public override IEnumerable <string> Process()
{
PileupCountList pc = new PileupCountList();
var format = options.GetSAMFormat();
var cm = new SmallRNACountMap(options.CountFile);
var srItems = SequenceRegionUtils.GetSequenceRegions(options.CoordinateFile, "miRNA", options.BedAsGtf);
srItems.ForEach(m =>
{
m.Seqname = m.Seqname.StringAfter("chr");
});
var srmap = srItems.GroupBy(m => m.Seqname).ToDictionary(m => m.Key, m => m.ToList());
StreamWriter swScript = null;
try
{
if (options.ExportIgvScript)
{
swScript = new StreamWriter(options.OutputFile + ".igv");
swScript.WriteLine("snapshotDirectory {0}", Path.GetDirectoryName(options.OutputFile).Replace('\\', '/'));
}
using (StreamWriter sw = new StreamWriter(options.OutputFile))
{
sw.WriteLine(@"##fileformat=VCFv4.2
##fileDate={0:yyyyMMdd}
##source={1}
##phasing=partial
##INFO=<ID=NS,Number=1,Type=Integer,Description=""Number of Samples With Data"">
##INFO=<ID=DP,Number=1,Type=Integer,Description=""Total Depth"">
##INFO=<ID=AF,Number=A,Type=Float,Description=""Allele Frequency"">
##INFO=<ID=FP,Number=1,Type=Float,Description=""Fisher Exact Test P-Value"">
##INFO=<ID=MN,Number=.,Type=String,Description=""miRNA name contains this position"">
##FILTER=<ID=FisherET,Description=""Fisher exact test Pvalue less than {2}"">
##FILTER=<ID=AltAlleFreq,Description=""Alternative allele frequency less than {3}"">
##FILTER=<ID=notMiRNA,Description=""Position not located in miRNA locus"">
##FORMAT=<ID=DP,Number=1,Type=Integer,Description=""Read Depth"">
##FORMAT=<ID=AD,Number=1,Type=Integer,Description=""Allelic Depth"">
#CHROM POS ID REF ALT QUAL FILTER INFO FORMAT {4}",
DateTime.Now,
"PileupCountBuilder",
options.FisherPValue,
options.MinimumAlternativeAlleleFrequency,
Path.GetFileNameWithoutExtension(options.InputFile));
using (var sr = SAMFactory.GetReader(options.InputFile, true))
{
int count = 0;
string line;
while ((line = sr.ReadLine()) != null)
{
count++;
if (count % 100 == 0)
{
if (Progress.IsCancellationPending())
{
throw new UserTerminatedException();
}
}
if (count % 100000 == 0)
{
Progress.SetMessage("{0} reads processed", count);
}
var parts = line.Split('\t');
var qname = parts[SAMFormatConst.QNAME_INDEX];
var seq = parts[SAMFormatConst.SEQ_INDEX];
//too short
if (seq.Length < options.MinimumReadLength)
{
continue;
}
SAMFlags flag = (SAMFlags)int.Parse(parts[SAMFormatConst.FLAG_INDEX]);
//unmatched
if (flag.HasFlag(SAMFlags.UnmappedQuery))
{
continue;
}
var cigar = parts[SAMFormatConst.CIGAR_INDEX];
//insertion/deletion
if (cigar.Any(m => m == 'I' || m == 'D'))
{
continue;
}
var sam = new SAMAlignedItem()
{
Qname = qname,
};
bool isReversed = flag.HasFlag(SAMFlags.QueryOnReverseStrand);
char strand;
if (isReversed)
{
strand = '-';
sam.Sequence = SequenceUtils.GetReverseComplementedSequence(seq);
}
else
{
strand = '+';
sam.Sequence = seq;
}
var loc = new SAMAlignedLocation(sam)
{
Seqname = parts[SAMFormatConst.RNAME_INDEX].StringAfter("chr"),
Start = int.Parse(parts[SAMFormatConst.POS_INDEX]),
Strand = strand,
