Mobile Menu

Which NGS DNA Library Prep Kit should you choose?

This feature was put together using content from Chapter 2 of the 2024 Sequencing Buyer’s Guide. For an in depth look at all aspects of sequencing, from sample prep to analysis, please download the full 100+ page sequencing guide for free.

In the ever-evolving landscape of genomics, the selection of an appropriate library preparation kit for next-generation sequencing (NGS) is a pivotal decision that can significantly impact the quality and reliability of the data generated. This article compares the specifications of DNA library prep kits from various solution providers to help you choose the right kit for your application

Unsurprisingly, as the number of sequencing platforms has rapidly expanded so too have the number of library preparation kits available. Many third-party kits are now being sold alongside core library preparation kits from sequencing instrument providers. The diverse range of products available ensures that researchers have a wealth of library preparation options, regardless of their sequencing requirements.

Additionally, the development of single-cell analysis techniques has transformed the sequencing space, with kits now being compatible with incredibly low input amounts for library generation. It is easier than ever to sequence precious samples at high sensitivity – essential for the analysis of rare and low abundance nucleic acid samples such as cell-free DNA. Many kits offer the ability to use a wide range of nucleic acid input amounts, meaning that one kit can be applied to a multitude of biological applications, including whole genome sequencing (WGS), transcriptome analysis, targeted sequencing of specific genomic loci and variant detection.


DNA library preparation

As DNA is the most well-established target for NGS analysis, a range of DNA library preparation kits have entered the market that are suited to various input quantities, genome types or applications. Like sequencing instruments, the kits can be divided into two main categories: those compatible with short-read sequencers and those designed for use with long-read platforms. Therefore, the selection process begins with choosing a kit that is complementary to the type of sequencing and instrument selected. Subsequently, a number of factors must be considered to ensure that the kit chosen can generate a robust library suitable for downstream sequencing and analysis.


It all adds up: factors to consider when purchasing a DNA library prep kit

Assay simplicity

In a world where high-throughput sequencing is becoming increasingly more common,the creation of NGS libraries with fewer steps, less reagents and simple instructions makes the sequencing process more manageable – especially when multiple samples are being processed in parallel. One prominent example of this is magnetic bead-based DNA purification, a labour-intensive step present in some kits that can result in errors in library preparation1. Therefore, selecting a kit with minimal pipetting steps or one with processes that can be automated (see below) may result in less errors and higher reproducibility.

One of the most prominent factors of complexity is time – researchers often desire an efficient workflow with minimal hands-on time required. In response to this demand, newer NGS library preparation products with streamlined workflows have been launched that can create functional libraries in just a couple of hours.

Automation

Preparing hundreds or thousands of samples at once is now a reality for some research labs. Luckily, many vendors – including Illumina, New England Biolabs and Qiagen – now offer automation solutions that aim to efficiently process samples and produce NGS libraries without human intervention. Such systems typically include liquid handling instruments, capable of autonomously running the full library preparation protocol. In addition to reducing hands-on time for researchers, automation also decreases contamination errors, improves sample quality and allows laboratories to scale-up their sequencing preparations as needed2.

DNA input

For some samples, the amount and type of DNA required for library preparation needs to be considered. Many kits are now compatible with low input amounts (often as little as 1 ng or less). Although this small amount may affect the quality or coverage of downstream sequencing data, this low input requirement is helpful when dealing with uncommon or low abundance samples. Additionally, there are several kits on the market that are specialised for dealing with damaged, low-quality DNA samples – such as the xGen™ ssDNA & Low-Input DNA Library Preparation Kit from Integrated DNA Technology (IDT). As a result, specialised products like these allow researchers to rescue valuable sequencing data from rare sources, including ancient DNA.

PCR vs No PCR

Library amplification via PCR is often a required step for many library preparation kits. Although PCR allows researchers to sequence samples with low DNA content, PCR may introduce GC bias, amplification bias and duplicates that may hinder downstream genome assembly or data analysis. To counteract this problem, many vendors have created PCR-free kits that offer reduced assay times and increased coverage across genomic regions that are traditionally challenging to sequence. One well-established example is Illumina’s TruSeq DNA PCR-Free kit, which shows impressive coverage improvement for G-rich, high GC and promoter regions when compared to data from their TruSeq DNA kit featuring PCR amplification3.

