Considerations when selecting an adapter design
Sequences for specific NGS platforms: During library preparation, adapters are attached (by ligation or PCR) to 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 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.
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 errors identified 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 unique dual sample index design for detection of low-frequency variants.
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. IDT’s 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.