NEBNext® Enzymatic 5hmC-seq (E5hmC-seq™) is a new method for the specific detection of 5hmC at the single base level.
5hmC is detected using a two-step enzymatic conversion workflow that minimizes damage to DNA and allows discrimination of 5hmC from both cytosine and 5mC, after Illumina® sequencing. The kit has an input range of 0.1–200 ng and includes NEBNext Ultra™ II library prep reagents and the E5hmC-seq Adaptor.
Note that index primers are not included and must be purchased separately (NEBNext Primers for Epigenetics, NEB #E3392, NEB #E3404).
High sensitivity of detection of 5hmC
0.1–200 ng input range
Even GC coverage
High-efficiency library preparation
E5hmC-seq and EM-seq data can be combined
Conversion module also available separately (NEB #E3365)
Typically, the modified cytosines 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) are detected by sequencing of Illumina libraries generated using the NEBNext EM-seq enzyme-based workflow or bisulfite conversion. However, these methods cannot differentiate between 5mC and 5hmC.
As with EM-seq, NEBNext Enzymatic 5hmC-seq (E5hmC-seq) uses an enzyme-based method to specifically detect 5hmC, without the DNA damage typical of bisulfite-based methods, and this sensitive method can be used with as little as 0.1 ng of input DNA.
In a two-step process, T4-BGT glucosylates 5hmC, providing protection from deamination by APOBEC in the next step. T4-BGT does not protect 5mC or unmethylated cytosines, which are deaminated by APOBEC to uracils. This is followed by amplification using a NEBNext master mix formulation of Q5U® (a modified version of Q5® High-Fidelity DNA Polymerase), and sequencing on the Illumina platform.
Bioinformatic analysis tools used for EM-seq and for bisulfite sequencing can also be used for E5hmC-seq. E5hmC-seq data can be subtracted from EM-seq data, thereby allowing determination of the precise location of individual 5mC and 5hmC sites.
Note that index primers are not included and must be purchased separately (NEBNext Primers for Epigenetics, NEB #E3392, NEB #E3404).
Figure 1: E5hmC-seq conversion method
Figure 2: E5hmC-seq produces high library yields across a broad input range
200–0.1 ng of human brain genomic DNA, sheared to 350 bp (Covaris® ME220) was used as input into the E5hmC-seq library protocol, using the number of PCR cycles shown. Library yields were determined using the Agilent® TapeStation® with High Sensitivity D1000 reagents. Values shown are the average of 4 technical replicates and error bars are standard deviation. E5hmC-seq consistently produces high-yield libraries across a wide range of inputs.
Figure 3: E5hmC-seq libraries have robust protection of 5hmC
Control T4 DNA that is fully hydroxymethylated at all cytosines was spiked in when preparing E5hmC-seq libraries using 200 ng to 0.1 ng of human brain DNA. DNA was sheared to 350 bp using the Covaris® ME220 instrument, E5hmC-seq libraries were prepared and sequenced on an Illumina® NovaSeq 6000 (2 x 150 bases). Approximately 1.9 billion (200 ng, 10 ng and 1 ng) or 715 million (0.5 ng and 0.1 ng) reads for each library were aligned to a composite human T2T, lambda and T4 reference genome using bwa-meth. Methylation information was extracted from the alignments using MethylDackel. Values shown are the average of two technical replicates and error bars show standard deviation. Percent 5hmC detected for control T4 DNA in the CpG, CHG and CHH contexts was ≥ 98.9%.
Figure 4: 5hmC detected by E5hmC-seq in human brain gDNA is consistent across inputs
200–0.1 ng of human brain genomic DNA was sheared to 350 bp (Covaris® ME220) and E5hmC-seq libraries were prepared and sequenced on an Illumina NovaSeq 6000 (2 x 150 bases). Approximately 1.9 billion (200 ng, 10 ng and 1 ng) or 715 million (0.5 ng and 0.1 ng) reads for each library were aligned to a composite human T2T, lambda and T4 reference genome using bwa-meth, and methylation information was extracted from the alignments using MethylDackel. Values shown are the average of two technical replicates and error bars show standard deviation. Detected 5hmC levels are similar between all inputs in the CpG, CHH and CHG contexts.