Multiplexing

Sample multiplexing (occasionally referred to as indexing) is the process of tagging each DNA fragment to identify which sample that DNA fragment originated from. By doing this, researchers can pool the libraries for multiple samples together and sequence several samples in parallel. After sequencing, the unique tag (often referred to as a barcode or index) can then be used to group the data into their respective samples before analysis takes place. Therefore, multiplexing is an attractive way to conduct high-throughput sequencing and save both time and money.

Cost

Ultimately, the best library preparation kit is one that produces the highest quality data for the lowest price. Hidden reagent costs, expenses associated with analysis, researcher time and kit usage (e.g., wasting resources due to reagent expiration) can all play a factor in the total cost per reaction – in addition to the initial expense of the library preparation kit itself. If the experiment requires a small number of samples, or if sequencing is not regularly required, labs may instead opt to outsource sequencing to NGS service providers.

In the following sections, we list the newest and most popular products from the leaders within the NGS library preparation space. Notably, kits for highly specialised starting materials (e.g., tumour tissue) and gene panels have been omitted for brevity. Instead, we focus on kits that are relevant to a wide variety of experimental setups. Although not covered here, many of the suppliers listed also sell more specialised sequencing products, such as Illumina’s AmpliSeq panels and Thermo Fisher Scientific’s Oncomine assays.

DNA library preparation kits for short-read systems

Owing to their immense popularity, the leaders of the short-read industry, Illumina, currently dominate the library preparation market (see Table 1). In addition to the various library preparation kits supplied by Illumina, many third-party companies also offer products that are compatible with Illumina sequencing instruments. In contrast, library preparation kits suitable for other short-read platforms (such as those offered by MGI and PacBio) can be purchased directly from the supplier.

Notably, some sequencing platforms (such as Singular Genomics’ G4 instrument and Element Biosciences’ AVITI system) offer library conversion kits, custom adapters and indices, which enable users to perform their library preparation using a wide range of kits from various third-party suppliers. This allows the sequencing instruments to easily slot into any pre-existing NGS workflows.

