public override void ConsumeCCSRead(PacBioCCSRead read, BWAPairwiseAlignment aln, List <Variant> variants)
{
var aligned = aln == null ? "FALSE" : "TRUE";
var end = String.Join(",", read.MutationsTested.ToString(), read.MutationsApplied.ToString(),
read.ProcessingTimeMS.ToString(), read.Barcode1.ToString(),
read.Barcode2.ToString());
string start = String.Join(",", read.Movie, read.HoleNumber.ToString(),
read.SnrA.ToString(), read.SnrC.ToString(), read.SnrG.ToString(),
read.SnrT.ToString(), aligned, read.ReadQuality.ToString(),
read.AvgZscore.ToString(), read.Sequence.Count.ToString(), read.NumPasses.ToString(),
read.ReadCountSuccessfullyAdded.ToString(), read.ReadCountBadZscore.ToString(),
read.ReadCountAlphaBetaMismatch.ToString(),
read.ReadCountOther.ToString());
if (aln == null)
{
start = start + ",NA,NA,NA,NA,NA,NA," + end;
}
else
{
var indels = variants.Count(z => z.Type == VariantType.INDEL);
var snps = variants.Count(z => z.Type == VariantType.SNP);
var total = variants.Count;
var length = aln.AlignedSAMSequence.RefEndPos - aln.AlignedSAMSequence.Pos;
start = start + "," + String.Join(",", length.ToString(), aln.AlignedSAMSequence.Pos, total.ToString(), indels.ToString(), snps.ToString(),
aln.AlignedSAMSequence.RName, end);
}
SW.WriteLine(start);
}
public override void ConsumeCCSRead(PacBioCCSRead read, BWAPairwiseAlignment aln, List <Variant> variants)
{
if (variants == null)
{
return;
}
foreach (var v in variants)
{
var common = String.Join(",", read.Movie, read.HoleNumber.ToString(), v.RefName,
v.StartPosition.ToString(), v.Length.ToString(), v.Type.ToString(), v.QV.ToString(), v.AtEndOfAlignment.ToString());
var snp = v as SNPVariant;
string unique;
if (snp != null)
{
unique = String.Join(",", snp.RefBP, snp.AltBP, "NA,NA,NA,NA,NA");
}
else
{
var indel = v as IndelVariant;
unique = string.Join(",", "NA,NA", indel.InsertionOrDeletion.ToString(),
indel.InHomopolymer.ToString(), indel.InsertedOrDeletedBases.ToString(),
indel.HomopolymerLengthInReference.ToString(), indel.HomopolymerBase);
}
SW.WriteLine(common + "," + unique);
}
}
public override void ConsumeCCSRead(PacBioCCSRead read, BWAPairwiseAlignment aln, List<Variant> variants)
{
var aligned = aln==null ? "FALSE" : "TRUE";
var end = String.Join (",", read.MutationsTested.ToString (), read.MutationsApplied.ToString (),
read.ProcessingTimeMS.ToString (), read.Barcode1.ToString(),
read.Barcode2.ToString ());
string start = String.Join (",", read.Movie, read.HoleNumber.ToString (),
read.SnrA.ToString (), read.SnrC.ToString (), read.SnrG.ToString (),
read.SnrT.ToString (), aligned, read.ReadQuality.ToString (),
read.AvgZscore.ToString (), read.Sequence.Count.ToString (), read.NumPasses.ToString (),
read.ReadCountSuccessfullyAdded.ToString(), read.ReadCountBadZscore.ToString(),
read.ReadCountAlphaBetaMismatch.ToString(),
read.ReadCountOther.ToString());
if (aln == null) {
start = start + ",NA,NA,NA,NA,NA,NA," + end;
} else {
var indels = variants.Count (z => z.Type == VariantType.INDEL);
var snps = variants.Count (z => z.Type == VariantType.SNP);
var total = variants.Count;
var length = aln.AlignedSAMSequence.RefEndPos - aln.AlignedSAMSequence.Pos;
start = start + "," + String.Join (",", length.ToString (), aln.AlignedSAMSequence.Pos, total.ToString (), indels.ToString (), snps.ToString (),
aln.AlignedSAMSequence.RName, end );
}
SW.WriteLine (start);
}
public override void ConsumeCCSRead(PacBioCCSRead read, BWAPairwiseAlignment aln, List<Variant> variants)
{
if (variants == null) {
return;
}
foreach (var v in variants) {
incorrectCounts [v.QV]++;
if (v.Type == VariantType.SNP) {
snpErrorCounts [v.QV]++;
} else {
var id = v as IndelVariant;
if (id.InsertionOrDeletion == IndelType.Deletion) {
deletionErrorCounts [v.QV]++;
} else if (id.InsertionOrDeletion == IndelType.Insertion) {
insertionErrorCounts [v.QV]++;
}
}
}
var refseq = aln.AlignedRefSeq;
var qseq = aln.AlignedQuerySeq as QualitativeSequence;
for (int i = 0; i < refseq.Count; i++) {
if (refseq [i] == qseq [i]) {
correctCounts [qseq.GetQualityScore (i)]++;
}
}
}
