Indexed Sequencing Overview for Paired End Flow Cells

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, 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.

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 iSeq 100, MiniSeq (with standard reagent kits), NextSeq systems, NovaSeq 6000 (with v1.5 reagents), NovaSeq X/X Plus, 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/X Plus. For HiSeq X, HiSeq 4000, and HiSeq 3000, the Index 2 sequencing primer is part of HP14, an indexing primer mix that contains primers for both index reads.

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. 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. 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.

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