Cigar = parts[SAMFormatConst.CIGAR_INDEX],
MismatchPositions = format.GetMismatchPositions(parts),
NumberOfMismatch = format.GetNumberOfMismatch(parts),
Sequence = seq
};
loc.ParseEnd(sam.Sequence);
sam.AddLocation(loc);
if (format.HasAlternativeHits)
{
format.ParseAlternativeHits(parts, sam);
}
var finished = pc.Add(sam, cm.GetCount(sam.Qname));
if (null == finished || 0 == finished.Count)
{
continue;
}
foreach (var fin in finished)
{
//if (fin.Chromosome.Equals("1") && fin.Position == 5160725)
//{
// Console.WriteLine(fin);
//}
var ft = fin.FisherExactTest();
if (ft.PValue <= options.FisherPValue)
{
var total = fin.Sum(m => m.Value);
var minallele = total * options.MinimumAlternativeAlleleFrequency;
if (ft.Sample2.Failed >= minallele)
{
List <GtfItem> srs;
List <string> ranges = new List <string>();
if (srmap.TryGetValue(sam.Locations[0].Seqname, out srs))
{
foreach (var seqr in srs)
{
if (seqr.Contains(fin.Position))
{
ranges.Add(seqr.GetNameLocation());
}
}
}
var alter = (from r in fin
where r.Key != fin.Reference
orderby r.Key
select r).ToList();
var str = string.Format("{0}\t{1}\t{2}\t{3}\t{4}\t{5}\t{6}\tNS={7};DP={8};AF={9};FP={10:0.##E0}{11}\tDP:AD\t{12}:{13},{14}",
fin.Chromosome,
fin.Position,
".",
fin.Reference,
(from r in alter
select r.Key.ToString()).Merge(","),
0,
ranges.Count == 0 ? "notMiRNA" : "PASS",
1,
total,
(from r in alter
select string.Format("{0:0.###}", r.Value * 1.0 / total)).Merge(","),
ft.PValue,
ranges.Count == 0 ? "" : ";" + ranges.Merge(","),
total,
ft.Sample2.Succeed,
(from r in alter
select r.Value.ToString()).Merge(","));
sw.WriteLine(str);
//Console.WriteLine(str);
if (swScript != null && ranges.Count > 0)
{
swScript.WriteLine(@"goto {0}:{1}
sort position
snapshot {0}_{2}_{1}.png", fin.Chromosome, fin.Position, ranges[0].Replace('(', '_').Replace(')', '_').Replace(':', '_'));
}
}
}
}
finished.Clear();
}
}
}
}
finally
{
if (swScript != null)
{
swScript.Close();
}
}
return(new string[] { options.OutputFile });
}
public void TestAdd()
{
SAMAlignedItem item1 = new SAMAlignedItem()
{
Sequence = "CTCTTAGATCGATGTGGTGCTCCGGAAAAAA",
};
item1.AddLocation(new SAMAlignedLocation(item1)
{
Seqname ="chr13",
Cigar = "5S21M5S",
MismatchPositions = "10T10",
Start = 39979942,
Sequence = "CTCTTAGATCGATGTGGTGCTCCGGAAAAAA"
});
SAMAlignedItem item2 = new SAMAlignedItem()
{
Sequence = "GATGTAGTGCTCCGGATTTTT"
};
item2.AddLocation(new SAMAlignedLocation(item2)
{
Seqname = "chr13",
Cigar = "21M",
MismatchPositions = "5T15",
Start = 39979947,
Sequence = "GATGTAGTGCTCCGGATTTTT"
});
List<PileupCount> all = new List<PileupCount>();
PileupCountList count = new PileupCountList();
var res1 = count.Add(item1, 2);
Assert.AreEqual(0, res1.Count);
var res2 = count.Add(item2, 3);
Assert.AreEqual(5, res2.Count);
all.AddRange(res2);
for (int i = 0; i < res2.Count; i++)
{
Assert.AreEqual(item1.Sequence[5 + i], res2[i].Reference);
Assert.AreEqual(item1.Sequence[5 + i], res2[i].First().Key);
Assert.AreEqual(2, res2[i].First().Value);
Assert.AreEqual(item1.Locations[0].Seqname, res2[i].Chromosome);
