Dark Cycle Sequencing

This section describes how to use dark cycle sequencing in the recipe.

Check the NextSeq 1000/2000 Sequencing System page on the Illumina Support Site to see if a custom recipe is available for your reagent kit. The are different custom recipes between XLEAP-SBS and standard SBS. If a recipe is not available, contact Illumina Technical Support.

Dark cycle sequencing is used to complete only the chemistry steps of a sequencing cycle. Check the Compatible Products page for your library prep kit on the Illumina Support Site to see if dark cycle sequencing is required.

For NextSeq 1000/2000 Control Software v1.3 and later, with Illumina Stranded Total RNA Prep with Ribo-Zero Plus and Illumina Stranded mRNA Prep, the software selects the appropriate dark cycle recipe if the Library Prep is specified in the sample sheet.

Use the following steps for dark cycle sequencing.

1. Download the recipe XML file from the NextSeq 1000/2000 Sequencing System page on the Illumina Support Site.
2. Edit the recipe XML file.
a. Identify the appropriate protocol section based on your read and indexed 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 indexed sequencing configuration is <Protocol Name="1 Read 0 Index" ProtocolType="1Read0Index" >.

b. 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" />.

c. Enter the dark cycle step on a new line for every dark cycle required.
3. Save the recipe XML file.

The following is a sample recipe with the dark cycle step:

<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" />
...

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. Return to Initiating a Standard SBS Sequencing Run.