E5hmC-seq : Detection of 5hmC at single base resolution™

Daniel J. Evanich, V. K. Chaithanya Ponnaluri, Vaishnavi Panchapakesa, Laura Blum,Matthew A. Campbell, Bradley W. Langhorst, Louise Williams | New England Biolabs, Inc.

Introduction

Results

DNA methylation is an epigenetic regulator of gene expression with important functions in development anddiseases such as cancer. Typically, the modified cytosines, 5-methylcytosine (5mC) and 5hydroxymethylcytosine (5hmC), are detected by sequencing Illumina libraries generated using an enzymebased workflow called NEBNext® EM-seq™ or bisulfite conversion. However, these methods cannot
differentiate between 5mC and 5hmC. Identification of specific 5hmC sites is important as there is increasing
interest in its role in regulating gene expression in embryonic stem cells and several neuronal cell types.
Methods currently exist to enable discrimination of 5mC and 5hmC (for example, oxBS-seq and TAB-seq),
however these are based on modifications of bisulfite sequencing, and suffer from reduced data quality due
to fragmentation and loss of DNA. Here we describe an enzymatic method that enables specific detection of5hmC, termed NEBNext Enzymatic 5hmC-seq (E5hmC-seq™).

Hydroxymethylation in Human Brain

The E5hmC-seq libraries had similar characteristics to EM-seq libraries, including expected insert sizes due
to intact DNA molecules, low duplication rates and minimal GC bias. Moreover, E5hmC-seq libraries werewell-correlated between inputs and replicates at higher sequencing depths. T4 phage DNA (all cytosines are5hmC) was used as an internal control, with 98-99% of cytosines identified as 5hmC. E5hmC-seq libraries
provide accurate measurements of 5hmC across a wide input range with expected insert sizes and minimal
GC bias. The ability to discriminate between 5mC and 5hmC will provide key insights into the role of thesecytosine modifications in development and disease.

E5hmC-seq: Enzymatic Detection of 5hmC Overview

CHG

CHH

100

Methylation(%)
Methylation

5hmC (%)

D

20
151050200 10 1 0.5 0.1

200 10 1 0.5 0.180604020200 10 1 0.5 0.10DNA Input (ng)

-5M5M -5M

5M -5M

5M -5M

5M -5M

5M

Calculatio.. Calculatio.. Calculatio.. Calculatio.. Calculatio..

CountsT4 phage

BCpG

CHG

CHH

200 10 1 0.5 0.1

200 10 1 0.5 0.1

200 10 1 0.5 0.1

100

5hmC levels detected in the CpG, CHG and CHH
contexts for the 200 ng to 0.1 ng human brain
E5hmC-seq libraries. (A) Global 5hmC levels inthe CpG context are approximately 20% and 0.4 to
0.6% for CHG and CHH contexts for all of the DNA
inputs. (B) 5hmC percentages for lambda controlDNA were < 0.5% indicating efficient deamination.
(C) 5hmC levels in the fully hydroxymethylated T4
phage control DNA are all ~99.0% indicating
efficient glucosylation and protection of 5hmC fromdeamination.
(D)
Countsofpercentage
hydroxymethylation per base in CpG context.
Methylation is binned per 3 percentage points.
Note that the majority of 5hmC is <60% per basewith minimal CpGs at 100% hydroxymethylation.
5hmC at 0% is not shown, but ranges from ~ 20%
to 40% of the total CpG for the 200 ng to 0.1 ng
inputs respectively.

7550250DNA Input (ng)LambdaC5hmC (%)

Methods

CpG

255hmC (%)

E5hmC-seq libraries were generated for 0.1 ng to 200 ng DNA isolated from human brain. Libraries wereprepared using NEBNext Ultra™ II reagents followed by two enzymatic steps to detect 5hmC. In the firststep, 5hmCs are glucosylated, which protects them from subsequent deamination by APOBEC. In contrast,
cytosines and 5mCs are deaminated resulting in their conversion to uracil and thymine, respectively. This
conversion allows discrimination of 5hmC from cytosine and 5mC. Finally, libraries were PCR amplified
using NEBNext Q5U® and sequenced on the Illumina® platform.

Human Brain

A

Input / Library1200ng
10ng
1ng
0.5ng
0.1ng
200ng_5h.. 10ng_5h.. 1ng_5hm.. 0.5ng_5h.. 0.1ng_5h..
DNA Inputs
CountCount
CountCount
Count-5M200 ng
5M -5M 10 ng5M -5M 1 ng5M -5M0.5 ng5M -5M0.1 ng5M

0.6
0.5
0.4
0.3
0.2
0.1
0.0

CpG

CHG

CHH

200 10 1 0.5 0.1

200 10 1 0.5 0.1

200 10 1 0.5 0.1

DNA Input (ng)E5hmC-seq Correlation and Coverage Analysis

E5hmC-seq Library Construction Workflow:• DNA was Covaris® sheared, end-repaired and ligated to E5hmC-seq adaptors• 5hmCs were glucosylated using UDP-glucose & T4-BGT. APOBEC thendeaminates 5mC and cytosine but not 5hmC.
• Libraries were amplified using NEBNext Q5U Master Mix.