Assert.AreEqual(item1.Pos + i, res2[i].Position);
}
item1.Locations[0].Seqname = "chr14";
var res3 = count.Add(item1, 2);
Assert.AreEqual(21, res3.Count);
all.AddRange(res3);
for (int i = 0; i < 16; i++)
{
if (i == 5)
{
Assert.AreEqual('T', res3[i].Reference);
Assert.True(res3[i].ContainsKey('G'));
Assert.AreEqual(2, res3[i]['G']);
Assert.True(res3[i].ContainsKey('A'));
Assert.AreEqual(3, res3[i]['A']);
}
else
{
Assert.AreEqual(item2.Sequence[i], res3[i].Reference);
Assert.AreEqual(item2.Sequence[i], res3[i].First().Key);
Assert.AreEqual(5, res3[i].First().Value);
}
}
for (int i = 16; i < 21; i++)
{
Assert.AreEqual(item2.Sequence[i], res3[i].First().Key);
Assert.AreEqual(3, res3[i].First().Value);
}
var res4 = count.Count;
Assert.AreEqual(21, res4.Count);
all.AddRange(res4);
for (int i = 0; i < res4.Count; i++)
{
Assert.AreEqual(item1.Sequence[5 + i], res4[i].First().Key);
Assert.AreEqual(2, res4[i].First().Value);
Assert.AreEqual(item1.Locations[0].Seqname, res4[i].Chromosome);
Assert.AreEqual(item1.Pos + i, res4[i].Position);
}
//all.ForEach(m => Output(m));
}
public override IEnumerable<string> Process()
{
PileupCountList pc = new PileupCountList();
var format = options.GetSAMFormat();
var cm = new SmallRNACountMap(options.CountFile);
var srItems = SequenceRegionUtils.GetSequenceRegions(options.CoordinateFile, "miRNA", options.BedAsGtf);
srItems.ForEach(m =>
{
m.Seqname = m.Seqname.StringAfter("chr");
});
var srmap = srItems.GroupBy(m => m.Seqname).ToDictionary(m => m.Key, m => m.ToList());
StreamWriter swScript = null;
try
{
if (options.ExportIgvScript)
{
swScript = new StreamWriter(options.OutputFile + ".igv");
swScript.WriteLine("snapshotDirectory {0}", Path.GetDirectoryName(options.OutputFile).Replace('\\', '/'));
}
using (StreamWriter sw = new StreamWriter(options.OutputFile))
{
sw.WriteLine(@"##fileformat=VCFv4.2
##fileDate={0:yyyyMMdd}
##source={1}
##phasing=partial
##INFO=<ID=NS,Number=1,Type=Integer,Description=""Number of Samples With Data"">
##INFO=<ID=DP,Number=1,Type=Integer,Description=""Total Depth"">
##INFO=<ID=AF,Number=A,Type=Float,Description=""Allele Frequency"">
##INFO=<ID=FP,Number=1,Type=Float,Description=""Fisher Exact Test P-Value"">
##INFO=<ID=MN,Number=.,Type=String,Description=""miRNA name contains this position"">
##FILTER=<ID=FisherET,Description=""Fisher exact test Pvalue less than {2}"">
##FILTER=<ID=AltAlleFreq,Description=""Alternative allele frequency less than {3}"">
##FILTER=<ID=notMiRNA,Description=""Position not located in miRNA locus"">
##FORMAT=<ID=DP,Number=1,Type=Integer,Description=""Read Depth"">
##FORMAT=<ID=AD,Number=1,Type=Integer,Description=""Allelic Depth"">
#CHROM POS ID REF ALT QUAL FILTER INFO FORMAT {4}",
DateTime.Now,
"PileupCountBuilder",
options.FisherPValue,
options.MinimumAlternativeAlleleFrequency,
Path.GetFileNameWithoutExtension(options.InputFile));
using (var sr = SAMFactory.GetReader(options.InputFile, true))
{
int count = 0;
string line;
while ((line = sr.ReadLine()) != null)
{
count++;
if (count % 100 == 0)
{
