1. Accel-NGS® Methyl-Seq DNA Library Kit
Library Preparation for Bisulfite-Converted DNA

2. Accel-NGS® Methyl-Seq DNA Library Kit
Applications
Whole Genome Bisulfite Sequencing (WGBS)
Reduced Representation Bisulfite Sequencing (RRBS)
Bisulfite-converted DNA enriched by MeDIP, ChIP, or other methods
Hybridization capture using NimbleGen™ SeqCap™ Epi Enrichment System
Ancient DNA samples that may contain uracil nucleotides as a result of damage
Sample Types
Genomic DNA
Formalin-fixed, paraffin-embedded (FFPE)
Circulating, cell-free DNA (cfDNA)
Fresh and frozen tissue
NimbleGen and SeqCap are trademarks of Roche NimbleGen, Inc.

3. Comparison of Methyl-Seq Workflows

4. Methyl-Seq Performance: Human WGBS
ACCEL-NGS METHYL-SEQ
TRADITIONAL WORKFLOW
RANDOM PRIMER
Bisulfite-Converted Library
Input (ng) 1
1000 50
PCR Cycles 11 10
Yield (nM)
> 4
GC Bias
Low
High
Post-bisulfite library construction enables high recovery of input DNA
Minimize the number of amplification cycles required
Create libraries from as little as 100 pg of DNA
Sequence-independent adapter ligation avoids bias
Cover AT-rich sequences produced by bisulfite conversion

5. Methyl-Seq Performance: Human WGBS
SAMPLE
METHOD
% READS ALIGNED
GENOME COVERAGE
% DUPLICATE READS
EST. LIBRARY SIZE (MILLIONS)
10 ng Human
Accel-NGS Methyl-Seq
86.4
8.9X
7.9
1,393

6. Methyl-Seq Performance: Arabidopsis WGBS
Arabidopsis thalania
Small genome model organism for methylation analysis
Methyl-Seq constructs higher complexity libraries and provides comprehensive coverage of CpX (CpG + CpH) sites
SAMPLE
% READS ALIGNED
GENOME COVERAGE
% DUPLICATE READS
EST. LIBRARY SIZE (MILLIONS)
RELATIVE LIBRARY SIZE
% CPX MISSING
% CPX COVERED > 10X
100 ng Arabidopsis
Accel-NGS Methyl-Seq
89.6
22X
1.9
714 1
0.56
92.2
Traditional
80.2
21X
2.7
604
0.85
0.57
88.1
Random Primer
71.4
16X
22.1 48
0.07
7.70
39.4
10 ng Arabidopsis
87.8
406
0.58
90.4
76.7
19X
11.9 70
0.17
83.9
71.9
22.2 45
0.11
5.2
45.2
1 ng Arabidopsis
83.3
18X
18.2 38
0.59
77.1
80.7
10X
62.3 6
0.16
2.00
17.0
73.4
12X
46.1 12
0.31
6.60
31.3

7. Methyl-Seq Performance: Arabidopsis WGBS
CpX Sites Uncovered
CpX Sites Covered ≥ 10X
(A) At all three inputs (100 ng, 10 ng, and 1 ng), the Accel-NGS® Methyl-Seq Kit and the Traditional Method leave a minimal amount of of CpX (CpG + CpH) sites uncovered. However, the Random Primer method exhibits at least 4% of CpX sites missing at every input tested. (B) The percentage of CpX sites covered at least 10X is greater than 90% for Methyl-Seq at 100 ng, but decreases to approximately 75% at 1 ng. The Traditional Method covers greater than 80% of CpX sites at least 10X for 100 ng and 10 ng inputs, but this coverage diminishes greatly at 1 ng. The Random Primer Method exhibits poor coverage of CpX sites at each input, with the percentage of CpX sites covered at least 10X at about 40% for all inputs.