Table 1: Details for selected DNA library preparation kits compatible with short-read sequencing platforms. Information was sourced from the supplier’s website or handbook associated with the product. Details provided were correct at the time of publication (January 2024), please check supplier’s website for the most up-to-date specifications and data. Suppliers are listed alphabetically. Abbreviations: RM – research mode, TCS – targeted capture sequencing, WES – whole exome sequencing, WGS – whole genome sequencing.
SupplierKitSystem CompatibilityAssay timeInput QuantityTarget Insert SizePCR RequiredApplications
AgilentSureSelect XT HS2 DNA Reagent KitIllumina instruments,
Element instruments
(library conversion required)
9 hours (TCS)10 – 200 ng of total DNA (from intact or highly fragmented FFPE samples)CustomisableYes  DNA targeted enrichment
Illumina Illumina DNA PCR-Free Prep Illumina: MiSeq,
MiSeqDx (RM),
NextSeq 500,
NextSeq 550,
NextSeq 550Dx (RM),
NovaSeq 6000,
NovaSeq X,
NovaSeq X Plus 
1.5 hours25 ng – 300 ng 450 bp +/- 75 bp NoDe novo assembly, WGS
Illumina DNA Prep Illumina: HiSeq 3000,
HiSeq 4000,
HiSeq X Five,
HiSeq X Ten,
iSeq 100,
MiniSeq,
MiSeq,
MiSeqDx (RM),
NextSeq 1000,
NextSeq 2000,
NextSeq 550,
NextSeq 550Dx (RM),
NovaSeq 6000,
NovaSeq X,
NovaSeq X Plus 
3-4 hoursSmall genomes (e.g. microbial): 1-500 ng DNA Large genomes (e.g. human): 100-500 ng DNA~350bp YesAmplicon sequencing, De novo assembly, WGS
Nextera XT DNA Library Preparation Kit Illumina: iSeq 100,
MiniSeq,
MiSeq,
NextSeq 1000,
NextSeq 2000,
NextSeq 500,
NextSeq 550 
5.5 hours1 ng 300 bp – 1.5 kb Yes16S rRNA sequencing, amplicon sequencing, De novo assembly, WGS
TruSeq DNA Nano Illumina: HiSeq 3000,
HiSeq 4000,
HiSeq X Five,
HiSeq X Ten,
MiniSeq,
MiSeq,
MiSeqDx (RM),
NextSeq 2000,
NextSeq 500,
NextSeq 550,
NovaSeq 6000,
NovaSeq X,
NovaSeq X Plus 
6 hours100 ng350 bp or 550 bp YesGenotyping, WGS
TruSeq DNA PCR-Free Illumina: HiSeq 3000,
HiSeq 4000,
HiSeq X Five,
HiSeq X Ten,
MiniSeq,
MiSeq,
MiSeqDx (RM),
NextSeq 1000,
NextSeq 2000,
NextSeq 500,
NextSeq 550,
NovaSeq 6000 
5 hours1 ug 350 bp or 550 bp NoGenotyping, WGS
Integrated DNA Technologies xGen DNA EZ Library Prep Kit Illumina instruments<2 hours100 pg – 1 μg 550 bp YesGenotyping, WES, WGS
xGen DNA Library MC Kit Illumina instruments  2 hours1 ng – 1 µg 350 bp   YesWGS, WES, metagenomic sequencing
xGen ssDNA & Low-Input DNA Library Prep Kit Illumina instruments2 hours10 pg – 250 ng 200 bp or 350 bp YesSequencing of low-quality degraded DNA/ ssDNA
MGIMGIEasy Duplex UMI Universal Library Prep Set MGI: DNBSEQ-G400,
DNBSEQ-G50,
DNBSEQ-T7 
7 hours10 ng – 1 µg  200 bp – 600 bp YesLow frequency variant detection
MGIEasy Exome FS Library Prep Set V2.0MGI: DNBSEQ-G4007 hours50 ng –400 ng200 bp – 600 bpYesWES
MGIEasy Fast FS DNA Library Prep Set MGI: DNBSEQ-G400,
DNBSEQ-G50,
DNBSEQ-T7 
2 – 2.3 hours1 ng – 200 ng 200 bp – 600 bp YesMicrobial genome sequencing, metagenome sequencing
MGIEasy Fast PCR-FREE FS Library Prep Set MGI: DNBSEQ-G400,
DNBSEQ-G50,
DNBSEQ-T7 
1.5 – 1.8 hours 50 ng – 300 ng 200 bp – 600 bp NoLow pass WGS, viral WGS, long amplicons sequencing
MGIEasy FS DNA Library Prep SetMGI: DNBSEQ-T7,
DNBSEQ-G400,
DNBSEQ-G50
5.5 hours5 ng – 400 ng200 bp – 600 bpYesWGS
MGIEasy PCR-Free Library Prep SetMGI: DNBSEQ-G400,
DNBSEQ-G50,
DNBSEQ-T7
3.5 hours80 ng –200 ng350 bp –400 bpNoWGS
