public Dictionary<string, BedItem> ReadBedItems()
{
Dictionary<string, BedItem> map = new Dictionary<string, BedItem>();
using (var gtf = new GtfItemFile(options.InputFile))
{
GtfItem item;
int count = 0;
while ((item = gtf.Next()) != null)
{
count++;
if ((count % 100000) == 0)
{
Progress.SetMessage("{0} gtf item processed", count);
}
BedItem loc;
if (!map.TryGetValue(item.GeneId, out loc))
{
loc = new BedItem();
loc.Name = item.GeneId;
loc.Seqname = item.Seqname;
loc.Start = item.Start;
loc.End = item.End;
loc.Strand = item.Strand;
map[item.GeneId] = loc;
continue;
}
map[item.GeneId].UnionWith(item);
}
}
map.Values.ToList().ForEach(m => m.Start--);
return map;
}
public override IEnumerable<string> Process()
{
var paramFile = options.OutputFile + ".param";
if (string.IsNullOrEmpty(options.ParamFile) || !Path.GetFullPath(options.ParamFile).Equals(Path.GetFullPath(paramFile)))
{
options.SaveToFile(options.OutputFile + ".param");
}
var bedfile = new BedItemFile<BedItem>(6);
var mirnas = new List<BedItem>();
if (File.Exists(options.MiRBaseFile))
{
Progress.SetMessage("Processing {0} ...", options.MiRBaseFile);
if (options.MiRBaseFile.EndsWith(".bed"))
{
mirnas = bedfile.ReadFromFile(options.MiRBaseFile);
mirnas.ForEach(m =>
{
m.Seqname = m.Seqname.StringAfter("chr");
m.Name = options.MiRBaseKey + ":" + m.Name;
});
}
else
{
using (var gf = new GtfItemFile(options.MiRBaseFile))
{
GtfItem item;
while ((item = gf.Next(options.MiRBaseKey)) != null)
{
BedItem loc = new BedItem();
loc.Seqname = item.Seqname.StringAfter("chr");
loc.Start = item.Start - 1;
loc.End = item.End;
loc.Name = options.MiRBaseKey + ":" + item.Attributes.StringAfter("Name=").StringBefore(";");
loc.Score = 1000;
loc.Strand = item.Strand;
mirnas.Add(loc);
}
}
}
Progress.SetMessage("{0} miRNA read.", mirnas.Count);
}
List<BedItem> trnas = new List<BedItem>();
if (File.Exists(options.UcscTrnaFile))
{
//reading tRNA from ucsc table without mitocondrom tRNA
Progress.SetMessage("Processing {0} ...", options.UcscTrnaFile);
trnas = bedfile.ReadFromFile(options.UcscTrnaFile);
trnas.ForEach(m => m.Seqname = m.Seqname.StringAfter("chr"));
//remove the tRNA not from 1-22, X and Y
trnas.RemoveAll(m => (m.Seqname.Length > 1) && !m.Seqname.All(n => char.IsDigit(n)));
//mitocondrom tRNA will be extracted from ensembl gtf file
trnas.RemoveAll(m => m.Seqname.Equals("M") || m.Seqname.Equals("MT"));
trnas.ForEach(m => m.Name = SmallRNAConsts.tRNA + ":" + m.Name);
Progress.SetMessage("{0} tRNA from ucsc read.", trnas.Count);
}
var others = new List<BedItem>();
if (File.Exists(options.EnsemblGtfFile))
{
//reading smallRNA/tRNA from ensembl gtf file
Progress.SetMessage("Processing {0} ...", options.EnsemblGtfFile);
using (var gf = new GtfItemFile(options.EnsemblGtfFile))
{
var biotypes = new HashSet<string>(SmallRNAConsts.Biotypes);
biotypes.Remove(SmallRNAConsts.miRNA);
GtfItem item;
int count = 0;
while ((item = gf.Next("gene")) != null)
{
string biotype;
if (item.Attributes.Contains("gene_biotype"))
{
