For enzymatic fragmentation and library preparation of FFPE DNA samples the NEBNext UltraShear FFPE DNA Library Prep Kit (NEB #E6655) is now available.
Enzymatic fragmentation of DNA as part of the library prep workflow provides many advantages compared to mechanical shearing. However, specialized fragmentation reagents are required for enzymatic shearing in order to maintain methylation marks on samples for methylome analysis or for use with challenging samples such as FFPE DNA.
NEBNext UltraShear™ is a mix of enzymes that has been designed and optimized to fragment these sample types upstream of library preparation. This improves library quality and allows retention of methylation marks.
View or download extensive performance data in our Data Supplement.
Compatible with methylation analysis workflows, including NEBNext® Enzymatic Methyl-seq (EM-seq™) (NEB #E7120)
Compatible with FFPE DNA
Fast workflow with minimal hands-on time
For methylation analysis, improves library yields, CpG coverage and sequencing metrics
For FFPE DNA, increases usable reads and coverage uniformity and decreases artificial mutation frequency
For enzymatic fragmentation and library preparation of FFPE DNA samples we recommend the NEBNext UltraShear FFPE DNA Library Prep Kit (NEB #E6655)
Note that for high-quality genomic DNA samples, for library prep with enzymatic fragmentation, we recommend NEBNext Ultra™ II FS DNA Library Prep for Illumina® (NEB #E7805, #E6177).
View or download extensive performance data in our Data Supplement.
Enzymatic methods for DNA fragmentation in NGS workflows enable streamlined protocols, improved performance and scalability. However, specialized fragmentation reagents are required for samples for methylation analysis, to ensure that methylation marks are not removed, and for FFPE DNA. NEBNext UltraShear is a novel enzyme mix designed for fragmentation of these sample types that has a fast workflow and improves library preparation and sequencing metrics for DNA methylation studies and FFPE DNA.
NEBNext UltraShear is compatible with NEBNext Enzymatic Methyl-seq (EM-seq) (NEB #E7120).
NEBNext UltraShear in FFPE DNA sequencing workflows:
Improved usable reads
Lower artificial mutation frequency
NEBNext UltraShear for methylated DNA sequencing e.g., EM-seq
Higher library yields
Improved sequencing metrics
Improved CpG Coverage
Note that for high quality genomic DNA library prep with enzymatic fragmentation, we recommend NEBNext Ultra II FS DNA Library Prep for Illumina (NEB #E7805, #E6177).
200 ng, 50 ng and 10 ng of NA12878 DNA spiked with control DNA (CpG methylated pUC19 DNA and unmethylated lambda DNA) were fragmented by either NEBNext UltraShear (20 minutes at 37°C) or Covaris® ME220 (350 bp protocol) followed by EM-seq library preparation. Library yields were quantified using Agilent® TapeStation® with the High Sensitivity D1000 ScreenTape®. EM-seq libraries fragmented by NEBNext UltraShear have higher yields than Covaris for the same number of PCR cycles for each input (200 ng = 4 cycles; 50 ng = 6 cycles; 10 ng = 8 cycles).
Figure 2: Improved CpG coverage in EM-seq libraries produced using NEBNext UltraShear
200 ng, 50 ng and 10 ng of NA12878 DNA spiked with control DNA (CpG methylated pUC19 DNA and unmethylated lambda DNA) was fragmented by either NEBNext UltraShear (20 minutes at 37°C) or Covaris ME220 (350 bp protocol) followed by EM-seq library preparation. Technical replicates were generated for each input amount. All libraries were sequenced on the same flowcell of an Illumina® NovaSeq® 6000 (2 x 100 bases). 725 million reads were sampled (seqtk) from each library for methylation analysis. Reads were adapter trimmed (fastp), aligned to the GRCh38 reference (bwa-meth), and duplicate marked (Picard MarkDuplicates) before calling methylation using MethylDackel. NEBNext UltraShear and Covaris fragmentation used ahead of the EM-seq workflow yielded a similar number of CpGs (~54 million) at minimum 1X coverage. At minimum 10X coverage, more CpGs are identified when NEBNext UltraShear is used, due to improved library diversity and coverage evenness.
Figure 3: NEBNext UltraShear with FFPE DNA improves usable reads
FFPE DNA was fragmented using NEBNext UltraShear (15 minutes at 37°C), Covaris ME220, Kapa EvoPlus® Kit, Kapa HyperPlus® Kit, Agilent SureSelect® Enzymatic Fragmentation Kit, NEBNext dsDNA Fragmentase® or NEBNext Ultra II FS. All samples were fragmented according to the respective protocols. Fragmentation with NEBNext dsDNA Fragmentase, Kapa kits and the Agilent kit was followed by a bead cleanup and library construction using the NEBNext Ultra II DNA library Prep Kit for Illumina. NEBNext UltraShear and Covaris-sheared samples were followed directly by use of the NEBNext Ultra II DNA Library Prep Kit for Illumina, and NEBNext Ultra II FS samples followed the recommended protocol for library prep. Each library was sequenced using the Illumina NextSeq® 500. 2 million (2 x 76 base) reads were used for this analysis. Reads were aligned to GRCh38 with Bowtie2 and analyzed using samtools flagstats and Picard CollectAlighmentSummaryMetrics. Percent of usable reads (mappable, proper pairs, and non-duplicates reads) were measured for each library and usable reads were averaged for technical replicates (bars represent error between two technical replicates) for all fragmentation methods. FFPE DNA libraries fragmented with NEBNext UltraShear had the highest percent of usable reads and had similar percent usable reads as high-quality DNA libraries (high-quality DNA had a comparable percent of usable reads across all fragmentation methods ≥ 96%; data not shown).
