NextSeq 500 FAQs

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  • Primers

  • 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.

    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.

  • Workflow

  • 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.

    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.

    A dilute solution of NaOCl is required for the automatic post-run wash. The required NaOCl is included in the reagent cartridge provided in NextSeq 500/550 and TG NextSeq 500/550 kits. 

    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.

    NextSeq Control Software (NCS) performs an automatic post-run wash after each successful sequencing run. The automatic post-run wash takes about 90 minutes.

    NCS v1.4 introduces two 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.

    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 550 System takes about 2 hours and 20 minutes.

    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.

    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.

  • Analysis

  • 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.

    The data set generated by the NextSeq 550 System is too large for on-instrument analysis. Data must be transferred to BaseSpace Sequence Hub or a local server for secondary analysis.

    You can transfer data from a sequencing run to BaseSpace Sequence Hub 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.

    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.