if (Progress.IsCancellationPending())
{
throw new UserTerminatedException();
}
}
if (count % 100000 == 0)
{
Progress.SetMessage("{0} reads processed", count);
}
var parts = line.Split('\t');
var qname = parts[SAMFormatConst.QNAME_INDEX];
var seq = parts[SAMFormatConst.SEQ_INDEX];
//too short
if (seq.Length < options.MinimumReadLength)
{
continue;
}
SAMFlags flag = (SAMFlags)int.Parse(parts[SAMFormatConst.FLAG_INDEX]);
//unmatched
if (flag.HasFlag(SAMFlags.UnmappedQuery))
{
continue;
}
var cigar = parts[SAMFormatConst.CIGAR_INDEX];
//insertion/deletion
if (cigar.Any(m => m == 'I' || m == 'D'))
{
continue;
}
var sam = new SAMAlignedItem()
{
Qname = qname,
};
bool isReversed = flag.HasFlag(SAMFlags.QueryOnReverseStrand);
char strand;
if (isReversed)
{
strand = '-';
sam.Sequence = SequenceUtils.GetReverseComplementedSequence(seq);
}
else
{
strand = '+';
sam.Sequence = seq;
}
var loc = new SAMAlignedLocation(sam)
{
Seqname = parts[SAMFormatConst.RNAME_INDEX].StringAfter("chr"),
Start = int.Parse(parts[SAMFormatConst.POS_INDEX]),
Strand = strand,
Cigar = parts[SAMFormatConst.CIGAR_INDEX],
MismatchPositions = format.GetMismatchPositions(parts),
NumberOfMismatch = format.GetNumberOfMismatch(parts),
Sequence = seq
};
loc.ParseEnd(sam.Sequence);
sam.AddLocation(loc);
if (format.HasAlternativeHits)
{
format.ParseAlternativeHits(parts, sam);
}
var finished = pc.Add(sam, cm.GetCount(sam.Qname));
if (null == finished || 0 == finished.Count)
{
continue;
}
foreach (var fin in finished)
{
//if (fin.Chromosome.Equals("1") && fin.Position == 5160725)
//{
// Console.WriteLine(fin);
//}
var ft = fin.FisherExactTest();
if (ft.PValue <= options.FisherPValue)
{
var total = fin.Sum(m => m.Value);
var minallele = total * options.MinimumAlternativeAlleleFrequency;
if (ft.Sample2.Failed >= minallele)
{
List<GtfItem> srs;
List<string> ranges = new List<string>();
if (srmap.TryGetValue(sam.Locations[0].Seqname, out srs))
{
foreach (var seqr in srs)
{
if (seqr.Contains(fin.Position))
{
ranges.Add(seqr.GetNameLocation());
}
}
}
var alter = (from r in fin
where r.Key != fin.Reference
orderby r.Key
select r).ToList();
var str = string.Format("{0}\t{1}\t{2}\t{3}\t{4}\t{5}\t{6}\tNS={7};DP={8};AF={9};FP={10:0.##E0}{11}\tDP:AD\t{12}:{13},{14}",
fin.Chromosome,
fin.Position,
".",
fin.Reference,
(from r in alter
select r.Key.ToString()).Merge(","),
0,
ranges.Count == 0 ? "notMiRNA" : "PASS",
1,
total,
(from r in alter
select string.Format("{0:0.###}", r.Value * 1.0 / total)).Merge(","),
ft.PValue,
ranges.Count == 0 ? "" : ";" + ranges.Merge(","),
total,
ft.Sample2.Succeed,
(from r in alter
select r.Value.ToString()).Merge(","));
sw.WriteLine(str);
//Console.WriteLine(str);
if (swScript != null && ranges.Count > 0)
{
swScript.WriteLine(@"goto {0}:{1}
sort position
snapshot {0}_{2}_{1}.png", fin.Chromosome, fin.Position, ranges[0].Replace('(', '_').Replace(')', '_').Replace(':', '_'));
}
}
}
}
finished.Clear();
}
}
}
}
finally
{
if (swScript != null)
{
swScript.Close();
}
}
return new string[] { options.OutputFile };
}
