> For the complete documentation index, see [llms.txt](https://help.connected.illumina.com/llms.txt). Markdown versions of documentation pages are available by appending `.md` to page URLs; this page is available as [Markdown](https://help.connected.illumina.com/annotation/core-functionality/methylation-annotation.md). # Methylation Annotation ### Introduction With the release of Illumina’s 5-base genome for methylation sequencing, Illumina Connected Annotation can annotate regions of interest using methylation data. Methylation annotation is generated independently of standard variant annotation—Illumina Connected Annotation. While performing methylation annotation, standard variant annotation will not be produced. Methylation annotation output, will be written in a file with the suffix `methylation.json.gz` to keep it separate from standard annotation. ### Input file When performing methylation annotation, Illumina Connected Annotation will accept a gVCF file as input that needs to contain some fields specific to methylation data: ``` ##INFO= ``` An example of a valid gVCF entry that can be used for methylation annotation is: ``` #CHROM POS ID REF ALT QUAL FILTER INFO FORMAT sample1 chr21 13204471 . A . PASS END=13204512;M5mC=...Z......................................,. GT:AD:DP:GQ:MIN_DP:PL:SPL:ICNT:M5mC:DPM5mC 0/0:19,0:19:42:15:0,42,588:0,45,255:19,0:0.2381:21 ``` In the INFO field, `M5mC` encodes methylation status per nucleotide for each sample (samples are comma-separated). Methylated cytosines are indicated by `Z` or `z`, while `.` indicates an unmethylated (or not reported) position. Each record covers the reference interval from `POS` through `END` (inclusive). In the example above, `M5mC` spans positions 13204471–13204512. In the sample column, the `M5mC` and `DPM5mC` fields report the methylation value and the corresponding read depth, respectively. ### Running Illumina Connected Annotation for methylation annotation To perform methylation annotation, Illumina Connected Annotation requires transcript annotation data (Ensembl or Refseq) to be present. The command line to run Illumina Connected Annotation for methylation annotation is as follows: ``` Annotator -i [input file path] -o [output file path name] -c [path to Cance folder] -r [path to genome reference file] --sd [path to supplementary data folder] --versions-config [path to version config json file] --enable-methylation-annotation true ``` {% hint style="info" %} Supplementary annotations are limited when performing methylation annotation. Currently, only CpG Islands supplementary data is supported. {% endhint %} ### Output file The output file will be a gzipped JSON file that contains methylation annotation information. The output will contain information of the genomic feature that are methylated. Example of the genome feature information produce in the output: ``` { "regionId": "ENST00000756859.1", "biotype": "lncrna", "samples": [ { "sampleId": "mate_len_200bp_100X", "averageCpGMethylation": 0.4166666666666667, "totalCpGCoverage": 12, "totalCpGPosition": 1.5 } ], "features": [ { "featureId": 3, "biotype": "intron", "samples": [ { "sampleId": "mate_len_200bp_100X", "averageCpGMethylation": 0.4166666666666667, "totalCpGCoverage": 12, "totalCpGPosition": 1.5 } ] } ] } ``` The table below describes the fields in the methylation annotation output: | Field | Description | | ----------------------- | ------------------------------------------------------------------------------------------------------------- | | `regionId` | Identifier of the genomic feature, such as transcript ID or CpG Island ID. | | `biotype` | The biotype of the genomic feature, such as transcript, intron, exon, etc. | | `samples` | Aggregate score for the corresponding genomic region for that particular sample. | | `features` | Detailed information for each genomic feature, for example exon, intron, etc. for that particular transcript. | | `averageCpGMethylation` | The average score obtained from the gVCF file aggregated for the genomic feature. | | `totalCpGCoverage` | The total depth of CpG sites for that genomic feature. | | `totalCpGPosition` | The total number of CpG sites that are methylated for the corresponding genomic feature. | {% hint style="info" %} Adding CpG Islands supplementary data annotation will produce output region with biotype `cpgIsland`. There will be more information in the output file for CpG Islands. Please refer to the [CpG Island data source section](/annotation/data-sources/cpg-island.md) for more details. {% endhint %} --- # Agent Instructions This documentation is published with GitBook. GitBook is the documentation platform designed so that both humans and AI agents can read, navigate, and reason over technical content effectively. Learn more at gitbook.com. ## Querying This Documentation If you need additional information that is not directly available in this page, you can query the documentation dynamically by asking a question. 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