Instrumentation > NextSeq 500/550 > FAQ

• At which cycle of NextSeq 500/550 are run metrics calculated?

• Can the NextSeq 500/550 v2.5 flow cell be used with the v2 Reagent cartridge and buffer cartridge?

• Can the output folder for a NextSeq 500/550 run be saved to the local computer?

• Common Questions Related to NextSeq 500/550 v2.5 Kit

• Common questions regarding custom recipes for the NextSeq 500/550

• Do NextSeq 500/550 v2.5 runs have different specifications than v2.0 reagent runs?

• How long does a NextSeq 500/550 system check take to complete?

• How long does it take to purge all reagents in a NextSeq 500/550 run?

• How many extra cycles are included in NextSeq 500/550 reagent cartridges?

• How to set up a NextSeq 500/550 to upload to a BaseSpace Enterprise account using NCS v.4.0.1

• Run setup modes for the NextSeq 500/550

• What do the NextSeq 500/550 status bar colors mean?

• What is the difference between NextSeq PhiX Control Kit and PhiX Control v3?

• What is the maximum read length supported in one direction on a NextSeq 500/550 run?

• Will the NextSeq 500/550 Control Software prevent run setup with read lengths fewer than 25 cycles?

The following diagram contains information on calculations made during the first 25 cycles during a NextSeq 500/550 run:

• When is the final cluster density calculated?

• When does template generation complete?

• When is phasing/prephasing calculated?

• When is clusters passing filter percentage determined?

• When is base calling determined?

• When is Q30 calculated (quality scoring)?

  

Yes, v2.5 kits include a v2.5 flow cell and v2 reagent and buffer cartridges.

Illumina does not recommend or support saving runs locally on NextSeq 500/550 due to potential negative effects on the computer. Instead, Illumina recommends saving to a network server/drive or an external hard drive.

Can NextSeq v2.5 flow cell be used with v2 Reagent Cartridge and Buffer Cartridge?

Yes, v2.5 kits include a v2.5 flow cell and v2 reagent and buffer cartridges.

In addition to starting intensities, are other run metrics expected to change?

Run specifications for the NextSeq v2.5 reagent kits, such as overall Q30 and output, are the same as for the v2 kits.

Can the NextSeq v2.5 reagent kit be used on the NextSeq 550Dx instrument?

Due to software incompatibility, NextSeq v2.5 reagent kits are for use only on the NextSeq 500/550 Research Use Only instrument at this time

Is there a visible change to the flow cell?

The flow cell part number and packaging labels are different; however there are no visible differences in the flow cell.

Can the v2 kit still be ordered?

NextSeq 500/550 v2 kits have been discontinued effective December 31, 2018; shipments continued through February 28, 2019. Illumina Advantage (TG) reagents versions can still be ordered.

Can the flow cell ID be used to distinguish between flow cell types?

No.

Are there any changes to loading?

Recommendation has changed for mid-output (MO) kits. NextSeq System Denature and Dilute Libraries Guide indicates 1.5 pM rather than 1.8 pM.

Are custom recipes for v2 kits usable with v2.5 kits?

No, custom recipes must be updated for v2.5 kits. NextSeq Control Software (NCS) v2.2 will not check if the recipe is correct (for v2 or v2.5) and will allow the run to continue to completion.

How can a custom recipe be obtained?

To obtain a custom recipe, contact your local Illumina Field Applications Scientist (FAS). For help contacting your FAS, contact Illumina Technical Support.

Can 'Local Run Manager' mode be used for run setup with a custom recipe?

No, 'Manual Mode' must be used for run setup in order to select a custom recipe.

Do dark cycles need to be added to the total cycle length entered into run setup?

No, dark cycles are accounted for in the custom recipe and do need to be added to the desired cycle length during run setup.

For information on using custom recipes during run setup, see Knowledge Base article How to Select Custom Recipes on the NextSeq 500/550 in NCS 4.0.X.

No. Run specifications for the NextSeq 500/550 v2.5 reagent kits, such as overall Q30 and output amount are the same as v2.0 kits.

The NextSeq 500/550 system checks take about 90 minutes to complete.

Purging reagents can take around 2-4 hours depending on the amount of reagents left in the NextSeq 500/550 cartridge. After the purge, a wash is performed, which takes an additional 1.5 hours.

There are extra cycles (17 or 18, depending of the kit) included in NextSeq 500/550 reagent kits that can be used for index reads or to extend insert read lengths.

• 75 cycle kits: 92 cycles total

• 150 cycle kits: 168 cycles total

• 300 cycle kits: 318 cycles total

NextSeq Control Software (NCS) v.4.0.1 allows for changing the Hosting Location for BaseSpace and to set a Private Domain for uploading run data using the following steps.

1. From the NCS home page, navigate to Manage Instrument.

1. Select System Configuration.

1. Then, select BaseSpace Sequence Hub Configuration.

1. Change the Hosting Location to the appropriate region and check the box for Private Domain. Enter the {Domain Name}. in the text box to designate the Enterprise Account Domain. Once done, select Save.

The NextSeq 500/550 run setup modes available depend on the version of the Windows operating system, and NextSeq Control Software (NCS) installed.

(Terms: LRM = Local Run Manager / BSSH = BaseSpace Sequencing Hub)

Windows 7 with NCS v2.2 or lower\*

Standalone Mode

• Set up the run in the control software.

• BSSH is not used. The data output of the run is only bcls; use bcl2fastq for FASTQ generation.

Prep Tab in BSSH

• If using BSSH for analysis (FASTQ generation, etc.), the run must be planned in Prep Tab.

\*LRM is not available on Windows 7-based systems.

Toggle between modes in Control Software (Manage Instrument / System Configuration).

Windows 10 with NCS v4+ and LRM v2

LRM Mode

• Create the run in LRM.

• The run can also be sent to BSSH.

Manual Mode

• Set up the run in the control software.

• A sample sheet is required if uploading the run to BSSH.

Toggle between modes during run setup (after selecting Sequence).

The NextSeq 500/550 instrument includes a light-emitting diode (LED) status bar beneath the touch screen monitor. The status bar provides an at-a-glance indication of the instrument state.

• Green: ready to sequence

• Blue: processing

• Orange: needs attention

• Yellow: wash is due within the next 24 hours

  

See the NextSeq 500 and NextSeq 550 Sequencing System Guide for more information.

• The PhiX library is the same in both kits, thus either kit can be used in any Illumina sequencing platforms.

• The only difference between kits is that the NextSeq PhiX Control Kit also includes Resuspension Buffer (RSB) whereas the PhiX Control v3 does not.

The maximum supported read length for NextSeq 500/550 runs is up to 151 reads in one direction. With this, the maximum total read length configuration is 2x151 (paired, 151 bp reads).

This type of run configuration is possible in standalone mode, PrepTab, and Local Run Manager. However, BaseSpace will not generate FASTQ files for reads that are less than 25 cycles.

Illumina does not recommended insert reads lengths shorter than 25 cycles.