> For the complete documentation index, see [llms.txt](https://knowledge.illumina.com/llms.txt). Markdown versions of documentation pages are available by appending `.md` to page URLs; this page is available as [Markdown](https://knowledge.illumina.com/library-preparation/general/library-preparation-general-reference_material-list/000002099.md).
# 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. Generally, 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. See [Guidelines for reverse complementing i5 sequences for demultiplexing](/software/general/software-general-reference_material-list/000001800.md) for guidance on the appropriate i5 index orientation to use depending on the sequencer and analysis workflow. **Index First Sequencing (MiSeq i100 Series Only)** The MiSeq i100 Series instrument 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 runs on the **NovaSeq 6000 (with v1 reagents**),\*\*\*\* **MiniSeq (with rapid reagent kits), and** **MiSeq (legacy platform)**. 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.
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 500/550, NextSeq 1000/2000, NovaSeq 6000 (with v1.5 reagents)**, and **NovaSeq X Series** 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 500/550, NextSeq 1000/2000, 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.
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.
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](https://support.illumina.com/downloads/indexed-sequencing-overview-15057455.html) guide for more information.
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| *For any feedback or questions regarding this article (Illumina Knowledge Article #2099), contact Illumina Technical Support* [*techsupport@illumina.com*](mailto:techsupport@illumina.com?subject=Question%2FFeedback%20Regarding%20Illumina%20Knowledge%20Article%20#000001800%20-%20Library%20Preparation%20\&body=Dear%20Illumina%20Technical%20Support,%0D%0A%0D%0A)*.* |
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