment page.

  • What power connections are required for the NextSeq system?

    The NextSeq instrument comes with a region-specific power cord. For more information, see the NextSeq System Site Prep Guide.

Primers

  • What has changed with the NextSeq 500/550 Kit v2?

    The NextSeq 500/550 Kit v2 provides an improved workflow during run setup. You no longer have to add sodium hypochlorite (NaOCl) and dual-index sequencing primers (BP13) manually before the run. All required reagents are included in the prefilled reagent cartridge v2.

    Reagents provided in the NextSeq 500/550 Kit v2 in combination with NCS v1.4 generate improved data quality and a higher yield of error-free reads, and enable the use of custom sequencing primers for dual-indexed runs.

  • Can I use a custom Index 2 primer on the NextSeq system?

    The release of NCS v1.4 and the NextSeq 500/550 Kit v2 together enable the use of a custom Index 2 primer. Using a custom Index 2 primer is not possible with previous kit or control software versions.

Libraries

  • What is the library loading volume and concentration required for sequencing on the NextSeq system?

    Using NextSeq Control Software v1.3, or later, the loading volume is 1.3 ml and the loading concentration is 1.8 pM.

    For more information, see NextSeq System Denature and Dilute Libraries Guide (15048776).

  • How many different libraries can be sequenced on a NextSeq flow cell?

    Libraries are transferred onto the flow cell from a single reservoir on the NextSeq reagent cartridge, reservoir #10. Therefore, any libraries that can be pooled can be sequenced together on the flow cell.

  • What are the supported insert sizes on the NextSeq system?

    Sequencing an insert size of 550 bp is supported with NextSeq v2 reagents. With NextSeq v1 reagents, an insert size of 350 bp is supported.

Reagents and Flow Cells

  • Can I use any kit version with NCS v1.4?

    Use the NextSeq 500/550 Kit v2 with NCS v1.4.

    The NextSeq 500 Kit (v1) is not compatible with NCS v1.4. If using the NextSeq 500 Kit (v1), do not upgrade your control software beyond NCS v1.3.

  • Are there compatibility requirements for using the NextSeq 500/550 Kit v2?

    Yes. Using the NextSeq 500/550 Kit v2 requires NextSeq Control Software (NCS) v1.4.

  • Are NextSeq flow cells available in single-read and paired-end versions?

    All NextSeq kits include reagents for paired-end sequencing and a paired-end flow cell. However, single-read runs are possible on a paired-end flow cell when the run is set up as a single-read run.

  • How many lanes are on a NextSeq flow cell?

    The NextSeq flow cell contains 4 physical lanes. However, libraries are loaded onto the flow cell from a single reservoir. You can sequence a single library or multiple pooled libraries on the flow cell.

  • How many tiles are imaged on a NextSeq flow cell?

    There are 2 types of flow cells available for the NextSeq system, the high-output flow cell and the mid-output flow cell. Both flow cells contain 4 lanes, but the lanes differ in width resulting in a different number of tiles.

    • Lanes on the high-output flow cell are imaged top and bottom in 3 swaths, or columns, for a total of 864 tiles.
    • Lanes on the mid-output flow cell are imaged top and bottom in 1 swath for a total of 288 tiles.

    For more information, see the NextSeq 500 System Guide or NextSeq 550 System Guide.

  • What kit sizes are available for the NextSeq system?

    NextSeq kits are available in three sizes:

    • The high-output kit is available in300 cycles (2 x 150), 150 cycles (2 x 75), and 75 cycles (1 x 75).
    • The mid-output kit is available in 300 cycles (2 x 150), 150 cycles (2 x 75).

    For output specifications, see NextSeq 500 specifications page

Workflow

  • Do NextSeq reagents use the same SBS chemistry as the HiSeq and MiSeq reagents?

    All Illumina sequencers use the Illumina patented reversible-terminator SBS chemistry. The NextSeq system employs the latest evolution in SBS technology: a novel 2-channel SBS method that supports reduced cycle time and data processing time.

  • How long does it take to complete a sequencing run on the NextSeq system?

    Run duration depends on the number of cycles performed and the type of flow cell used. Run duration is the same for the NextSeq 500 system and the NextSeq 550 system. For more information, see the NextSeq System specifications page.

  • Is NaOCl required for the NextSeq instrument wash?

    A dilute solution of NaOCl is required for the automatic post-run wash.The required NaOCl is included in the reagent cartridge provided in the NextSeq 500/550 Kit v2 and the TG NextSeq 500/550 Kit. However, if you are using the original NextSeq 500 Kit (v1), load 3 ml NaOCl in reservoir #28 before loading the reagent cartridge.

    Requirements for NaOCl differ for manual instrument washes depending on the version of control software you are using:

    • The Manual Post-Run Wash option introduced with NCS v1.4 requires diluted NaOCl.
    • The manual wash option provided with NCS v1.3 or earlier versions does not require NaOCl.

  • How long does it take to perform an instrument wash on the NextSeq system?

    The NextSeq software performs an automatic post-run wash after each successful sequencing run. The automatic post-run wash takes about 90 minutes.

    NCS v1.4 introduces 2 manual washes: the Quick Wash and the Manual Post-Run Wash.

    • Quick Wash—Uses a Tween 20 solution and takes about 20 minutes.
    • Manual Post-Run Wash—Uses a Tween 20 solution with diluted NaOCl and takes about 90 minutes.

    When you initiate a manual wash with NCS v1.3, the software checks that an automatic post-run wash was performed after the last run.

    • If a wash was not performed, the manual wash takes about 90 minutes.
    • If an automatic post-run wash was performed, the manual wash takes 20 minutes.

  • How long does the cluster generation step take on the NextSeq system?

