NEBNext® Sample Preparation for Next Generation Sequencing
Preparation of high quality libraries at high yield is a critical first step in next generation sequencing workflows. NEBNext® reagents are a series of highly pure reagents that facilitate sample preparation of DNA and RNA and target enrichment of DNA for next generation sequencing. NEBNext kits are carefully designed to improve yields and library diversity using a broad range of input amounts from a wide variety of sample types. This has been most recently expanded with the addition of a novel enzyme-based DNA fragmentation system in the NEBNext Ultra II FS DNA Library Prep Kit (NEB #E7805), which enables reliable high-yield high-quality library construction with as little as 100 pg of input DNA.
Another recent technology addition to the NEBNext product line is NEBNext Direct® for target enrichment. This unique hybridization-based technology enables high sensitivity variant calling with the speed and precision previously only feasible using multiplex PCR-based approaches. The NEBNext Direct Cancer HotSpot Panel (NEB #E7000) provides highly specific hybridization-based capture of 190 common cancer targets from 50 genes, while the NEBNext Direct BRCA1/BRCA2 Panel (NEB #E6627) generates 100% coverage of all protein coding regions in BRCA1 and BRCA2 genes.
NEBNext® Sample Preparation for Next Generation Sequencing includes these areas of focus:
- Illumina® Library Preparation
- RNA for Illumina®
- DNA for Illumina®
- NEBNext® Ultra™ II DNA Library Prep
- NEBNext® Ultra™ II RNA Library Prep
- Ion Torrent™ DNA Library Preparation
- DNA for Ion Torrent™
- DNA Library Preparation
- RNA Library Preparation
- DNA Fragmentation
- FFPE DNA Repair
- NEBNext FFPE DNA Repair Mix
- NEBNext® Automation
FAQs for NEBNext® Sample Preparation for Next Generation Sequencing
Protocols for NEBNext® Sample Preparation for Next Generation Sequencing
- Appendix A for use with NEBNext Ultra II RNA First Strand Synthesis Module (E7771)
- Appendix A for use with Ultra™ II RNA and Ultra II Directional RNA (E7760, E7765, E7770, E7775)
- Guidelines for Running Samples on the Illumina MiSeq (E7000)
- Guidelines for Running Samples on the Illumina MiSeq (E6627)
- Guidelines for Setting up PCR Reactions (E6627)
- Guidelines for Setting Up PCR Reactions (NEB #E7000X only)
- Index Pooling Guidelines - 96 Single Index Kit (E6609)
- Index Pooling Guidelines (E6627)
- Index Pooling Guidelines (NEB #E6631)
- Index Pooling Guidelines (NEB #E7000)
- Protocol for a PCR reaction using NEBNext® Ultra™ II Q5® Master Mix (M0544)
- Protocol for Directional RNA-seq Workflows and NEBNext® Ultra™ II RNA First Strand Synthesis Module (E7771)
- Protocol for NEBNext Direct BRCA1/BRCA2 Panel (E6627)
- Protocol for NEBNext Direct® Cancer HotSpot Panel (NEB #E7000)
- Protocol for Non-directional RNA-seq Workflows and NEBNext® Ultra™ II RNA First Strand Synthesis Module (E7771)
- Protocol for use with FFPE RNA, NEBNext® rRNA Depletion Kit (Human/Mouse/Rat) (E6310) and NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina® (E7760, E7765)
- Protocol for use with FFPE RNA, NEBNext® rRNA Depletion Kit (Human/Mouse/Rat) (E6310) and NEBNext® Ultra™ II RNA Library Prep Kit for Illumina® (E7770, E7775)
- Protocol for use with NEBNext® Ultra™ II DNA Library Prep Kit for Illumina® (E7645, E7103)
- Protocol for use with NEBNext® Ultra™ II End Repair/dA-Tailing Module (E7546)
- Protocol for use with NEBNext® Poly(A) mRNA Magnetic Isolation Module (E7490) and NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina® (E7760, E7765)
- Protocol for use with NEBNext® Poly(A) mRNA Magnetic Isolation Module (E7490) and NEBNext® Ultra™ II RNA Library Prep Kit for Illumina® (E7770, E7775)
- Protocol for use with NEBNext® rRNA Depletion Kit (Human/Mouse/Rat) (E6310) and NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina® (E7760, E7765)
- Protocol for use with NEBNext® rRNA Depletion Kit (Human/Mouse/Rat) (E6310) and NEBNext® Ultra™ II RNA Library Prep Kit for Illumina® (E7770, E7775)
- Protocol for use with Purified mRNA or rRNA Depleted RNA and NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina® (E7760, E7765)
- Protocol for use with Purified mRNA or rRNA Depleted RNA and NEBNext® Ultra™ II RNA Library Prep Kit for Illumina® (E7770, E7775)
- Protocol for use with rRNA Depleted FFPE RNA and NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina® (E7760, E7765)
- Protocol for use with rRNA Depleted FFPE RNA and NEBNext® Ultra™ II RNA Library Prep Kit for Illumina® (E7770, E7775)
- Setting up the PCR Reactions < 96 Samples and 96 Samples - 96 Single Index Kit (E6609)
- Improved methods for site-directed mutagenesis using Gibson Assembly™ Master Mix
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Addressing Challenges in Microbiome DNA Analysis
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Improving Enzymatic DNA Fragmentation for Next Generation Sequencing Library Construction
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The Quantitation Question: How does accurate library quantitation influence sequencing?
The determination of the number of sequencing-ready molecules present after library preparation is an important step in the next generation sequencing (NGS) workflow and has a strong influence on the success of both a sequencing run and a sequencing-based experiment.
Other Tools & Resources
Application Notes
Feature Articles
- Luck, A.N., Anderson, K.G., McClung, C.M., VerBerkmoes, N.C., Foster, J.M., Michalski, M.L. and Slatko, B.E. 2015. Tissue-specific transcriptomics and proteomics of a filarial nematode and its Wolbachia endosymbiont BMC Genomics. , PubMedID: 26559510, DOI:
Publications related to NEBNext® Sample Preparation for Next Generation Sequencing
Legal Information
While NEB develops and validates its products for various applications, the use of this product may require the buyer to obtain additional third party intellectual property rights for certain applications.
For more information about commercial rights, please contact NEB's Global Business Development team at gbd@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.
Videos
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NEBNext Ultra II Directional RNA Workflow
Learn more about the streamlined workflow for the NEBNext Ultra II Directional RNA Library Prep Kit.
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Size Selection and Cleanup with NEBNext Ultra II and SPRI beads
This video walks you through the size selection and cleanup steps using the NEBNext Ultra II DNA
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Tips for Optimizing DNA Inputs in NGS Library Construction
Optimizing DNA inputs for NGS library prep is an important step, if you want to ensure a high-quality library. Start by following these tips.
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Tips for Optimizing RNA Inputs in NGS Library Construction
Watch as Deyra explains how to optimize your RNA inputs for successful NGS library preparation.
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NEBNext Direct Workflow
View our tutorial for help visualizing NEBNext Direct’s unique and enabling workflow.
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NEBNext Direct® Target Enrichment
Learn about the innovative NEBNext Direct technology for target enrichment for next generation sequencing.
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How Library Preparation Affects Sequencing Accuracy
In this webinar, Laurence Etwiller and Jennifer Ong discuss their recent publications, and how library preparation affects sequencing accuracy and variant calling. View full Q & A summary.
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Behind the paper: DNA damage is a pervasive cause of sequencing errors, directly confounding variant identification
NEB researchers published a paper in Science highlighting DNA damage as a prevalent source of errors in public cancer databases. Learn about how addressing this damage could improve the detection of low-frequency disease variants.
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Challenges and Opportunities for Next Generation Sequencing Target Enrichment
Target enrichment offers several advantages over more comprehensive genomic profiling for a variety of scientific and applications. During this webinar, we explore the advantages and limitations of target enrichment, and discuss how NEBNext Direct®, a novel solution for selective enrichment of genomic targets, addresses these challenges.
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NEBNext Ultra II DNA Library Prep Protocol
This video walks you through DNA Library Preparation using the NEBNext Ultra II DNA Library Prep Kit. The video also includes tips for optimization as well as safe stopping points.
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NEBNext for Core Lab Facilities
Are you running a core sequencing facility? Watch as Eileen shares a few reasons that NEBNext products are particularly well suited to sequencing core labs.
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12 Quick Tips for NGS Library Preparation
These 12 quick tips for NGS library preparation are a great refresher if you're a seasoned pro, or a great introduction if you're a beginner.