public override void ConsumeCCSRead(PacBioCCSRead read, BWAPairwiseAlignment aln, List<Variant> variants)
{
int i = 1;
foreach (var v in read.ZScores) {
var s = String.Join (",", read.Movie, read.HoleNumber, i.ToString (), v.ToString ());
SW.WriteLine (s);
i++;
}
}
public override void ConsumeCCSRead(PacBioCCSRead read, BWAPairwiseAlignment aln, List<Variant> variants)
{
var prefix = String.Join (",",
read.Movie,
read.HoleNumber.ToString ());
SW.WriteLine (prefix + ",A," + read.SnrA);
SW.WriteLine (prefix + ",C," + read.SnrC);
SW.WriteLine (prefix + ",G," + read.SnrG);
SW.WriteLine (prefix + ",T," + read.SnrT);
}
public override void ConsumeCCSRead(PacBioCCSRead read, BWAPairwiseAlignment aln, List <Variant> variants)
{
int i = 1;
foreach (var v in read.ZScores)
{
var s = String.Join(",", read.Movie, read.HoleNumber, i.ToString(), v.ToString());
SW.WriteLine(s);
i++;
}
}
public override void ConsumeCCSRead(PacBioCCSRead read, BWAPairwiseAlignment aln, List <Variant> variants)
{
var prefix = String.Join(",",
read.Movie,
read.HoleNumber.ToString());
SW.WriteLine(prefix + ",A," + read.SnrA);
SW.WriteLine(prefix + ",C," + read.SnrC);
SW.WriteLine(prefix + ",G," + read.SnrG);
SW.WriteLine(prefix + ",T," + read.SnrT);
}
public CCSDataSet(Sequence ccs, List<Sequence> subreads, BWAPairwiseAlignment aln)
{
// Call Variants
var results = VariantCaller.LeftAlignIndelsAndCallVariants (aln);
Variants = results.Item2;
Variants.ForEach (p => {
p.StartPosition += aln.AlignedSAMSequence.Pos;
p.RefName = aln.Reference;
});
// Get the ungapped reference
var ccs_aligned = results.Item1.SecondSequence;
// Generate an ungapped reference
var refseq = results.Item1.PairwiseAlignedSequences.First().FirstSequence;
var reference = new Sequence (DnaAlphabet.Instance,
refseq.Where (bp => bp != '-').ToArray (),
false)
{ ID = aln.Reference + "/" + aln.AlignedSAMSequence.Pos + "-" + aln.AlignedSAMSequence.RefEndPos };
var reference_rc = reference.GetReverseComplementedSequence () as Sequence;
Reads = subreads.Select(z => new AlignedSequence(z, reference, reference_rc)).Where( x => (Math.Abs((x.Sequence.Length - reference.Count)) / (double) reference.Count) < 0.2).ToList();
Reads.Insert (0, new AlignedSequence (reference, reference, reference_rc));
ccs.ID = "CCS";
Reads.Insert(1, new AlignedSequence(ccs, reference, reference_rc));
// Now to rejigger the alignments
int[] metaOffSets = Enumerable.Range(0, (int)reference.Count).
Select (i => Reads.Max( r => r.GetInsertsBeforePosition(i))).
ToArray();
Reads.ForEach(r => {
r.ReJiggerAlignment(metaOffSets);
Console.WriteLine(r.Sequence);});
CCS = ccs;
Reference = reference;
Aln = aln;
}
public override void ConsumeCCSRead(PacBioCCSRead read, BWAPairwiseAlignment aln, List<Variant> variants)
{
if (variants == null) {
return;
}
foreach (var v in variants) {
var common = String.Join (",", read.Movie, read.HoleNumber.ToString (), v.RefName,
v.StartPosition.ToString (), v.Length.ToString (), v.Type.ToString (), v.QV.ToString (), v.AtEndOfAlignment.ToString ());
var snp = v as SNPVariant;
string unique;
if (snp != null) {
unique = String.Join (",", snp.RefBP, snp.AltBP, "NA,NA,NA,NA,NA");
} else {
var indel = v as IndelVariant;
unique = string.Join (",", "NA,NA", indel.InsertionOrDeletion.ToString (),
indel.InHomopolymer.ToString (), indel.InsertedOrDeletedBases.ToString (),
indel.HomopolymerLengthInReference.ToString (), indel.HomopolymerBase);
}
SW.WriteLine (common + "," + unique);
}
}
public override void ConsumeCCSRead(PacBioCCSRead read, BWAPairwiseAlignment aln, List <Variant> variants)
{
if (variants == null)
{
return;
}
foreach (var v in variants)
{
incorrectCounts [v.QV]++;
if (v.Type == VariantType.SNP)
{
snpErrorCounts [v.QV]++;
}
else
{
var id = v as IndelVariant;
if (id.InsertionOrDeletion == IndelType.Deletion)
{
deletionErrorCounts [v.QV]++;
}
else if (id.InsertionOrDeletion == IndelType.Insertion)
{
insertionErrorCounts [v.QV]++;
}
}
}
var refseq = aln.AlignedRefSeq;
var qseq = aln.AlignedQuerySeq as QualitativeSequence;
for (int i = 0; i < refseq.Count; i++)
{
