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 Protocol with T4 DNA Ligase (M0202)
- Ligation of 3´ and 5´ Adaptors (E6120)
- NEBNext dA-Tailing Module Protocol (E6053)
- NEBNext End Repair Module Protocol (E6050)
- NEBNext Quick Ligation Module Protocol (E6056)
- PCR Amplification (E6120)
- Quick Ligation Protocol (M2200)
- Reverse Transcription (E6120)
- Protocol for NEBNext® Ultra™ II Non-Directional RNA Second Strand Synthesis Module (E6111)
- Transformation Protocol
- Protocol Phusion® High-Fidelity PCR Master Mix with HF Buffer
- PCR Optimization with Phusion® High-Fidelity PCR Kit
- Protocol for a Routine PCR with Phusion® High-Fidelity PCR Kit
- PCR Optimization of the Control Template using Phusion® High-Fidelity PCR Kit
- NEBNext End Prep (E7442)
- Protocol for use with NEBNext Ultra DNA Library Prep Kit for Illumina (E7370)
- Protocol for use with NEBNext DNA Library Prep Master Mix Set for Illumina (E6040)
- Setting up the PCR Reaction - NEBNext Multiplex Oligos for Illumina (Dual Index Primers Set 1) (E7600)
- 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
- Refer to the manual for a list of NEBNext library prep kits that contain protocols for use of NEBNext adaptors and primers.
- Setting up the PCR Reaction - NEBNext Multiplex Oligos for Illumina (Dual Index Primers Set 2) (E7780)
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.
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.
- A Robust, Streamlined, Enzyme-based DNA Library Preparation Method Amenable to a Wide Range of DNA Inputs (2019)
- EM-seq™ Enables Accurate and Precise Methylome Analysis of Challenging DNA Samples (2019)
- NEBNext® Ultra™II FS DNA: A Robust Enzyme-based DNA Library Preparation Method Compatible with Plant and Animal Samples (2019)
- Uncovering the Cannabis sativa Methylome Through Enzymatic Methyl-seq (2019)
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