New England Biolabs  NEBNext Enzymatic Methyl-seq KitIllumina instruments7.5 hours10 ng – 200 ngCustomisableYesMethylome Analysis
NEBNext Fast DNA Fragmentation & Library Prep Set for Ion TorrentIon Torrent instruments <2 hours10 ng – 1 μg Enzymatic Fragmentation; CustomisableYes WGS, WES 
NEBNext Fast DNA Library Prep Set for Ion TorrentIon Torrent instruments <2 hours 10 ng – 1 μg  CustomisableYes WGS, WES 
NEBNext FFPE DNA Library Prep KitIllumina instruments2.8 – 3.8 hours 5 ng – 250 ngCustomisableYes FFPE DNA
NEBNext Ultra II DNA Library Prep Kit for IlluminaIllumina instruments 1.7 – 3.2 hours 500 pg – 1 µg  CustomisableYes WGS, WES, ChIP-seq
NEBNext Ultra II FS DNA Library Prep Kit for IlluminaIllumina instruments1.3 – 3.2 hours100 pg – 500 ngEnzymatic Fragmentation; CustomisableYes WGS, WES 
NEBNext Ultra II DNA PCR-free Library Prep Kit for IlluminaIllumina instruments<2 hours250 ng – 1 µg400 bp peak size rangeNoWGS, WES
NEBNext Ultra II FS DNA PCR-free Library Prep Kit for IlluminaIllumina instruments<2 hours50 ng – 500 ngEnzymatic Fragmentation; 300 bp, 350 bp, 450 bp peak size rangesNoWGS, WES
NEBNext UltraExpress DNA Library Prep Kit  Illumina instruments110 minutes10 ng – 200 ngCustomisable (200 bp peak size by default)YesWGS, WES
NEBNext UltraExpress FS DNA Library Prep KitIllumina instruments 105 minutes10 ng – 200 ng  Enzymatic Fragmentation; Customisable (150 bp peak size by default)Yes WGS, WES 
NEBNext Ultrashear FFPE DNA Library Prep KitIllumina instruments3.25 – 4.25 hours5 ng – 250 ngEnzymatic Fragmentation; CustomisableYes FFPE DNA
PacBio Onso DNA Library Prep kitPacBio: Onso3 hours10 ng – 1 μg Application specificPCR-free and amplified workflows availableWGS, WES, targeted sequencing
Onso fragmentation DNA library prep kit PacBio: Onso 3 hours10 ng – 500 ngApplication specificPCR-free and amplified workflows availableWGS, WES, targeted sequencing
QiagenQIAseq FX DNA Library Kit Illumina instruments2.5 hours20 pg – 1 µg FlexibleOptionalWGS, WES, metagenomic sequencing
QIAseq Ultralow Input Library Kit Illumina instruments  2.5 hours10 pg –100 ng  Not specifiedOptionalSequencing of low-input/ degraded DNA
RocheKAPA EvoPlus Kits Illumina instruments  2 hours10 ng – 500 ng FlexibleOptionalWES, WGS
KAPA HyperPlus Kits Illumina instruments  2.5 hours 1 ng – 1 µg FlexibleOptionalWES, WGS
KAPA HyperPrep Kits Illumina instruments2.75 hours1 ng – 1 µg Not specifiedOptional  Amplicon sequencing, WES, WGS,
Thermo Fisher Scientific Collibri PCR-free PS DNA Library Prep Kits for Illumina Systems Illumina: iSeq,
HiSeq1000/1500/2000/
2500/3000/4000/X,
MiSeq,
MiniSeq,
NextSeq 500/550,
NovaSeq 6000
2 hours500 ng – 1 μg 150 bp – 1000 bp No WGS
Collibri PS DNA Library Prep Kits for Illumina Systems Illumina: iSeq,
HiSeq1000/1500/2000/
2500/3000/4000/X, MiSeq,
MiniSeq,
NextSeq 500/550, NovaSeq 6000
~2.5 hours1 ng – 1 μg 150 bp –1000 bp YesWGS
Ion Plus Fragment Library Kit Ion Torrent: Ion PGM,
Ion Proton,
Ion S5,
Ion S5 XL,
Ion GeneStudio™ S5 Series 
~2 hours100 ng or 1 µg 100 bp –600 bp Yes WGS
Twist BioscienceLibrary Preparation Enzymatic Fragmentation Kit 2.0 Illumina instruments3 hours1 ng – 500 ng    FlexibleYesTargeted sequencing, WGS
Mechanical Fragmentation Library Preparation Kit Illumina instruments3 hours1 ng – 500 ng   200 bp – 250 bpYesTargeted sequencing, WGS
cfDNA Library Preparation KitIllumina instruments2 hours1 ng – 20 ng300 bp – 400 bpYesTargeted sequencing, WGS


