/// <summary>
/// Converts Flag to string.
/// In a string FLAG, each character represents one bit with
///p=0x1 (paired), P=0x2 (properly paired), u=0x4 (unmapped),
///U=0x8 (mate unmapped), r=0x10 (reverse), R=0x20 (mate reverse)
///1=0x40 (first), 2=0x80 (second), s=0x100 (not primary),
///f=0x200 (failure) and d=0x400 (duplicate).
/// </summary>
/// <param name="flag">Sequence Flag.</param>
/// <returns>String of flag.</returns>
private string GetFlagDesc(SAMFlags flag)
{
string str = string.Empty;
if ((flag & SAMFlags.PairedRead) == SAMFlags.PairedRead)
{
str = str + "p";
}
if ((flag & SAMFlags.MappedInProperPair) == SAMFlags.MappedInProperPair)
{
str = str + "P";
}
if ((flag & SAMFlags.UnmappedQuery) == SAMFlags.UnmappedQuery)
{
str = str + "u";
}
if ((flag & SAMFlags.UnmappedMate) == SAMFlags.UnmappedMate)
{
str = str + "U";
}
if ((flag & SAMFlags.QueryOnReverseStrand) == SAMFlags.QueryOnReverseStrand)
{
str = str + "r";
}
if ((flag & SAMFlags.MateOnReverseStrand) == SAMFlags.MateOnReverseStrand)
{
str = str + "R";
}
if ((flag & SAMFlags.FirstReadInPair) == SAMFlags.FirstReadInPair)
{
str = str + "1";
}
if ((flag & SAMFlags.SecondReadInPair) == SAMFlags.SecondReadInPair)
{
str = str + "2";
}
if ((flag & SAMFlags.NonPrimeAlignment) == SAMFlags.NonPrimeAlignment)
{
str = str + "s";
}
if ((flag & SAMFlags.QualityCheckFailure)
== SAMFlags.QualityCheckFailure)
{
str = str + "f";
}
if ((flag & SAMFlags.Duplicate) == SAMFlags.Duplicate)
{
str = str + "d";
}
return str;
}
/// <summary>
/// Converts Flag to string.
/// In a string FLAG, each character represents one bit with
///p=0x1 (paired), P=0x2 (properly paired), u=0x4 (unmapped),
///U=0x8 (mate unmapped), r=0x10 (reverse), R=0x20 (mate reverse)
///1=0x40 (first), 2=0x80 (second), s=0x100 (not primary),
///f=0x200 (failure) and d=0x400 (duplicate).
/// </summary>
/// <param name="flag">Sequence Flag.</param>
/// <returns>String of flag.</returns>
private string GetFlagDesc(SAMFlags flag)
{
string str = string.Empty;
if ((flag & SAMFlags.PairedRead) == SAMFlags.PairedRead)
{
str = str + "p";
}
if ((flag & SAMFlags.MappedInProperPair) == SAMFlags.MappedInProperPair)
{
str = str + "P";
}
if ((flag & SAMFlags.UnmappedQuery) == SAMFlags.UnmappedQuery)
{
str = str + "u";
}
if ((flag & SAMFlags.UnmappedMate) == SAMFlags.UnmappedMate)
{
str = str + "U";
}
if ((flag & SAMFlags.QueryOnReverseStrand) == SAMFlags.QueryOnReverseStrand)
{
str = str + "r";
}
if ((flag & SAMFlags.MateOnReverseStrand) == SAMFlags.MateOnReverseStrand)
{
str = str + "R";
}
if ((flag & SAMFlags.FirstReadInPair) == SAMFlags.FirstReadInPair)
{
str = str + "1";
}
if ((flag & SAMFlags.SecondReadInPair) == SAMFlags.SecondReadInPair)
{
str = str + "2";
}
if ((flag & SAMFlags.NonPrimeAlignment) == SAMFlags.NonPrimeAlignment)
{
str = str + "s";
}
if ((flag & SAMFlags.QualityCheckFailure)
== SAMFlags.QualityCheckFailure)
{
str = str + "f";
}
if ((flag & SAMFlags.Duplicate) == SAMFlags.Duplicate)
{
str = str + "d";
}
return(str);
}
public List <ChromosomeCountSlimItem> Build(string fileName)
{
if (File.Exists(options.CategoryMapFile))
{
Progress.SetMessage("Reading name map file " + options.CategoryMapFile + " ...");