Figure 5: E5hmC-seq provides even GC coverage
200–0.1 ng of human brain genomic DNA was sheared to 350 bp (Covaris ME220) and E5hmC-seq libraries were prepared and sequenced on an Illumina NovaSeq 6000 (2 x 150 bases). Approximately 1.9 billion (200 ng, 10 ng and 1 ng) or 715 million (0.5 ng and 0.1 ng) reads for each library were aligned to a composite human T2T, lambda and T4 reference genome using bwa-meth. GC coverage was analyzed using Picard and the distribution of normalized coverage across different GC contents of the genome (0–100%) was plotted. The GC content distribution of the human T2T genome is plotted as a histogram. E5hmC-seq libraries have uniform GC coverage across the full input range.
Figure 6: E5hmC-seq exhibits high CpG coverage across a range of inputs
200–0.1 ng of human brain genomic DNA was sheared to 350 bp (Covaris ME220) and E5hmC-seq libraries were prepared and sequenced on an Illumina NovaSeq 6000 (2 x 150 bases). Approximately 1.9 billion (200 ng, 10 ng and 1 ng) or 715 million (0.5 ng and 0.1 ng) reads for each library were aligned to a composite human T2T, lambda and T4 reference genome using bwa-meth. Methylation information was extracted from the alignments using MethylDackel and reported in methylkit format across all three contexts. Using the CpG specific file a cumulative coverage plot was generated for CpG sites covered using E5hmC-seq libraries across all inputs. The T2T genome covers a maximum of 67.8 million CpGs when the top and bottom strands are counted independently. E5hmC-seq covered over 56 million CpG sites for 0.5 ng to 200 ng inputs and roughly 48 million CpG sites for 0.1 ng input libraries.
Figure 7: E5hmC-seq libraries are well correlated at higher sequencing depths
200 ng, 10 ng and 1 ng E5hmC-seq libraries were sequenced to a depth of 1.9 billion total 150-base reads and correlations were plotted using methylKit at 1X minimum coverage (~ 56.5 million CpGs were common to all libraries). Correlations for the 200 ng and 10 ng input libraries were ≥ 0.81 between replicates. E5hmC-seq libraries for 200 ng, 10 ng and 1 ng were progressively downsampled to ~1.5 billion, 1.1 billion, 700 million and 300 million total reads, and correlation analysis was performed. We observed lower correlations across inputs at 300 million and 700 million reads compared to correlations using 1.1, 1.5 and 1.9 billion reads. This demonstrates the need for deeper sequencing of E5hmC-seq libraries due to the lower abundance of 5hmC signal in the sample.
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
E3350S
Multi-temperature
NEBNext® Enzymatic 5hmC-seq Kit
E3350-2
-20
Control DNA CpG unmethylated Lambda
E7123AVIAL
-20
1 x 0.024 ml
2 ng/µl
Control DNA 5hmC T4
E3349AVIAL
-20
1 x 0.024 ml
1.5 ng/µl
NEBNext® Ultra II End Prep Reaction Buffer
E7647AVIAL
-20
1 x 0.168 ml
Not Applicable
NEBNext® Ultra II End Prep Enzyme Mix
E7646AVIAL
-20
1 x 0.072 ml
Not Applicable
NEBNext® Ultra™ II Ligation Master Mix
E7648AVIAL
-20
1 x 0.72 ml
Not Applicable
NEBNext® Ligation Enhancer
E7374AVIAL
-20
1 x 0.024 ml
Not Applicable
NEBNext® Carrier DNA
E3351AVIAL
-20
1 x 0.024 ml
Not Applicable
Elution Buffer
E7124AVIAL
-20
1 x 2.1 ml
Not Applicable
NEBNext® Glucosylation Reaction Buffer
E3352AVIAL
-20
1 x 0.12 ml
Not Applicable
