/// <summary>
/// Output bed file of regions. Each region spans both probes and the target interval
/// Note that the BED format uses:
/// 0-based start position (inclusive) and 1-based end position (inclusive)
/// which is equivalent to saying:
/// 0-based start position (inclusive) and 0-based end position (exclusive)
/// </summary>
public static void WriteRegionBed(NexteraManifest manifest, string outputPath, GenomeMetadata genome)
{
using (BgzipOrStreamWriter writer = new BgzipOrStreamWriter(outputPath))
{
WriteRegionBed(manifest, writer, genome);
}
}
public static void WriteTargetBed(NexteraManifest manifest, BgzipOrStreamWriter writer, GenomeMetadata genome)
{
List<NexteraManifest.ManifestRegion> tempRegions = manifest.Regions;
if (genome != null)
{
tempRegions = new List<NexteraManifest.ManifestRegion>(manifest.Regions);
Dictionary<string, int> chromsomeIndexLookup = new Dictionary<string, int>();
//generate chromsome index lookup and sort
for (int chromosomeIndex = 0; chromosomeIndex < genome.Sequences.Count; chromosomeIndex++)
{
GenomeMetadata.SequenceMetadata sequence = genome.Sequences[chromosomeIndex];
chromsomeIndexLookup[sequence.Name] = chromosomeIndex;
}
tempRegions.Sort((a, b) => a.CompareTo(b, chromsomeIndexLookup));
}
foreach (NexteraManifest.ManifestRegion region in tempRegions)
{
TargetInterval interval = region.GetTargetInterval();
writer.WriteLine(string.Join("\t", new[]
{
interval.ReferenceName,
(interval.Begin - 1).ToString(CultureInfo.InvariantCulture),
interval.End.ToString(CultureInfo.InvariantCulture),
region.Name //region name is needed for PUMA metrics outputs to generate .coverage.csv file
}));
}
}
public static void WriteRegionBed(NexteraManifest manifest, BgzipOrStreamWriter writer, GenomeMetadata genome)
{
List<NexteraManifest.ManifestRegion> tempRegions = manifest.Regions;
if (genome != null)
{
tempRegions = new List<NexteraManifest.ManifestRegion>(manifest.Regions);
Dictionary<string, int> chromsomeIndexLookup = new Dictionary<string, int>();
//generate chromsome index lookup and sort
for (int chromosomeIndex = 0; chromosomeIndex < genome.Sequences.Count; chromosomeIndex++)
{
GenomeMetadata.SequenceMetadata sequence = genome.Sequences[chromosomeIndex];
chromsomeIndexLookup[sequence.Name] = chromosomeIndex;
}
tempRegions.Sort((a, b) => a.CompareTo(b, chromsomeIndexLookup));
}
foreach (NexteraManifest.ManifestRegion region in tempRegions)
{
writer.WriteLine(string.Format("{0}\t{1}\t{2}", region.Chromosome, region.Start - 1, region.End));
}
}
/// <summary>
/// Outputs the copy number calls to a text file.
/// </summary>
/// <param name="outVcfPath">File to write to.</param>
/// <param name="segments">List of segments to write out.</param>
public static void WriteSegments(string outVcfPath, List<CanvasSegment> segments, string reference, string sampleName,
List<string> extraHeaders, bool reportPloidy, PloidyInfo ploidy, bool reportAllSites = false, bool reportGermlineGenotype = false)
{
string cnvtype = null;
string filter = null;
// report GT for resequencing workflow and MCC for tumour-normal workflow
if (reportGermlineGenotype && reportPloidy)
{
throw new Exception("WriteSegments VCF file output error: reportGermlineGenotype and reportPloidy can not be both true");
}
using (BgzipOrStreamWriter writer = new BgzipOrStreamWriter(outVcfPath))
{
// Write the VCF header:
writer.WriteLine("##fileformat=VCFv4.1");
writer.WriteLine("##source=Isas," + CanvasCommon.CanvasVersionInfo.NameString + " " + CanvasCommon.CanvasVersionInfo.VersionString);
writer.WriteLine("##reference={0}", Path.Combine(reference, "genome.fa"));
if (extraHeaders != null)
{
foreach (string header in extraHeaders)
{
writer.WriteLine(header);
}
}
GenomeMetadata genome = new GenomeMetadata();
genome.Deserialize(Path.Combine(reference, "GenomeSize.xml"));
foreach (GenomeMetadata.SequenceMetadata chromosome in genome.Sequences)
{
writer.WriteLine("##contig=<ID={0},length={1}>", chromosome.Name, chromosome.Length);
