NEBNext Enzymatic Methyl-seq (EM-seq™) is a high-performance enzyme-based alternative to bisulfite conversion for the identification of 5mC and 5hmC. Unlike bisulfite conversion, this highly efficient method minimizes DNA damage, resulting in superior detection of methylated cytosines, with fewer sequencing reads.
The new NEBNext Enzymatic Methyl-seq v2 Kit has a wider input range (as low as 100 pg) and a faster, more streamlined workflow than the original EM-seq kit (NEB #E7120).
The NEBNext Enzymatic Methyl-seq v2 Kit includes conversion reagents, library prep reagents and the EM-seq Adaptor. Multiple sets of the required index primers (NEBNext LV Unique Dual Index Primers) are available separately, enabling greater flexibility in multiplexing.
Superior sensitivity of detection of 5mC and 5hmC
0.1 ng - 200 ng input range
Detection of more CpGs with fewer sequencing reads
Even GC coverage
High performance library preparation and larger library insert sizes
Index primers supplied separately
Enzymatic fragmentation of DNA compatible with EM-seq workflows can be achieved using NEBNext UltraShear® (NEB #M7634).
View or download extensive EM-seq v2 performance data in our Data Supplement.
For conversion only, the NEBNext Enzymatic Methyl-seq v2 Conversion Module (NEB #E8020) is also available.
For specific detection of 5hmC, the NEBNext Enzymatic E5hmC-seq Kit (NEB #E3350) is available.
Product Information
View or download extensive EM-seq v2 performance data in our Data Supplement.
NEBNext Enzymatic Methyl-seq is a high-performance enzyme-based alternative to bisulfite conversion for methylome analysis using Illumina® sequencing. With the expanded input range of the latest NEBNext Enzymatic Methyl-seq v2 Kit, as little as 100 pg of input DNA can be used. The protocol has also been streamlined to minimize cleanup steps and reduce workflow time.
Libraries are prepared using the supplied NEBNext Ultra II reagents and the optimized EM-seq Adaptor. Index primers are available separately, as NEBNext LV Unique Dual Index Primers (NEB #E3390, E3392, E3400, E3402, E3404, E3406, E3408).
EM-seq is a two-step enzymatic conversion process to detect modified cytosines. In the first step, TET2 and T4-BGT protect modified cytosines from downstream deamination. TET2 enzymatically oxidizes 5mC and 5hmC, and T4-BGT glucosylates 5hmC.
The second enzymatic step uses APOBEC to deaminate unmodified cytosines to uracils, but 5mC and 5hmC protected in the first step are not deaminated.
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. The consistently high conversion performance and minimized DNA damage with the EM-seq protocol, in combination with highly efficient Ultra II library prep, result in superior detection of CpGs with fewer sequencing reads.
Bioinformatic analysis tools used for bisulfite sequencing can also be used for EM-seq.
NEBNext UltraShear (NEB #M7634) has been optimized for enzymatic fragmentation of DNA compatible with EM-seq workflows.
For specific detection of 5hmC, the NEBNext Enzymatic E5hmC-seq Kit (NEB #E3350) is now also available.
Figure 1: EM-seq™ conversion method
The EM-seq™ v2 workflow accommodates a wider input range than the original EM-seq workflow, with a 100-fold lower minimum input amount. The v2 workflow is more streamlined, has one fewer cleanup step and is faster. Note that NEBNext LV UDI primers are not included in the kit and are available separately. Figure 2: EM-seq™ v2 exhibits high CpG coverage across a range of inputs
EM-seq™ v2 libraries were prepared from 200–0.1 ng of NA12878 DNA (sheared to 350 bp using Covaris® ME220), spiked with unmethylated lambda and CpG-methylated pUC19. Libraries were sequenced on an Illumina® NovaSeq® 6000 (2 x 150 bases). Approximately 910 million reads for each library were aligned to a composite human T2T, lambda and pUC19 reference genome using bwa-meth. The T2T genome covers a maximum of 67.8 million CpGs when the top and bottom strands are counted independently. EM-seq covered over 56 million CpG sites for 200–1 ng inputs and roughly 45 million CpG sites for 0.1 ng input libraries. Figure 3: NEBNext EM-seq v2 identifies more CpGs than WGBS and the original EM-seq, at lower sequencing coverage depth
EM-seq v2 (NEB #E8015), EM-seq (NEB #E7120) and WGBS libraries were prepared from 200 ng and 10 ng of NA12878 DNA (sheared to ~350 bp), spiked with unmethylated lambda and CpG-methylated pUC19. Libraries were sequenced on an Illumina NovaSeq 6000. For accurate comparison of the original EM-seq and WGBS data with EM-seq v2 data, we evaluated data from approximately 625 million 100 base reads for each library aligned to a composite human T2T, lambda and pUC19 reference genome using bwa-meth.
The T2T genome covers a maximum of 67.8 million CpGs when the top and bottom strands are counted independently. EM-seq v2 and EM-seq covered over 54 million CpG sites for both 200 ng and 10 ng inputs; however, WGBS libraries covered only 46 million and 39 million for 200 ng and 10 ng inputs respectively at 1X coverage. The dashed lines represent coverage of (A) 10X and (B) 8X. The table lists the percentage of CpG sites covered by different libraries at (A) 10X and (B) 8X coverage level. Figure 4: EM-seq™ v2 produces high library yields across a broad input range
200–0.1 ng of NA12878 genomic DNA, sheared to 350 bp (Covaris® ME220) was used as input into the EM-seq™ v2 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 two technical replicates and error bars show standard deviation. EM-seq v2 consistently produces high-yield libraries across a wide range of inputs. Figure 5: EM-seq™ v2 provides even GC coverage
EM-seq™ v2 libraries were prepared from 200–0.1 ng of NA12878 DNA (sheared to 350 bp using Covaris® ME220), spiked with unmethylated lambda and CpG-methylated pUC19. Libraries were sequenced on an Illumina® NovaSeq® 6000 (2 x 150 bases). Approximately 910 million reads for each library were aligned to a composite human T2T, lambda and pUC19 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 for reads mapping to human genome. EM-seq v2 libraries have uniform GC coverage across the input range.
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In the United States and Europe, this product cannot be used in, or incorporated into, any blood-based, sequencing-based screening diagnostic assay (including product or service-based offerings) for the early detection of colorectal, gastric, lung, liver, ovarian, breast, prostate, esophageal, thyroid, uterine, pancreatic, bladder or kidney cancer indications.
This product and/or its manufacture and/or its use either alone or in combination with other product(s) are protected by one or more of the following patents and related pending patents: U.S. Pat. Nos. 9,121,061, 9,896,726, 10,227,646, 10,260,088, 10,619,200, 11,001,876, and 11,124,825; European Pat. Nos. EP2825645 and EP3368688; JP6224689; and CN108699598.
This product is licensed for research and commercial use from Bio-Rad Laboratories, Inc., under U.S. Pat. No. 8,470,573 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. For more information, please email busdev@neb.com.
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