Figure 4: NEBNext UltraShear with FFPE DNA reduces artificial mutations
FFPE DNA was fragmented using NEBNext UltraShear (15 minutes at 37°C), Covaris ME220, Kapa EvoPlus Kit, Kapa HyperPlus Kit, Agilent SureSelect Enzymatic Fragmentation Kit, NEBNext dsDNA Fragmentase or NEBNext Ultra II FS. All samples were fragmented according to the recommended protocols. Fragmentation with NEBNext dsDNA Fragmentase, Kapa® kits and the Agilent kit was followed by a bead cleanup and NEBNext Ultra II DNA library Prep Kit for Illumina. NEBNext UltraShear and Covaris-sheared samples were followed directly by use of the NEBNext Ultra II DNA Library Prep Kit for Illumina, and NEBNext Ultra II FS samples followed the recommended protocol for library prep. Each library was sequenced using the Illumina NextSeq 500. 2 million (2 x 76 base) reads were used for this analysis. Reads were aligned to GRCh38 with Bowtie2. Artificial C to T mutations were calculated with Tasmanian tool for read 1 and 2 and averaged for technical replicates (bars represent error between two technical replicates). The libraries fragmented with NEBNext UltraShear resulted in the lowest C to T artificial mutation frequency compared to other fragmentation methods for FFPE DNA both reads (R1= Read 1 and R2= Read 2).
Figure 5: NEBNext UltraShear fragments high-quality genomic DNA in a time-dependent manner
50 ng human DNA (NA12878) was fragmented for 5–45 minutes at 37°C followed by 15 minutes at 65°C. Fragmentation occurs during the 37°C incubation step of NEBNext UltraShear. The average fragmentation size and pattern (High Sensitivity D5000 ScreenTape on Agilent TapeStation) is based on fragmentation time.
This product is related to the following categories:
The following reagents are supplied with this product:
NEB #
Component Name
Component #
Stored at (°C)
Amount
Concentration
M7634S
-20
NEBNext UltraShear®
M7634SVIAL
-20
1 x 0.096 ml
Not Applicable
NEBNext UltraShear™ Reaction Buffer
B9042SVIAL
-20
1 x 0.336 ml
Not Applicable
500 mM DTT
B1079SVIAL
-20
1 x 0.048 ml
Not Applicable
M7634L
-20
NEBNext UltraShear®
M7634LVIAL
-20
1 x 0.384 ml
Not Applicable
NEBNext UltraShear™ Reaction Buffer
B9042LVIAL
-20
2 x 0.672 ml
Not Applicable
500 mM DTT
B1079LVIAL
-20
1 x 0.192 ml
Not Applicable
Properties & Usage
Materials Required but not Supplied
1X TE (10 mM Tris pH 8.0, 1 mM EDTA)
0.2 ml thin wall PCR tubes
Magnetic rack/stand (NEB #S1515S; Alpaqua® #A001322 or equivalent)
PCR machine
Vortex
Microcentrifuge
Bioanalyzer®, TapeStation® or other fragment analyzer and associated consumables
80% Ethanol
For use with NEBNext UltraShear Protocol:
SPRIselect™ Reagent Kit (Beckman Coulter, Inc. #B23317), AMPure® XP Beads (Beckman Coulter, Inc. #A63881) or Monarch®® PCR & DNA Cleanup Kit (NEB# T1030S/L) are recommended for Section 1.
For use with NEBNext Ultra II End Repair/dA-Tailing Module Protocol:
NEBNext Ultra II End Repair/dA-Tailing Module (NEB #E7546S/L) for Section 2.
Recommended Material Not Included: NEBNext Ultra II Ligation Module (NEB #E7595) and NEBNext Multiplex Oligos (www.neb.com/oligos).
For use with NEBNext Enzymatic Methyl-seq Protocol:
NEBNext Enzymatic Methyl-seq Kit (NEB #E7120S/L) for Section 3.
Formamide (Sigma #F9037-100 ml) or optional 0.1 N NaOH. Formamide is preferred. If using NaOH, please see NEBNext Enzymatic Methyl-seq Kit (NEB #E7120) FAQs
Quality Control tests are performed on each new lot of NEB product to meet the specifications designated for it. Specifications and individual lot data from the tests that are performed for this particular product can be found and downloaded on the Product Specification Sheet, Certificate of Analysis, data card or product manual. Further information regarding NEB product quality can be found here.
Specifications & Change Notifications
The Specification sheet is a document that includes the storage temperature, shelf life and the specifications designated for the product. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]
The Certificate of Analysis (COA) is a signed document that includes the storage temperature, expiration date and quality controls for an individual lot. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]_[Lot Number]
Products and content are covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB). The use of trademark symbols does not necessarily indicate that the name is trademarked in the country where it is being read; it indicates where the content was originally developed. The use of this product may require the buyer to obtain additional third-party intellectual property rights for certain applications. For more information, please email busdev@neb.com.
This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.
New England Biolabs (NEB) is committed to practicing ethical science – we believe it is our job as researchers to ask the important questions that when answered will help preserve our quality of life and the world that we live in. However, this research should always be done in safe and ethical manner. Learn more.
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