Jun 11, 2026
Direct enzymatic fragmentation of genomic DNA with NEBNext UltraShear for use with Illumina 5-Base DNA Library Prep without post-shearing cleanup
- Christian Pflueger1,2,
- Jahnvi Pflueger3,
- Ryan Lister1,2
- 1University of Western Australia;
- 2Harry Perkins Institute of Medical Research;
- 3Illumina
Protocol Citation: Christian Pflueger, Jahnvi Pflueger, Ryan Lister 2026. Direct enzymatic fragmentation of genomic DNA with NEBNext UltraShear for use with Illumina 5-Base DNA Library Prep without post-shearing cleanup. protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vzwnq2vx1/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: June 11, 2026
Last Modified: June 11, 2026
Protocol Integer ID: 318902
Keywords: Enzymatic fragmentation, 5-base library preparation, DNA methylation, Ultrashear, base dna library preparation workflow, direct enzymatic fragmentation, enzymatic fragmentation, direct enzymatic fragmentation of genomic dna, enzymatic shearing, enzymatic shearing for qc, end repair step of the illumina, end repair for the illumina, base dna library prep, μl dna input volume, ultrashear fragmentation, base dna prep, workflow for illumina, end repair step, genomic dna, throughput library preparation, mechanical covaris shearing, starting end repair, mechanical shearing
Abstract
This protocol describes a modified workflow for Illumina 5-Base DNA Prep in which mechanical Covaris shearing is replaced by enzymatic fragmentation using NEBNext UltraShear. The workflow is designed to support mid- to high-throughput library preparation by removing the bottleneck of mechanical shearing. NEBNext UltraShear fragmentation is performed in a final volume of 45 μL, which is directly compatible with the 45 μL DNA input volume required for the End Repair step of the Illumina 5-Base DNA Library Preparation Workflow. An optional AMPure cleanup may be performed after enzymatic shearing for QC or buffer exchange, but this cleanup is optional prior to starting end repair for the Illumina 5-Base workflow.
Guidelines
- This protocol was developed for purified genomic DNA and is intended to be used along with the Illumina 5-base DNA Prep Product Documentation (Document # 200073497 v00)
- Part 1 of the protocol details how to benchmark each lot of NEBNext UltraShear enzyme before using it for production-scale library preparation.
- Do not rely solely on the manufacturer's example fragmentation time. Fragmentation efficiency can vary by enzyme lot, DNA source, DNA quality, and workflow conditions.
- For this workflow, the desired fragmentation profile of DNA is in the 100-1000 bp range, with minimal fragments in the 1000-5000 bp range.
- Part 2 of the protocol details how to proceed directly into Illumina 5-Base DNA Prep after performing NEBNext UltraShear fragmentation
- Optional post-shearing AMPure / Illumina Purification Bead cleanup of 1.5x can be used for method development and QC but is not required for the streamlined workflow.
Materials
All reagents are provided in the Illumina 5-base DNA prep kit unless mentioned otherwise. Refer to the Illumina DNA Prep Product Documentation for user-supplied consumables and equipment information
- Purified genomic DNA
- Genomic DNA Methylation Control (GDMC)
- NEBNext UltraShear Reaction Buffer (part of NEBNext UltraShear Cat # M7634)
- NEBNext UltraShear enzyme (part of NEBNext UltraShear Cat # M7634)
- Nuclease-free water
- 0.2 mL PCR tubes or PCR plate
- Thermocycler
- Ice bucket
- Illumina Purification Beads (IPB)
- AMPure XP beads or equivalent magnetic purification beads, optional
- Magnetic rack, optional
- Freshly prepared 80% ethanol, optional
- Low-bind tubes or plate for elution, optional
- Illumina 5-Base DNA Prep kit
- Illumina index adapters compatible with the 5-Base DNA Prep workflow
- ProNex Size-Selective Purification System (Promega Cat # NG2001)
- Qubit dsDNA HS assay or equivalent
- Agilent D5000 ScreenTape or equivalent fragment analysis method (Agilent, ladder: catalog # 5067-5590, D5000 Reagents: 5067-5589 and D5000 ScreenTape: 5067-5588)
Safety warnings
Critical: Benchmarking is essential. In this test, the selected condition was 30 min at 37°C, whereas the manufacturer's example data might have suggested a shorter incubation time.
