Indexed Sequencing Overview for Paired End Flow Cells

Background Illumina sequencing platforms allow for sequencing most dual-index libraries, though the workflow used to sequence the i5 Index Read varies depending on the platform. Legacy platforms prime i5 Index Read with use of a grafted oligo. In contrast, newer platforms prime i5 Index Read with a solution-based primer. This affects the sequencing orientation of the i5 Index Read, either forward or reverse-complement, and can affect demultiplexing if the Index is not listed in the correct orientation. When planning sequencing runs in Local Run Manager or BaseSpace Run Planning, Illumina recommends inputting the i5 Index sequence in the forward orientation as the software automatically reverse-complements the i5 Index sequence when necessary. Index First Sequencing (MiSeq i100 Series Only) The MiSeq i100 Series instruments uses the Index First sequencing workflow by default. Under this workflow, both the i7 and i5 Index reads are sequenced before Sequencing Reads 1 and 2. Both Indexes are sequenced in the forward orientation similar to Workflow A, though they are sequenced using solution-based primers, similar to Workflow B. Therefore, Index First sequencing represents a hybrid of both workflows.

Workflow A (forward strand):

The chemistry applied to the Index 2 Read during a paired-end dual-indexed run on the NovaSeq 6000 (with v1 reagents),**** MiniSeq (with rapid reagent kits), MiSeq (legacy platform), HiSeq 2500, or HiSeq 2000 is specific to the paired-end flow cell. Reading the i5 index requires seven additional chemistry-only cycles. This step uses the resynthesis mix, a paired-end reagent, during the Index 2 Read process.

1. Read 1—Read 1 follows the standard Read 1 sequencing protocol using SBS reagents. The Read 1 sequencing primer is annealed to the template strand during the cluster generation step.

2. Index Read preparation—The Read 1 product is removed and the Index 1 (i7) sequencing primer is annealed to the same template strand.

3. Index 1 (i7) Read—Following Index Read preparation, the Index 1 (i7) Read is performed. The read length depends on the system and run parameters, see Maximum read length for Illumina sequencing platforms for limits.

4. Index 2 (i5) Read—The Index 1 (i7) Read product is removed and the template anneals to the grafted P5 primer on the surface of the flow cell. The run proceeds through an additional 7 chemistry-only cycles (no imaging occurs), followed by Index 2 (i5) Read sequencing in the forward orientation.

5. Paired End turnaround—The original template strand is used to regenerate the complementary strand followed by limited cycle re-amplification. Then, the original template strand is removed to allow hybridization of the Read 2 sequencing primer.

6. Read 2—Read 2 follows the standard paired-end sequencing protocol using SBS reagents.

Workflow B (reverse complement):

A dual-indexed sequencing run on the MiSeq i100 Series (Read-First workflow), iSeq 100, MiniSeq (with standard reagent kits), NextSeq systems, NovaSeq 6000 (with v1.5 reagents), NovaSeq X Series, HiSeq X, or HiSeq 3000/4000 performs the Index 2 Read after the Read 2 resynthesis step. This workflow requires a dedicated Index 2 (i5) primer sequence, which sequences the index 2 (i5) in the reverse complement orientation compared to the Workflow A Illumina platforms. The Index 2 sequencing primer is part of the dual-indexing primer mix for iSeq 100, MiniSeq (with standard reagents kit), NextSeq systems, NovaSeq 6000 (with v1.5 reagents), and NovaSeq X Series.

1. Read 1—Read 1 follows the standard Read 1 sequencing protocol using SBS reagents. The Read 1 sequencing primer is annealed to the template strand during the cluster generation step.

2. Index Read preparation—The Read 1 product is removed and the Index 1 (i7) sequencing primer is annealed to the same template strand.

3. Index 1 (i7) Read—Following Index Read preparation, the Index 1 (i7) Read is performed.

   1. The read length depends on the system and run parameters, see Maximum read length for Illumina sequencing platforms for limits.

4. Paired End turnaround—The Index 1 Read product is removed and the original template strand is used to regenerate the complementary strand followed by limited cycle re-amplification. Then, the original template strand is removed to allow hybridization of the Index 2 Read sequencing primer.

5. Index 2 (i5) Read—Following paired end turnaround, the Index 2 (i5) Read is performed. As sequencing occurs after Paired-end Turnaround, the sequence is read in the reverse-complement orientation.

   1. The read length depends on the system and run parameters, see Maximum read length for Illumina sequencing platforms for limits.

6. Read 2 preparation—The Index 2 Read product is removed and the Read 2 sequencing primer is annealed to the same template strand.

7. Read 2—Read 2 follows the standard paired-end sequencing protocol using SBS reagents.

Refer to the Indexed Sequencing on Illumina Systems guide for more information.

For any feedback or questions regarding this article (Illumina Knowledge Article #2099), contact Illumina Technical Support [email protected].