Questions & Answers

Expand All


What is the difference between v1 and v2 kits? Can they be used together?

The different index kit versions (v1 and v2) are not chemically different. The v2 kits were introduced to provide more index combinations. When Set A though Set D are combined, up to 384 unique index combinations are possible. The v1 and v2 index kits provide some of the same indexes. For a list of indexes included in each kit, see Contents & Storage.

If you plan to scale up to multiplex more than 96 samples, the v2 index kits are recommended.

With the introduction of the Nextera XT Index Kit v2, catalog no. FC-131-1002 has been obsolesced (Nextera XT Index Kit - 96 indexes, 384 samples).

What applications are recommended for the Nextera XT kit?

The Nextera XT kit is recommended for small genomes, PCR amplicons greater than 300 bp, plasmids, microbial genomes, concatenated amplicons, and double-stranded cDNA.

Which indexes are included in each kit?

For a list of indexes, see Product Compatibility.

Can I use IDT for Illumina Nextera DNA UD Indexes with this kit?

Yes, this kit is compatible with IDT for Illumina Nextera DNA UD Indexes. However, the indexes are not compatible with bead-based normalization. Use the standard or manual normalization workflow with IDT for Illumina Nextera DNA UD Indexes. For more information, see Best Practices for Standard and Bead-Based Normalization in Nextera XT DNA Library Preparation Kits.


What is the estimated overall time for preparing libraries with the Nextera XT kit?

The estimated time for 8 samples is approximately 90 minutes for the prep and 70 minutes for the bead based normalization (BBN). The estimated time for 96 samples is approximately 3 hours for the prep and 90 minutes for the BBN.

What are the main differences between the protocols for the Nextera kit and the newer Nextera XT kit?

The Nextera XT kit introduces faster tagmentation, easy-to-use workflow, and optional bead-based normalization with only 1 ng of input DNA. In comparison, the Nextera DNA kit is optimized for larger genomes and uses 50 ng of input DNA with no bead-normalization reagents. The reagents provided in the Nextera DNA Library Prep Kit and the Nextera XT Library Prep Kit have been optimized for each workflow and are not interchangeable.

What are the considerations when working with environmental samples, or sample types that might have inhibitors of the tagmentation reaction?

Certain sample types are more likely to contain potential inhibitors of enzymatic reactions. For a list of potential inhibitors, see  Nextera XT Library Prep: Tips and Troubleshooting (Pub. No. 770-2015-015).

What is the recommended genome size for whole-genome sequencing with the Nextera XT kit?

Illumina recommends using the Nextera XT Library Prep Kit for microbial genomes < 5 Mb. For larger genomes, use the Nextera DNA, TruSeq DNA PCR-Free, or TruSeq Nano DNA library prep kits.

What are the recommended amplicon sizes for the Nextera XT kit?

Smaller amplicon inputs into Nextera XT preps typically yield smaller insert size ranges. To maximize recovery of smaller fragments out of the AMPureXP cleanup we recommend > 300 bp to ensure even coverage across the length of the DNA fragment. An expected drop off in sequencing coverage about 50 bp from each distal end of a fragment may be seen. This is because the tagmentation reaction cannot add an adapter right at the distal end of a fragment. For PCR amplicon sequencing this can be easily averted by simply designing your amplicons to be ~100 bases larger than the desired insert to be sequenced.

Are there any protocol differences based on different amplicon lengths?

For amplicons greater than 500bp, Illumina recommends using a 0.6x AMPure XP cleanup (30 μl volume of beads). For amplicons less than 500bp, Illumina recommends using a 1.8x AMPure XP cleanup (90 μl volume of beads) to maximize yield.

Is there a lower limit of the number of amplicons to use in my pool?

To obtain higher quality sequencing data, Illumina recommends sequencing samples with high diversity and avoiding monotemplate stretches during sequencing. Low diversity can occur with pools of one or a few amplicons. Additionally, it is important to maintain color balance for each base of the read or index read being sequenced, otherwise sequencing could fail due to registration failure. Please refer to the MiSeq System User Guide (part # 15027617) for recommendations on low diversity libraries or the Nextera XT User Guide (part # 15031942) for information on low plexity pooling guidelines.

How do I pool only a few samples and get good sequencing results?

For recommended index combinations, see Index Adapters Pooling Guide.

Where can I find more information about normalization?

Illumina library prep protocols include many features designed to increase ease-of-use and reduce total hands-on time. Library normalization is the process of obtaining equal amounts of library from different samples before loading the libraries onto the flow cell.

The Nextera XT Index Kit v2 (Set A-D) and the Nextera XT 24 and 96 Index Kits can be used with the bead-based normalization workflow. The IDT for Illumina Nextera DNA UD Indexes are not compatible with bead-based normalization. Use the standard or manual normalization workflow with IDT for Illumina Nextera DNA UD Indexes. For more information, see Best Practices for Standard and Bead-Based Normalization in Nextera XT DNA Library Preparation Kits.

How do I know what the best index combinations are for my library prep?

When creating a sample sheet in Illumina Experiment Manager (IEM), the software alerts you when improper combinations are used. Thus, creating your sample sheet before beginning library prep and pooling is highly recommended.

Excepting HiSeq 2500, IEM does not provide notification for color balance problems. For guidance, see the Index Adapters Pooling Guide.

Always pool samples with valid index combinations to avoid image registration failures. provides bulletins on library pooling, including pooling guidelines for the NextSeq and MiniSeq systems.