Critical: Do not perform AMPure cleanup at this point when using the streamlined workflow. The full 45 μL UltraShear reaction can be transferred directly into the End Repair reaction.
Before start
- Thaw NEBNext UltraShear Reaction Buffer and NEBNext UltraShear enzyme on ice.
- Mix NEBNext UltraShear Reaction Buffer thoroughly before use.
- Keep UltraShear enzyme and master mix reactions on ice before starting the incubation.
- Pre-program and pre-warm thermocyclers with heated lid for fragmentation and heat inactivation.
- For benchmarking, prepare a fragmentation time course rather than a single incubation time (Refer to section 3 for more details)
Part 1: Protocol for Benchmarking Fragmentation conditions
Prepare genomic DNA input
Quantify purified genomic DNA using Qubit dsDNA HS assay or equivalent.
Assess DNA quality using TapeStation, Femto Pulse, Bioanalyzer, gel electrophoresis, or an equivalent method.
For each reaction, prepare 100 ng genomic DNA with GDMC in nuclease-free water as per manufacturer’s instructions.
Adjust the DNA with GDMC and water volume to 27 μL per reaction.
Note: This protocol uses 100 ng genomic DNA input per reaction. Adjustments may be required for
other input amounts.
Assemble NEBNext UltraShear fragmentation reaction
For each 45 μL reaction, combine the reagents shown below on ice
Mix gently but thoroughly.
Briefly spin down the reaction.
Keep reactions on ice until all samples are ready to load into the thermocycler.
| Reagent | Volume per reaction | |
| Genomic DNA plus nuclease-free water | 27 µL | |
| NEBNext UltraShear Reaction Buffer | 14 µL | |
| NEBNext UltraShear enzyme | 4 µL | |
| Total | 45 µL |
Critical: The 45 µL reaction volume is intentional because it matches the DNA input volume for the
Illumina 5-Base DNA Prep End Repair step.
Benchmark Ultrashear fragmentation time
Prepare a fragmentation time course before committing to production-scale library preparation.
Suggested initial time course: 15, 20, 25, and 30 min at 37°C.
Note: A broader time course, such as 10-30 min or 15-45 min,
may be useful when testing a new enzyme lot, new DNA source, or degraded sample
type.
For each time point, prepare an individual 45 μL fragmentation reaction.
Incubate reactions at 37°C.
At the end of each time point, immediately transfer the reaction to a thermocycler held at 65°C.
Heat-inactivate at 65°C for 15 min.
Keep heat-inactivated reactions on ice or at 4°C until cleanup or QC.
IPB / AMPure XP cleanup after fragmentation for benchmarking on Fragment Analyser
Note: this clean up is useful during benchmarking but is not required for the streamlined direct-entry workflow
Bring IPB / AMPure XP beads to room temperature and mix thoroughly.
Add 1.5x AMPure beads to each fragmented DNA reaction.
Mix thoroughly and incubate according to the bead manufacturer’s instructions.
Place on a magnetic rack until the solution clears.
Remove and discard the supernatant without disturbing the beads.
Wash beads twice with freshly prepared 80% ethanol.
Air-dry beads briefly. Do not over-dry.
Elute in 47 μL nuclease-free water or suitable low-EDTA buffer.
Use 1 μL for D5000 TapeStation QC.
Reserve 45 μL for use into the Illumina 5-Base DNA Prep End Repair step.
Evaluate fragmentation profile
Run 1 μL of each cleaned-up benchmarking reaction on a D5000 ScreenTape or equivalent.
Assess the fraction of DNA in the 100-1000 bp region.
Assess the fraction of DNA in the 1000-5000 bp region.
Interpret DNA in the 1000-5000 bp region as under-fragmented material.
