static public int ProcessReferenceFASTA(string fastaPathA, string fastaPathB)
{
GenericRead chrA = new GenericRead();
GenericRead chrB = new GenericRead();
long CountAB = 0;
long CountA = 0;
long CountB = 0;
long CountNeither = 0;
using (FastaReader readerA = new FastaReader(fastaPathA))
using (FastaReader readerB = new FastaReader(fastaPathB))
{
readerA.GetNextEntry(ref chrA); // Discard chrM from new output
while (true)
{
bool result = readerA.GetNextEntry(ref chrA);
if (!result) break;
readerB.GetNextEntry(ref chrB);
if (chrA.Bases.Length != chrB.Bases.Length) throw new Exception();
for (int baseIndex = 0; baseIndex < chrA.Bases.Length; baseIndex++)
{
bool isUniqueA = chrA.Bases[baseIndex] < 'a';
bool isUniqueB = chrB.Bases[baseIndex] < 'a';
if (isUniqueA && isUniqueB)
{
CountAB++;
}
else if (isUniqueA && !isUniqueB)
{
CountA++;
}
else if (!isUniqueA && isUniqueB)
{
CountB++;
}
else
{
CountNeither++;
}
}
Console.WriteLine("After {0}: {1},{2},{3},{4}", chrA.Name,
CountAB, CountA, CountB, CountNeither);
double percentAgreement = 100 * (CountAB + CountNeither) / (double)(CountAB + CountA + CountB + CountNeither);
Console.WriteLine("Percent agreement: {0:F2}", percentAgreement);
}
}
return 0;
}
static public void CheckUniqueness()
{
string fastaPath = @"D:\Genomes\Homo_sapiens\UCSC\hg19\Sequence\WholeGenomeFasta\genome.fa";
string[] Reads = new string[]
{
"AACCCTAACCCAACCCTAACCCTAACCCTAACCCT", // 10097 B
"ACCCTAACCCAACCCTAACCCTAACCCTAACCCTA", // 10098 B
"AGAGGACAACGCAGCTCCGCCCTCGCGGTGCTCTC", // 10553 A
"TTTTTTCCTATACATACATACCCATGATAAAGTTT" // 30763880 A
};
using (FastaReader readerA = new FastaReader(fastaPath))
{
GenericRead chrA = new GenericRead();
while (true)
{
bool result = readerA.GetNextEntry(ref chrA);
if (!result) break;
Console.WriteLine(chrA.Name);
string bases = chrA.Bases.ToUpperInvariant();
// Search for each:
for (int readIndex = 0; readIndex < Reads.Length; readIndex++)
{
int pos = -1;
while (true)
{
pos = bases.IndexOf(Reads[readIndex], pos + 1);
if (pos == -1) break;
Console.WriteLine("{0}\t{1}\t{2}\t{3}", readIndex, Reads[readIndex], chrA.Name, pos);
}
pos = -1;
string revComp = Illumina.SecondaryAnalysis.Utilities.GetReverseComplement(Reads[readIndex]);
while (true)
{
pos = bases.IndexOf(revComp, pos + 1);
if (pos == -1) break;
Console.WriteLine("{0}\t{1}\t{2}\t{3}\tRevComp", readIndex, Reads[readIndex], chrA.Name, pos);
}
}
}
}
Console.WriteLine(">>>Done.");
}
public void Main(string fastaPath, string outputPath)
{
Console.WriteLine("{0} Start", DateTime.Now);
Console.WriteLine("Load FASTA file at {0}, write kmer-flagged output to {1}", fastaPath, outputPath);
// Make multiple passes over the file, to manage memory usage. In each pass, we'll accumulate a dictionary
// of up to N kmers, and we'll flag as non-unique all occurrences of those kmers. In subsequent passes,
// we know we can ignore positions that were already flagged as non-unique. We know that we're complete when
// we make a pass where the dictionary doesn't fill up.
this.PassIndex = 0;
this.IncompletePositions = 1; // This will be set to the number of positions whose uniqueness status is UNKNOWN.
