NextSeq 550 FAQs

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  • Primers

  • Yes. There are three reservoirs on the NextSeq reagent cartridge reserved for custom primers: #7 for a custom Read 1 primer, #8 for a custom Read 2 primer, and #9 for a custom Index 1 primer or custom Index 2 primer.

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

  • Analysis

  • 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.

    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.

    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 scan output default file format is GTC, which allows for direct analysis using the BlueFuse Multi software. If you prefer to use GenomeStudio for analysis, you can configure the instrument to generate IDAT files for export.