# Guidelines for reverse complementing i5 sequences for demultiplexing

### **Article Quick Reference Table**This table describes how to enter the i5 sequence, Forward (F) or Reverse Complement (RC), depending on the instrument and software used.**Notes**

* LRM: Local Run Manager
* BSSH: BaseSpace Sequence Hub
* IRM: Illumina Run Manager

| Instrument        | BSSH Run Planning\* or BSSH Planned Run Requeue | On Instrument Setup / Analysis | Standalone bcl2fastq or BSSH FASTQ Generation | BCL Convert App                                           | Standalone BCL Convert |
| ----------------- | ----------------------------------------------- | ------------------------------ | --------------------------------------------- | --------------------------------------------------------- | ---------------------- |
| MiSeq             | F                                               | F (LRM)                        | F                                             | F                                                         | F                      |
| MiniSeq\*\*\*     | F                                               | F (LRM)                        | RC                                            | <p>F (v2 Sample Sheet)\*\*</p><p>RC (v1 Sample Sheet)</p> | RC                     |
| iSeq 100          | F                                               | F (LRM)                        | RC                                            | <p>F (v2 Sample Sheet)\*\*</p><p>RC (v1 Sample Sheet)</p> | RC                     |
| NextSeq 500/550   | F                                               | F (LRM)                        | RC                                            | <p>F (v2 Sample Sheet)\*\*</p><p>RC (v1 Sample Sheet)</p> | RC                     |
| NextSeq 1000/2000 | F                                               | F (DRAGEN BCL Convert)         | RC                                            | F                                                         | F                      |
| NovaSeq 6000      | F                                               | NA                             | RC                                            | <p>F (v2 Sample Sheet)\*\*</p><p>RC (v1 Sample Sheet)</p> | RC                     |
| NovaSeq X/X Plus  | F                                               | F (DRAGEN BCL Convert / IRM)   | RC                                            | F                                                         | F                      |
| MiSeq i100 Series | F                                               | F (DRAGEN BCL Convert / IRM)   | <p>F (index-first)<br>RC (read-first)</p>     | F                                                         | F                      |

\*BSSH Run Planning does not have the option to generate a sample sheet for standalone BCL convert analysis. To use BSSH Run Planning to create a v2 sample sheet for iSeq 100, MiniSeq, NextSeq 500/550, and NovaSeq 6000 instruments for Standalone BCL Convert, select the Library kit option "Not Specified" and input the i5 index in the reverse complement orientation.

\*\*The v2 sample sheet must include the "IndexOrientation,Forward" in the Header, for analysis software to reverse complement the i5 sequences. If this line is not present, the i5 index should be in the reverse complement orientation in the sample sheet.

\*\*\*MiniSeq Rapid Kits: Always forward orientation

**Full Article**

### \*\*Instrument Sequencing Orientation:\*\*When performing dual indexing, all Illumina sequencers read the first index (i7) in the forward orientation; however, the second index (i5) is read in different orientations depending on the instrument and chemistry, in particular, differences between when the paired-end turnaround and i5 read take place:

* On the MiSeq, the MiniSeq Rapid kits and the MiSeq i100 (index-first sequencing) the i5 sequence is read in the **forward orientation**.
* On the iSeq 100, MiniSeq, NextSeq 500/550, NextSeq 1000/2000, NovaSeq 6000/6000Dx, the NovaSeq X/X Plus, and the MiSeq i100 Series (read-first sequencing) the i5 sequence is read in the **reverse complement orientation**.

Most Illumina sample sheet generation software assume that the **i5 sequence is always in the forward orientation** and will automatically create the sample sheet with the i5 index orientation in the correct orientation. However, there are exceptions for Standalone FASTQ generation software (bcl2fastq and BCL convert) which perform less processing. There are also special considerations when pairing an instrument with a fastq generation software that was not written for that instrument. Below describes the possible scenarios.

