public List <GtfItem> ReadGtfItems()
{
List <GtfItem> result = new List <GtfItem>();
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);
}
item.Name = item.Attributes.StringAfter("locus_tag=").StringBefore(";");
result.Add(item);
}
}
return(result);
}
public override IEnumerable <string> Process()
{
var paramFile = options.OutputFile + ".param";
options.SaveToFile(options.OutputFile + ".param");
var bedfile = new BedItemFile <BedItem>(6);
Progress.SetMessage("building chromosome name map ...");
var mitoName = "M";
Dictionary <string, string> chrNameMap = new Dictionary <string, string>();
var ff = new FastaFormat(int.MaxValue);
var faiFile = options.FastaFile + ".fai";
if (File.Exists(faiFile))
{
using (StreamReader sr = new StreamReader(faiFile))
{
string line;
while ((line = sr.ReadLine()) != null)
{
var name = line.Split('\t')[0];
chrNameMap[name] = name;
if (name.StartsWith("chr"))
{
chrNameMap[name.StringAfter("chr")] = name;
}
if (!name.StartsWith("chr"))
{
chrNameMap["chr" + name] = name;
}
if (name.Equals("chrMT") || name.Equals("MT"))
{
mitoName = "MT";
}
if (name.Equals("chrM") || name.Equals("M"))
{
mitoName = "M";
}
}
}
}
else
{
using (StreamReader sr = new StreamReader(options.FastaFile))
{
Sequence seq;
while ((seq = ff.ReadSequence(sr)) != null)
{
var name = seq.Name;
chrNameMap[name] = name;
if (name.StartsWith("chr"))
{
chrNameMap[name.StringAfter("chr")] = name;
}
if (!name.StartsWith("chr"))
{
chrNameMap["chr" + name] = name;
}
if (name.Equals("chrMT") || name.Equals("MT"))
{
mitoName = "MT";
}
if (name.Equals("chrM") || name.Equals("M"))
{
mitoName = "M";
}
}
}
}
var longMitoName = chrNameMap[mitoName];
Progress.SetMessage("mitochondral chromosome name = {0}", longMitoName);
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 readed.", 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"));
var removed = trnas.Where(m => (m.Seqname.Length > 1) && !m.Seqname.All(n => char.IsDigit(n))).ToList();
if (removed.Count != trnas.Count)
{
//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 = GetTRNAName(m.Name));
Progress.SetMessage("{0} tRNA from ucsc readed.", trnas.Count);
if (File.Exists(options.UcscMatureTrnaFastaFile))
{
var seqs = SequenceUtils.Read(options.UcscMatureTrnaFastaFile);
foreach (var seq in seqs)
{
var tRNAName = GetTRNAName(seq.Name);
trnas.Add(new BedItem()
{
Seqname = seq.Name,
Start = 0,
End = seq.SeqString.Length,
Strand = '+',
Name = tRNAName,
Sequence = seq.SeqString
});
}
}
}
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 = mitoName;
loc.Start = item.Start - 1;
loc.End = item.End;
loc.Name = string.Format(SmallRNAConsts.mt_tRNA + ":" + longMitoName + ".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.Equals("MT"))
{
seqName = mitoName;
}
//ignore all smallRNA coordinates on scaffold or contig.
//if (seqName.Length > 5)
//{
// continue;
//}
var gene_name = item.Attributes.StringAfter("gene_name \"").StringBefore("\"");
var lowGeneName = gene_name.ToLower();
if (lowGeneName.StartsWith("rny") || lowGeneName.Equals("y_rna"))
{
biotype = "yRNA";
}
BedItem loc = new BedItem();
loc.Seqname = seqName;
loc.Start = item.Start - 1;
loc.End = item.End;
//if (lowGeneName.EndsWith("_rrna") && loc.Length < 200)
//{
// biotype = "rRNA";
//}
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);
foreach (var bi in all)
{
if (chrNameMap.ContainsKey(bi.Seqname))
{
bi.Seqname = chrNameMap[bi.Seqname];
}
}
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:" + SmallRNAConsts.rRNADB_KEY + 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();
Progress.SetMessage("{0} : {1}", pir, locs.Count);
GenomeUtils.SortChromosome(locs, m => m.Seqname, m => (int)m.Start);
foreach (var loc in locs)
{
sw.WriteLine(bedfile.GetValue(loc));
}
}
}
