Custom recipes on the NextSeq 1000/2000

A custom recipe can enable dark cycle sequencing on the NextSeq 1000/2000. Dark cycle sequencing is used to complete only the chemistry steps of a sequencing cycle (omitting the imaging). Custom recipes are enabled on the NextSeq 1000/2000 beginning with NextSeq 1000/2000 control software version 1.2.

If using NextSeq 1000/2000 control software version:

• v1.2, must select the custom recipe from the dropdown menu during run setup.

• v1.3 or later, the custom recipe for Illumina Stranded mRNA Prep ligation and Illumina Stranded Total RNA is automatically selected when the library prep kit is defined during online run setup in Run Planning.

Custom recipe templates are available on the NextSeq 1000/2000 Sequencing System Product Files page. Custom recipes for P1 and P2 flow cells are cross-compatible between the NextSeq 1000 and NextSeq 2000 platforms. Custom recipes for the P3 and P4 flow cells are only compatible with the NextSeq 2000.

Instructions for editing a recipe to include Dark Cycle Sequencing at the beginning of a read can be found in the NextSeq 1000/2000 Product Documentation.

Contact Illumina Technical Support or your local Field Applications Scientist if dark cycles need to be inserted into the middle of the read.

Edit the Recipe File

1. Locate desired XML starting recipe.

• Starting recipe can be located on the instrument at usr/local/illumina/recipes/ [LO, MO, HO, or XO].

  • LO = Low Output P1 flow cell, MO = Mid Output P2 flow cell, HO = High Output P3 flow cell, XO = P4 flow cell.

• Custom recipe templates can also be found on the NextSeq 1000/2000 Sequencing System Product Files page.

1. Copy starting recipe and save as *AnyName.*xml.

2. Identify the appropriate protocol section based on your read and index sequencing configuration. There are six different possible protocols per custom recipe that can be edited.

• For example, the protocol for a single Read 1 with no index sequencing configuration is [Protocol Name="1 Read 0 Index" ProtocolType="1Read0Index"]

• Before [ReadRef ReadName="Read 1"/] and [ReadRef ReadName="Read 2"/], enter the following dark cycle step on a new line [DarkCycle ChemistryName="Dark Cycle Before First Base" /]

• Enter the dark cycle step on a new line for every dark cycle required.

1. Save the recipe XML file.

• Save to the usr/local/illumina/recipes directory. This directory is owned by the Root account, and the recipe will need to be placed there using the command prompt. See Knowledge Base Article Moving Custom Recipes using the Command Line Terminal.

The following is a sample recipe with dark cycle: Protocol Name="1 Read 0 Index" ProtocolType="1Read0Index" >

ChemistryRef ChemistryName="Start" / ChemistryRef ChemistryName="Prime Cartridge" /> ChemistryRef ChemistryName="BIX Mixing" /> ChemistryRef ChemistryName="Prime Cartridge" /> ChemistryRef ChemistryName="ExAmp Transfer" /> ChemistryRef ChemistryName="ExAmp Mixing" /> ChemistryRef ChemistryName="Prime Cartridge" /> Obdd ChemistryName="Library Denaturation and Dilution" /> ChemistryRef ChemistryName="Prime Cartridge" /> Obcg ChemistryName="Cluster Generation" /> ChemistryRef ChemistryName="SBS Prime" /> ChemistryRef ChemistryName="Read Prep" /> DarkCycle ChemistryName="Dark Cycle Before First Base" /> ReadRef ReadName="Read 1" /> SetThermalZoneTemp Enable="false" Zone="FlowCellHeater" />/Protocol> Protocol Name="1 Read 1 Index" ProtocolType="1Read1Index" >

ChemistryRef ChemistryName="Start" /> ChemistryRef ChemistryName="2min 60C Vacuum Hold" /> ...

Attach the recipe to the run

1. In the Run Setup in the control software, select Choose under custom Recipe.

2. Navigate to the updated recipe XML file.

3. Select Open.

4. Continue with run Setup, and select Prep.

RunParameters.xml file will display Recipe Name and Version used on sequencing run. For example:

Figure 1: Runparameters.xml file from NextSeq 1000 and 2000 P1: Illumina Stranded Total RNA with RiboZero Plus BaseSpace Demo Data.

For any feedback or questions regarding this article (Illumina Knowledge Article #3421), contact Illumina Technical Support [email protected].