HashSet<string> finishedChromosomes = new HashSet<string>(); // For speed, keep track of chromosomes that are already fully processed.
while (IncompletePositions > 0)
{
IncompletePositions = 0;
PassIndex++;
Kmers.Clear();
this.GenomePosition = 0;
int chromosomeIndex = -1;
using (FastaReader reader = new FastaReader(fastaPath))
{
GenericRead fastaEntry = new GenericRead();
while (reader.GetNextEntry(ref fastaEntry))
{
chromosomeIndex++;
// Speedup option: Skip over chromosome that's already been fully processed:
if (finishedChromosomes.Contains(fastaEntry.Name))
{
GenomePosition += fastaEntry.Bases.Length;
continue;
}
ProcessOneChromosome(fastaEntry, chromosomeIndex);
if (IncompletePositions == 0 && !finishedChromosomes.Contains(fastaEntry.Name))
{
finishedChromosomes.Add(fastaEntry.Name);
}
} // loop over chromosomes
// Now let's go back and make a note of the various positions that are now definitively known
// to be unique kmers!
int UniqueCount = 0;
foreach (string key in Kmers.Keys)
{
long oldPos = Kmers[key];
if (oldPos < 0) continue;
UniqueCount++;
int tempPos = 0;
for (int tempIndex = 0; tempIndex < ChromosomeNonUniqueFlags.Count; tempIndex++)
{
if (tempPos + ChromosomeNonUniqueFlags[tempIndex].Length > oldPos)
{
// Note: Assumption is that OldPos - tempIndex will always be an int
// since no single chromosome's length is longer than maxint.
ChromosomeFinishedFlags[tempIndex].Set((int)(oldPos - tempPos), true);
break;
}
tempPos += ChromosomeNonUniqueFlags[tempIndex].Length;
}
}
Console.WriteLine("{0} >>>Pass {1}: flagged {2} unique kmers", DateTime.Now, PassIndex, UniqueCount);
Console.WriteLine();
}
} // Pass loop
Console.WriteLine("{0} Flagging complete", DateTime.Now);
this.WriteOutputs(fastaPath, outputPath);
Console.WriteLine("{0} Output written to {1}", DateTime.Now, outputPath);
}
private void WriteOutputs(string fastaPath, string outputPath)
{
int chromosomeIndex = -1;
using (FastaReader reader = new FastaReader(fastaPath))
using (FastaWriter writer = new FastaWriter(outputPath))
{
GenericRead fastaEntry = new GenericRead();
while (reader.GetNextEntry(ref fastaEntry))
{
chromosomeIndex++;
StringBuilder baseBuilder = new StringBuilder();
BitArray nonUniqueFlags = ChromosomeNonUniqueFlags[chromosomeIndex];
for (int chromPos = 0; chromPos < fastaEntry.Bases.Length; chromPos++)
{
if (nonUniqueFlags[chromPos])
{
baseBuilder.Append(char.ToLowerInvariant(fastaEntry.Bases[chromPos]));
}
else
{
baseBuilder.Append(char.ToUpperInvariant(fastaEntry.Bases[chromPos]));
}
}
writer.WriteEntry(fastaEntry.Name, baseBuilder.ToString());
}
}
}
/// <summary>
/// Bin alignments.
/// </summary>
/// <param name="referenceFile">Reference fasta file.</param>
/// <param name="binSize">Desired number of alignments per bin.</param>
/// <param name="possibleAlignments">BitArrays of possible alignments.</param>
/// <param name="observedAlignments">BitArrays of observed alignments.</param>
/// <param name="predefinedBins">Pre-defined bins. null if not available.</param>
/// <returns>A list of bins.</returns>
static List<GenomicBin> BinCounts(string referenceFile, int binSize, CanvasCoverageMode coverageMode, NexteraManifest manifest,
Dictionary<string, BitArray> possibleAlignments,
Dictionary<string, HitArray> observedAlignments,
Dictionary<string, Int16[]> fragmentLengths,
Dictionary<string, List<GenomicBin>> predefinedBins,
string outFile)
{
bool debugGCCorrection = false; // write value of GC bins and correction factor
Dictionary<string, GenericRead> fastaEntries = new Dictionary<string, GenericRead>();
List<string> chromosomes = new List<string>();
Int16 meanFragmentSize = 0;
Int16 meanFragmentCutoff = 3;
if (coverageMode == CanvasCoverageMode.GCContentWeighted)
meanFragmentSize = MeanFragmentSize(fragmentLengths);
using (FastaReader reader = new FastaReader(referenceFile))
{
GenericRead fastaEntry = new GenericRead();
// Loop through each chromosome in the reference.
