Methylation Analysis Report
Platform: NovaSeq-S4-2x150
Kit: Zymo EZ DNA
Methylation Gold
Project ID: XXXXX-XX
Species: Human
Figure 1. Flowchart of Methylation Pipeline
Figure 2. Per Position Sequence Content
Figure 3. M-bias Plot
Next, the high-quality reads are aligned to the corresponding
reference genome through the advanced DRAGEN Methylation pipeline
tailored for Whole Genome Bisulfite Sequencing (WGBS) data. When
designated, duplicate reads are filtered out, and methylation calling is
conducted within the same pipeline. Simultaneously, a genome wide
Cytosine Report compatible with MethylKit is generated for each
individual sample. Detailed mapping statistics are visually depicted in
Figure 4, while further numerical insights can be found in Table 1.
Similarly, methylation statistics are summarized in Figure 5, with
supplementary specifics shown in Table 2.
Figure 4. Pie Chart Depicting Different Alignment Types
Sequence pairs analysed in total | 1246888 |
Paired-end alignments with a unique best hit | 664864 |
Pairs without alignments under any condition | 549413 |
Pairs that did not map uniquely | 32611 |
Genomic sequence context not extractable (edges of chromosomes) | 1 |
Table 1. Allignment Stats
Figure 5. Bar Chart Displaying Percentage of Methylation in Various Contextst
Total C’s analysed | 41019163 |
Methylated C’s in CpG context | 1448307 |
Methylated C’s in CHG context | 228669 |
Methylated C’s in CHH context | 923127 |
Methylated C’s in Unknown context | 32153 |
Unmethylated C’s in CpG context | 604275 |
Unmethylated C’s in CHG context | 8653986 |
Unmethylated C’s in CHH context | 29160799 |
Unmethylated C’s in Unknown context | 63034 |
Percentage methylation (CpG context)% | 70.6% |
Percentage methylation (CHG context)% | 2.6% |
Percentage methylation (CHH context)% | 3.1% |
Methylated C’s in Unknown context% | 33.8% |
Table 2. Methylation Statistics Across Different Contexts
For each pair of samples in each group, the differential methylation analysis is performed by extracting relevant cytosine context (i.e. CpG, CHH, or CHG) from the genome-wide cytosine report. From the resulting files, first, the reads with coverage of less than X are filtered out to retain high confidence, and methylation counts. The counts are then normalized. For further analysis bases covered in all the samples are retained. For CpG context only, destrand=TRUE is set which merge reads on both strands of a CpG dinucleotide to achieve better coverage.
Figure 6. CpG PCA Plot Contrasting Two Conditions
Figure 7. CpG Clustering Plot
Figure 8 Pie Chart Displaying Annotations of DMRs
We offer additional analyses, including region-based DMR detection
and DMR identification in non-CpG contexts. These analyses follow the
same methodology as explained in the previous section.
Moreover, we take great satisfaction in providing customized analyses
and producing publication-ready figures that contribute to your research
objectives. Our dedicated team is ready to collaborate closely with you,
ensuring that both the analysis and visuals we deliver are in perfect
alignment with your research goals. We invite you to discuss any
customized analysis requests with our project manager, who will expertly
guide you through the process of conceptualization, planning, and
execution of the specialized analyses you may require.
This
individualized approach empowers us to cater to your distinct needs,
resulting in profound insights and impactful figures that drive your
research forward.
Table3: Key Files Included in Your Delivery Folder
File Path | Description |
---|---|
00.Fastq | Fastq Files |
01.FastqQualityCheck/multiqc_report.html | Aggregated QC report |
01.FastqQualityCheck/Dragen_Stats/*csv | Various Dragen Stats |
02.BamFiles/*.bam | Duplicate marked alignment file |
03.CX_Report/*.CX_report.txt.gz | Cytosine Report |
04.DE_analysis/C**/ | DE Analysis and associated plots for CpG and others |
Sample | Group | ID |
---|---|---|
Sample1 | A | ID_1 |
Sample2 | A | ID_2 |
Sample3 | A | ID_3 |
Sample4 | B | ID_4 |
Sample5 | B | ID_5 |
Sample6 | B | ID_6 |
Software | Version |
---|---|
DRAGEN Methylation Pipeline | 3.6.3 |
FASTQ Toolkit | 2.2.5 |
FastQC | v0.11.9 |
Bowtie2 | 2.4.1 |
Bismark | 0.22.3 |
MethylKit | 1.12.0 |
R | 3.6 |
Reference genome | Hg38 |
Genome annotation file | NCBI Refseq (As recommended by Methylkit) |
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Email: custom-services@admerahealth.com
Phone:
908-222-0533