### \*\*Index Orientation in Sample Sheet:\*\*The following provides guidance on when to reverse complement the i5 sequences when analyzing iSeq 100, MiniSeq, NextSeq 500/550, NextSeq 1000/2000, NovaSeq 6000/6000Dx, NovaSeq X/X Series, or MiSeq i100 Series data.

### **Generating FASTQ files from a v1 Sample Sheet**

* **Manually creating a v1 sample sheet** for use with **bcl2fastq**, or uploading the sample sheet to **BaseSpace as a manual mode run (BaseSpace FASTQ Generation):** enter the i5 sequence in the orientation the i5 sequence is read (See above). Runs started in manual mode have their data and supplied sample sheet directly passed to bcl2fastq/BaseSpace FASTQ Generation with no further processing.
* **Local Run Manager (LRM):** i5 sequences must be entered into the software in the **forward orientation**. LRM will detect the instrument type from the run files and create a final sample sheet with the i5 indexes in the correct orientation for that platform.
  * If the sequence is reverse complemented this will only be shown in the SampleSheetUsed.csv file or the SampleSheet.csv file created by LRM located in the Analysis folder.
* **BSSH BCL Convert app:** The BCL convert app in BaseSpace can be launched with a v1 Sample Sheet. For the iSeq 100, MiniSeq, NextSeq 500/550, and NovaSeq 6000 the i5 index should be listed in the orientation that the instrument sequences the read (see table). If using a v1 Sample Sheet in the BCL Convert app for the NextSeq 1000/2000, NovaSeq X/X Plus, and MiSeq i100, the index orientation should be in the forward orientation.

### **Generating FASTQ files from a v2 Sample Sheet**

* #### **BCL convert app and BSSH Run Planning:**
  * NextSeq 1000/2000, NovaSeq X/X Plus, and MiSeq i100 Series i5 indexes will correctly be output in the forward orientation for later downstream processing. Custom kits should also have the i5 indexes entered in the forward orientation.
  * For all other platforms, Run Planning will add a line to the Header section of the v2 sample sheet (IndexOrientation,Forward) and the i5 will be written in the forward orientation and processed correctly downstream.
* #### **Standalone BCL Convert:**
  * Enter the **reverse complement of the i5 sequence** for iSeq 100, MiniSeq, NextSeq 500/550, and the NovaSeq 6000.
  * The MiSeq platform is sequenced in the forward orientation and the i5 index in the sample sheet should be in the **forward orientation.**
  * For the NextSeq 1000/2000, NovaSeq X/X Plus and MiSeq i100 Series use the **i5 in the forward orientation**. The RunInfo.xml files for the NextSeq 1000/2000, NovaSeq X/X Plus and MiSeq i100 Series contain a flag telling BCL Convert to convert the sample sheet i5 sequence to the reverse complement automatically, as necessary.
* #### **Illumina Run** **Manager (IRM)**
  * On the NovaSeq X/X Plus and MiSeq i100 expects i5 indexes to be input in the **Forward orientation.**

### **Color Balance Considerations**When assessing index sets for color balance, the sequence *observed* by the instrument must be assessed, regardless of the orientation used for the sample sheet. For the i7 index, always evaluate color balance using the "i7 bases for sample sheet" column orientation. For the i5 index, assess color balance using the forward orientation for MiSeq runs, MiniSeq Rapid Kits, and MiSeq i100 index-first runs and the reverse-complement orientation for all other instruments.

### **Important Caveats for MiniSeq Rapid Kits**

* The MiniSeq Rapid kits implemented a recipe change that uses the grafted oligo for the i5 index priming, so it is read in the forward orientation similar to a MiSeq.
* If running in manual mode and using bcl2fastq or BCL Convert, or uploading the sample sheet to BaseSpace as a manual mode run and attaching a sample sheet, enter the i5 sequence in the **forward direction**. Runs uploaded in manual mode have their data directly passed to bcl2fastq with no further processing.
* For Local Run Manager (LRM), input the sequences in the **reverse complement** when setting up the run so the software automatically creates the **forward orientation** as needed for analysis. This is the opposite of most of our guidance and is only for the MiniSeq Rapid kits.

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