while (reader.GetNextEntry(ref fastaEntry))
{
chromosomes.Add(fastaEntry.Name);
fastaEntries[fastaEntry.Name] = fastaEntry;
fastaEntry = new GenericRead();
}
}
// calculate GC content of the forward read at every position along the genome
Dictionary<string, byte[]> readGCContent = new Dictionary<string, byte[]>();
if (coverageMode == CanvasCoverageMode.GCContentWeighted)
{
byte gcCap = (byte)numberOfGCbins;
List<ThreadStart> normalizationTasks = new List<ThreadStart>();
foreach (KeyValuePair<string, Int16[]> fragmentLengthsKVP in fragmentLengths)
{
string chr = fragmentLengthsKVP.Key;
GenericRead fastaEntry = fastaEntries[chr];
normalizationTasks.Add(new ThreadStart(() =>
{
// contains GC content of the forward read at every position for current chr
byte[] gcContent = new byte[fastaEntry.Bases.Length];
int gcCounter = 0;
// Iteratively calculate GC content of "reads" using fasta genome reference
for (int pos = 0; pos < fastaEntry.Bases.Length - meanFragmentSize * meanFragmentCutoff - 1; pos++)
{
Int16 currentFragment = 0;
if (fragmentLengthsKVP.Value[pos] == 0)
currentFragment = meanFragmentSize;
else
currentFragment = Convert.ToInt16(Math.Min(fragmentLengthsKVP.Value[pos], meanFragmentSize * meanFragmentCutoff));
for (int i = pos; i < pos + currentFragment; i++)
{
switch (fastaEntry.Bases[i])
{
case 'C':
case 'c':
case 'G':
case 'g':
gcCounter++;
break;
default:
break;
}
}
if (gcCounter < 0)
gcCounter = 0;
gcContent[pos] = (byte)Math.Min(100 * gcCounter / currentFragment, gcCap);
gcCounter = 0;
}
lock (readGCContent)
{
readGCContent[chr] = gcContent;
}
}));
}
Console.WriteLine("{0} Launching normalization tasks.", DateTime.Now);
Console.Out.Flush();
//Parallel.ForEach(normalizationTasks, t => { t.Invoke(); });
Illumina.SecondaryAnalysis.Utilities.DoWorkParallelThreads(normalizationTasks);
Console.WriteLine("{0} Normalization tasks complete.", DateTime.Now);
Console.Out.Flush();
}
// populate observed and expected read GC bin vectors
float[] observedVsExpectedGC = new float[0];
if (coverageMode == CanvasCoverageMode.GCContentWeighted)
observedVsExpectedGC = ComputeObservedVsExpectedGC(observedAlignments, readGCContent, manifest, debugGCCorrection, outFile);
Dictionary<string, List<GenomicBin>> perChromosomeBins = new Dictionary<string, List<GenomicBin>>();
List<ThreadStart> binningTasks = new List<ThreadStart>();
foreach (KeyValuePair<string, GenericRead> fastaEntryKVP in fastaEntries)
{
string chr = fastaEntryKVP.Key;
if (!possibleAlignments.ContainsKey(chr)) continue;
if (predefinedBins != null && !predefinedBins.ContainsKey(chr)) continue;
BinTaskArguments args = new BinTaskArguments();
args.FastaEntry = fastaEntryKVP.Value;
args.Chromosome = chr;
args.PossibleAlignments = possibleAlignments[chr];
args.ObservedAlignments = observedAlignments[chr];
args.CoverageMode = coverageMode;
perChromosomeBins[chr] = predefinedBins == null ? new List<GenomicBin>() : predefinedBins[chr];
args.Bins = perChromosomeBins[chr];
args.BinSize = binSize;
if (coverageMode == CanvasCoverageMode.GCContentWeighted)
args.ReadGCContent = readGCContent[chr];
else
args.ReadGCContent = null;
args.ObservedVsExpectedGC = observedVsExpectedGC;
binningTasks.Add(new ThreadStart(() => { BinCountsForChromosome(args); }));
}
Console.WriteLine("{0} Launch BinCountsForChromosome jobs...", DateTime.Now);
Console.Out.WriteLine();
//Parallel.ForEach(binningTasks, t => { t.Invoke(); });
Illumina.SecondaryAnalysis.Utilities.DoWorkParallelThreads(binningTasks);
Console.WriteLine("{0} Completed BinCountsForChromosome jobs.", DateTime.Now);
Console.Out.WriteLine();
List<GenomicBin> finalBins = new List<GenomicBin>();
foreach (string chr in chromosomes)
{
if (!perChromosomeBins.ContainsKey(chr)) continue;
finalBins.AddRange(perChromosomeBins[chr]);
}
return finalBins;
}