B
Number CpGs (millions)A
CpG coverage

7060

200 ng10 ng
1 ng
0.5 ng0.1 ng50403020100051015202530

35404550

55

60

65

1.9 billion reads

1.5 billion reads

1.1 billion reads

700 million reads

Observed Coverage Depth

Library Construction
E5hmC-seq libraries:• 200 ng, 10 ng, 1 ng, 0.5 ng & 0.1 ng of human brain genomic DNA were spiked with the control DNAs:
unmethylated lambda and T4 phage DNA (all cytosines are 5hmC modified)• Libraries were constructed following the E5hmC-seq workflow• Libraries were sequenced using an Illumina NovaSeq® 6000 using 2x 150 base reads. Read numbers used inanalysis were:• 1.9 billion reads for 200 ng, 10 ng and 1 ng inputs• 715 million reads for the 1 ng and 0.1 ng inputs

Sequencing and Data Analysis
Align(bwa-meth)Trim

Deduplicate

MethylDackel

PicardmethylKit(A) At 1X depth, the 200 ng to 0.5 ng libraries detected
56 million CpGs and the 0.1 ng input detected ~48
million CpGs. Coverage was calculated using 1.9 billion
reads for the 200 ng - 1 ng inputs and 715 million reads
for the 0.5 and 0.1 ng DNA inputs. (B) Pearson
correlations of sequencing reads for 200 ng, 10 ng and1 ng DNA inputs. Correlations were made using 1.9billion, 1.5 billion, 1.1 billion and 700 million reads.
Approximately 56 million CpGs at 1X coverage wereconsidered for correlation analysis at each read input.

Coverage of Genomic Features• Reads were adaptor trimmed (fastp) then aligned to a human T2T composite genomes using bwa-meth• 5hmC information was extracted from the alignments using MethylDackel and levels were evaluated independently
for each chromosome• methylKit data was used for Pearsons correlation at 1x minimum coverage• Picard was used to mark duplicates as well as calculate library insert size and GC bias

Results
E5hmC-seq Library Profiles and Metrics for Human Brain
AB

Library Yields

50454035302520
151050DNA input (ng)

200

10

10.50.1

PCR cycles

48111214715 million
reads

Percent

Library Yield (nM)

60
40

Coverage of genomic features. Left Panel: 1.9 billion reads for the 200 ng to 1 ng inputs. Right Panel: 715
million reads for the 0.5 ng and 0.1 ng DNA inputs. The number of features with coverage ≥ 5X is indicated.
Coverage of genomic feature are represented with one point per region with the vertical position representing
the average coverage of the feature. Points are staggered horizontally to avoid excess overlapping.

200100Useablereads

CpG Islands

Percent

80

Library Insert Size

60
40

Coverage (X)

0.002

0100

Effective
coverage

200 ng10 ng
1 ng
0.5 ng0.1 ng

0.003

0.001
0.000

80
60
40
20
00200

400

600

800

200

101

0.50.1DNA Input (ng)Insert Size (bp)DE5hmC-seq library profiles and metrics for human brain. E5hmC-seq libraries were made using 200 - 0.1 ng ofhuman brain genomic DNA. (A) Average library yields from four replicate libraries per input. Error bars are +/standard deviation. (B) Read numbers, mapping, duplication and useable read percentages. Reads werealigned to the human T2T genome. (C) Library insert sizes are consistent across DNA inputs.

0.20

1.0

0.15

0.8
0.10
0.5
0.05

0.3
0.0

0.00
20

40

60

80

Average Fraction of Windows

1.3

0Feature annotations are from NCBI’s RefSeq browser. CpG islands were defined based on the UCSC genome
browser. Heatmaps generated using deepTools showing the distribution of coverage in 2 kb and 1 kb windows
around transcription start sites (TSS) and CpG islands, respectively. Dark blues indicate high coverage andlight blue/white indicate little or no coverage. The heatmaps show that E5hmC-seq has even coverage at all
DNA inputs across these genomic features.

Conclusions

GC bias plot

1.5

Normalized Coverage

Transcription Start Sites

200.004

Frequency

1.9 billion
reads

80CC

100

Duplication

200 ng
10 ng
1 ng0.5 ng
0.1 ng100Percent GC

GC coverage was analyzed using Picard and the distribution of normalized coverage across different GC
contents of the genome (0 – 100%) was plotted. E5hmC-seq libraries exhibit uniform GC coverage over arange of inputs.

E5hmC-seq libraries:• streamlined and user-friendly protocol• expected insert sizes• minimal GC bias indicating uniform GC
coverage over a range of inputs• accurate measurements of hydroxymethylation
across inputs from 200 ng – 0.1 ng• E5hmC-seq libraries have high coverage ofdiverse genomic feature types

E5hmC-seq data subtracted from EM-seq data (5mC and 5hmC) gives a complete representationof 5mC and 5hmC

Acknowledgements
We thank Dora Posfai, Kristen Augulewicz, and Harry Bell for their technical support and members of NEBNext for productive discussions.