Process
Genome Indexing
Description
This process involves transforming the genome graph from GFA format into two fully converted genome graphs: one depleted of C bases and another depleted of G bases. Additionally, if desired, you may include a spike-in genome in FASTA format to estimate the conversion rate in a single step further.
Warning
Please use the same VG version for both genome indexing and alignment. Use different versions may cause VG to crash.
Example Usage
methylGrapher PrepareGenome -gfa YourGFAFilePath -lp LambdaPhageFastaFilePath -prefix OutputPrefix -t ThreadsUsed
Required arguments
-gfa <gfa_file_path> -prefix <output_prefix>
Optional arguments
-lp <lambda_phage_genome_path> -t <threads_used> (default: 1)
Deduplication
Refer to FastUniq documentation for more details.
Main
Description
Run Align and MethylCall in a single command. The output will be stored in the working directory specified by the user. The index prefix is used to locate the genome index files generated by the PrepareGenome step.
Example Usage
methylGrapher Main -t ThreadsUsed -work_dir TheWorkingDir -index_prefix PrepareGenomeOutputPrefix
Arguments
Combination of Align and MethylCall.
Align
Description
VG Giraffe alignment, please provide work directory and index prefix.
Example Usage
methylGrapher Align -t ThreadsUsed -work_dir TheWorkingDir -index_prefix PrepareGenomeOutputPrefix
Required arguments
-work_dir <working_directory>
-index_prefix <index_prefix>
-fq1 <fastq_file_path (read1)>
-fq2 <fastq_file_path (read2)>
Optional arguments
-t <threads_used> (default: 1)
-directional <Y/N> (default: Y)
-compress <Y/N> (default: N)
MethylCall
Description
Methylation call from vg giraffe alignment result.
Example Usage
methylGrapher MethylCall -t ThreadsUsed -work_dir TheWorkingDir -index_prefix PrepareGenomeOutputPrefix
Required arguments
-work_dir <working_directory>
-index_prefix <index_prefix>
Optional arguments
-minimum_identity <minimum_identity> (default: 20)
-minimum_mapq <minimum_mapq> (default: 0)
-discard_multimapped (default: Y)
-batch_size <batch_size> (default: 4096)
-t <threads_used> (default: 1)
-cg_only <Y/N> (default: Y), only output methylation call in CG context
-genotyping_cytosine <Y/N> (default: N)
genotyping_cytosine option is available starting from v0.2.0. The index built before v0.2.0 does not support this option. If you want to use this option, please re-build the index using methylGrapher v0.2.0 or later. It tries to use the unconverted bases to figure out a cytosine in the reference genome graph is actually a cytosine or thymine. If it is homozygous thymine, then methylation call would be a false positive 0%. If enabled, it will also output a table of cytosine genotyping result in the working directory. The false positive cytosine methylation call will be removed from graph.methyl.
ConversionRate
Description
Estimate the conversion rate from the spike-in genome.
Example Usage
methylGrapher ConversionRate -work_dir TheWorkingDir -index_prefix PrepareGenomeOutputPrefix
Required arguments
-work_dir <working_directory>
-index_prefix <index_prefix>
MergeCpG
Description
Merge cytosine methylation call (graph.methyl) into CpG methylation call. During graph indexing, all CpG locations are identified and stored in a separate TSV file. The graph CpG locations are stored under {index_prefix}cpg.tsv, with CpG id and both cytosine location on graph coordinate. MergeCpG function will merge the cytosine methylation call (graph.methyl) into CpG methylation call using graph CpG id.
Example Usage
methylGrapher MergeCpG -work_dir TheWorkingDir -index_prefix PrepareGenomeOutputPrefix
Required arguments
-work_dir <working_directory>
-index_prefix <index_prefix>