Are there any additional components I must order to complete the prep?

Completing Nextera XT library prep requires ordering the library prep kit and corresponding index kits: Nextera XT DNA Library Prep Kits (catalog # FC-131-1024, FC-131-1096) and corresponding Nextera XT Index Kits (catalog # FC-131-1001, FC-131-1002, FC-131-2001, FC-131-2002, FC-131-2003, FC-131-2004) or the IDT for Illumina Nextera DNA UD Indexes (catalog # 20027213).

The indexes are optimized for the Nextera XT DNA Library Prep Kits and are not interchangeable with the Nextera DNA Library Prep Kit. The IDT for Illumina Nextera DNA UD Indexes can only be used with the standard or manual normalization workflow.

The index plate fixture (catalog # FC-130-1005) is not included in the library prep kits or index kits, and must also be purchased separately for tubed indexed kits.

Library Evaluation

How do I make sure that I have an accurate amount of input DNA?

The Nextera XT protocol is optimized for 1 ng of input DNA total. Illumina strongly recommends quantifying the starting genomic material. Nextera XT library prep kits use an enzymatic DNA fragmentation step and thus can be more sensitive to DNA input compared to mechanical fragmentation methods. The ultimate success of the assay strongly depends on using an accurately quantified amount of input DNA library. Therefore, the correct quantitation of the DNA library is essential.

To obtain an accurate quantification of the DNA library, quantify the starting DNA library using a fluorometric-based method specific for duplex DNA, such as the Qubit dsDNA BR Assay system. Use 2 μl of each DNA sample with 198 μl of the Qubit working solution for sample quantification. Avoid methods that measure total nucleic acid content (eg, nanodrop or other UV absorbance methods) because common contaminants such as ssDNA, RNA, and oligos are not substrates for the Nextera XT assay.

Are there any performance differences (in terms of sequencing coverage) for GC rich amplicons?

Although most amplicons of interest will not likely be high GC-content, please note that coverage of high GC-content amplicons may be more variable as compared to other amplicons.


Can I sequence Nextera XT libraries on any Illumina sequencing platform?

Yes, the Nextera XT DNA Library Prep Kit is compatible with any Illumina sequencing instrument. When sequencing on a HiSeq X or HiSeq 3000/4000 patterned flow cell, Nextera XT DNA libraries might perform with relatively lower %PF due to their longer fragment size. However, these instruments typically provide more data due to the higher output.

Do I need any additional reagents for sequencing? When do I need the TruSeq Dual Index Sequencing Primer Box?

A TruSeq Dual Index Sequencing Primer Box PE (PE-121-1003) or TruSeq Dual Index Sequencing Primer Box SR (FC-121-1003) is needed to sequence Nextera XT libraries on a HiSeq 2500 using TruSeq v3 chemistry. Use the PE box with paired-end flow cells and the SR box with single-read flow cells. Other HiSeq 2500 chemistry and other systems  do not require this additional box.

What is the final library concentration I need before prepping for sample loading onto an Illumina sequencing system?

For most sequencing systems, 2–4 nM of final library is required to denature and dilute libraries in preparation for sequencing. For details, see the denature and dilute libraries guide for your instrument.

Can I run TruSeq HT libraries with Nextera libraries on the same lane or same flow cell? Are the indexes the same or different?

Illumina does not support, and strongly advises against, running libraries prepared with different library prep kits in the same lane of a flow cell. Running libraries prepared by different library prep kits in different lanes of the same flow cell or spiking in Illumina PhiX control library in the same lane as any user-prepared libraries is supported.

If different library types are run in different lanes, dual index recipes and the Dual Index Primer Box are required. The indexes for TruSeq HT and Nextera are unique and not shared.

How does dual indexing work?

For paired-end flow cells, dual indexing now requires 23 additional cycles of sequencing - eight cycles for the Index 1 (i7) Read, eight cycles for the Index 2 (i5) Read, plus seven non-imaging, chemistry-only cycles at the beginning of the Index 2 (i5) Read. For single-read flow cells, dual indexing only requires 16 additional cycles of sequencing - eight cycles for the Index 1 (i7) Read and eight cycles for the Index 2 (i5) Read.


What analysis tools are available for Nextera XT?

The appropriate analysis tool depends on the application. MiSeq Reporter includes various analysis workflows, including amplicon, assembly, metagenomics, and resequencing. BaseSpace Sequence Hub also offers various apps for de novoassembly, metagenomics, resequencing, and so on.

Are there any differences in analysis for Nextera XT samples?

A new PCR Amplicon analysis workflow is available in MiSeq Reporter v1.3 (MSR) for the MiSeq system. The PCR Amplicon workflow requires specifying a manifest which is created in the Illumina Experiment Manager. A manifest is a list of all the targeted regions and their chromosome start and end positions. The manifest specifies regions of interest (ROIs) for the aligner and variant caller, which results in faster analysis times and visualization of results specific for only the ROIs. Note that CASAVA is not compatible with Manifest files. Please refer to the Illumina Experiment Manager User Guide (part # 15031335) for more information on sample sheet and manifest creation for Nextera XT libraries. The MiSeq software (MCS 1.2/MSR 1.3) can be downloaded from the Downloads tab of the Nextera XT Support page.

Are the Illumina adapter sequences available?

Illumina adapter sequences are provided in the Illumina Customer Sequence Letter.