The NextSeq instrument comes with a region-specific power cord. For more information, see the NextSeq System Site Prep Guide.
For instrument specifications, see the Site Prep/Lab Environment page.
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.
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).
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.
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.
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.
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.
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.
NextSeq kits are available in three sizes:
For output specifications, see NextSeq 500 specifications page.
No, a wash is not required after a scan. However, if the system has been dry for 7 days, a wash is required before proceeding to another scan.
Supported BeadChips include the CytoSNP-850K, HumanCytoSNP-12, and HumanKaryomap-12.
One BeadChip can be scanned at a time.
The NextSeq software performs an automatic post-run wash on the NextSeq 550 system after a successful sequencing run.
Array scanning is enabled out of the box on the NextSeq 550 system.
Scanning takes approximately 40 minutes per BeadChip. The CytoSNP-850K scans at a rate of approximately 5 minutes per sample. The HumanCyto-SNP12 and HumanKaryomap-12 scan at a rate of 3.5 minutes per sample.
Yes; however, do not store the BeadChip adapter without a BeadChip on the imaging compartment stage on the NextSeq 550 system.
NextSeq 550 array data quality is comparable to the iScan system. For instance, both the iScan system and NextSeq 550 system can achieve > 99% SNP calls with a > 99% concordance (R^2) for the Infinium CytoSNP-850k BeadChip.
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:
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.
Following the sequencing run, the NCS v2.0 software initiates an automatic post-run wash on the NextSeq 550 system, which takes about 90 minutes. When the wash is complete, the Home button becomes active and you can begin an array scan.
You can start an array scan when the NextSeq 550 system completes the automatic post-run wash following a sequencing run.
Make sure that the BeadChip is seated flat on the adapter and that the BeadChip is free of dust. Use canned air or other compressed dusting method to clear the debris.
No, the BeadChip and flow cell fit in the same imaging compartment stage and use the same cameras.
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.
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.
You can start a sequencing run when the NextSeq 550 system completes the array scan.
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.
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.
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.
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.
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.
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.
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.
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.
The operating system on the NextSeq computer is Windows 7.
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.
NCS v2.0 adds support for scanning BeadChip workflows. Scanning BeadChips on a NextSeq 500 system requires a hardware change.
No. Always perform a wash when a wash is due. It is not possible to proceed to scanning or sequencing until a wash is performed. Instrument washes are required every 7 days, even when the instrument is used for array scans, since the instrument is in a dry state. Regular washes help maintain the instrument fluidics system.
To perform a scan on the NextSeq 550, you need Decode (*.dmap) files, a manifest (*.bpm) file, and a cluster (*.egt) file for the BeadChip you are using. The Decode files are unique for each BeadChip barcode. The manifest and cluster files are unique to the BeadChip product type.
Array scanning requires about 400 MB of free space on the NextSeq 550 hard drive. NCS v2.0 automatically manages disk space.
Use the Decode File Client Utility to download Decode files directly from Illumina servers using standard HTTP protocol. You can download the Decode File Client and install it on a computer with access to the network location for the Decode file (DMAP) folder.
These files are preloaded in the software, and the location of the files is specified on the BeadChip Scan Configuration screen. From the NCS Home screen, select Manage Instrument, System Configuration, and then BeadChip Scan Configuration. Manifest files use the *.bpm file format, and cluster files use the *.egt file format.
For more information, see the NextSeq 550 System Guide.
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.
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:
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.
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.
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.