nameMap = new MapItemReader(0, 1).ReadFromFile(options.CategoryMapFile).ToDictionary(m => m.Key, m => m.Value.Value);
}
var result = new List <ChromosomeCountSlimItem>();
var queries = new Dictionary <string, SAMChromosomeItem>();
var chromosomes = new Dictionary <string, ChromosomeCountSlimItem>();
Regex chromosomeRegex = null;
Func <string, bool> acceptChromosome;
if (string.IsNullOrEmpty(options.ChromosomePattern))
{
acceptChromosome = m => true;
}
else
{
chromosomeRegex = new Regex(options.ChromosomePattern);
acceptChromosome = m => chromosomeRegex.Match(m).Success;
}
Progress.SetMessage("Parsing alignment file " + fileName + " ...");
using (var sr = SAMFactory.GetReader(fileName, true))
{
int count = 0;
int waitingcount = 0;
string line;
while ((line = sr.ReadLine()) != null)
{
if (count % 1000 == 0)
{
if (Progress.IsCancellationPending())
{
throw new UserTerminatedException();
}
}
if (count % 100000 == 0 && count > 0)
{
Progress.SetMessage("{0} candidates from {1} reads", waitingcount, count);
}
count++;
var parts = line.Split('\t');
SAMFlags flag = (SAMFlags)int.Parse(parts[SAMFormatConst.FLAG_INDEX]);
//unmatched
if (flag.HasFlag(SAMFlags.UnmappedQuery))
{
continue;
}
var seqname = GetName(parts[SAMFormatConst.RNAME_INDEX]);
if (!acceptChromosome(seqname))
{
continue;
}
var qname = parts[SAMFormatConst.QNAME_INDEX];
SAMChromosomeItem query;
if (!queries.TryGetValue(qname, out query))
{
query = new SAMChromosomeItem();
query.Qname = qname;
queries[qname] = query;
if (options.KeepSequence)
{
query.Sequence = parts[SAMFormatConst.SEQ_INDEX];
if (flag.HasFlag(SAMFlags.QueryOnReverseStrand))
{
query.Sequence = SequenceUtils.GetReverseComplementedSequence(query.Sequence);
}
}
}
query.Chromosomes.Add(seqname);
ChromosomeCountSlimItem item;
if (!chromosomes.TryGetValue(seqname, out item))
{
item = new ChromosomeCountSlimItem();
item.Names.Add(seqname);
chromosomes[seqname] = item;
result.Add(item);
}
item.Queries.Add(query);
waitingcount++;
}
Progress.SetMessage("Finally, there are {0} candidates from {1} reads", waitingcount, count);
}
foreach (var query in queries.Values)
{
query.Chromosomes = query.Chromosomes.Distinct().OrderBy(m => m).ToList();
}
foreach (var sam in chromosomes.Values)
{
sam.Queries = sam.Queries.Distinct().OrderBy(m => m.Qname).ToList();
}
if (!string.IsNullOrEmpty(options.PreferPrefix))
{
foreach (var query in queries.Values)
{
if (query.Chromosomes.Any(l => l.StartsWith(options.PreferPrefix)))
{
var chroms = query.Chromosomes.Where(l => l.StartsWith(options.PreferPrefix)).ToArray();
foreach (var chrom in chroms)
{
chromosomes[chrom].Queries.Remove(query);
query.Chromosomes.Remove(chrom);
}
}
}
result.RemoveAll(l => l.Queries.Count == 0);
}
return(result);
}
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 virtual bool AcceptFlags(SAMFlags flags)
{
return !flags.HasFlag(SAMFlags.UnmappedQuery);
}
protected override List <T> DoBuild <T>(string fileName, out List <QueryInfo> totalQueries)
{
var result = new List <T>();
_format = _options.GetSAMFormat();
totalQueries = new List <QueryInfo>();
using (var sr = SAMFactory.GetReader(fileName, true))
{
int count = 0;
int waitingcount = 0;
string line;
