Considerations when selecting an adapter design

IDT provides xGen Dual Index UMI Adapters, xGen Stubby Adapter and UDI Primer Pairs, 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 Primer Pairs, 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.

* For bisulfite sequencing, see our FAQ for additional information.

Applications

The Lotus DNA Library Prep Kit produces high-quality next generation sequencing (NGS) libraries that are suitable for many applications including:

• Whole genome sequencing (WGS)
• PCR-free sequencing
• Detection of germline inherited single nucleotide variations (SNVs) and insertions/deletions (indels)
• Hybridization capture of target sequences (e.g., the exome or transcripts of interest)
• Low frequency somatic variation detection of SNVs and indels
• RNA-seq starting with full-length, double-stranded cDNA input
• Metagenomic sequencing

Method

This kit involves minimal enzymatic incubations and bead-based purification steps, thereby reducing sample handling and overall library preparation time to under 2 hours. The major steps are depicted in Figure 1 and are summarized as follows:

• Enzymatic preparation. Fragmentation, end-repair, and dA-tailing of dsDNA are all performed in a single reaction.
• Ligation of either full-length or stubby P5 and P7 adapters. When using full-length adapters, the final PCR step is optional and can be used to increase library yield. However, stubby adapters (sometimes called truncated adapters) require amplification with primers to incorporate sample indexing sequences and to add the P5 and P7 sequences.
• Optional PCR amplification. Choose to amplify your libraries based on adapter and DNA input used. A bead-based cleanup removes oligonucleotides and small fragments after PCR.

Kit overview

The Lotus kit comes with all the buffers, reagents, and enzymes needed for fragmentation, adapter ligation, and an optional PCR (to add index sequences, depending on adapter type, or to increase the amount of DNA for sequencing). You supply the DNA and add adapters that fit your goals. Table 1 provides additional specifications for using this kit.

Table 1. Specifications of the Lotus DNA Library Prep Kit.

Adapters

Complete your library prep with our selection of ready-to-use xGen Dual Index UMI Adapters or xGen Stubby Adapter and UDI Primer Pairs (Table 2). We also offer many high-quality, custom adapters and index primers that are application-specific and ideal for use with the Lotus kit.

Table 2. How to choose IDT adapters for common applications when using the Lotus DNA Library Prep Kit.

Complete workflow for hybridization capture experiments

Lotus DNA Library Prep Kit paired with adapters from IDT create libraries that are compatible with xGen hybridization capture probes and reagents for when you only need to perform targeted sequencing (Figure 2). Here is a list of the key components you will need:

• xGen Hybridization and Wash Kit
• xGen Universal Blockers—TS Mix or 10 bp TS Mix
• xGen Lockdown Panels and Probe Pools
• xGen Gene Capture Pools
• xGen Library Amplification Primers