Select the shortest incubation time that provides adequate fragments in the desired 100-1000 bp region and sufficiently reduces the under-fragmented 1000-5000 bp fraction.
Example result: A test time course using input genomic DNA showed progressive reduction of under-sheared DNA with increasing UltraShear incubation time. In this test, 30 min at 37°C gave the best profile among the tested conditions and was selected for subsequent library preparation.
Critical: Benchmarking is essential. In this test, the selected condition was 30 min at 37°C, whereas the manufacturer’s example data might have suggested a shorter incubation time.
| A | B | C | D | E | |
| Time | Avg size 100-1000 bp | % total 100-1000 bp | Avg size 1000-5000 bp | % total 1000-5000 bp | |
| 10 min | 568 bp | 36.60% | 1964 bp | 43.55% | |
| 15 min | 531 bp | 52.43% | 1648 bp | 30.78% | |
| 20 min | 508 bp | 59.10% | 1578 bp | 24.80% | |
| 25 min | 489 bp | 65.19% | 1529 bp | 18.98% | |
| 30 min | 480 bp | 65.56% | 1532 bp | 17.77% |
Benchmarking Fragmentation time results
Figure 1. Overlay of D5000 TapeStation profiles for NEBNext UltraShear time-course reactions. The 30 min condition showed the lowest residual high-molecular-weight fraction among tested conditions while preserving a broad library-prep-compatible distribution.
Figure 2. NEB example fragmentation profile. This illustrates why initial fragmentation times may appear adequate based on manufacturer guidance, but enzyme lot benchmarking by users remains important.
Part 2: Protocol for using Illumina 5-Base DNA Prep directly after UltraShear Fragmentation
Direct UltraShear fragmentation for Illumina 5-Base DNA Prep
Prepare 100 ng genomic DNA and GDMC per sample in 27 μL nuclease-free water.
Add 14 μL NEBNext UltraShear Reaction Buffer.
Add 4 μL NEBNext UltraShear enzyme.
Mix gently and briefly spin down.
Incubate at 37°C for the optimized fragmentation time.
Heat-inactivate at 65°C for 15 min.
After heat inactivation, place reactions on ice or proceed directly to Illumina 5-Base DNA Prep End Repair.
Critical: Do not perform AMPure cleanup at this point when using the streamlined workflow. The full 45 μL UltraShear reaction can be transferred directly into the End Repair reaction.
Begin Illumina 5-Base DNA Prep at End Repair
Use the full 45 μL heat-inactivated UltraShear reaction as the DNA input for the Illumina 5-Base DNA Prep End Repair step.
Continue with the Illumina 5-Base DNA Prep protocol from the End Repair step.
Refer to the current Illumina 5-Base DNA Prep product documentation for reagent names, reagent volumes, incubation conditions, and cleanup conditions.
Note: The Illumina 5-Base protocol should be treated as the source of truth for all downstream library preparation steps after fragmentation.
Continue Illumina 5-Base DNA Prep workflow
Perform End Repair according to the Illumina 5-Base DNA Prep protocol
Perform Adapter Ligation according to the Illumina 5-Base DNA Prep protocol.
Perform DNA methylation conversion using the Illumina 5-Base DNA Prep conversion reagents and conditions.
Perform PCR with indexed adapters according to the Illumina 5-Base DNA Prep protocol.
Perform cleanup with Illumina Purification Beads according to the Illumina 5-Base DNA Prep protocol.
Quantify final libraries using Qubit dsDNA HS assay or equivalent.
Assess final library size distribution using Agilent TapeStation, Bioanalyzer, Fragment Analyzer, or equivalent.
Perform optional ProNex size selection for desired fragment distribution (refer to details below).
Pool libraries according to required sequencing depth and calculated library molarity.