In 2023, several new products were launched in the short-read DNA library preparation area. One notable highlight is the release of New England Biolab’s new NEBNext UltraExpress™ DNA and RNA Kits, compatible with Illumina platforms. Announced in November 2023, the kits are designed with speed in mind – with DNA and RNA libraries being successfully generated within 2 hours and one day respectively4. The kits also follow a simplified protocol using the same adapter concentration and PCR cycle numbers regardless of input amount used, making library preparation easier whilst maintaining sample input flexibility.

Additionally, 2023 was a milestone year for PacBio with the launch of their Onso short-read platform. The novel system was launched in August last year alongside two library preparation kits, designed to create libraries from high-molecular weight or fragmented/damaged samples. The system aims to generate high (Q40+) accuracy data that supports all major short-read applications.



DNA library preparation kits for long-read systems

Over in the long-read arena, the industry leaders – Oxford Nanopore Technologies and PacBio – have a number of library preparation kits available to purchase directly (see Table 2). Interestingly, third-party suppliers of products for long-read library preparation are less common than in short-read sequencing. However, one notable outlier is New England Biolabs, which have partnered with Oxford Nanopore Technologies on their NEBNext® Companion Module for use in Oxford Nanopore Technologies’ ligation sequencing protocols.

Table 2: Details for selected DNA library preparation kits compatible with long-read sequencing platforms. Information was sourced from the supplier’s website or handbook associated with the product. Details provided were correct at the time of publication (January 2024), please check supplier’s website for the most up-to-date specifications and data. Abbreviations: WES – whole exome sequencing, WGS – whole genome sequencing.
SupplierKitSystem CompatibilityAssay timeInput QuantityApplications
IlluminaIllumina Complete Long Read Prep, HumanIllumina: NovaSeq 6000, NovaSeq 6000Dx,
NovaSeq X,
NovaSeq X Plus
7.5 – 8.5 hours50 ng DNAWGS
Oxford Nanopore Technologies  16S Barcoding KitOxford Nanopore: MinION Mk1B, MinION Mk1C, GridION, Flongle25 minutes (plus PCR time)10 ng gDNABacterial 16S rRNA sequencing, metagenomics, targeted sequencing
Ligation Sequencing KitOxford Nanopore: Flongle, MinION Mk1B, MinION Mk1C, GridION, PromethION1 hour1000 ng gDNA or 100-200 fmol for ampliconsWGS, methylation analysis, metagenomics, single-cell sequencing, targeted sequencing
Rapid PCR Sequencing KitOxford Nanopore: MinION Mk1B, MinION Mk1C, GridION, PromethION, Flongle15 minutes (plus PCR time)1 ng – 5 ng gDNAWGS, metagenomics
Rapid Sequencing KitOxford Nanopore: Flongle MinION Mk1B MinION Mk1C GridION PromethION10 minutes50 ng – 100 ng gDNAWGS, metagenomics, methylation analysis
Ultra-long DNA Sequencing KitOxford Nanopore: MinION Mk1B, MinION Mk1C, GridION, PromethION~ 3.5 hours (plus overnight elution)6M cellsWGS, methylation analysis
PacBioSMRTbell prep kit 3.0PacBio: Revio and Sequel II/IIe4 hours300 ng – 5 µgMetagenomics, methylation analysis, targeted sequencing, WGS


Illumina threw their hat into the ring of long-read sequencing in 2023, with the debut of their novel Complete Long Read Prep kit. The novel technology makes long-read sequencing possible on their powerful NovaSeq instruments, which are commonly used for short-read sequencing. Following a simple one-day protocol, users can generate sequencing reads of 5-7 kb (with some reads >10 kb) that enable complete coverage of genomic regions that may be missed using short-read technology alone.

Last year also saw PacBio announce their collaboration with leading library preparation automation partners, ensuring that their HiFi long-read sequencing can be carried out faster and with higher throughput. The sequencing giants informed customers that several automation providers – Hamilton, Integra, Revvity and Tecan – have successfully created fully automated protocols for PacBio’s Revio and Sequel instruments, enabling users to easily scale up their sequencing to thousands of genomes per year5.


References

1.     Hess, J. F. et al. Library preparation for next generation sequencing: A review of automation strategies. Biotechnol. Adv. 41, 107537 (2020).

2.     Socea, J. N., Stone, V. N., Qian, X., Gibbs, P. L. & Levinson, K. J. Implementing laboratory automation for next-generation sequencing: benefits and challenges for library preparation. Front Public Health 11, 1195581 (2023).

3.     Illumina Inc. TruSeqTM DNA PCR-Free. https://www.illumina.com/content/dam/illumina-marketing/documents/products/datasheets/datasheet_truseq_dna_pcr_free_sample_prep.pdf (2017).

4.     New England Biolabs. New England Biolabs® launches new NEBNext UltraExpressTM DNA and RNA Kits for faster, easier NGS library prep workflows. https://www.neb.com/en-gb/about-neb/news-and-press-releases/new-england-biolabs-launches-new-nebnext-ultraexpress-dna-and-rna-kits-for-faster-easier-ngs-library-prep-workflows (2023).

5.     PacBio announces collaboration with leading library preparation automation partners. PacBio https://www.pacb.com/press_releases/pacbio-announces-collaboration-with-leading-library-preparation-automation-partners/ (2023).