Considerations when selecting an adapter design
IDT provides xGen™ UDI-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 important 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 sequencing instrument: 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 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 adapters from IDT are base-balanced to prevent GC bias and color-balanced for 2 and 4-color Illumina instruments. 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 enable precise quantification, since PCR duplicates will share the same insert and UMI tag. UMI deduplication is useful for RNA-seq gene expression analysis and other quantitative sequencing methods. UMIs have also 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 (Figure 2). 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 detection of low-frequency variants. IDT Custom NGS Adapters can also be configured with UMIs.