8. Methyl-Seq: cfDNA and Cancer Burden
PNAS, vol 110, no 47 (2013) pp

9. Methyl-Seq: cfDNA and Cancer Burden
5 ng of cfDNA for bisulfite-converted WGBS libraries
10 million mapped reads to detect hypomethylation levels
Experimental design considerations
Acquire positive cfDNA samples
Need to be less than 3 days old
From patients with diagnosed cancer
Identifying known positive controls
“Healthy” cfDNA control samples
Cancer-derived samples are compared to an average of “healthy” controls
Not all healthy controls are fit
Does obesity, alcoholism, age, inflammation….change methylation levels?

10. Detecting Genome-Wide Hypomethylation with 10 Million Reads
Circos plot represents hypomethylation status on chromosomes 1-22 between 5 healthy controls and sample 8 (Metastatic colorectal adenocarcinoma with liver metastasis, 2 cm primary).

11. Methylation Density: Tumor cfDNA Samples
PATHOLOGY
% HYPOMETHYLATION 1
Fallopian tube high grade papillary serous carcinoma pT3c N1 with 2 nodes involved by micrometasasis
0.4% 2
5 cm ovarian ‘borderline’ serous content (cancer-like)
1.1% 3
Recurrent pT2, pN0 mammary carcinoma, 2.15 cm
2.4% 4
pT1/pN1 pancreatic adenocarcinoma with neoadjuvant therapy
3.6% 5
Metastatic colon cancer to the liver (previously treated)
4.4% 6
14 cm ovarian ‘borderline’ serous content (cancer-like)
18.0% 7
Colon-cancer, non-resectable Adenocarcinoma T4a by imaging 8
Metastatic colorectal adenocarcinoma with liver metastasis, 2 cm primary
43.4%
Percent hypomethylation was calculated by comparing the tumor samples’ methylation density (MD) by 1 Mb genome windows/bins to average of 5 healthy controls. Bins were assigned as hypomethylated if the MD was >3, SD lower than the average MD.

12. Reduced Representation Bisulfite Sequencing
RRBS is a form of targeted BS-Seq
MspI digest followed by selection of a specific range of fragment sizes enriches for CpG islands
Alternative workflow for RRBS with Accel-NGS® Methyl-Seq
Traditional method requires dsDNA for library preparation, so size selection (orange box) selects for bp inserts ( bp library molecules).
Methyl-Seq library construction is compatible with ssDNA, so size selection selects for bp inserts ( bp fragments).
Size selection must occur before bisulfite conversion because conversion will fragment DNA, affecting fragment size selection. Improper size selection may affect CpG island coverage.

13. Targeted Methylation Sequencing from 1 ng
INPUT
METHOD
% ALIGNED
% ON TARGET
% DUPLICATION
MEAN COVERAGE
% COVERED > 2X
% COVERED > 20x
NONE COVERED
100 ng
SWIFT 90 73
6.5
49X
98.6
78.6
0.8
1 µg
Kapa 80
9.4
51X
81.1
10 ng 91 77
26.0
35X
98.5
71.0 87 78 1X
24.7
0.2
47.7
1 ng
62.0 8X
93.6
2.3
1.0
Coverage metrics were analyzed for inputs of 1 µg and 100 ng, quantities that are within specification for Kapa and Swift library preparation, respectively. Additionally, lower inputs of 10 ng (Kapa and Swift) and 1 ng (Swift only) were also analyzed. A substantial increase in duplicate reads and decrease in genome coverage can be observed for Kapa libraries at 10 ng. However, the Swift kit performs well at 10 ng, with the performance metrics at 1 ng comparable to the 10 ng metrics from the Kapa kit.

14. Differentially Methylated Region (DMR) Analysis from 10 ng
DMR analysis was performed for 10 ng libraries from both Kapa and Swift, comparing DNA from an H1 ES cell line and a B-lymphocyte cell line (NA12878). This figure illustrates the 294,130 DMRs called from the Swift Methyl-Seq kit (37,799 hypomethylated and 256,331 hypermethylated). In contrast, the 10 ng Kapa libraries resulted in only 464 total DMR calls (not shown).

15. Accel-NGS® Methyl-Seq DNA Library Kit Ordering Information
PRODUCT NAME
REACTIONS
CATALOG NO.
Accel-NGS Methyl-Seq DNA Library Kit 12
DL-ILMMS-12 48
DL-ILMMS-48
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