xGen™ NGS Adapters & Indexing Primers for Illumina^® sequencing

Considerations when selecting an adapter and primer design

IDT provides extensive solutions for stocked indexing solutions. Adapters are available with single index, unique dual index (UDI), and unique molecular identifiers (UMI) found in the IDT xGen UDI-UMI indexes (Figure 2). In addition, the adapters can be designed to include:

• Index lengths of 8 nt or 10 nt
• Combinatorial or unique dual indexes (CDI or UDI)
• Multiplex capacity up to 1536 unique indexes
• Indexing primers designed for performing enzymatic library normalization using our proprietary Normalase technology.

In addition to the stocked products, IDT provides the ability for even greater flexibility and control, by using the Custom Adapter Configurator tool to guide you through designing your own Custom NGS Adapters.

Sample indexing

Sample indexes (or indices) enable multiple samples to be sequenced together (i.e., multiplexed) on the same instrument flow cell or chip. Each sample index, typically 8–10 bases, is specific to a given sample library and is used for de-multiplexing during data analysis to assign individual sequence reads to the correct sample. Predesigned indexes from IDT are balanced to prevent GC bias and color-balanced for 2- and 4-color Illumina instruments. Adapters and primers may contain single or dual sample indexes depending on the number of libraries combined and the level of specification desired. Illumina recommends using unique dual indexes (UDIs) as a method to mitigate errors introduced by index-hopping. UDIs are particularly important when using instruments with patterned flow cells, such as the NovaSeq^® system from Illumina.

IDT provides several indexing options with its Custom NGS Adapters. These include both IDT and Illumina 8- and 10-base index series, as well as the ability to upload your own index files.

Molecular barcoding

Unique molecular identifiers (UMIs) provide reliable error correction. UMIs are short sequences that incorporate a unique barcode onto each molecule within a given sample library.

• UMIs are used to identify PCR duplicates. PCR duplicates created during library amplification steps will share the same insert and UMI tag.
• UMIs have also been shown to reduce the rate of false-positive variant calls and increase variant detection. By incorporating individual barcodes on each original DNA fragment, variant alleles present in the original sample (true variants) can be distinguished from errors introduced during library preparation, target enrichment, or sequencing. Any identified errors can be removed by bioinformatics methods before final data analysis.

Adapters that contain UMIs, such as the xGen UDI-UMI Adapters, are available with a UDI design for identification of low-frequency variants. IDT Custom NGS Adapters can also be configured with UMIs.

Adapter manufacturing

Stringent manufacturing methods are critical for producing high-quality NGS adapters. Substandard manufacturing practices can lead to low-purity adapters or adapter cross-contamination, either of which will negatively impact sequencing results. The IDT proprietary TruGrade™ process uses state-of-the-art synthesis and purification methods, designed specifically for NGS adapters. IDT also offers GMP^‡ ISO 13485 grade adapter manufacturing for clinical research applications.

In addition to stringent manufacturing methods, IDT also performs functional NGS testing on each new batch of stocked adapter and indexing primer products to ensure they produce sequencing libraries that meet our high-quality standards.

Our leadership in NGS adapter synthesis made us the clear choice when Illumina sought a partner to develop the next generation of indexed adapters to improve sample multiplexing. These adapters contain Illumina’s unique dual indexes (UDIs), which mitigate sample misassignment due to index hopping. We manufacture UDI adapters for Illumina and are also licensed to sell your own custom designs containing these UDI sequences.

For more information, or to order custom adapters, please contact us.

Choose from a variety of options that personalize the adapters to your NGS workflow for Illumina sequencing. Order Custom NGS Adapters when you need to:

• Scale up your experiments—get the delivery format (volume, plate, single-use) and volume-pricing that best suit your NGS study.
• Expand your set of barcodes to increase sample throughput—augment limited sets of stocked adapters. Our design tool will support uploading up to 2000 custom index sequences!
• Supplement commercial library kit components—create your own custom library prep workflow that fits your experimental design.
  

The Custom Adapter Configurator tool helps you design the adapters best suited to your experiments by walking you through these choices:

• Library prep method
• Configuration (SI, CDI, UDI, UDI-UMI)
• Attachment style
• Indexing strategy
• Number of indexes
• Size (reaction number or nmol)
• Delivery format (tubes, plates)
• Methylated/unmethylated
• TruGrade^TM processing
  

Table 2. Suggested barcode configuration based on NGS application.