}
writer.WriteLine("##ALT=<ID=CNV,Description=\"Copy number variable region\">");
writer.WriteLine("##FILTER=<ID=q10,Description=\"Quality below 10\">");
writer.WriteLine("##FILTER=<ID=L10kb,Description=\"Length shorter than 10kb\">");
writer.WriteLine("##INFO=<ID=SVTYPE,Number=1,Type=String,Description=\"Type of structural variant\">");
writer.WriteLine("##INFO=<ID=END,Number=1,Type=Integer,Description=\"End position of the variant described in this record\">");
writer.WriteLine("##INFO=<ID=CNVLEN,Number=1,Type=Integer,Description=\"Number of reference positions spanned by this CNV\">");
if (reportGermlineGenotype)
writer.WriteLine("##FORMAT=<ID=GT,Number=1,Type=String,Description=\"Genotype\">");
writer.WriteLine("##FORMAT=<ID=RC,Number=1,Type=Float,Description=\"Mean counts per bin in the region\">");
writer.WriteLine("##FORMAT=<ID=BC,Number=1,Type=Float,Description=\"Number of bins in the region\">");
writer.WriteLine("##FORMAT=<ID=CN,Number=1,Type=Integer,Description=\"Copy number genotype for imprecise events\">");
if (reportPloidy)
writer.WriteLine("##FORMAT=<ID=MCC,Number=1,Type=Integer,Description=\"Major chromosome count (equal to copy number for LOH regions)\">");
writer.WriteLine("#CHROM\tPOS\tID\tREF\tALT\tQUAL\tFILTER\tINFO\tFORMAT\t" + sampleName);
SanityCheckChromosomeNames(genome, segments);
foreach (GenomeMetadata.SequenceMetadata chromosome in genome.Sequences)
{
foreach (CanvasSegment segment in segments)
{
if (segment.Chr.ToLowerInvariant() != chromosome.Name.ToLowerInvariant()) continue;
int referenceCN = 2;
if (ploidy != null) referenceCN = ploidy.GetReferenceCopyNumber(segment);
filter = null;
bool isReferenceCall = false;
if (segment.CopyNumber == referenceCN) isReferenceCall = true;
if (reportPloidy && segment.CopyNumber == 2 && segment.MajorChromosomeCount.HasValue && segment.MajorChromosomeCount != 1) isReferenceCall = false; // If we're reporting ploidy and there's LOH, this isn't a reference call.
// We can skip reporting of reference sites:
if (!reportAllSites && isReferenceCall)
continue;
if (segment.QScore < 10)
filter = "q10";
if (segment.End - segment.Begin < 10000)
{
if (filter != null)
filter = filter + ";L10kb";
else
filter = "L10kb";
}
if (filter == null)
filter = "PASS";
if (segment.CopyNumber < referenceCN)
cnvtype = "LOSS";
else if (segment.CopyNumber > referenceCN)
cnvtype = "GAIN";
else
cnvtype = "REF";
// The Dude abides... from vcf 4.1 spec:
// If any of the ALT alleles is a symbolic allele (an angle-bracketed ID String “<ID>”) then the padding base is required and POS denotes the
// coordinate of the base preceding the polymorphism.
writer.Write("{0}\t{1}\tCanvas:{2}:{0}:{3}-{4}\t", segment.Chr, isReferenceCall ? segment.Begin + 1 : segment.Begin, cnvtype, segment.Begin + 1, segment.End);
writer.Write("N\t{0}\t{1}\t{2}\t", isReferenceCall ? "." : "<CNV>", segment.QScore, filter);
if (segment.copyNumber != referenceCN)
writer.Write("SVTYPE=CNV;");
else if (!isReferenceCall)
writer.Write("SVTYPE=LOH;");
if (segment.copyNumber != referenceCN || !isReferenceCall)
writer.Write("END={0};CNVLEN={1}", segment.End, segment.End - segment.Begin);
else
writer.Write("END={0}", segment.End);
// FORMAT field
if (reportGermlineGenotype)
writer.Write("\tGT:RC:BC:CN", segment.End);
else
writer.Write("\tRC:BC:CN", segment.End);
if (reportPloidy && segment.MajorChromosomeCount.HasValue) writer.Write(":MCC");
// writing GT for resequencing workflow
if (reportGermlineGenotype)
{
writer.Write("\t{0}/{1}:", segment.MajorChromosomeCount, segment.CopyNumber);
}
else
writer.Write("\t");
writer.Write("{1}:{2}:{3}", segment.End, Math.Round(segment.MeanCount, 0, MidpointRounding.AwayFromZero), segment.BinCount, segment.CopyNumber);
// writing MCC for tumour-normal workflow
if (reportPloidy && segment.MajorChromosomeCount.HasValue)
{
writer.Write(":{0}", segment.MajorChromosomeCount);
}
writer.WriteLine();
}
}
}
}
/// <summary>
/// Outputs the copy number calls to a text file.
