Library preparation for the Illumina sequencing platform requires fragmentation of DNA followed by 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 and FFPE DNA. Our most recent NEBNext Ultra II FS DNA kits include a new fragmentation reagent, which is also 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 for any GC content DNA. The high-efficiency Ultra II ligation reagents and Q5 Ultra II PCR master mix complete the library prep workflow. The result is a fast, high-yield, high-quality library prep workflow that is completely scalable.
FAQs for DNA for Illumina®
Protocols for DNA for Illumina®
- 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
Other Tools & Resources
Ultra II FS DNA Library Preparation Workflow
ChIP DNA Library Preparation Workflow for Illumina
Ultra II DNA Library Preparation for Illumina
DNA Product Details for Illumina
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.
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.