Next Generation Sequencing Library Preparation

Product Overview​

Successful Next Generation Sequencing (NGS) hinges on several factors, but one of the most impactful of them is the choice of library preparation workflow. As NGS matures into an essential analysis method, libraries of the highest quality are required, in yields sufficient for a wide range of applications and throughputs, from single-cell and cfDNA to PCR-free whole-genome analysis. Automation compatibility is a major consideration, too, as liquid-handling robots are becoming more accessible and commonplace.

NEBNext UltraExpress® and NEBNext® Ultra™ II: Faster workflows for better libraries

For 15 years, NEB has addressed these challenges by offering solutions that streamline sample prep workflows, minimize inputs and improve library yield and quality. With over 100 products, the NEBNext portfolio includes sample prep solutions for DNA sequencing and RNA sequencing from a wide range of sample types. The NEBNext workflow lies at the heart of the portfolio, and products are available in a convenient kit format, or as separate modules – offering flexible library prep workflows that are easily scaled and automated. Our new NEBNext UltraExpress workflows take speed and convenience even further, generating high-quality libraries in less time with a single protocol (with a single adaptor concentration and number of PCR cycles) for all samples within the input range. The flagship NEBNext Ultra II workflow sets the standard for high-quality NGS library preparation for complex and precious samples.

NEBNext reagents are currently available for next generation sequencing sample preparation on Illumina®, Oxford Nanopore Technologies® (ONT) and Ion Torrent® sequencing platforms, and library quantitation for Illumina and Ultima Genomics®.

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Give NEBNext a try with your own samples.

We offer samples of many of our NEBNext kits, modules and oligos.

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Find out how colleagues are using NEBNext

NEBNext reagents has been cited in >35,000 peer-reviewed publications, as well as preprints and protocols.

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Need help with product selection?

If you need assistance navigating the ever-growing NEBNext product line, NEBNext Selector tool can help guide your product selection.

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Looking for a custom format?

NEBNext reagents can be ordered in bulk or customized formats for advanced research and development projects.

Featured Products

  • NEBNext® Low-bias Small RNA Library Prep Kit
  • NEBNext UltraExpress® FS DNA Library Prep Kit
  • NEBNext UltraExpress® DNA Library Prep Kit
  • NEBNext UltraExpress® RNA Library Prep Kit
  • NEBNext® Ultra™ II Directional RNA Library Prep with Sample Purification Beads
  • NEBNext® Enzymatic Methyl-seq v2 Conversion Module
  • NEBNext® Enzymatic Methyl-seq v2 Kit

Next Generation Sequencing Library Preparation includes these subcategories:

Library Preparation for Illumina
Library Preparation for Ion Torrent
Library Preparation for Oxford Nanopore Technologies
Infectious Diseases & ARTIC Sequencing
DNA Fragmentation
Library Preparation for Ultima Genomics
Library Preparation for Element Biosciences
RNA Depletion & mRNA Enrichment
Methylome Analysis
Microbiome DNA Enrichment
NEBNext® Multiplex Oligos (Adaptors & Primers)
FFPE DNA
Library Preparation for MGI®
NGS Library Quantitation
Automation for NEBNext® NGS Library Prep
Small RNA Library Preparation

Application Notes for Next Generation Sequencing Library Preparation

  • Obtain superior NGS library performance with lower input amounts using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina
  • Increased transcription detection with the NEBNext Single Cell/Low Input RNA Library Prep Kit
  • Monarch HMW DNA Extraction Kit improves sample preparation for Oxford Nanopore Technologies sequencing of malaria parasites
  • High-yield, Scalable Library Preparation with the NEBNext Ultra™ II FS DNA Library Prep Kit
  • Improving NGS library performance with lower input amounts using the NEBNext Ultra II RNA Library Prep Kit for Illumina (non-directional) – Advances in non-strand-specific RNA library construction in a ribosomal RNA depletion workflow
  • Improving NGS library performance with lower input amounts using the NEBNext™ Ultra II RNA Library Prep Kit for Illumina (non-directional)

Tools & Resources

Feature Articles

Improving Enzymatic DNA Fragmentation for Next Generation Sequencing Library Construction

Among the available options for DNA fragmentation, enzymatic fragmentation is demonstrably gentler on the sample, yielding less damage at any scale. The new NEBNext® Ultra™ II FS DNA Library Prep Kit for Illumina® includes a single step for enzymatic fragmentation, end repair, and dA tailing.

