Overview

This resource lists the adapter sequences for Illumina library prep kits.

The library prep kit support pages on the Illumina support site provide additional resources. These resources include software, training, and compatible products. Always check support pages for the latest versions.

When dual indexing, the orientation of the Index 2 (i5) sequence varies by platform. The orientation depends on whether the Index 2 primer is grafted to the flow cell or is in solution.

MiSeq and HiSeq 2000/2500 i5 adapters:

• Adapters are grafted to the flow cell and read in the forward orientation.
• Enter the i5 bases on the sample sheet in forward orientation.

iSeq 100, MiniSeq, NextSeq 500/550, NextSeq 1000/2000, HiSeq 3000/4000, NovaSeq 6000 (v1.5 reagents), and NovaSeq X/X Plus i5 adapters:

• Adapters are in solution and read in the reverse complement orientation.
• If manually creating a sample sheet to be analyzed in bcl2fastq, enter the i5 bases in reverse orientation.
• If using BaseSpace Run Planning, Illumina Experiment Manager, BaseSpace Prep Tab, or Local Run Manager, enter the i5 bases on the sample sheet in forward orientation. The software automatically creates the reverse complement for analysis.
• If using DRAGEN/BCL Convert for NextSeq 1000/2000 or NovaSeq X/X Plus using a manually created v2 sample sheet, enter the i5 bases in forward orientation. DRAGEN/BCL Convert automatically uses the reverse complement for analysis. For all other platforms, use the reverse complement orientation of the i5 sequence.

For Illumina kits where adapter trimming is recommended, the sequence of the adapter to be trimmed is listed.

When read length exceeds the DNA insert size, sequences corresponding to the library adapters can be present at the 3’ end of the reads. Trimming the adapter sequence from the FASTQ file improves alignment accuracy and performance in secondary analysis. Reads start at the beginning of the DNA insert. Therefore, adapter sequences will not be present at the 5’ end of reads.

For Illumina Experiment Manager, BaseSpace Run Planning, BaseSpace Prep Tab, and Local Run Manager, enter the sequence for adapter trimming in the sample sheet generated for Illumina kits. For specific trimming settings, or for guidance on creating custom kits, refer to the software support pages on the Illumina support site.