Dual indexing in general, which uses both i7 (Index 1) and i5 (Index 2) indices, enables multiple RNA-Seq libraries to be multiplexed in a single sequencing lane or run. Using dual indexing, the combination of both i5 and i7 indices determines the unique barcode combination for sample identification and increases the accuracy of read identification when the sequencing data is demultiplexed.
There are two types of dual indexing: combinatorial dual indexing, and unique dual indexing (Fig. 1).
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Figure 1 | Comparison of combinatorial and unique dual indexing strategies. Regardless of the strategy used, each library (A – F) must have a unique combination of i5 (purple, 5001-5006) and i7 indices (yellow, 7001 – 7006). When using combinatorial dual indexing, the same i5 and i7 indices can be used for different libraries as long as the other index is unique (e.g., libraries B and C both have i5 index 5002, but different i7 indices (7002 or 7003)). For truly unique dual indexing, each i5 and i7 index can be used only once, for a single library. In this way only a maximum of 96 uniquely dual-indexed libraries can be multiplexed in a single sequencing lane or run.
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