if (refseq [i] == qseq [i])
{
correctCounts [qseq.GetQualityScore(i)]++;
}
}
}
public abstract void ConsumeCCSRead(PacBioCCSRead read, BWAPairwiseAlignment aln, List <Variant> variants);
public abstract void ConsumeCCSRead(PacBioCCSRead read, BWAPairwiseAlignment aln, List<Variant> variants);
public static void Main(string[] args)
{
try {
PlatformManager.Services.MaxSequenceSize = int.MaxValue;
PlatformManager.Services.DefaultBufferSize = 4096;
PlatformManager.Services.Is64BitProcessType = true;
if (args.Length > 3)
{
Console.WriteLine("Too many arguments");
DisplayHelp();
}
else if (args.Length < 2)
{
Console.WriteLine("Not enough arguments");
DisplayHelp();
}
else if (args [0] == "h" || args [0] == "help" || args [0] == "?" || args [0] == "-h")
{
DisplayHelp();
}
else
{
string bam_name = args [0];
string out_dir = args [1];
string ref_name = args.Length > 2 ? args [2] : null;
if (!File.Exists(bam_name))
{
Console.WriteLine("Can't find file: " + bam_name);
return;
}
if (ref_name != null && !File.Exists(ref_name))
{
Console.WriteLine("Can't find file: " + ref_name);
return;
}
if (Directory.Exists(out_dir))
{
Console.WriteLine("The output directory already exists, please specify a new directory or delete the old one.");
return;
}
Directory.CreateDirectory(out_dir);
List <CCSReadMetricsOutputter> outputters = new List <CCSReadMetricsOutputter> ()
{
new ZmwOutputFile(out_dir),
new ZScoreOutputter(out_dir),
new VariantOutputter(out_dir),
new SNROutputFile(out_dir),
new QVCalibration(out_dir)
};
ISequenceParser reader;
if (bam_name.EndsWith(".fastq", StringComparison.OrdinalIgnoreCase))
{
reader = new FastQCCSReader();
}
else
{
reader = new PacBioCCSBamReader();
}
BWAPairwiseAligner bwa = null;
bool callVariants = ref_name != null;
if (callVariants)
{
bwa = new BWAPairwiseAligner(ref_name, false);
}
// Produce aligned reads with variants called in parallel.
var reads = new BlockingCollection <Tuple <PacBioCCSRead, BWAPairwiseAlignment, List <Variant> > >();
Task producer = Task.Factory.StartNew(() =>
{
try
{
Parallel.ForEach(reader.Parse(bam_name), y => {
var z = y as PacBioCCSRead;
try {
BWAPairwiseAlignment aln = null;
List <Variant> variants = null;
if (callVariants)
{
aln = bwa.AlignRead(z.Sequence) as BWAPairwiseAlignment;
if (aln != null)
{
variants = VariantCaller.CallVariants(aln);
variants.ForEach(p => {
p.StartPosition += aln.AlignedSAMSequence.Pos;
p.RefName = aln.Reference;
});
}
}
var res = new Tuple <PacBioCCSRead, BWAPairwiseAlignment, List <Variant> >(z, aln, variants);
reads.Add(res);
}
catch (Exception thrown) {
Console.WriteLine("CCS READ FAIL: " + z.Sequence.ID);
Console.WriteLine(thrown.Message);
}
});
} catch (Exception thrown) {
Console.WriteLine("Could not parse BAM file: " + thrown.Message);
while (thrown.InnerException != null)
{
Console.WriteLine(thrown.InnerException.Message);
thrown = thrown.InnerException;
}
}
reads.CompleteAdding();
});
// Consume them into output files.
foreach (var r in reads.GetConsumingEnumerable())
{
foreach (var outputter in outputters)
{
outputter.ConsumeCCSRead(r.Item1, r.Item2, r.Item3);
}
}
// throw any exceptions (this should be used after putting the consumer on a separate thread)
producer.Wait();
// Close the files
outputters.ForEach(z => z.Finish());
}
}
catch (DllNotFoundException thrown) {
Console.WriteLine("Error thrown when attempting to generate the CCS results.");
Console.WriteLine("A shared library was not found. To solve this, please add the folder" +
" with the downloaded files libbwasharp and libMonoPosixHelper" +
"to your environmental variables (LD_LIBRARY_PATH on Ubuntu, DYLD_LIBRARY_PATH on Mac OS X).");
Console.WriteLine("Error: " + thrown.Message);
Console.WriteLine(thrown.StackTrace);
}
catch (Exception thrown) {
Console.WriteLine("Error thrown when attempting to generate the CCS results");
Console.WriteLine("Error: " + thrown.Message);
Console.WriteLine(thrown.StackTrace);
while (thrown.InnerException != null)
{
Console.WriteLine("Inner Exception: " + thrown.InnerException.Message);
thrown = thrown.InnerException;
}
}
}