Library preparation for the major next generation sequencing 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™ or SOLiD™ 4 library construction, can be used directly in the ligation step. For Illumina® libraries and some libraries intended for the 454™ platform, 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 for Illumina, SOLiD and Ion Torrent platforms can be selected via gel electrophoresis before amplification by the polymerase chain reaction (PCR). 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.
Ion Torrent™ and SOLiD™ 4 are trademarks owned by Life Technologies, Inc.
Illumina® is a registered trademark of Illumina, Inc.
454™ is a trademark of Roche.
FAQs for DNA Library Preparation
Protocols for DNA Library Preparation
- Control Reaction Protocol for PreCR Repair Mix
- Data Analysis - NEBNext Library Quant Kit (E7630)
- Double Digest Protocol with Standard Restriction Enzymes
- Expected Results - NEBNext Library Quant Kit (E7630)
- Experimental Considerations - NEBNext Library Quant Kit (E7630)
- Guidelines for Running Samples on the Illumina MiSeq (E7000)
- Guidelines for Setting up PCR Reactions (E6627X only)
- Guidelines for Setting Up PCR Reactions (E7000X only)
- Index Pooling Guidelines - 96 Single Index Kit (E6609)
- Ligation Protocol with T4 DNA Ligase (M0202)
- Loop-mediated Isothermal Amplification (LAMP)
- NEBNext FFPE DNA Repair Mix (M6630) - Protocol for use with Other User-supplied Library Construction Reagents
- NEBNext Library Quant Kit Protocol - NEBNext Library Quant Kit (E7630)
- NEBNext Library Quant Kit Quick Protocol (E7630)
- Optimizing Restriction Endonuclease Reactions
- PCR Using NEBNext® High-Fidelity 2X PCR Master Mix (M0541)
- Protocol for a PCR reaction using NEBNext® Q5® Hot Start HiFi PCR Master Mix (M0543)
- Protocol for a PCR reaction using NEBNext® Ultra™ II Q5® Master Mix (M0544)
- 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)
- Protocol for Digestion Prior to droplet digital PCR (ddPCR)
- Protocol for Direct Digestion of gDNA during droplet digital PCR (ddPCR)
- Protocol for NEBNext Direct BRCA1/BRCA2 Panel (E6627)
- Protocol for NEBNext Direct® Cancer HotSpot Panel (NEB #E7000)
- Protocol for use with NEBNext FFPE DNA Repair Mix (M6630) and NEBNext DNA Library Prep Master Mix Set for Illumina (E6040)
- Protocol for use with NEBNext Ultra DNA Library Prep Kit for Illumina (E7370)
- 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)
- Quick Ligation Protocol (M2200)
- Sequential Reaction Protocol for PreCR Repair Mix
- Setting up the PCR Reactions < 96 Samples and 96 Samples - 96 Single Index Kit (E6609)
- Standard Reaction Protocol for PreCR Repair Mix
- Transformation Protocol
Improving Enzymatic DNA Fragmentation for Next Generation Sequencing Library Construction
Other Tools & Resources
DNA Library Preparation Workflow
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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