var miRNA_bed = FileUtils.ChangeExtension(options.OutputFile, ".miRNA.bed");
Progress.SetMessage("Saving miRNA coordinates to " + miRNA_bed + "...");
using (var sw = new StreamWriter(miRNA_bed))
{
var pir = SmallRNAConsts.miRNA;
var locs = all.Where(m => m.Name.StartsWith(pir)).ToList();
Progress.SetMessage("{0} : {1}", pir, locs.Count);
GenomeUtils.SortChromosome(locs, m => m.Seqname, m => (int)m.Start);
foreach (var loc in locs)
{
sw.WriteLine(bedfile.GetValue(loc));
}
}
Progress.SetMessage("Saving smallRNA miss1 coordinates to " + options.OutputFile + ".miss1 ...");
using (var sw = new StreamWriter(options.OutputFile + ".miss1"))
{
foreach (var pir in SmallRNAConsts.Biotypes)
{
if (pir == SmallRNABiotype.lincRNA.ToString() || pir == SmallRNABiotype.lncRNA.ToString())
{
continue;
}
var locs = all.Where(m => m.Name.StartsWith(pir)).ToList();
locs.RemoveAll(l => l.Name.Contains(SmallRNAConsts.rRNADB_KEY));
Progress.SetMessage("{0} : {1}", pir, locs.Count);
GenomeUtils.SortChromosome(locs, m => m.Seqname, m => (int)m.Start);
foreach (var loc in locs)
{
sw.WriteLine(bedfile.GetValue(loc));
}
}
}
Progress.SetMessage("Saving smallRNA miss1 coordinates to " + options.OutputFile + ".miss0 ...");
using (var sw = new StreamWriter(options.OutputFile + ".miss0"))
{
foreach (var pir in SmallRNAConsts.Biotypes)
{
if (pir != SmallRNABiotype.lincRNA.ToString() && pir != SmallRNABiotype.lncRNA.ToString() && pir != SmallRNABiotype.rRNA.ToString())
{
continue;
}
var locs = all.Where(m => m.Name.StartsWith(pir)).ToList();
if (pir == SmallRNABiotype.rRNA.ToString())
{
locs.RemoveAll(l => !l.Name.Contains(SmallRNAConsts.rRNADB_KEY));
}
Progress.SetMessage("{0} : {1}", pir, locs.Count);
GenomeUtils.SortChromosome(locs, m => m.Seqname, m => (int)m.Start);
foreach (var loc in locs)
{
sw.WriteLine(bedfile.GetValue(loc));
}
}
}
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()));
}
var result = new List <string>(new[] { options.OutputFile });
var fasta = Path.ChangeExtension(options.OutputFile, ".fasta");
if ((File.Exists(options.UcscTrnaFile) && File.Exists(options.UcscMatureTrnaFastaFile)) || File.Exists(options.RRNAFile))
{
result.Add(fasta);
using (var sw = new StreamWriter(fasta))
{
string line;
using (var sr = new StreamReader(options.FastaFile))
{
while ((line = sr.ReadLine()) != null)
{
sw.WriteLine(line);
}
}
if (File.Exists(options.UcscTrnaFile) && File.Exists(options.UcscMatureTrnaFastaFile))
{
using (var sr = new StreamReader(options.UcscMatureTrnaFastaFile))
{
while ((line = sr.ReadLine()) != null)
{
sw.WriteLine(line);
}
}
}
if (File.Exists(options.RRNAFile))
{
using (var sr = new StreamReader(options.RRNAFile))
{
while ((line = sr.ReadLine()) != null)
{
sw.WriteLine(line);
}
}
}
}
}
var faFile = options.OutputFile + ".fa";
Progress.SetMessage("Extracting sequence from " + options.FastaFile + "...");
var b2foptions = new Bed2FastaProcessorOptions()
{
GenomeFastaFile = options.FastaFile,
InputFile = options.OutputFile,
OutputFile = faFile,
KeepChrInName = false,
};
if (!File.Exists(options.UcscMatureTrnaFastaFile))
{
b2foptions.AcceptName = m => m.StartsWith(SmallRNAConsts.miRNA) || m.StartsWith(SmallRNAConsts.mt_tRNA) || m.StartsWith(SmallRNAConsts.tRNA);
}
else
{
b2foptions.AcceptName = m => m.StartsWith(SmallRNAConsts.miRNA) || m.StartsWith(SmallRNAConsts.mt_tRNA);
}
new Bed2FastaProcessor(b2foptions)
{
Progress = this.Progress
}.Process();
if (File.Exists(options.UcscMatureTrnaFastaFile))
{
Progress.SetMessage("Extracting sequence from " + options.UcscMatureTrnaFastaFile + " ...");
using (var sw = new StreamWriter(faFile, true))
{
foreach (var tRNA in trnas)
{
if (!string.IsNullOrEmpty(tRNA.Sequence))
{
sw.WriteLine(">{0}", tRNA.Name);
sw.WriteLine("{0}", tRNA.Sequence);
}
}
}
}
return(result);
}