/// </summary>
public static void WriteSegments(string outVcfPath, List<CanvasSegment> segments, string wholeGenomeFastaDirectory, string sampleName,
List<string> extraHeaders, PloidyInfo ploidy, int qualityThreshold = 10)
{
using (BgzipOrStreamWriter writer = new BgzipOrStreamWriter(outVcfPath))
{
// Write the VCF header:
writer.WriteLine("##fileformat=VCFv4.1");
writer.WriteLine($"##source={CanvasVersionInfo.NameString} {CanvasVersionInfo.VersionString}");
writer.WriteLine($"##reference={Path.Combine(wholeGenomeFastaDirectory, "genome.fa")}");
foreach (string header in extraHeaders ?? new List<string>())
{
writer.WriteLine(header);
}
GenomeMetadata genome = new GenomeMetadata();
genome.Deserialize(Path.Combine(wholeGenomeFastaDirectory, "GenomeSize.xml"));
foreach (GenomeMetadata.SequenceMetadata chromosome in genome.Sequences)
{
writer.WriteLine($"##contig=<ID={chromosome.Name},length={chromosome.Length}>");
}
string qualityFilter = $"q{qualityThreshold}";
writer.WriteLine("##ALT=<ID=CNV,Description=\"Copy number variable region\">");
writer.WriteLine($"##FILTER=<ID={qualityFilter},Description=\"Quality below {qualityThreshold}\">");
writer.WriteLine("##FILTER=<ID=L10kb,Description=\"Length shorter than 10kb\">");
writer.WriteLine("##INFO=<ID=SVTYPE,Number=1,Type=String,Description=\"Type of structural variant\">");
writer.WriteLine("##INFO=<ID=END,Number=1,Type=Integer,Description=\"End position of the variant described in this record\">");
writer.WriteLine("##INFO=<ID=CNVLEN,Number=1,Type=Integer,Description=\"Number of reference positions spanned by this CNV\">");
writer.WriteLine("##FORMAT=<ID=RC,Number=1,Type=Float,Description=\"Mean counts per bin in the region\">");
writer.WriteLine("##FORMAT=<ID=BC,Number=1,Type=Float,Description=\"Number of bins in the region\">");
writer.WriteLine("##FORMAT=<ID=CN,Number=1,Type=Integer,Description=\"Copy number genotype for imprecise events\">");
writer.WriteLine("##FORMAT=<ID=MCC,Number=1,Type=Integer,Description=\"Major chromosome count (equal to copy number for LOH regions)\">");
writer.WriteLine("#CHROM\tPOS\tID\tREF\tALT\tQUAL\tFILTER\tINFO\tFORMAT\t" + sampleName);
SanityCheckChromosomeNames(genome, segments);
foreach (GenomeMetadata.SequenceMetadata chromosome in genome.Sequences)
{
foreach (CanvasSegment segment in segments)
{
if (!segment.Chr.Equals(chromosome.Name, StringComparison.OrdinalIgnoreCase)) continue;
int referenceCopyNumber = ploidy?.GetReferenceCopyNumber(segment) ?? 2;
CnvType cnvType = segment.GetCnvType(referenceCopyNumber);
// From vcf 4.1 spec:
// If any of the ALT alleles is a symbolic allele (an angle-bracketed ID String “<ID>”) then the padding base is required and POS denotes the
// coordinate of the base preceding the polymorphism.
string alternateAllele = cnvType.ToAltId();
int position = (alternateAllele.StartsWith("<") && alternateAllele.EndsWith(">")) ? segment.Begin : segment.Begin + 1;
writer.Write($"{segment.Chr}\t{position}\tCanvas:{cnvType.ToVcfId()}:{segment.Chr}:{segment.Begin + 1}-{segment.End}\t");
writer.Write($"N\t{alternateAllele}\t{segment.QScore}\t{segment.Filter}\t", alternateAllele, segment.QScore, segment.Filter);
if (cnvType != CnvType.Reference)
writer.Write($"SVTYPE={cnvType.ToSvType()};");
writer.Write($"END={segment.End}");
if (cnvType != CnvType.Reference)
writer.Write($";CNVLEN={segment.End - segment.Begin}");
// FORMAT field
writer.Write("\tRC:BC:CN", segment.End);
if (segment.MajorChromosomeCount.HasValue)
{
writer.Write(":MCC");
}
writer.Write("\t{1}:{2}:{3}", segment.End, Math.Round(segment.MeanCount, 0, MidpointRounding.AwayFromZero), segment.BinCount, segment.CopyNumber);
if (segment.MajorChromosomeCount.HasValue)
{
writer.Write(":{0}", segment.MajorChromosomeCount);
}
writer.WriteLine();
}
}
}
}