Advances in next generation sequencing: How researchers at NEB are working to improve our understanding of the genome, epigenome and transcriptome.

NEB scientists develop sequencing technologies to analyze genome-wide methylation, transcriptional start sites, full-length transcriptomes, chromatin accessibility, and much more.

Brochures

Sequencing to Insight: Real-world applications powered by NEBNext® Poster
NEBNext Ultra II Reagents & Kits Brochure
NEBNext For RNA Sample Prep Brochure
NEBNext RNA Depletion Brochure
NEBNext Direct Genotyping Solution Brochure
NEBNext® for DNA Sample Prep Brochure
NEBNext for Ion Torrent™ Brochure

Usage Guidelines

NEB E3406 LV UDI IlluminaLRM

Posters

Induro™, a Novel Reverse Transcriptase for Nanopore Direct RNA Sequencing with Significantly Improved RNA 5′ Coverage
Streamlined, Express DNA Library Preparation Methods Meet Requirements of High-throughput Library Construction
NEBNext UltraExpress™ RNA: A fast and flexible workflow for stranded RNA-seq library preparation
NEBNext® solutions for challenging methods and samples
E5hmC-seq™: Detection of 5hmC at single base resolution
E5hmC-seq: Detection of 5hmC at single-base resolution
Streamlining RNA and DNA library construction methods to meet the challenges of high-throughput sequencing
Matched fresh frozen and FFPE patient tissues reveal the enhanced sensitivity and data quality of a novel DNA library prep method
Streamlined whole-genome and targeted sequencing workflows for prevalent RNA viruses: SARS-CoV-2, Respiratory Syncytial Virus, and Influenza A
Methods for sequencing cell-free DNA and cell-free RNA from human plasma
An end-to-end enzymatic solution for methylomes from low-input DNA
Germline variant calling with EM-seq data
A sample-to-results workflow for Respiratory Syncytial Virus (RSV) whole-genome sequencing
Accurate detection of small non-coding RNAs using NEBNext Low-bias Small RNA Library Prep Kit
Integration of whole exome sequencing and RNA-seq facilitates somatic mutation detection in tumor tissue samples
High-throughput methyl-binding domain-based DNA enrichment and sequencing
Real-world applications powered by NEBNext® Poster
Rapid, robust and sensitive DNA, RNA and small RNA library construction methods for evaluation of various samples and species

Legal Information

Products and content are covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB). The use of trademark symbols does not necessarily indicate that the name is trademarked in the country where it is being read; it indicates where the content was originally developed. All other trademarks are the property of their respective owners. The use of this product may require the buyer to obtain additional third-party intellectual property rights for certain applications. For more information, please email busdev@neb.com.

This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.

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Product Listing

NEB offers a wide range of DNA polymerases, and through our commitment to research, ensures the development of innovative, high quality tools for DNA amplification, qPCR and PCR.

