# Manual Upgrade

The NextSeq 500/550 Integration v2.5.0 integration package requires the NextSeq 500/550 v1.3 workflow available from IPP v2.10. If you do not want to upgrade through the IPP, you can manually upgrade the workflow configuration from NextSeq 500/550 v1.2 workflow to v1.3 workflow by following instructions in this guide to generate samplesheet compatible with LRM v4.0.

## Generate NextSeq 500/550 Samplesheet Compatible with LRM v4.0

To support the generation of LRM v4.0-compatible samplesheet, you must modify the **Denature and Dilute** step, and replacement of template files.

### Modify Denature and Dilute Step

**Automation**

1. From Configuration, select the Automation tab.
2. Rename the **Generate bcl2fastq2 NextSeq Samplesheet** automation to **Generate NextSeq Samplesheet**.

<figure><img src="https://2084401275-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2FfjuebS41N49G1Eh55hP7%2Fuploads%2Fgit-blob-c07f361f33a1c2c4d28ef04d5e02bdbc166e2c5c%2Fnextseq-generate-nextseq-samplesheet-automation.png?alt=media" alt=""><figcaption></figcaption></figure>

{% hint style="info" %}
The default command line for the automation remains unchanged.
{% endhint %}

**Master Step Fields**

1. From Configuration, select the Custom Fields tab.
2. Select the **Denature and Dilute** Master Step.
3. Add the following master step fields:

   | **Field Name**       | **Field Type** | **Field Constraints/Options** | **Preset Values/Additional Options and Drop-Down Items**                                                     |
   | -------------------- | -------------- | ----------------------------- | ------------------------------------------------------------------------------------------------------------ |
   | Comment              | Multiline Text |                               |                                                                                                              |
   | Genome Folder        | Text           |                               |                                                                                                              |
   | Manifest             | Text           |                               |                                                                                                              |
   | Samplesheet Template | Text Dropdown  | Required Field                | <p>Presets</p><ul><li>NextSeq\_Samplesheet.csv</li><li>NextSeq\_ReverseComplement\_Samplesheet.csv</li></ul> |
   | Taxonomy Database    | Text           |                               |                                                                                                              |
4. Remove the following master step fields:
   * Adapter
   * Adapter Read 2
   * Mask Adapter
   * Mask Adapter Read 2
   * SampleSheet Template
5. Update the following master step fields:

   | **Field Name**  | **Changes Required**                                                                                                                                                                                                                                                       | **Screenshot**                                                                                                                                                                                                                                                                                              |
   | --------------- | -------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- | ----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- |
   | Experiment Name | <ul><li>Update Required Field to <strong>Yes</strong></li></ul>                                                                                                                                                                                                            | <div><figure><img src="https://2084401275-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2FfjuebS41N49G1Eh55hP7%2Fuploads%2Fgit-blob-afc276194859d6f5d6c7c5655c8ad14d9ddd7d1f%2Fnextseq-experiment-name-master-step-field.png?alt=media" alt=""><figcaption></figcaption></figure></div> |
   | Workflow        | <ul><li><p>Update Dropdown presets to</p><ul><li>Assembly</li><li>DNA Amplicon</li><li>DNA Enrichment</li><li>GenerateFASTQ</li><li>Library QC</li><li>16S Metagenomics</li><li>PCR Amplicon</li><li>Resequencing</li><li>RNA Fusion</li><li>Small RNA</li></ul></li></ul> | <div><figure><img src="https://2084401275-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2FfjuebS41N49G1Eh55hP7%2Fuploads%2Fgit-blob-aba16d81f1b51be5b1032e6063f7d6317b3f16ef%2Fnextseq-workflow-master-step-field.png?alt=media" alt=""><figcaption></figcaption></figure></div>        |

**Group of Defaults**

1. From Configuration, select the Custom Fields tab.
2. Select the **Denature and Dilute** Master Step.
3. Add the following Group of Defaults:

   | **Group of Defaults** | **Details**                                                         | **Screenshot**                                                                                                                                                                                                                                                                                      |
   | --------------------- | ------------------------------------------------------------------- | --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- |
   | RNA Fusion            | <ul><li>Read 1 Cycles = 251</li><li>Workflow = RNA Fusion</li></ul> | <div><figure><img src="https://2084401275-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2FfjuebS41N49G1Eh55hP7%2Fuploads%2Fgit-blob-1c3a977b80e27a5edb189187d2f4bfeb1c64f972%2Fnextseq-rna-fusion-gp-of-defaults.png?alt=media" alt=""><figcaption></figcaption></figure></div> |
   | Small RNA             | <ul><li>Read 1 Cycles = 251</li><li>Workflow = Small RNA</li></ul>  | <div><figure><img src="https://2084401275-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2FfjuebS41N49G1Eh55hP7%2Fuploads%2Fgit-blob-454459584ce3ce675d362619870689a9d5ebb3ea%2Fnextseq-small-rna-gp-of-defaults.png?alt=media" alt=""><figcaption></figcaption></figure></div>  |
4. Update the following Group of Defaults:

   | **Group of Defaults** | **Changes Required**                                                                                                              | **Screenshot**                                                                                                                                                                                                                                                                                            |
   | --------------------- | --------------------------------------------------------------------------------------------------------------------------------- | --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- |
   | Custom Amplicon       | <ul><li>Rename to <em>DNA Amplicon</em></li><li>Update <strong>Workflow</strong> field to <em>DNA Amplicon</em></li></ul>         | <div><figure><img src="https://2084401275-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2FfjuebS41N49G1Eh55hP7%2Fuploads%2Fgit-blob-903d66e390f19c6a7e8579b0091854ba8d3aa888%2Fnextseq-dna-amplicon-gp-of-defaults.png?alt=media" alt=""><figcaption></figcaption></figure></div>     |
   | Enrichment            | <ul><li>Rename to <em>DNA Enrichment</em></li><li>Update <strong>Workflow</strong> field to <em>DNA Enrichment</em></li></ul>     | <div><figure><img src="https://2084401275-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2FfjuebS41N49G1Eh55hP7%2Fuploads%2Fgit-blob-438b217ca4184a59313228fc8e23d1b4ae227a7c%2Fnextseq-dna-enrichment-gp-of-defaults.png?alt=media" alt=""><figcaption></figcaption></figure></div>   |
   | Generate FASTQ        | <ul><li>Rename to <em>GenerateFASTQ</em></li></ul>                                                                                | <div><figure><img src="https://2084401275-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2FfjuebS41N49G1Eh55hP7%2Fuploads%2Fgit-blob-18ff3aa93022d7f20c92ca4f432f5ee23d6bb22a%2Fnextseq-generatefastq-gp-of-defaults.png?alt=media" alt=""><figcaption></figcaption></figure></div>    |
   | Library QC            | <ul><li>Update <strong>Workflow</strong> field to <em>Library QC</em></li></ul>                                                   | <div><figure><img src="https://2084401275-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2FfjuebS41N49G1Eh55hP7%2Fuploads%2Fgit-blob-c944a5bdd7ff330512e9c2841c4c1b6cbeb64799%2Fnextseq-library-qc-gp-of-defaults.png?alt=media" alt=""><figcaption></figcaption></figure></div>       |
   | Metagenomics          | <ul><li>Rename to <em>16S Metagenomics</em></li><li>Update <strong>Workflow</strong> field to <em>16S Metagenomics</em></li></ul> | <div><figure><img src="https://2084401275-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2FfjuebS41N49G1Eh55hP7%2Fuploads%2Fgit-blob-9938b879db457f0fb0d74555078fc9a928670fb9%2Fnextseq-16s-metagenomics-gp-of-defaults.png?alt=media" alt=""><figcaption></figcaption></figure></div> |

**Step File Placeholders**

1. From Configuration, select the Lab Work tab.
2. Select the **Denature and Dilute** Master Step.
3. Rename the following Step File Placeholders under Record Details milestone:
   * from **bcl2fastq SampleSheet** to **Samplesheet**
   * from **bcl2fastq SampleSheet Generation Log** to **Samplesheet Generation Log**

<figure><img src="https://2084401275-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2FfjuebS41N49G1Eh55hP7%2Fuploads%2Fgit-blob-aeb1d1929c5d2784d47c90a53a1738de9214d85f%2Fnextseq-dnd-step-file-placeholder.png?alt=media" alt=""><figcaption></figcaption></figure>

### Replace Template Files

Replace the following template files at */opt/gls/clarity/extensions/conf/driverfiletemplates/*:

* BCL2FASTQ\_Reverse\_Complement\_Samplesheet.csv with NextSeq\_ReverseComplement\_Samplesheet.csv

{% file src="<https://2084401275-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2FfjuebS41N49G1Eh55hP7%2Fuploads%2Fgit-blob-7ac2777fd758c421a8c44a746b2a3ab26a9b3c34%2FNextSeq_ReverseComplement_Samplesheet.csv?alt=media&token=8b34b076-b3c5-4b50-a93a-490a83929fe9>" %}

* BCL2FASTQ\_Samplesheet.csv with NextSeq\_Samplesheet.csv

{% file src="<https://2084401275-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2FfjuebS41N49G1Eh55hP7%2Fuploads%2Fgit-blob-3090415e7823c4213e10c8d65916e7b78f40e471%2FNextSeq_Samplesheet.csv?alt=media&token=b611a67e-a60c-4f08-8f2b-8493a439d8e7>" %}

{% hint style="info" %}
Contact Illumina Support to replace the files if required.
{% endhint %}