while ((line = sr.ReadLine()) != null)
{
count++;
if (count % 1000 == 0)
{
if (Progress.IsCancellationPending())
{
throw new UserTerminatedException();
}
}
var parts = line.Split('\t');
var qname = parts[SAMFormatConst.QNAME_INDEX];
var qi = new QueryInfo(qname);
totalQueries.Add(qi);
SAMFlags flag = (SAMFlags)int.Parse(parts[SAMFormatConst.FLAG_INDEX]);
if (!_filter.AcceptFlags(flag))
{
continue;
}
var mismatchCount = _format.GetNumberOfMismatch(parts);
var seq = parts[SAMFormatConst.SEQ_INDEX];
qi.Mismatch = mismatchCount;
qi.Length = seq.Length;
//too many mismatchs
if (!_filter.AcceptMismatch(mismatchCount))
{
continue;
}
if (!_filter.AcceptQueryName(qname))
{
continue;
}
if (!_filter.AcceptLength(seq.Length))
{
continue;
}
var cigar = parts[SAMFormatConst.CIGAR_INDEX];
if (!_filter.AcceptCigar(cigar))
{
continue;
}
var seqname = parts[SAMFormatConst.RNAME_INDEX].StringAfter("chr");
var start = int.Parse(parts[SAMFormatConst.POS_INDEX]);
var end = SAMUtils.ParseEnd(start, cigar);
bool isReversed = flag.HasFlag(SAMFlags.QueryOnReverseStrand);
char strand;
if (isReversed)
{
strand = '-';
}
else
{
strand = '+';
}
var sam = new T();
var loc = new SAMAlignedLocation(sam)
{
Seqname = seqname,
Start = start,
End = end,
Strand = strand,
};
if (!_filter.AcceptLocus(loc))
{
continue;
}
if (isReversed)
{
seq = SequenceUtils.GetReverseComplementedSequence(seq);
}
sam.Qname = qname;
sam.Sequence = seq;
loc.AlignmentScore = _format.GetAlignmentScore(parts);
loc.Cigar = cigar;
loc.NumberOfMismatch = mismatchCount;
loc.MismatchPositions = _format.GetMismatchPositions(parts);
if (_format.HasAlternativeHits)
{
_format.ParseAlternativeHits(parts, sam);
}
result.Add(sam);
waitingcount++;
if (waitingcount % 100 == 0)
{
Progress.SetMessage("{0} feature reads from {1} reads", waitingcount, count);
}
}
}
return(result);
}
public virtual bool AcceptFlags(SAMFlags flags)
{
return(!flags.HasFlag(SAMFlags.UnmappedQuery));
}
protected override List <T> DoBuild <T>(string fileName, out List <QueryInfo> totalQueries)
{
var result = new List <T>();
_format = _options.GetSAMFormat();
totalQueries = new List <QueryInfo>();
using (var sr = SAMFactory.GetReader(fileName, true))
{
int count = 0;
int waitingcount = 0;
string line;
while ((line = sr.ReadLine()) != null)
{
if (count % 1000 == 0)
{
if (Progress.IsCancellationPending())
{
throw new UserTerminatedException();
}
}
if (count % 100000 == 0 && count > 0)
{
Progress.SetMessage("{0} candidates from {1} reads", waitingcount, count);
}
count++;
var qname = line.StringBefore("\t");
//Console.WriteLine("line = {0}", line);
//Console.WriteLine("query = {0}", qname);
var qi = new QueryInfo(qname);
totalQueries.Add(qi);
var parts = line.Split('\t');
SAMFlags flag = (SAMFlags)int.Parse(parts[SAMFormatConst.FLAG_INDEX]);
//unmatched
if (flag.HasFlag(SAMFlags.UnmappedQuery))
{
continue;
}
//too many mismatchs
var mismatchCount = _format.GetNumberOfMismatch(parts);
var seq = parts[SAMFormatConst.SEQ_INDEX];
qi.Mismatch = mismatchCount;
qi.Length = seq.Length;
qi.NoPenaltyMutation = 0;
if (_options.T2cAsNoPenaltyMutation)
{
}
if (mismatchCount > _options.MaximumMismatch)
{
continue;
}
if (!AcceptQueryName(qname))