UDP-Glucose
E3353AVIAL
-20
1 x 0.024 ml
Not Applicable
T4-BGT
E3354AVIAL
-20
1 x 0.024 ml
Not Applicable
Stop Reagent
E7132AVIAL
-20
1 x 0.024 ml
Not Applicable
Deamination Reaction Buffer
E3356AVIAL
-20
1 x 0.096 ml
Not Applicable
Recombinant Albumin
E3357AVIAL
-20
1 x 0.024 ml
Not Applicable
APOBEC
E7133AVIAL
-20
1 x 0.024 ml
Not Applicable
NEBNext® E5hmC-seq™ Adaptor
E3366AVIAL
-20
1 x 0.06 ml
Not Applicable
NEBNext® Q5U® Master Mix
E3369AVIAL
-20
1 x 1.08 ml
Not Applicable
NEBNext® Sample Purification Beads
E3355S
25
NEBNext® Sample Purification Beads
E3355AVIAL
25
1 x 5.2 ml
Not Applicable
E3350L
Multi-temperature
NEBNext® Enzymatic 5hmC-seq Kit
E3350-3
-20
Control DNA CpG unmethylated Lambda
E7123AAVIAL
-20
1 x 0.096 ml
2 ng/µl
Control DNA 5hmC T4
E3349AAVIAL
-20
1 x 0.096 ml
1.5 ng/µl
NEBNext® Ultra II End Prep Reaction Buffer
E7647AAVIAL
-20
1 x 0.672 ml
Not Applicable
NEBNext® Ultra II End Prep Enzyme Mix
E7646AAVIAL
-20
1 x 0.288 ml
Not Applicable
NEBNext® Ultra™ II Ligation Master Mix
E7648AAVIAL
-20
3 x 0.96 ml
Not Applicable
NEBNext® Ligation Enhancer
E7374AAVIAL
-20
1 x 0.096 ml
Not Applicable
NEBNext® Carrier DNA
E3351AAVIAL
-20
1 x 0.096 ml
Not Applicable
Elution Buffer
E7124AAVIAL
-20
1 x 8.6 ml
Not Applicable
NEBNext® Glucosylation Reaction Buffer
E3352AAVIAL
-20
1 x 0.48 ml
Not Applicable
UDP-Glucose
E3353AAVIAL
-20
1 x 0.096 ml
Not Applicable
T4-BGT
E3354AAVIAL
-20
1 x 0.096 ml
Not Applicable
Stop Reagent
E7132AAVIAL
-20
1 x 0.096 ml
Not Applicable
Deamination Reaction Buffer
E3356AAVIAL
-20
1 x 0.384 ml
Not Applicable
Recombinant Albumin
E3357AAVIAL
-20
1 x 0.096 ml
Not Applicable
APOBEC
E7133AAVIAL
-20
1 x 0.096 ml
Not Applicable
NEBNext® E5hmC-seq™ Adaptor
E3366AAVIAL
-20
1 x 0.24 ml
Not Applicable
NEBNext® Q5U® Master Mix
E3369AAVIAL
-20
1 x 4.32 ml
Not Applicable
NEBNext® Sample Purification Beads
E3355L
25
NEBNext® Sample Purification Beads
E3355AAVIAL
25
1 x 20.64 ml
Not Applicable
Properties & Usage
Materials Required but not Supplied
Covaris® instrument and the required tubes or other fragmentation equipment
NEBNext Primers for Epigenetics (Unique Dual Index Set 2B) NEB #E3392S (24 reactions) or Set 3 NEB #E3404S (96 reactions)
PCR strip tubes or 96-well plates
Formamide (Sigma #F9037-100 ml), Hi-Di™ Formamide (Thermo Fisher Scientific® #4401457) or 0.1 N NaOH. Formamide is preferred. If using NaOH, please see FAQ on NEB #E3350 FAQ page.
80% Ethanol
1X TE (10 mM Tris-HCl pH 8.0, 1 mM EDTA), low TE (10 mM Tris-HCl pH 8.0, 0.1 mM EDTA) or 10 mM Tris-HCl pH 7.5 or 8.0
Nuclease-free Water
Magnetic rack/stand, such as NEBNext Magnetic Separation Rack (NEB #S1515)
Metal cooling block, such as Diversified Biotech® (#CHAM-1000)
PCR machine
Agilent® Bioanalyzer®, TapeStation® or other fragment analyzer and associated consumables
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]
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.
Licenses
This product is licensed for research and commercial use from Bio-Rad Laboratories, Inc., under U.S. Pat. Nos. 6,627,424, 7,541,170, 7,670,808, 7,666,645, and corresponding patents in other countries. No rights are granted for use of the product for Digital PCR or real-time PCR applications, with the exception of quantification in Next Generation Sequencing workflows.
