WGBS Report — DEMO-WGBS

1. Project Overview

Total Samples

6

biological replicates

Total Input Reads

4.20B

across all samples

Avg Mapping Rate

95.80%

DRAGEN mapping

Avg Dup Marked

9.47%

duplicate reads

Avg Coverage

22.06x

genome-wide mean

Mean CpG Methylation

75.21%

from methyl metrics

Study Design: WGBS libraries were processed with DRAGEN Methylation v4.4.4001 using the GRCr8 reference. Quality control and summary metrics were consolidated with MultiQC. Differential methylation analysis was performed with MethylKit v1.32.0 using a 10% methylation difference and q-value 0.05 thresholds.

2. Sample Information

Sample	Group	Species
Sample1	Treatment	GRCr8
Sample2	Treatment	GRCr8
Sample3	Treatment	GRCr8
Sample4	WT	GRCr8
Sample5	WT	GRCr8
Sample6	WT	GRCr8

3. Methods

Library processing and trimming: Adapter and quality trimming were performed using Trim Galore v0.6.10 (Cutadapt v4.9) with parameters --clip_r1 5 --clip_r2 5 --three_prime_clip_r1 5 --three_prime_clip_r2 5 --nextseq 20. QC summaries were consolidated with MultiQC.

Alignment and methylation calling: Reads were aligned to the GRCr8 reference genome using DRAGEN Methylation v4.4.4001 in directional mode. Duplicate reads were marked in BAM files, and methylation calls were generated to produce genome-wide cytosine reports compatible with MethylKit.

Differential methylation analysis: MethylKit v1.32.0 was used to identify differentially methylated bases with Genomation v1.38.0 annotations. Thresholds were 10% methylation difference and q-value ≤ 0.05.

4. Quality Control (QC)

Key QC metrics below are summarized from the MultiQC report in 01.QualityCheck/. These include total input reads, mapping rate, duplication, insert length, and base quality (Q30).

Sample	Total Input Reads	Mapped Reads	Properly Paired	Dup Marked	Insert Len Median	Q30 Bases	Avg Coverage	Genome ≥10x	Genome ≥20x
Sample1	686.5M	95.45%	93.42%	9.84%	127	97.00%	21.35x	80.71%	55.22%
Sample2	739.5M	95.49%	93.16%	11.89%	139	96.69%	23.61x	82.82%	61.70%
Sample3	693.9M	94.60%	91.81%	9.71%	130	96.78%	21.75x	81.85%	57.22%
Sample4	711.0M	95.63%	93.29%	8.83%	127	96.91%	22.19x	82.68%	60.80%
Sample5	646.9M	96.51%	94.44%	8.43%	134	96.84%	20.91x	81.62%	56.16%
Sample6	718.2M	97.10%	95.64%	8.12%	121	97.06%	22.52x	82.87%	61.44%

M-bias

CpG M-bias for all samples (R1 solid, R2 dashed). Y-axis fixed at 0–100.

Non-CpG M-bias (mean of CHG+CHH) for all samples (R1 solid, R2 dashed). Y-axis fixed at 0–100.

How to interpret: M-bias plots show methylation rates by read position. A stable profile across the read indicates minimal position-specific bias. Deviations at read ends often reflect end-repair or adapter/trim artifacts; those bases can be clipped if needed.

5. Mapping

Reads were aligned to GRCr8 with DRAGEN Methylation v4.4.4001. Duplicate reads were marked in the BAM files. Alignment files are provided in 02.BamFiles/.

Avg Properly Paired

93.63%

paired reads

Avg Insert Length

130

median bp

Avg Q30 Bases

96.88%

base quality

6. Methylation

Genome-wide cytosine methylation metrics are summarized below. CpG methylation is high (expected for vertebrate genomes), while CHG/CHH methylation remains low.

Sample	CpG %	CHG %	CHH %	Total C Analyzed
Sample1	74.35%	1.55%	1.96%	7,036,152,983
Sample2	74.86%	1.64%	2.07%	8,202,773,082
Sample3	73.63%	1.59%	1.98%	7,247,735,834
Sample4	75.42%	1.41%	1.78%	7,307,879,960
Sample5	76.57%	1.50%	1.87%	7,118,154,821
Sample6	76.45%	1.58%	1.98%	7,253,512,282

Summary: Mean CpG methylation across samples is 75.21%. CHG and CHH methylation averages are 1.54% and 1.94%, respectively.

7. Differential Methylation

Differential methylation analysis was performed with MethylKit (q-value 0.05, 10% methylation difference threshold). Plots below summarize clustering, PCA, and annotation of differentially methylated bases.

Clustering

All CpG sites.

Differential CpG sites.

PCA

All CpG sites.

Differential CpG sites.

Annotation

Annotation distribution for differential CpG sites.

Top Differentially Methylated Bases

Hypermethylated (Top 10, highest positive meth.diff):

Chr	Start	End	Meth.Diff	Annotation
7	43996561	43996561	96.92	intron
7	44106340	44106340	93.48	intron
2	235218112	235218112	92.45	intron
1	250614430	250614430	91.67	intergenic
7	43778584	43778584	87.18	intron
3	83351625	83351625	86.96	intergenic
7	44072369	44072369	86.92	intron
2	235329569	235329569	86.71	intergenic
7	43818888	43818888	85.92	intron
17	21160348	21160348	85.71	promoter

Hypomethylated (Top 10, most negative meth.diff):

Chr	Start	End	Meth.Diff	Annotation
3	62292563	62292563	-95.83	intron
3	62554926	62554926	-94.67	intergenic
3	62519637	62519637	-94.12	intron
3	62392615	62392615	-93.62	intergenic
3	62410909	62410909	-93.02	intergenic
3	62561856	62561856	-92.31	intergenic
3	62291888	62291888	-91.07	intron
3	62780256	62780256	-90.94	intergenic
3	62502663	62502663	-90.80	intergenic
7	43847516	43847516	-89.18	intron

8. File Structure

Directory	Description
`01.QualityCheck/`	DRAGEN QC, trimming stats, MultiQC report
`02.BamFiles/`	Alignment files (duplicate-marked) and indexes
`03.CytosineReport/`	Genome-wide cytosine methylation reports
`04.DM_Analysis/`	Differential methylation analysis outputs

9. References

Tools and versions used in this report:

Tool	Version	Purpose
DRAGEN Methylation	v4.4.4001	Alignment and methylation calling
Trim Galore	v0.6.10	Adapter/quality trimming
Cutadapt	v4.9	Adapter trimming engine
MultiQC	Report	QC aggregation
MethylKit	v1.32.0	Differential methylation
Genomation	v1.38.0	Genomic annotation

Whole Genome Bisulfite Sequencing (WGBS) Report