{
continue;
}
//too short
if (seq.Length < _options.MinimumReadLength)
{
continue;
}
//too long
if (seq.Length > _options.MaximumReadLength)
{
continue;
}
var cigar = parts[SAMFormatConst.CIGAR_INDEX];
////insertion/deletion
//if (cigar.Any(m => m == 'I' || m == 'D'))
//{
// continue;
//}
bool isReversed = flag.HasFlag(SAMFlags.QueryOnReverseStrand);
char strand;
if (isReversed)
{
strand = '-';
seq = SequenceUtils.GetReverseComplementedSequence(seq);
}
else
{
strand = '+';
}
var score = _format.GetAlignmentScore(parts);
var sam = new T()
{
Qname = qname,
Sequence = seq
};
var seqname = parts[SAMFormatConst.RNAME_INDEX];
var loc = new SAMAlignedLocation(sam)
{
Seqname = seqname,
Start = int.Parse(parts[SAMFormatConst.POS_INDEX]),
Strand = strand,
Cigar = cigar,
NumberOfMismatch = mismatchCount,
AlignmentScore = score,
MismatchPositions = _format.GetMismatchPositions(parts)
};
loc.ParseEnd(sam.Sequence);
sam.AddLocation(loc);
if (_format.HasAlternativeHits)
{
_format.ParseAlternativeHits(parts, sam);
}
result.Add(sam);
waitingcount++;
}
Progress.SetMessage("Finally, there are {0} candidates from {1} reads", waitingcount, count);
}
return(result);
}
public override IEnumerable <string> Process()
{
var countFiles = options.GetCountFiles();
countFiles.Sort((m1, m2) => m1.Name.CompareTo(m2.Name));
var countMap = new Dictionary <string, Dictionary <string, int> >();
int fileIndex = 0;
foreach (var file in countFiles)
{
fileIndex++;
Progress.SetMessage("Reading {0}/{1}: {2} ...", fileIndex, countFiles.Count, file.File);
var queries = new HashSet <string>();
using (var sr = SAMFactory.GetReader(file.File, true))
{
int count = 0;
string line;
while ((line = sr.ReadLine()) != null)
{
count++;
if (count % 1000 == 0)
{
if (Progress.IsCancellationPending())
{
throw new UserTerminatedException();
}
}
var parts = line.Split('\t');
SAMFlags flag = (SAMFlags)int.Parse(parts[SAMFormatConst.FLAG_INDEX]);
//unmatched
if (flag.HasFlag(SAMFlags.UnmappedQuery))
{
continue;
}
queries.Add(parts[SAMFormatConst.QNAME_INDEX]);
}
}
var countDic = new Dictionary <string, int>();
countMap[file.Name] = countDic;
var cm = new MapItemReader(0, 1, informationIndex: 2).ReadFromFile(file.AdditionalFile);
foreach (var query in queries)
{
var count = cm[query];
countDic[count.Information] = int.Parse(count.Value);
}
Progress.SetMessage("{0} reads mapped.", queries.Count);
}
var uniques = (from c in countMap.Values
from seq in c.Keys
select seq).Distinct().ToArray();
var uniqueCounts = (from seq in uniques
let totalCount = (from c in countMap.Values
where c.ContainsKey(seq)
select c[seq]).Sum()
select new { Sequence = seq, Count = totalCount }).OrderByDescending(m => m.Count).ToArray();
using (var sw = new StreamWriter(options.OutputFile))
{
sw.WriteLine("Sequence\t" + (from cf in countFiles select cf.Name).Merge("\t"));
foreach (var uc in uniqueCounts)
{
var seq = uc.Sequence;
sw.Write(seq);
foreach (var cf in countFiles)
{
var map = countMap[cf.Name];
int count;
if (map.TryGetValue(seq, out count))
{
sw.Write("\t{0}", count);
}
else
{
sw.Write("\t0");
}
}
sw.WriteLine();
}
}
Progress.End();
return(new string[] { Path.GetFullPath(options.OutputFile) });
}