  • EpiMark® Methylated DNA Enrichment Kit
  • EpiMark® N6-Methyladenosine Enrichment Kit
  • EpiMark® Hot Start Taq DNA Polymerase
  • NEBNext UltraExpress® DNA Library Prep Kit
  • NEBNext UltraExpress® FS DNA Library Prep Kit
  • NEBNext UltraExpress® RNA Library Prep Kit
  • NEBNext UltraShear®
  • NEBNext UltraShear® FFPE DNA Library Prep Kit
  • NEBNext UltraShear® Long Read
  • NEBNext® Circularization Module for MGI®
  • NEBNext® Companion Module for Oxford Nanopore Technologies® Direct RNA Sequencing
  • NEBNext® Companion Module for Oxford Nanopore Technologies® Ligation Sequencing
  • NEBNext® Companion Module v2 for Oxford Nanopore Technologies® Ligation Sequencing
  • NEBNext® Enzymatic 5hmC-seq Conversion Module
  • NEBNext® Enzymatic Methyl-seq v2 Conversion Module
  • NEBNext® FFPE DNA Library Prep Kit
  • NEBNext® FFPE DNA Repair Mix
  • NEBNext® FFPE DNA Repair v2 Module
  • NEBNext® Flu A Integrated Indexing Primer Module
  • NEBNext® FS DNA Library Prep Kit for MGI®
  • NEBNext® Immune Sequencing Kit (Human)
  • NEBNext® Immune Sequencing Kit (Mouse)
  • NEBNext® Library Quant Kit for Illumina®
  • NEBNext® Library Quant Kit for Ultima Genomics®
  • NEBNext® Low-bias Small RNA Library Prep Kit
  • NEBNext® LV Unique Dual Index Primers Set 2B
  • NEBNext® LV Unique Dual Index Primers Set 3
  • NEBNext® Microbiome DNA Enrichment Kit
  • NEBNext® Multiplex Oligos for Illumina® (Unique Dual Index UMI Adaptors RNA Set 1)
  • NEBNext® Multiplex Oligos for Illumina® (96 Index Primers)
  • NEBNext® Multiplex Oligos for Illumina® (96 Unique Dual Index Primer Pairs Set 2)
  • NEBNext® Multiplex Oligos for Illumina® (96 Unique Dual Index Primer Pairs Set 3)
  • NEBNext® Multiplex Oligos for Illumina® (96 Unique Dual Index Primer Pairs)
  • NEBNext® Multiplex Oligos for Illumina® (Unique Dual Index UMI Adaptors DNA Set 1)
  • NEBNext® Multiplex Oligos for MGI® (Dual Index Primer Pairs Set 1)
  • NEBNext® Multiplex Small RNA Library Prep Kit for Illumina® (Index Primers 1-48)
  • NEBNext® Multiplex Small RNA Library Prep Set for Illumina® (Set 1)
  • NEBNext® Multiplex Small RNA Library Prep Set for Illumina® (Set 2)
  • NEBNext® Multiplex Small RNA Library Prep Set for SOLiD™ (Set 1)
  • NEBNext® Poly(A) mRNA Magnetic Isolation Module
  • NEBNext® Q5® Hot Start HiFi PCR Master Mix
  • NEBNext® Quick Ligation Reaction Buffer
  • NEBNext® RNA Depletion Core Reagent Set
  • NEBNext® RNA Library Prep Kit for MGI®
  • NEBNext® rRNA Depletion Kit (Bacteria)
  • NEBNext® rRNA Depletion Kit (Bacteria) with RNA Sample Purification Beads
  • NEBNext® rRNA Depletion Kit v2 (Human/Mouse/Rat) with RNA Sample Purification Beads
  • NEBNext® Single Cell Lysis Module
  • NEBNext® Single Cell/Low Input RNA Library Prep Kit for Illumina®
  • NEBNext® Small RNA Library Prep Set for Illumina® (Multiplex Compatible)
  • NEBNext® Ultra™ End Repair/dA-Tailing Module
  • NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina®
  • NEBNext® Ultra™ II Directional RNA Library Prep with Sample Purification Beads
  • NEBNext® Ultra™ II Directional RNA Second Strand Synthesis Module
  • NEBNext® Ultra™ II DNA Library Prep Kit for Illumina®
  • NEBNext® Ultra™ II End Repair/dA-Tailing Module
  • NEBNext® Ultra™ II FS DNA Library Prep with Sample Purification Beads
  • NEBNext® Ultra™ II FS DNA Module
  • NEBNext® Ultra™ II Ligation Module
  • NEBNext® Ultra™ II RNA First Strand Synthesis Module
  • NEBNext® ARTIC SARS-CoV-2 Companion Kit (Oxford Nanopore Technologies®)
  • NEBNext® Enzymatic 5hmC-seq Kit
  • NEBNext® Enzymatic Methyl-seq v2 Kit
  • NEBNext® High Input Poly(A) mRNA Isolation Module
  • NEBNext® LV Unique Dual Index Primers Set 1
  • NEBNext® LV Unique Dual Index Primers Set 2
  • NEBNext® LV Unique Dual Index Primers Set 2A
  • NEBNext® LV Unique Dual Index Primers Set 4
  • NEBNext® LV Unique Dual Index Primers Set 5
  • NEBNext® Multiplex Oligos for Illumina® (96 Unique Dual Index Primer Pairs Set 4)
  • NEBNext® Multiplex Oligos for Illumina® (96 Unique Dual Index Primer Pairs Set 5)
  • NEBNext® Multiplex Oligos for Illumina® (Unique Dual Index UMI Adaptors DNA Set 2)
  • NEBNext® Multiplex Oligos for Illumina® (Unique Dual Index UMI Adaptors DNA Set 3)
  • NEBNext® Multiplex Oligos for Illumina® (Unique Dual Index UMI Adaptors DNA Set 4)
  • NEBNext® Ultra™ II FS DNA PCR-free Library Prep with Sample Purification Beads
  • NEBNext® Ultra™ II DNA PCR-free Library Prep Kit for Illumina®
  • NEBNext® Ultra™ II FS DNA PCR-free Library Prep Kit for Illumina®
  • NEBNext UltraExpress® DNA Library Prep Kit
  • NEBNext UltraExpress® FS DNA Library Prep Kit
  • NEBNext UltraExpress® RNA Library Prep Kit
  • NEBNext® ARTIC SARS-CoV-2 RT-PCR Module
  • NEBNext® Globin & rRNA Depletion Kit (Human/Mouse/Rat)
  • NEBNext® Magnetic Separation Rack
  • NEBNext® Multiplex Oligos for Illumina® (96 Index Primers)
  • NEBNext® Poly(A) mRNA Magnetic Isolation Module
  • NEBNext® RNA Depletion Core Reagent Set
  • NEBNext® rRNA Depletion Kit (Bacteria)
  • NEBNext® rRNA Depletion Kit (Bacteria) with RNA Sample Purification Beads
  • NEBNext® rRNA Depletion Kit v2 (Human/Mouse/Rat)
  • NEBNext® rRNA Depletion Kit v2 (Human/Mouse/Rat) with RNA Sample Purification Beads
  • NEBNext® Single Cell Lysis Module
  • NEBNext® Single Cell/Low Input cDNA Synthesis & Amplification Module
  • NEBNext® Single Cell/Low Input RNA Library Prep Kit for Illumina®
  • NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina®
  • NEBNext® Ultra™ II Directional RNA Library Prep with Sample Purification Beads
  • NEBNext® Ultra™ II Directional RNA Second Strand Synthesis Module
  • NEBNext® Ultra™ II DNA Library Prep Kit for Illumina®
  • NEBNext® Ultra™ II DNA Library Prep with Sample Purification Beads
  • NEBNext® Ultra™ II End Repair/dA-Tailing Module
  • NEBNext® Ultra™ II FS DNA Library Prep Kit for Illumina
  • NEBNext® Ultra™ II FS DNA Library Prep with Sample Purification Beads
  • NEBNext® Ultra™ II Ligation Module
  • NEBNext® Ultra™ II Q5® Master Mix
  • NEBNext® Ultra™ II RNA First Strand Synthesis Module
  • NEBNext® Ultra™ II RNA Library Prep Kit for Illumina®
  • NEBNext® Ultra™ II RNA Library Prep with Sample Purification Beads
  • NEBNext® ARTIC SARS-CoV-2 Companion Kit (Oxford Nanopore Technologies®)
  • NEBNext® Ultra™ II FS DNA PCR-free Library Prep with Sample Purification Beads
  • NEBNext® Ultra™ II DNA PCR-free Library Prep Kit for Illumina®
  • NEBNext® Ultra™ II FS DNA PCR-free Library Prep Kit for Illumina®
  • NEBNext® Fast DNA Library Prep Set for Ion Torrent™
  • NEBNext UltraShear® Long Read
  • NEBNext® Companion Module for Oxford Nanopore Technologies® Direct RNA Sequencing
  • NEBNext® Companion Module for Oxford Nanopore Technologies® Ligation Sequencing
  • NEBNext® Companion Module v2 for Oxford Nanopore Technologies® Ligation Sequencing
  • NEBNext® ARTIC SARS-CoV-2 Companion Kit (Oxford Nanopore Technologies®)
  • NEBNext® ARTIC SARS-CoV-2 FS Library Prep Kit (Illumina®)
  • NEBNext® ARTIC SARS-CoV-2 RT-PCR Module
  • NEBNext® Flu A Integrated Indexing Primer Module
  • NEBNext® Immune Sequencing Kit (Human)
  • NEBNext® Immune Sequencing Kit (Mouse)
  • NEBNext® RSV Primer Module
  • NEBNext® ARTIC SARS-CoV-2 Companion Kit (Oxford Nanopore Technologies®)
  • NEBNext UltraShear®
  • NEBNext UltraShear® Long Read
  • NEBNext® dsDNA Fragmentase®
  • NEBNext® Ultra™ II FS DNA Library Prep Kit for Illumina
  • NEBNext® Ultra™ II FS DNA Library Prep with Sample Purification Beads
  • NEBNext® Ultra™ II FS DNA Module
  • NEBNext® dsDNA Fragmentase® Reaction Buffer v2
  • NEBNext® Enzymatic Methyl-seq Kit
  • NEBNext® Library Quant Kit for Ultima Genomics®
  • NEBNext® Enzymatic Methyl-seq v2 Kit
  • NEBNext UltraExpress® DNA Library Prep Kit
  • NEBNext UltraExpress® FS DNA Library Prep Kit
  • NEBNext UltraExpress® RNA Library Prep Kit
  • NEBNext UltraShear® FFPE DNA Library Prep Kit
  • NEBNext® FFPE DNA Library Prep Kit
  • NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina®
  • NEBNext® Ultra™ II Directional RNA Library Prep with Sample Purification Beads
  • NEBNext® Ultra™ II DNA Library Prep Kit for Illumina®
  • NEBNext® Ultra™ II FS DNA Library Prep Kit for Illumina
  • NEBNext® Ultra™ II FS DNA Library Prep with Sample Purification Beads
  • NEBNext® Globin & rRNA Depletion Kit (Human/Mouse/Rat)
  • NEBNext® Globin & rRNA Depletion Kit (Human/Mouse/Rat) with RNA Sample Purification Beads
  • NEBNext® Magnetic Separation Rack
  • NEBNext® Poly(A) mRNA Magnetic Isolation Module
  • NEBNext® RNA Depletion Core Reagent Set
  • NEBNext® rRNA Depletion Kit (Bacteria)
  • NEBNext® rRNA Depletion Kit (Bacteria) with RNA Sample Purification Beads
  • NEBNext® rRNA Depletion Kit v2 (Human/Mouse/Rat)
  • NEBNext® rRNA Depletion Kit v2 (Human/Mouse/Rat) with RNA Sample Purification Beads
  • EpiMark® Methylated DNA Enrichment Kit
  • EpiMark® N6-Methyladenosine Enrichment Kit
  • EpiMark® Hot Start Taq DNA Polymerase
  • NEBNext® Enzymatic 5hmC-seq Conversion Module
  • NEBNext® Enzymatic Methyl-seq Kit
  • NEBNext® Enzymatic Methyl-seq v2 Conversion Module
  • NEBNext® LV Unique Dual Index Primers Set 2B
  • NEBNext® LV Unique Dual Index Primers Set 3
  • NEBNext® Enzymatic 5hmC-seq Kit
  • NEBNext® Enzymatic Methyl-seq v2 Kit
  • NEBNext® Magnetic Separation Rack
  • NEBNext® Microbiome DNA Enrichment Kit
  • NEBNext® LV Unique Dual Index Primers Set 2B
  • NEBNext® LV Unique Dual Index Primers Set 3
  • NEBNext® Multiplex Oligos for Illumina (Unique Dual Index UMI Adaptors RNA Set 1)
