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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.
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 the NextSeq System Denature and Dilute Libraries Guide.
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.
The NextSeq v2.5 flow cells use an updated FC chemistry that improves stability and robustness. This chemistry allows us to ship the flow cells at ambient temperature; once received, store the flow cells at 2°C to 8°C.
Yes, NextSeq Control Software - NCS v2.2.0 or later is required to run the NextSeq v2.5 Reagent Kits.
No, the NextSeq 550Dx instruments require NCS 3.0, which is not compatible with the NextSeq 500/550 v2.5 kits.
Yes NCS 2.2.0 is customer installable. NCS 2.2.0 will be pushed to registered BaseSpace users, if configured for automatic updates. Otherwise, download the software from the Product Files page.
The flow cell packaging is updated to reflect the NextSeq v2.5 naming but the flow cell itself looks the same.
No, the NextSeq v2.5 flow cells follow the same protocol as the NextSeq v2 kits and have been optimized to use the same loading concentration.
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.
Yes. Using NextSeq 500/550 v2.5 reagents requires NextSeq Control Software (NCS) v2.2.0.
NextSeq 500/550 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 four 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 two types of flow cells available for the NextSeq system: the high-output flow cell and the mid-output flow cell. Both flow cells contain four lanes of different width, resulting in a different number of tiles.
NextSeq reagent kits are available in three sizes:
For output specifications, see NextSeq Series Specifications.
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:
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.
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.
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.
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.
NCS v1.4 is not compatible with the NextSeq 500 Kit (v1). Use NCS v1.3, or earlier, with v1 kits.
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 Sequence Hub. 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 five cycles of the sequencing run. To detect a cluster during template generation, there must be at least one base other than G in the first five 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 the instrument is connected to BaseSpace Sequence Hub, use the Prep tab to record library and index information and specify run parameters. When runs are set up on the Prep tab, a sample sheet is not required.
The NextSeq 550 System can perform up to a 150-cycle paired-end run (2 × 150) using available NextSeq 500/550 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 control computer is Windows 7.
A 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 Sequence Hub, BCL files are automatically converted to FASTQ files after data transfer is complete. If BCL files are saved to a local server, use bcl2fastq2 Conversion Software to convert base calls into FASTQ file format.
NCS v2.0 adds support for scanning BeadChip workflows. Scanning BeadChips on a NextSeq 500 system requires a hardware change.
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.
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: