Library preparation for the Illumina® sequencing platform requires fragmentation of DNA, repair of 3´ and 5´ ends to form blunt-ended, phosphorylated molecules, and the addition of a non-templated dA-tail before ligation to an adaptor. If necessary to achieve sufficient yields, the final step is PCR amplification of the library. For more detail, select a workflow tab below.
NEBNext® kits are available for sample preparation of genomic DNA, ChIP DNA, methylated DNA, microbiome DNA, and FFPE DNA. These are complemented by a wide range of index primers, for multiplexing. Setting a new standard, our NEBNext Ultra™ II FS DNA kits include a new fragmentation reagent, which is combined with end-repair and dA-tailing reagents, enabling these steps to be performed in the same tube, without any clean-ups (and therefore without any sample loss). The same fragmentation protocol is used for any input amount from 100 pg to 500 ng, and DNA of any GC content for added ease. The high-efficiency Ultra II ligation reagents and Q5® Ultra II PCR master mix complete the library prep workflow, resulting in a fast, high-yield, high-quality library prep workflow that is completely scalable.
NEBNext Enzymatic Methyl-seq, an enzyme-based alternative to bisulfite sequencing employs Ultra II reagents and enables superior detection of 5mC and 5hmC, from fewer sequencing reads. Details on this workflow can be found below.
- Ligation of 3´ and 5´ Adaptors (E6120)
- Ligation Protocol with T4 DNA Ligase (M0202)
- NEBNext dA-Tailing Module Protocol (E6053)
- NEBNext End Prep (E7442)
- NEBNext End Repair Module Protocol (E6050)
- NEBNext Quick Ligation Module Protocol (E6056)
- PCR Amplification (E6120)
- PCR Optimization of the Control Template using Phusion® High-Fidelity PCR Kit
- PCR Optimization with Phusion® High-Fidelity PCR Kit
- Please see manual (NEB #E6000) for protocols.
- Please see manual (NEB #E6100) for protocols
- Please see manual (NEB #E6240) for protocols
- Please see manual (NEB #E7445) for protocols
- Protocol for a Routine PCR with Phusion® High-Fidelity PCR Kit
- Protocol for NEBNext® Ultra™ II Non-Directional RNA Second Strand Synthesis Module (E6111)
- Protocol for use with NEBNext DNA Library Prep Master Mix Set for Illumina (E6040)
- Protocol for use with NEBNext Ultra DNA Library Prep Kit for Illumina (E7370)
- Protocol Phusion® High-Fidelity PCR Master Mix with HF Buffer
- Quick Ligation Protocol (M2200)
- Refer to the manual for a list of NEBNext library prep kits that contain protocols for use of NEBNext adaptors and primers.
- Reverse Transcription (E6120)
- Setting up the PCR Reaction - NEBNext Multiplex Oligos for Illumina (Dual Index Primers Set 1) (E7600)
- Setting up the PCR Reaction - NEBNext Multiplex Oligos for Illumina (Dual Index Primers Set 2) (E7780)
- Transformation Protocol
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.
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 [email protected].
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.
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 for DNA quantification and assessing purity.
This video walks you through the size selection and cleanup steps using the NEBNext Ultra II DNA
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.
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.
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.
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.
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.