Sample Pooling: How to Choose Appropriate Indexed Sequencing Adapters

Low-Plex Sample Pooling:

In order to get the best sequencing results possible, it is critical to select appropriate indexed adapters. What does this mean exactly and why is it so important?

The Illumina HiSeq and MiSeq sequencing instruments use sequencing by synthesis (SBS) technology in conjunction with fluorescence-based imaging of every nucleotide. In other words, when your sample is being “read” on a HiSeq or MiSeq, it is actually being copied base-by-base within the flowcell using fluorescently-tagged nucleotide bases. After each individual base addition the flowcell is imaged, and clusters emitting fluorescence are counted. Illumina instruments use two fluorescent lasers: green for bases G and T and red for bases A and C.

For each lane of a flowcell with more than one Illumina indexed adapter, it is important that at each base both the red and green laser are activated. DNA and RNA samples are highly diverse, so for data reads this mechanism is inconsequential. Indexes, however, are intentionally uniform. Therefore, in order to assure that indexes of low-plex lanes (2-4 unique indexes) are accurately sequenced it is crucial to select adapter combinations that result in each laser reading a base during each cycle. For instance, if selecting two indexes to be sequenced together:

example of appropriate Index pairing for 2-plex pool

Figure 1. Appropriate Index pairing for 2-plex pool. The color of each base represents the color of the Illumina system laser that registers bases of this type. Notice how at each position of the six-base-pair index of index AD006 is a different color from each base of index AD0012 (represented by the vertical blue line between bases of each index). Each laser is thus able to read a base during each cycle.

example of Ineffective Index pairing for 2-plex pool.

Figure 2. Ineffective Index pairing for 2-plex pool. The color of each base represents the color of the Illumina system laser that registers bases of this type. Thease two indexes would result in poor Index Sequencing reads due to the fact that at positions 1,3 and 4 only the red laser would read a base and at 2 only the green laser (represented by red X between bases).

Keep these rules in mind when selecting indexes for low-plex pooling (2-4 libraries per lane). Failure to select adapters with appropriate laser color balance could result in low quality scores or even Index Read sequencing failure.

For more information see the Illumina Library Prep Pooling Guidelines

High-Plex Sample Pooling:

It is important that each index of a high-plex pool is unique enough to account for potential mismatches. When selecting indexed sequencing adapters for pooling of five or more libraries it is important to select indexes with at least 3 bases difference (edit distance).