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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), MiSeq, HiSeq 2500, or HiSeq 2000 is specific to the paired-end flow cell. Seven additional chemistry-only cycles are required to read the i5 index. This step uses the resynthesis mix, a paired-end reagent, during the Index 2 Read process.
  1. 1.
    Read 1Read 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. 2.
    Index Read preparationThe Read 1 product is removed and the Index 1 (i7) sequencing primer is annealed to the same template strand.
  3. 3.
    Index 1 (i7) ReadFollowing Index Read preparation, the Index 1 (i7) Read performs up to 20 cycles of sequencing.
  4. 4.
    Index 2 (i5) ReadThe 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 up to 20 cycles of sequencing.
  5. 5.
    Read 2 resynthesisThe Index Read product is removed and the original template strand is used to regenerate the complementary strand. Then, the original template strand is removed to allow hybridization of the Read 2 sequencing primer.
  6. 6.
    Read 2Read 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, NextSeq 500/550, NextSeq 1000/2000, NovaSeq 6000 (with v1.5 reagents), HiSeq X, or HiSeq 3000/4000 performs the Index 2 Read after the Read 2 resynthesis step. This workflow requires a reverse complement of the Index 2 (i5) primer sequence compared to the primer sequence used on other Illumina platforms. The Index 2 sequencing primer is part of the dual-indexing primer mix for iSeq 100, MiniSeq, and NextSeq. 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. 1.
    Read 1Read 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. 2.
    Index Read preparationThe Read 1 product is removed and the Index 1 (i7) sequencing primer is annealed to the same template strand.
  3. 3.
    Index 1 (i7) ReadFollowing Index Read preparation, the Index 1 (i7) Read performs eight cycles of sequencing.
  4. 4.
    Read 2 resynthesisThe Index 1 Read product is removed and the original template strand is used to regenerate the complementary strand. Then the original template strand is removed to allow hybridization of the Index 2 (i5) sequencing primer.
  5. 5.
    Index 2 (i5) ReadFollowing Read 2 resynthesis, the Index 2 (i5) Read performs eight cycles of sequencing.
  6. 6.
    Read 2 preparationThe Index 2 Read product is removed and the Read 2 sequencing primer is annealed to the same template strand.
  7. 7.
    Read 2Read 2 follows the standard paired-end sequencing protocol using SBS reagents.
Refer to the Indexed Sequencing Overview Guide for more information.
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