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

• IEM: Illumina Experiment Manager

• BSSH: BaseSpace Sequence Hub

Instrument	Run setup	Standalone bcl2fastq or BSSH FASTQ Generation	Standalone BCL Convert
MiSeq	F (LRM, IEM)	F	F
MiniSeq*	F (LRM, Preptab, IEM)	RC	RC
iSeq 100	F (LRM, IEM)	RC	RC
NextSeq 500/550	F (LRM, Preptab, IEM)	RC	RC
NextSeq 1000/2000	F (local, BSSH Run Planning)	RC	F
NovaSeq 6000 (v1.5 kits)	RC (local), F (BSSH Run Planning)	RC	RC
NovaSeq X/X Plus	F (local, BSSH Run Planning)	RC	F
MiSeq i100 Series	F (local, BSSH Run Planning)	F (index-first) RC (read-first)	F

*MiniSeq Rapid Kits: Always forward orientation

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When performing dual indexing, all Illumina sequencers read the first index (i7) in the same direction; 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, HiSeq 2000/2500, 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 500/500, NextSeq 1000/2000, HiSeq 3000/4000/X systems, NovaSeq 6000 (v1.5 reagents), the NovaSeq X/X Plus and the MiSeq i100 Series (read-first sequencing) the i5 sequence is read in the reverse complement orientation. See the section below for information on when to use which orientation of the i5 sequence for these platforms depending on the analysis software used.

When demultiplexing, the final sample sheet provided to the FASTQ generation software needs to have the i5 index in the correct orientation for that platform. However, most Illumina sample sheet generation software handles this automatically. This means that these software work on the assumption that your i5 sequence is always in the forward orientation and will automatically create the reverse complement of the sequences provided for the appropriate instrument type.

The following provides guidance on when to reverse complement and when to not reverse complement the i5 sequences when analyzing iSeq, MiniSeq, NextSeq 500/550, NextSeq 1000/2000, HiSeq 3000/4000/X, NovaSeq 6000 (v1.5 reagents), NovaSeq X/X Plus, or MiSeq i100 Series (index-first sequencing) data.

• When manually creating a v1 sample sheet for these systems with bcl2fastq, or uploading the sample sheet to BaseSpace as a manual mode run (BaseSpace FASTQ Generation) and attaching a sample sheet, enter the reverse complement of the i5 sequence. Runs uploaded in manual mode have their data directly passed to bcl2fastq with no further processing.

  • The NextSeq 1000/2000, NovaSeq 6000, and NovaSeq X/X Plus also support attaching a v2 sample sheet in manual mode to BSSH for auto launching BCL Convert instead. For generation and use of v2 sample sheets for BCL Convert, see the section on BaseSpace Run Planning below.

• When using BCL Convert either locally or in BaseSpace enter the reverse complement of the i5 sequence for the above platforms except for the NextSeq 1000/2000, NovaSeq X/X Plus and MiSeq i100 Series, which uses 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. This flag is not recognized by bcl2fastq, and RunInfo.xml files for other platforms do not contain the flag.

  • See exception for when creating a v2 sample sheet in BSSH Run Planning below.

• When using Illumina Experiment Manager (IEM), the sequences displayed are what is entered into the final sample sheet. Kit definitions have the i5 in the forward orientation. The i5 sequences will be appropriately reverse complemented and displayed in the IEM interface based on instrument/reagent type.

• For BaseSpace Sequence Hub Preptab or Local Run Manager (LRM) sequences are always displayed in the forward orientation when setting up the run and the software automatically creates the reverse complement as needed for analysis. If the sequence is reverse complemented this will only be shown in the SampleSheetUsed.csv file (Preptab and LRM) or the SampleSheet.csv file created by LRM at the analysis start.

For IEM, Preptab, or LRM, this holds true for all built-in kits as well as custom kits: i5 sequences must be entered into these software in the forward orientation in all circumstances and for all instrument types.

• 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.

• IEM will use the instrument/reagent type selected in the setup wizard to generate the final sample sheet with the i5 sequences in the correct orientation.

• Preptab will always reverse complement the provided i5 sequences as it is used with NextSeq and MiniSeq data.

For Run Planning in BaseSpace, the output is always a v2 sample sheet for BCL Convert:

• 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 other supported platforms (such as the NovaSeq 6000), 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.

• If generating v2 sample sheets for instruments other than the ones listed, confirm the final sample sheet has the i5 index in the correct orientation as described above.

Important Caveats for MiniSeq Rapid Kits

• The MiniSeq Rapid made 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.

Color Balance ConsiderationsWhen 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 asses 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 and MiniSeq runs with rapid reagents, and the reverse-complement orientation for all other instruments.

For any feedback or questions regarding this article (Illumina Knowledge Article #1800), contact Illumina Technical Support [e-mail].