This function calls cytosine methylation and stores calls in BAM files.
Arguments
- input.bam.file
input BAM file location string.
- output.bam.file
output BAM file location string.
- genome
reference (genomic) sequences file location string or an output of
preprocessGenome.- nthreads
non-negative integer for the number of additional HTSlib threads to be used during file decompression (default: 1).
- verbose
boolean to report progress and timings (default: TRUE).
Value
list object with simple statistics of processed ("nrecs") records and calls made ("ncalled"). Even though "ncalled" can be less than "nrecs" (e.g., because not all reads are mapped), all records from the input BAM are written to the output BAM.
Details
The function makes cytosine methylation calls for short-read methylation (bisulfite/enzymatic) sequencing alignments from input BAM file and writes them in the XM tag of the output BAM file. Calls are made on the basis of reference (e.g., genomic) sequence and observed sequence and cytosine context of reads. Data reading/processing is done by means of HTSlib, therefore it is possible to significantly (>5x) speed up the calling using several (4-8) HTSlib decompression threads.
Methylation calling with this function is only possible for sequencing data obtained using either bisulfite or other similar sequencing method (enzymatic methylation sequencing). Cytosine methylation in long-read, native DNA sequencing alignments should be called using other, appropriate tools.
It is a requirement that the genomic strand the read was aligned to is known. This information is typically stored in XG tag of Bismark/Illumina BAM files, or in YD tag of BWA-meth alignment files, or in ZS tag of BSMAP alignment files. `epialleleR` is aware of that and will use the whichever tag is available.
The sequence context of cytosines (h/H for "CHH", x/X for "CHG", z/Z for "CG") is determined based on the actual (observed) sequence of the read. E.g., if read "ACGT" was aligned to the forward strand of reference sequence "ACaaGT" with the CIGAR string "2M2D2M" (2 bases match, 2 reference bases are deleted, 2 bases match), then methylation call string will be ".Z.." (in contrast to the reference's one of ".H...."). This makes cytosine calls nearly identical to ones produced by Bismark Bisulfite Read Mapper and Methylation Caller or Illumina DRAGEN Bio IT Platform, however with one important distinction: `epialleleR` reports sequence context of cytosines followed by unknown bases ("CNN") as "H.." instead of "U.." (unknown; as for example Illumina DRAGEN Bio IT Platform does). Similarly, forward strand context of "CNG" is reported as "X..", forward strand context of "CGN" -> "Z..", reverse strand context of "NNG" -> "..H", reverse strand context of "CNG" -> "..X", reverse strand context of "NCG" -> "..Z". Both lowercase and uppercase ACGTN symbols in reference sequence are allowed and correctly recognised, however all the other symbols (e.g., extended IUPAC symbols, MRSVWYHKDB) within sequences are converted to N.
As a reference sequence, the function expects either location of
(preferably `bgzip`ped) FASTA file or an object obtained by
preprocessGenome. The latter is recommended if methylation
calling is to be performed on multiple BAM files.
The alignment records of the output BAM file will contain additional XM tag with the methylation call string for every mapped read which did not have XM tag available. Besides that, XG tag with reference sequence strand ("CT" or "GA") is added to such reads in case it wasn't present.
Please note that for the purpose of methylation calling, the very same
reference genome must be used for both alignment (when BAM is produced) and
calling cytosine methylation by callMethylation method.
Exception is thrown if reference sequence header of BAM file doesn't match
reference sequence data provided (this matching is performed on the basis
of names and lengths of reference sequences).
See also
preprocessGenome for preloading reference sequences
and `epialleleR` vignettes for the description of usage and sample data.
Bismark Bisulfite Read Mapper and Methylation Caller, bwa-meth for fast and accurate alignment of long bisulfite-seq reads, BSMAP: whole genome bisulfite sequence MAPping program, or info on Illumina DRAGEN Bio IT Platform.
Examples
callMethylation(
input.bam.file=system.file("extdata", "test", "dragen-se-unsort-xg.bam", package="epialleleR"),
output.bam.file=tempfile(pattern="output-", fileext=".bam"),
genome=system.file("extdata", "test", "reference.fasta.gz", package="epialleleR")
)
#> Reading reference genome file
#> [0.001s]
#> Making methylation calls
#> [0.021s]
#> $nrecs
#> [1] 100
#>
#> $ncalled
#> [1] 100
#>