Library preparation for the major next generation sequencing (NGS) platforms requires the ligation of specific adaptor oligos to fragments of the DNA to be sequenced. First, DNA is fragmented to the optimal length determined by the downstream platform. Because DNA fragmentation does not result in homogeneous, blunt-ended fragments, end repair is needed to ensure that each molecule is free of overhangs, and contains 5′ phosphate and 3′ hydroxyl groups. Libraries to be used in blunt-ended adaptor ligation, including Ion Torrent™ library construction, can be used directly in the ligation step. For Illumina® libraries, incorporation of a non-templated deoxyadenosine 5′-monophosphate (dAMP) onto the 3′ end of blunted DNA fragments, a process known as dA-tailing, is necessary. dA-tails prevent concatamer formation during downstream ligation steps, and enable DNA fragments to be ligated to adaptors with complementary dT-overhangs. The desired adaptor-ligated DNA size can be achieved via bead-based size selection before optional PCR amplification. New England Biolabs supplies reagents for DNA library preparation for the leading sequencing platforms. For more information, choose the DNA Library Construction Workflow tab below.
- Control Reaction Protocol for PreCR Repair Mix
- Ligation Protocol with T4 DNA Ligase (M0202)
- Quick Ligation Protocol (M2200)
- Sequential Reaction Protocol for PreCR Repair Mix
- Standard Reaction Protocol for PreCR Repair Mix
- Transformation Protocol
- PCR Using NEBNext® High-Fidelity 2X PCR Master Mix (M0541)
- Optimizing Restriction Endonuclease Reactions
- Protocol for blunting ends by 3' overhang removal and fill-in of 3' recessed (5' overhang) ends using DNA Polymerase I, Large (Klenow) Fragment (M0210)
- Protocol for blunting ends by 3’ overhang removal and 3’ recessed (5’ overhang) end fill-in using T4 DNA Polymerase (M0203)
- Protocol for Cre Recombinase (M0298)
- Loop-mediated Isothermal Amplification (LAMP)
- Protocol for a PCR reaction using NEBNext® Q5® Hot Start HiFi PCR Master Mix (M0543)
- Protocol for use with NEBNext Ultra DNA Library Prep Kit for Illumina (E7370)
- Protocol for use with NEBNext FFPE DNA Repair Mix (M6630) and NEBNext DNA Library Prep Master Mix Set for Illumina (E6040)
- NEBNext FFPE DNA Repair Mix (M6630) - Protocol for use with Other User-supplied Library Construction Reagents
- Data Analysis - NEBNext Library Quant Kit (E7630)
- Experimental Considerations - NEBNext Library Quant Kit (E7630)
- NEBNext Library Quant Kit Protocol - NEBNext Library Quant Kit (E7630)
- NEBNext Library Quant Kit Quick Protocol (E7630)
- Expected Results - NEBNext Library Quant Kit (E7630)
- Protocol for use with NEBNext® Ultra™ II End Repair/dA-Tailing Module (E7546)
- Protocol for a PCR reaction using NEBNext® Ultra™ II Q5® Master Mix (M0544)
- Setting up the PCR Reactions < 96 Samples and 96 Samples - 96 Single Index Kit (E6609)
- Index Pooling Guidelines - 96 Single Index Kit (E6609)
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.
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.
Enzymatic Methyl-seq: The Next Generation of Methylome Analysis
- A Robust, Streamlined, Enzyme-based DNA Library Preparation Method Amenable to a Wide Range of DNA Inputs (2019)
- A Single-tube, Low Input Protocol for Long Read Sequencing (2019)
- EM-seq™ Enables Accurate and Precise Methylome Analysis of Challenging DNA Samples (2019)
- NEBNext Direct® Custom Ready Panels Overcome Challenges Associated with Targeted Re-sequencing (2019)
- NEBNext® Ultra™II FS DNA: A Robust Enzyme-based DNA Library Preparation Method Compatible with Plant and Animal Samples (2019)
- Vladimir Potapov, Jennifer L. Ong. 2017. Examining Sources of Error in PCR by Single-Molecule Sequencing. PLOS One. , PubMedID: 28683110, DOI:
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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.
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.
Get a quick overview of NGS library preparation from an NEB scientist.
This video will help you decide if you need to do a size selection or a simple clean-up for the NEBNext Ultra DNA or RNA.