    Cluster generation is the first step in the sequencing run in which single DNA molecules are bound to the surface of the flow cell, and then amplified to form clusters. Cluster generation on the NextSeq system takes about 2 hours and 20 minutes.

  • How is scanning enabled on the NextSeq 500 system?

    NextSeq 500 customers can purchase a package to upgrade to a NextSeq 550 system to enable scanning. The package includes a hardware change and a software update.

  • How does the data quality compare between the NextSeq 550 system and NextSeq 500 system?

    The NextSeq 500 system and NextSeq 550 system offer the same high sequencing data quality. There is no change to output or quality specifications between runs on the different systems.

Software

  • What’s new in NextSeq Control Software (NCS) v1.4?

    NCS v1.4 enables the use of the NextSeq 500/550 Kit v2, supports BaseSpace run monitoring in standalone mode, introduces a reagent purge feature, can proceed to sequencing automatically after a successful pre-run check, and includes a Quick Wash option. 

  • Can I continue to use the NextSeq 500 Kit (v1) after upgrading to NCS v1.4?

    NCS v1.4 is not compatible with the NextSeq 500 Kit (v1). Use NCS v1.3, or earlier, with v1 kits.

  • Can I use Sequencing Analysis Viewer (SAV) to view sequencing data from a NextSeq system?

    Yes. Use SAV 1.8.36, or later to view sequencing data from a NextSeq system. You can download SAV for use on a networked computer or use SAV in BaseSpace. SAV 1.8.36 is also compatible with HiSeq and MiSeq run data.

  • How many cycles are required for template generation on the NextSeq system?

    Template generation, the process of identifying defining cluster positions over the entire flow cell surface, is performed during the first 5 cycles of the sequencing run. To detect a cluster during template generation, there must be at least 1 base other than G in the first 5 cycles.

  • Which version of Illumina Experiment Manager (IEM) is compatible with the NextSeq system?

    A sample sheet is not required for a sequencing run unless you are using the system in standalone mode. Use IEM version 1.8.2, or later, to create a sample sheet.

    If you are connected to BaseSpace, use the BaseSpace Prep tab to record library and indexing information, and to specify other run parameters. When runs are set up on the Prep tab, a sample sheet is not required.

  • How many cycles of sequencing are supported on the NextSeq system?

    The NextSeq system can perform up to a 150-cycle paired-end run (2 x 150) using available NextSeq kits. Kits are available in sizes of 300 cycles, 150 cycles, and 75 cycles. Each kit includes additional cycles for index reads.

  • Is a sample sheet required for a sequencing run on the NextSeq system?

    When connected to BaseSpace, a sample sheet is not required. Library and indexing information is entered on the BaseSpace Prep tab before the run, and the information is passed to the NextSeq system. Available run names appear on the instrument screen during the run setup steps.

    However, if the instrument is configured to run in standalone mode (not connected to BaseSpace), use Illumina Experiment Manager (IEM) v1.8.2, or later, to create a sample sheet.

  • What operating system runs on the NextSeq computer?

    The operating system on the NextSeq computer is Windows 7.

  • What is the output file format from a sequencing run on the NextSeq system?

    The NextSeq system generates base call (BCL) files aggregated by lane with a BCL file for each lane, for each cycle. The aggregate file contains the base call and associated quality score for every cluster.

    When using BaseSpace, BCL files are automatically converted to FASTQ files when data transfer is complete. If BCL files are saved to a local server, use bcl2fastq 2.0 to convert base calls from a NextSeq run. The FASTQ converter must be run on a Linux server.

  • What’s new in NextSeq Control Software (NCS) v2.0?

    NCS v2.0 adds support for scanning BeadChip workflows. Scanning BeadChips on a NextSeq 500 system requires a hardware change. 

  • What is bcl2fastq2?

    The bcl2fastq2 Conversion Software converts base call (BCL) files generated on the NextSeq system. If you plan to use a third-party data analysis solution outside of BaseSpace, configure the system to operate in standalone mode and use the bcl2fastq2 Conversion Software. For more information, see bcl2fastq Conversion Software.

    Only version 2.0, or later, is compatible with NextSeq data.

Analysis

  • What happens if my data connection is interrupted during a run on the NextSeq system?

    In the event that data transfer is interrupted during a run, data are stored temporarily on the instrument computer until the connection is restored. When the connection is restored, transferring of data resumes automatically.

    If the connection is not restored before the end of the run, data must be removed from the instrument computer manually before a subsequent run can begin.

  • Does the NextSeq system perform data analysis on the instrument?

    The data set generated by the NextSeq system is too large for on-instrument analysis. Data must be transferred to BaseSpace or a local server for secondary analysis.

  • Do I need a dedicated server to analyze run data from the NextSeq system?

    You can transfer data from a sequencing run to BaseSpace for analysis and storage. Additionally, you can configure the NextSeq system to transfer data to a local server and perform analysis using third-party software.

  • How are base calling and quality scoring performed on the NextSeq system?

    Base calling and quality scoring are performed by an updated implementation of Real-Time Analysis (RTA), called RTA v2, which includes important differences from RTA on other Illumina sequencing systems. For example, all processes are performed in memory to maximize processing speed, and configuration files and output file formats are different. For more information, see the NextSeq 500 System Guide or NextSeq 550 System Guide.

    The workflow includes the following steps:

    • Template generation—Defines the position of each cluster on the flow cell.
    • Imaging—In real time, images are passed in memory to RTA v2.
    • Base calling—Determines a base for every cluster at a specific cycle. After a base is called, RTA v2 assigns a quality score. The result is written to base call (BCL) files, which are aggregated in one file per lane per cycle.