  • NEBNext® Multiplex Oligos for Illumina® (96 Unique Dual Index Primer Pairs Set 2)
  • NEBNext® Multiplex Oligos for Illumina® (96 Unique Dual Index Primer Pairs Set 3)
  • NEBNext® Multiplex Oligos for Illumina® (96 Unique Dual Index Primer Pairs)
  • NEBNext® Multiplex Oligos for Illumina® (Dual Index Primers Set 2)
  • NEBNext® Multiplex Oligos for Illumina® (Unique Dual Index UMI Adaptors DNA Set 1)
  • NEBNext® Multiplex Oligos for Illumina® (Dual Index Primers Set 1)
  • NEBNext® LV Unique Dual Index Primers Set 1
  • NEBNext® LV Unique Dual Index Primers Set 2
  • NEBNext® LV Unique Dual Index Primers Set 2A
  • NEBNext® LV Unique Dual Index Primers Set 4
  • NEBNext® LV Unique Dual Index Primers Set 5
  • NEBNext® Multiplex Oligos for Illumina® (96 Unique Dual Index Primer Pairs Set 4)
  • NEBNext® Multiplex Oligos for Illumina® (Unique Dual Index UMI Adaptors DNA Set 2)
  • NEBNext® Multiplex Oligos for Illumina® (Unique Dual Index UMI Adaptors DNA Set 3)
  • NEBNext® Multiplex Oligos for Illumina® (Unique Dual Index UMI Adaptors DNA Set 4)
  • NEBNext UltraShear®
  • NEBNext UltraShear® FFPE DNA Library Prep Kit
  • NEBNext® FFPE DNA Library Prep Kit
  • NEBNext® FFPE DNA Repair Mix
  • NEBNext® FFPE DNA Repair v2 Module
  • NEBNext® Magnetic Separation Rack
  • NEBNext® Circularization Module for MGI®
  • NEBNext® FS DNA Library Prep Kit for MGI®
  • NEBNext® Multiplex Oligos for MGI® (Dual Index Primer Pairs Set 1)
  • NEBNext® RNA Library Prep Kit for MGI®
  • NEBNext® Library Dilution Buffer
  • NEBNext® Library Quant DNA Standards
  • NEBNext® Library Quant Kit for Illumina®
  • NEBNext® Library Quant Kit for Ultima Genomics®
  • NEBNext Direct® Genotyping Solution
  • NEBNext® Enzymatic Methyl-seq Conversion Module
  • NEBNext® Enzymatic Methyl-seq Kit
  • NEBNext® Multiplex Oligos for Illumina® (96 Index Primers)
  • NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina®
  • NEBNext® Ultra™ II Directional RNA Library Prep with Sample Purification Beads
  • NEBNext® Ultra™ II Directional RNA Second Strand Synthesis Module
  • NEBNext® Ultra™ II DNA Library Prep with Sample Purification Beads
  • NEBNext® Ultra™ II FS DNA Library Prep Kit for Illumina
  • NEBNext® Ultra™ II FS DNA Library Prep with Sample Purification Beads
  • NEBNext® Ultra™ II FS DNA Module
  • NEBNext® Ultra™ II Q5® Master Mix
  • NEBNext® Ultra™ II RNA First Strand Synthesis Module
  • NEBNext® Ultra™ II FS DNA PCR-free Library Prep with Sample Purification Beads
  • NEBNext® Ultra™ II DNA PCR-free Library Prep Kit for Illumina®
  • NEBNext® Ultra™ II FS DNA PCR-free Library Prep Kit for Illumina®
  • NEBNext UltraShear®
  • NEBNext® Low-bias Small RNA Library Prep Kit
  • NEBNext® Multiplex Small RNA Library Prep Kit for Illumina® (Index Primers 1-48)
  • NEBNext® Multiplex Small RNA Library Prep Set for Illumina® (Set 1)
  • NEBNext® Multiplex Small RNA Library Prep Set for Illumina® (Set 2)
  • NEBNext® Small RNA Library Prep Set for Illumina® (Multiplex Compatible)
  • NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina®
  • NEBNext® Ultra™ II DNA Library Prep Kit for Illumina
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