Re: [Bioc-devel] VRanges-class positive strandness and locateVariants() strandawareness

[Robert Castelo]

one option for me is just to add a metadata column with the strand of 
the overlapping feature. however, i'm interested to fully understand the 
rationale behind this aspect of the design of the VRanges object.

a VRanges object unrolls variants in a VCF file per alternative allele 
and sample. variants in VCF files are obtained from tallying reads 
aligned on a reference genome. so, my understanding is that the 
reference allele is the allele of the reference genome against which the 
reads were aligned while the alternate allele(s) are allele calls 
different from the reference. from this perspective, my interpretation 
is that ref and alt alleles have already a strand, which is the strand 
of the reference chromosome against which the reads were aligned to. i'm 
interested in this interpretation of the strand of the variants because 
i'm interested in the interpretation of sequence-features containing the 
reference and the alternate alleles, such as differences in a binding 
site with the reference and the alternate allele.

if we relax the meaning of elements in a VRanges object to, not only 
variants x allele x sample, but to variants x allele x sample x 
annotated-feature, then i think it would make sense to have the 
strand-specific annotation in the strand slot of the VRanges object.

while this idea may be good or not for a number of reasons, i'm now 
mostly interested in knowing whether i'm misinterpreting the design of 
VRanges objects, and maybe variant calling in general or i'm in a more 
or less right path in using a VRanges object to hold variant annotations.


thanks!!!

robert.

On 06/11/2015 04:30 AM, Michael Lawrence wrote:
I guess it depends on what the strand should mean. Would having a
negative strand indicate that the ref/alt should be complemented? I'm
not sure it's a good idea to conflate the strand of the variant itself
with the strand of an overlapping feature.

On Wed, Jun 10, 2015 at 1:17 PM, Robert Castelo<robert.cast...@upf.edu>  wrote:
my understanding was that VRanges is a container for variants and variant
annotations and strand is just one annotation more. when we use
locateVariants() a variant can be annotated to multiple transcripts where in
one overlaps an exon, in another an intron and so on. In all those
transcripts annotations there is a strand annotation, the strand of the
transcript. if the transcript is annotated in the negative strand of the
reference chromosome then the annotation of a transcript region to a variant
is going to be also on the negative strand.

both locateVariants() and predictCoding() return GRanges objects with strand
annotations according to the transcripts being annotated. I thought it made
sense in VariantFiltering to use VRanges objects as a  container for
variants and annotations and, for this reason, I would like to carry on the
strand annotation into the VRanges object. Is there a strong reason for a
VRanges object, derived from GRanges, not to have strand?


On 6/10/15 6:26 PM, Michael Lawrence wrote:

VRanges is supposed to enforce strand. The goal is to use "*" always,
for simplicity and consistency with the result of readVcf(). Is there
a use case for negative strand variants?

On Wed, Jun 10, 2015 at 5:54 AM, Robert Castelo<robert.cast...@upf.edu>
wrote:

Michael,

regarding our email exchange three weeks ago, I found a couple of places
in
VariantAnnotation that IMO need to be updated to avoid enforcing strand
on
VRanges.

these places occur when constructing and validating VRanges objects,
concretely at:

1. file R/methods-VRanges-class.R at the VRanges class constructor:

VRanges<-
    function(seqnames = Rle(), ranges = IRanges(),
             ref = character(), alt = NA_character_,
             totalDepth = NA_integer_, refDepth = NA_integer_,
             altDepth = NA_integer_, ..., sampleNames = NA_character_,
             softFilterMatrix = FilterMatrix(matrix(nrow = length(gr),
ncol =
0L),
               FilterRules()),
             hardFilters = FilterRules())
{
    gr<- GRanges(seqnames, ranges,
                  strand = .rleRecycleVector("*", length(ranges)), ...)
[...]

that precludes setting the strand at construction time:

library(VariantAnnotation)
VRanges(seqnames="chr1", ranges=IRanges(1, 5), ref="T", alt="C",
strand="-")
Error in GRanges(seqnames, ranges, strand = .rleRecycleVector("*",
length(ranges)),  :
    formal argument "strand" matched by multiple actual arguments


2. R/AllClasses.R at the VRanges class validity function
.valid.VRanges():

.valid.VRanges.strand<- function(object) {
    if (any(object@strand == "-"))
      paste("'strand' must always be '+' or '*'")
}

[...]

