Indexing FAQs

• For single-read flow cells, dual indexing introduces 16 additional cycles of sequencing: 8 cycles for the Index 1 (i7) Read and 8 cycles for the Index 2 (i5) Read.
• For paired-end flow cells, dual indexing introduces 23 additional cycles of sequencing: 8 cycles for the Index 1 (i7) Read, 8 cycles for the Index 2 (i5) Read, and 7 nonimaging, chemistry-only cycles at the beginning of the i5 Read.

There are several advantages of using dual indexes:

• Dual indexes increase the accuracy of sample identification by adding another identifier to minimize issues caused by index-hopping events
• Dual-indexed libraries allow higher multiplexing and the ability to run more samples per lane
• Dual-indexed libraries are sequenced with 2 additional reads, termed Index 1 (i7) Read and Index 2 (i5) read

Index hopping (or index switching) is a known phenomenon where sequencing reads are assigned to incorrect indexes, potentially leading to misalignment of reads or incorrect assumptions in downstream analyses. Using unique dual indexes minimizes index hopping and is recommended for sensitive applications.

The following steps may help you mitigate these issues:

• Use dual-indexing kits, rather than single-indexing kits, in library preparation
• Increase the percentage of PhiX spike-in, which will reduce the number of adjacent clusters with indexes
• Lower the amount of library loaded to decrease the cluster density

• For single reads, only one end of the nucleic acid fragment is sequenced at your indicated length (e.g., 50-, 75-, or 100-bp reads)
• For paired-end reads, nucleic acid fragments are sequenced at the indicated length first from one end, then from the other, allowing the detection of insertions, deletions, and genomic rearrangements

Yes, paired-end sequencing can be done with single-indexed libraries. This is because single vs. dual-indexed adapters have nothing to do with sequencing due to how the sequencing is performed. Sequencing is done using Read Primer 1 or Read Primer 2, both of which are present in all libraries. You can also do single reads even if you use dual-indexed kits.

Yes, both index sequences can be obtained from single-read runs. Please select the appropriate workflow based on the type of flow cell (e.g., single-read or paired-end) so that the correct chemistry is used. If you are using a single-read flow cell, the TruSeq® Dual Index Sequencing Primer Box, Single-Read (Illumina, Cat. # FC-121-1003) is necessary to sequence both index reads.

Any samples can be multiplexed. However, to get the best results, you should consider the following:

• Fragment lengths from each sample must match. Libraries of different lengths will hybridize to the flow cell at different efficiencies, which can lead to uneven sample coverage.
• All samples must use the same barcoding strategy (e.g., do not mix and match single-indexed samples with dual-indexed samples).
• All samples must be barcoded with the same type of kit (e.g., you cannot mix TruSeq® barcoded samples with Nextera® barcoded samples).
• Illumina-equivalent indexes must be known if using non-Illumina library prep kits. Alternately, you must know the index sequences and use them as a custom barcode.

For HiSeq®/MiSeq® instruments, Illumina uses a red laser/LED to sequence bases A and C, and a green laser/LED to sequence bases G and T. For each cycle, both the red and green channel need to be read to ensure proper image registration (e.g., A or C and G or T must be in each cycle). If this color balance is not maintained, sequencing the index read could fail.

For NovaSeq^TM, NextSeq®, and MiniSeq^TM instruments that use two-color chemistry, valid index combinations must include some indexes that do not start with ‘GG' in the first two cycles.