Library QC before pooling and recommended ProNex size selection
Libraries generated from Covaris-sheared DNA and from NEBNext UltraShear directly into the Illumina 5-Base workflow produce comparable library profiles after Illumina Purification Bead cleanup, where UltraShear prepared libraries exhibit a broader fragment size distribution. In the example below, lanes B1-E1 are libraries prepared from Covaris M220-sheared DNA targeting ~450 bp input fragments. Lanes F1-G1 are libraries generated from 30 min NEBNext UltraShear fragmentation at 37°C, and then processed directly into the Illumina 5-Base End Repair step without post-shearing cleanup. All shown libraries have been processed through the Illumina 5-Base workflow and cleanup. Libraries prepared via Ultrashear fragmentation tend to have a broader fragment distribution. To achieve a tighter fragment distribution optimal for a 2x151 cycle sequencing run (~450-800 bp), we performed a double-sided ProNex Size selection as detailed below.
Figure 3. Example D5000 ScreenTape gel image comparing Covaris-sheared 5-Base libraries and direct UltraShear 5-Base libraries after IPB clean up, prior to ProNex size selection. B1-E1: Covaris M220-sheared libraries. F1-G1: 30 min 37°C NEBNext UltraShear direct-entry libraries. The direct-entry UltraShear libraries show slightly wider size distribution pre-ProNex library profiles compared to the Covaris-sheared controls.
Pool libraries according to the desired number of reads per sample.
Perform double-sided ProNex cleanup on the pooled library.
Use a 0.95x first-sided selection followed by a 0.25x second-sided selection.
Elute the final pooled library in an appropriate volume for sequencing submission.
Quantify the size-selected pool by Qubit dsDNA HS assay or equivalent.
Assess the final pooled library size distribution by TapeStation or equivalent.
Expected results
- Successful UltraShear fragmentation should generate a broad fragment distribution enriched in the 100-1000 bp range.
- The 1000-5000 bp region can be used as a practical indicator of under-fragmented DNA.
- A well-performing condition should reduce high-molecular-weight carryover while preserving sufficient fragment yield for Illumina 5-Base library preparation.
- In the example benchmarking experiment, 30 min at 37°C gave the best tested balance between enrichment of 100-1000 bp fragments and reduction of 1000-5000 bp under-sheared DNA.
- After Illumina 5-Base library preparation and Illumina Purification Bead cleanup, Ultrashear fragmented libraries are expected to show a wider size distribution as compared to Covaris-sheared libraries, as assessed by D5000 TapeStation or equivalent. Therefore, a double-sided Pronex cleanup is recommended for tighter fragment size distribution for optimal loading and 300 cycle sequencing.
Troubleshooting
Problem: Excess DNA remains in the 1000-5000 bp region.
Possible cause: Fragmentation time is too short, enzyme lot is less active than expected, or DNA quality/source differs from benchmark material.
Suggested solution: Increase the 37°C fragmentation time and repeat the time course. Can also refer to NEB’s FAQs for improving fragmentation conditions.
Problem: Fragment profile is too small.
Possible cause: Fragmentation time is too long or enzyme activity is higher than expected.
Suggested solution: Repeat the time course using shorter incubation times.
Problem: Low library yield after Illumina 5-Base DNA Prep.
Possible cause: Low DNA recovery, over-fragmentation, bead cleanup loss, or inaccurate input quantification.
Suggested solution: Confirm DNA input by Qubit, review fragmentation profile, and avoid unnecessary cleanup before End Repair.
Problem: Poor reproducibility between runs.
Possible cause: Inconsistent reaction setup, enzyme handling, thermocycler ramp differences, or enzyme lot variation.
Suggested solution: Prepare a master mix where appropriate, keep reactions cold before incubation, use consistent thermocyclers with pre-heated lids and pre-warmed to the programs, and benchmark new UltraShear enzyme lots.
Protocol references
- Illumina 5-Base DNA Prep product documentation: https://support-docs.illumina.com/LP/5-baseDNAPrep/200073497_00_illumina-5-base-dna-prep-prod-doc.pdf
- NEBNext UltraShear manual: https://www.neb.com/en-au/-/media/nebus/files/manuals/manualm7634.pdf
- Pronex size selection manual: https://www.promega.com.au/resources/protocols/technical-manuals/500/pronex-size-selective-purification-system-protocol/