.valid.VRanges<- function(object)
{
    c(.valid.VRanges.ref(object),
      .valid.VRanges.alt(object),
      .valid.VRanges.strand(object),
      .valid.VRanges.depth(object))
}

that prompts an error when variants annotated on the negative strand are
detected:

library(VariantAnnotation)
example(VRanges)
strand(vr)<- "-"
c(vr)
Error in validObject(.Object) :
    invalid class “VRanges” object: 'strand' must always be '+' or '*'


cheers,

robert.

On 05/22/2015 09:49 PM, Michael Lawrence wrote:

This changed recently. VariantAnnotation in devel no longer enforces a
strand on VRanges, or at least it allows the "*" case.


On Fri, May 22, 2015 at 11:33 AM, Robert Castelo<robert.cast...@upf.edu
<mailto:robert.cast...@upf.edu>>  wrote:

      Hi,

      I have encountered myself in a strange situation when using the
      function locateVariants() from VariantAnnotation with an input
      VRanges object. The problem is that some of the expected coding
      annotations are not showing up when using locateVariants() with
      default parameters.

      After investigating this situation I think I found the reason,
which
      does not look like a bug but I would like that you give me some
      clarification about the logic behind using locateVariants() with
      VRanges objects.

      The documentation of the VRanges-class says that in this class of
      objects "The strand is always constrained to be positive (+).". I
      guess there may be a good reason for this but I could not find it
in
      the documentation or googling about it.

      This means that when you coerce a CollapsedVCF object (obtained,
for
      example, from a VCF file via readVcf()) to a VRanges object, even
      though variants in the VCF may have no strand, they get a positive
      strand in the VRanges object.

      The problem arises then, when you call locateVariants() with this
      VRanges object, because features on the negative strand are never
      going to overlap with the variants since, by default, the argument
      ignore.strand=FALSE.

      Let me illustrate this with a toy example. Consider the SNP
      rs1129038
      (http://www.ncbi.nlm.nih.gov/projects/SNP/snp_ref.cgi?rs=1129038)
at
      chr15:28356859 with allels A/G. It is located on the 3' UTR of the
      gene HERC2 coded on the negative strand of the human reference
      genome. Let's build a toy VRanges object having this variant:

      library(VariantAnnotation)
      vr<- VRanges(seqnames="chr15",
                     ranges=IRanges(28356859, 28356859),
                     ref="A", alt="G",
                     refDepth=5, altDepth=7,
                     totalDepth=12, sampleNames="A")
      strand(vr)
      factor-Rle of length 1 with 1 run
         Lengths: 1
         Values : +
      Levels(3): + - *

      Let's build now its CollapsedVCF counterpart by using the
      corresponding coercion method and set the strand to "*":

      vcf<- asVCF(vr)
      strand(vcf)<- "*"

      Now run locateVariants() on both objects with UCSC annotations:

      library(TxDb.Hsapiens.UCSC.hg19.knownGene)
      txdb<- TxDb.Hsapiens.UCSC.hg19.knownGene

      locateVariants(vcf, txdb, region=AllVariants())
      GRanges object with 2 ranges and 9 metadata columns:
             seqnames               ranges strand | LOCATION  LOCSTART
      LOCEND   QUERYID        TXID         CDSID
      <Rle>  <IRanges>  <Rle>  |<factor>  <integer>  <integer>  <integer>
      <character>  <IntegerList>
         [1]    chr15 [28356859, 28356859]      * | threeUTR        50 50
              1       55386
         [2]    chr15 [28356859, 28356859]      * | threeUTR        50 50
              1       55387
                  GENEID       PRECEDEID        FOLLOWID
      <character>  <CharacterList>  <CharacterList>
         [1]        8924
         [2]        8924
         -------
         seqinfo: 1 sequence from an unspecified genome; no seqlengths