biotype = item.Attributes.StringAfter("gene_biotype \"").StringBefore("\"");
}
else if (item.Attributes.Contains("gene_type"))
{
biotype = item.Attributes.StringAfter("gene_type \"").StringBefore("\"");
}
else
{
continue;
}
if (File.Exists(options.UcscTrnaFile) && biotype.Equals(SmallRNAConsts.tRNA))
{
continue;
}
if (biotype.Equals("Mt_tRNA"))
{
count++;
var gene_name = item.Attributes.Contains("gene_name") ? item.Attributes.StringAfter("gene_name \"").StringBefore("\"") : item.GeneId;
BedItem loc = new BedItem();
loc.Seqname = "MT";
loc.Start = item.Start - 1;
loc.End = item.End;
loc.Name = string.Format(SmallRNAConsts.tRNA + ":chrMT.tRNA{0}-{1}", count, gene_name.StringAfter("-"));
loc.Score = 1000;
loc.Strand = item.Strand;
trnas.Add(loc);
}
else if (biotypes.Contains(biotype))
{
string seqName;
if (item.Seqname.ToLower().StartsWith("chr"))
{
seqName = item.Seqname.Substring(3);
}
else
{
seqName = item.Seqname;
}
if (seqName.Equals("M"))
{
seqName = "MT";
}
//ignore all smallRNA coordinates on scaffold or contig.
if (seqName.Length > 5)
{
continue;
}
var gene_name = item.Attributes.StringAfter("gene_name \"").StringBefore("\"");
BedItem loc = new BedItem();
loc.Seqname = seqName;
loc.Start = item.Start - 1;
loc.End = item.End;
loc.Name = biotype + ":" + gene_name + ":" + item.GeneId;
loc.Score = 1000;
loc.Strand = item.Strand;
others.Add(loc);
}
}
}
}
var all = new List<BedItem>();
all.AddRange(mirnas);
all.AddRange(trnas);
all.AddRange(others);
if (File.Exists(options.RRNAFile))
{
var seqs = SequenceUtils.Read(options.RRNAFile);
foreach(var seq in seqs)
{
all.Add(new BedItem()
{
Seqname = seq.Name,
Start = 0,
End = seq.SeqString.Length,
Strand = '+',
Name = "rRNA:" + seq.Name
});
}
}
Progress.SetMessage("Saving smallRNA coordinates to " + options.OutputFile + "...");
using (var sw = new StreamWriter(options.OutputFile))
{
foreach (var pir in SmallRNAConsts.Biotypes)
{
var locs = all.Where(m => m.Name.StartsWith(pir)).ToList();
GenomeUtils.SortChromosome(locs, m => m.Seqname, m => (int)m.Start);
foreach (var loc in locs)
{
sw.WriteLine(bedfile.GetValue(loc));
}
}
}
Progress.SetMessage("Extracting sequence from " + options.FastaFile + "...");
new Bed2FastaProcessor(new Bed2FastaProcessorOptions()
{
GenomeFastaFile = options.FastaFile,
InputFile = options.OutputFile,
OutputFile = options.OutputFile + ".fa",
KeepChrInName = false,
AcceptName = m => m.StartsWith(SmallRNAConsts.miRNA) || m.StartsWith(SmallRNAConsts.tRNA),
})
{
Progress = this.Progress
}.Process();
var summaryFile = options.OutputFile + ".info";
Progress.SetMessage("Writing summary to " + summaryFile + "...");
using (var sw = new StreamWriter(summaryFile))
{
sw.WriteLine("Biotype\tCount");
all.ConvertAll(m => m.Name).Distinct().GroupBy(m => m.StringBefore(":")).OrderByDescending(m => m.Count()).ToList().ForEach(m => sw.WriteLine("{0}\t{1}", m.Key, m.Count()));
}
return new string[] { options.OutputFile };
}
public Block(BedItem parent)
{
this.parent = parent;
}
public Block(BedItem parent)
{
this.parent = parent;
}
