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Custom NGS Adapters

Adapter and indexing primer selections for DNA library prep

Choose from a variety of options that personalize the adapters to your NGS workflow. 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.
  • 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 processing

Ordering

Use the Custom Adapter Configurator tool to guide you through choices of adapter format and options. These include library prep method, configuration, attachment style, indexing strategy and number of indexes, amount, delivery format, and whether to include modifications or TruGrade processing. The tool allows you to select adapters for immediate purchase through our online cart or request customer support for assistance.

TruSeq–Compatible Full-length Adapters

Each sequence design includes duplexed adapters, delivered in Duplex Buffer (IDT), and provided in tubes.

* Price per number of adapters selected. Addition of methylation and/or TruGrade processing will incur an additional fee.

TruSeq–Compatible Stubby Adapter and Indexing Primers

Each sequence design includes duplexed adapters, delivered in Duplex Buffer (IDT) and provided in tubes, as well as a primer mix delivered in IDTE (1X TE), pH 8, available in plates.

* Price per number of adapter-primer sets selected. Addition of methylation and/or TruGrade processing will incur an additional fee.

Nextera–Compatible Indexing Primers

Each sequence design includes a primer mix delivered in IDTE (1X TE), pH 8, available in plates.

* Price per number of indexes selected. Addition of TruGrade processing will incur an additional fee.

Additional fees for methylated bases

Additional fees for TruGrade processing

If ordering >384 sequences, please use this request form.

(Visit the IDT Usage, warranty & licensing page for specific information about Illumina trademarks.)

The Custom Adapter Configurator guides you through selection of several adapter design options. Here are some details about those choices:

Formats compatible with NGS library preparation kits: The commercial kits used to prepare DNA sequences for NGS library construction use different mechanisms for attaching adapters to the sample DNA fragments. These typically involve either ligation or tagmentation (using transposase). You will select one of the following styles dependent on the DNA sample prep kit you plan to use to generate your library:

TruSeq™–Compatible Full-length Adapters and TruSeq™– Compatible Stubby Adapter and Indexing Primers are for ligation-based library preparation. These adapters are compatible with Illumina TruSeq DNA Sample Prep Kits, the NEBNext® Ultra™ DNA Library Prep Kit, and Kapa HyperPrep Kits.

Nextera™–Compatible Indexing Primers are for transposase-based library preparation. These primers are compatible with Nextera DNA Library Prep Kits.

Configuration: Primers and adapters are often designed to include short sequence elements—indexed barcodes or indexes—that allow identification of individual DNA fragments within samples and samples from pooled sample mixes. These barcode indexes may also help prevent sample mis-assignment and resolve index hopping. Types of barcode indexes include: single index (SI), unique dual index identifier (UDI), combinatorial dual index (CDI), and adapters with both a unique dual index identifier and unique molecular identifier (UDI-UMI).

Select the barcode complexity based on your NGS sequencing application (Table 1).

Table 1. Suggested barcode configuration based on NGS application.

ApplicationBest choiceAlternatives
Germline characterization
SNPs, indels, crude CNV; no concern of sample crosstalk
UDISI, CDI, or UDI-UMI
Germline characterization where sample crosstalk is important
Translational research, inherited genetics, infectious disease
UDIUDI-UMI
Low-level mutation detection, down to ~1% frequency
Cancer, mosaicism, other diseases where somatic mutations are important
UDI-UMI 

Attachment Style: One of 2 different TruSeq adapter structures—TruSeq™–Compatible Full-length Adapters (indexed duplex adapters) or the TruSeq™–Compatible Stubby Adapter and Indexing Primers (universal duplex adapters + indexed primer mix)—are attached to DNA library fragments. Typically, the duplex adapters are ligated directly to sample DNA fragments. When using the TruSeq™–Compatible Stubby Adapter, a PCR step then adds the indexed primers (included with the adapters) to the ligated stubby-Y adapters. Figure 1 provides a comparison of these structures.

Nextera™–Compatible Indexing Primers are added onto sample DNA fragments using a PCR reaction targeting short adapter sequences incorporated at both ends by a previous transposase-driven reaction, called tagmentation. Figure 1 shows this interaction.

Figure 1. Adapter structures.

Indexing strategy and number of indexes: Including a short index sequence (8–10 bases) in your adapters/primer mix enables multiple samples to be sequenced together (i.e., multiplexed) on the same instrument flow cell or chip. Each sample index 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.

Select from barcode indexes of 8 or 10 bases, with sequences designed by IDT or Illumina (IDT 8, IDT 10, ILMN 8, ILMN 10). See the Resources tab below, for links to the IDT 8, IDT 10, and ILMN 8 index sequence files. The selected IDT 8, IDT 10, ILMN 8, or ILMN 10 index sequences will be provided once your Custom NGS Adapters order has been placed.

Alternatively, you can upload up to 2000 custom index sequences.

Choose to have the barcode indexes numbered consecutively or with numbering starting at predefined index positions.

Size (reaction number or nmol): Receive adapter amounts based on final mass (2, 8, or 24 nmol), or TruSeq/Nextera reaction number (16 or 96 reactions).

Adapter or primersConcentration (μM)1 reaction (nmol)
TruSeq™–Compatible Full-length Adapters150.075
TruSeq™–Compatible Stubby Adapter150.075
TruSeq™–Compatible Primers100.050
Nextera™–Compatible Indexing Primers100.025

Delivery format: Select to have your adapters delivered in tubes, plates, or single-use plates. Adapters and primer mixes arrive on dry ice. TruSeq™–Compatible Full-length Adapters and the TruSeq™–Compatible Stubby Adapter are suspended in Nuclease-free Duplex Buffer (30 mM HEPES, pH 7.5; 100 mM potassium acetate; IDT). TruSeq™–Compatible Indexing Primers and Nextera™–Compatible Indexing Primers are suspended in IDTE (1X TE Solution, pH 8.0; IDT). 

Methylated/unmethylated: Unmethylated adapters will suffice for most applications. Methylated adapters are used with epigenetics studies that require bisulfate conversion.

TruGrade processing: Sensitive NGS applications can detect even low levels of barcode crosstalk. Barcode crosstalk can cause sample misassignment. There are multiple sources of barcode crosstalk, including manufacturing cross-contamination of adapter oligos. By including TruGrade processing, you can reduce levels of cross-contamination to as low as 0.01%.

Find more details on the IDT TruGrade webpage and in the DECODED Newsletter article, Tips for minimizing sample misassignment during multiplex NGS.

IDT has a long-standing reputation as a leader and pioneer in custom oligo manufacturing. Adapters are custom oligos, and IDT has been supplying high-quality adapters for NGS applications for over a decade. 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.

Sensitive NGS applications can detect even low levels of barcode crosstalk. Barcode crosstalk can cause sample misassignment. There are multiple sources of barcode crosstalk, including manufacturing cross-contamination of adapter oligos. IDT’s proprietary TruGrade process uses state-of-the-art synthesis and purification methods, designed specifically for NGS adapters. TruGrade processing can reduce levels of cross-contamination to as low as 0.01%.

Find more details on the IDT TruGrade webpage and in the DECODED Newsletter article, Tips for minimizing sample misassignment during multiplex NGS.

IDT also offers GMP grade adapter manufacturing for clinical applications.

IDT ensures the accuracy of each and every oligonucleotide by ESI mass spectrometry. While ESI-MS can’t always discriminate one barcode from another (sometimes molecules with very similar masses cannot be effectively resolved), this attention to quality provides an extra layer that insures the adapter sequence ordered is the sequence provided.