      locateVariants(vr, txdb, region=AllVariants())
      GRanges object with 1 range and 9 metadata columns:
             seqnames               ranges strand |   LOCATION  LOCSTART
      LOCEND   QUERYID      TXID         CDSID
      <Rle>  <IRanges>  <Rle>  |<factor>  <integer>  <integer>  <integer>
      <integer>  <IntegerList>
         [1]    chr15 [28356859, 28356859]      + | intergenic<NA>  <NA>
              1<NA>
                  GENEID                         PRECEDEID
FOLLOWID
      <character>  <CharacterList>  <CharacterList>
         [1]<NA>  100132565,100289656,100616223,...            2567
         -------
         seqinfo: 1 sequence from an unspecified genome; no seqlengths

      Note that while we get the 3' UTR annotation from the strandless
VCF
      object we do not get it from the VRanges object with the positive
      strand. To make my point clear: this positive strand shows up when
      you coerce a strandless VCF object to a VRanges one, because
      positive strandness seems to be the convention for VRanges objects:

      as(vcf, VRanges)
      VRanges object with 1 range and 1 metadata column:
             seqnames               ranges strand         ref
      alt     totalDepth       refDepth       altDepth
      <Rle>  <IRanges>  <Rle>  <character>  <characterOrRle>  <integerOrRle>
      <integerOrRle>  <integerOrRle>
         [1]    chr15 [28356859, 28356859]      +           A
         G             12              5              7
               sampleNames softFilterMatrix |      QUAL
      <factorOrRle>  <matrix>  |<numeric>
         [1]             A                  |<NA>
         -------
         seqinfo: 1 sequence from an unspecified genome; no seqlengths
         hardFilters: NULL


      Of course, if I run locateVariants() with the argument
      ignore.strand=TRUE, then I get the expected annotation:

      locateVariants(vr, txdb, region=AllVariants(), ignore.strand=TRUE)
      GRanges object with 2 ranges and 9 metadata columns:
             seqnames               ranges strand | LOCATION  LOCSTART
      LOCEND   QUERYID        TXID         CDSID
      <Rle>  <IRanges>  <Rle>  |<factor>  <integer>  <integer>  <integer>
      <character>  <IntegerList>
         [1]    chr15 [28356859, 28356859]      + | threeUTR       677
      677         1       55386
         [2]    chr15 [28356859, 28356859]      + | threeUTR       677
      677         1       55387
                  GENEID       PRECEDEID        FOLLOWID
      <character>  <CharacterList>  <CharacterList>
         [1]        8924
         [2]        8924
         -------
         seqinfo: 1 sequence from an unspecified genome; no seqlengths


      So, my question is, given that VRanges objects are enforced to have
      a positive strand, would not be better to have ignore.strand=TRUE
as
      default in locateVariants?

      Alternatively, I would recommend that locateVariants() issues a
      warning, or maybe an error, when the input object is VRanges and
      ignore.strand=FALSE.

      Finally, out of curiosity, why a VRanges object enforces the
      positive strand in all its genomic ranges? Would not be better just
      taking the strand of the CollapsedVCF object when coercing the
      CollapsedVCF object to VRanges?


      thanks!!


      robert.

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--
Robert Castelo, PhD
Associate Professor
Dept. of Experimental and Health Sciences
Universitat Pompeu Fabra (UPF)
Barcelona Biomedical Research Park (PRBB)
Dr Aiguader 88
E-08003 Barcelona, Spain
telf: +34.933.160.514
fax: +34.933.160.550

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Bioc-devel@r-project.org mailing list
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--
Robert Castelo, PhD
Associate Professor
Dept. of Experimental and Health Sciences
Universitat Pompeu Fabra (UPF)
Barcelona Biomedical Research Park (PRBB)
Dr Aiguader 88
E-08003 Barcelona, Spain
telf: +34.933.160.514
fax: +34.933.160.550

_______________________________________________
Bioc-devel@r-project.org mailing list
https://stat.ethz.ch/mailman/listinfo/bioc-devel