Considerations when selecting an adapter design
IDT provides xGen Dual Index UMI Adapters, xGen Stubby Adapter and Unique Dual Index Primers, and a Custom Adapter Configurator tool that guides you through design of Custom NGS Adapters. Below we describe some of the design considerations you will want to take into account.
Sequences for specific NGS platforms: During library preparation, adapters are attached (by ligation, PCR, or tagmentation) to the fragments of each sample library. Adapters include platform-specific sequences for fragment recognition by the sequencer: for example, the P5 and P7 sequences (Figure 1) enable library fragments to bind to the flow cells of Illumina platforms. Each NGS instrument provider uses a specific set of sequences for this purpose. IDT manufactures adapters for all major NGS platforms.
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 6–10 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. Adapters may contain single or dual sample indexes depending on the number of libraries combined and the level of accuracy 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.
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 the highest levels of error correction and accuracy. UMIs are short sequences, often with degenerate bases, that incorporate a unique barcode onto each molecule within a given sample library. UMIs have been shown to reduce the rate of false-positive variant calls and increase sensitivity of 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 Dual Index UMI Adapters, are available with a UDI design for detection 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 grade adapter manufacturing for clinical applications.
Choose IDT adapter products based on the flexibility your experimental workflow requires. Click on the xGen Dual Index UMI Adapters—Tech Access, the xGen Stubby Adapter and UDI Primers, or the Custom NGS Adapters headings in Table 1 to review product offerings. The Custom NGS Adapters provide a step-by-step tool to help you configure adapters best suited for your research.