public ProteinWithAppliedVariants(string variantBaseSequence, Protein protein, IEnumerable <SequenceVariation> appliedSequenceVariations, string individual)
: base(variantBaseSequence, protein.Accession, organism: protein.Organism, geneNames: new List <Tuple <string, string> >(protein.GeneNames),
oneBasedModifications: protein.OneBasedPossibleLocalizedModifications.ToDictionary(x => x.Key, x => x.Value),
proteolysisProducts: new List <ProteolysisProduct>(protein.ProteolysisProducts), name: protein.Name, fullName: protein.FullName,
isDecoy: protein.IsDecoy, isContaminant: protein.IsContaminant,
databaseReferences: new List <DatabaseReference>(protein.DatabaseReferences), sequenceVariations: new List <SequenceVariation>(protein.SequenceVariations),
disulfideBonds: new List <DisulfideBond>(protein.DisulfideBonds), databaseFilePath: protein.DatabaseFilePath)
{
Protein = protein;
AppliedSequenceVariations = appliedSequenceVariations != null?appliedSequenceVariations.ToList() : new List <SequenceVariation>();
Individual = individual;
}
public ProteinWithAppliedVariants(string variantBaseSequence, Protein protein, IEnumerable <SequenceVariation> appliedSequenceVariations,
IEnumerable <ProteolysisProduct> applicableProteolysisProducts, IDictionary <int, List <Modification> > oneBasedModifications, string individual)
: base(variantBaseSequence,
protein.Accession + (appliedSequenceVariations == null ? "" : "_" + CombineSimpleStrings(appliedSequenceVariations)),
organism: protein.Organism,
geneNames: new List <Tuple <string, string> >(protein.GeneNames),
oneBasedModifications: oneBasedModifications.ToDictionary(x => x.Key, x => x.Value),
proteolysisProducts: new List <ProteolysisProduct>(applicableProteolysisProducts),
name: protein.Name + (appliedSequenceVariations == null ? "" : " variant:" + CombineDescriptions(appliedSequenceVariations)),
fullName: protein.FullName + (appliedSequenceVariations == null ? "" : " variant:" + CombineDescriptions(appliedSequenceVariations)),
isDecoy: protein.IsDecoy,
isContaminant: protein.IsContaminant,
databaseReferences: new List <DatabaseReference>(protein.DatabaseReferences),
sequenceVariations: new List <SequenceVariation>(protein.SequenceVariations),
disulfideBonds: new List <DisulfideBond>(protein.DisulfideBonds),
databaseFilePath: protein.DatabaseFilePath)
{
Protein = protein;
AppliedSequenceVariations = appliedSequenceVariations != null?appliedSequenceVariations.ToList() : new List <SequenceVariation>();
Individual = individual;
}
/// <summary>
/// Protein construction that clones a protein but assigns a different base sequence
/// For use in SILAC experiments
/// </summary>
/// <param name="originalProtein"></param>
/// <param name="silacSequence"></param>
/// <param name="silacAccession"></param>
public Protein(Protein originalProtein, string silacSequence)
{
BaseSequence = silacSequence;
Accession = originalProtein.Accession;
NonVariantProtein = originalProtein.NonVariantProtein;
Name = originalProtein.Name;
Organism = originalProtein.Organism;
FullName = originalProtein.FullName;
IsDecoy = originalProtein.IsDecoy;
IsContaminant = originalProtein.IsContaminant;
DatabaseFilePath = originalProtein.DatabaseFilePath;
SampleNameForVariants = originalProtein.SampleNameForVariants;
GeneNames = originalProtein.GeneNames;
ProteolysisProducts = originalProtein.ProteolysisProducts;
SequenceVariations = originalProtein.SequenceVariations;
AppliedSequenceVariations = originalProtein.AppliedSequenceVariations;
OriginalNonVariantModifications = originalProtein.OriginalNonVariantModifications;
OneBasedPossibleLocalizedModifications = originalProtein.OneBasedPossibleLocalizedModifications;
DatabaseReferences = originalProtein.DatabaseReferences;
DisulfideBonds = originalProtein.DisulfideBonds;
SpliceSites = originalProtein.SpliceSites;
DatabaseFilePath = originalProtein.DatabaseFilePath;
}
/// <summary>
/// Protein construction with applied variations
/// </summary>
/// <param name="variantBaseSequence"></param>
/// <param name="protein"></param>
/// <param name="appliedSequenceVariations"></param>
/// <param name="applicableProteolysisProducts"></param>
/// <param name="oneBasedModifications"></param>
/// <param name="sampleNameForVariants"></param>
public Protein(string variantBaseSequence, Protein protein, IEnumerable <SequenceVariation> appliedSequenceVariations,
IEnumerable <ProteolysisProduct> applicableProteolysisProducts, IDictionary <int, List <Modification> > oneBasedModifications, string sampleNameForVariants)
: this(variantBaseSequence,
VariantApplication.GetAccession(protein, appliedSequenceVariations),
organism : protein.Organism,
geneNames : new List <Tuple <string, string> >(protein.GeneNames),
oneBasedModifications : oneBasedModifications != null ? oneBasedModifications.ToDictionary(x => x.Key, x => x.Value) : new Dictionary <int, List <Modification> >(),
proteolysisProducts : new List <ProteolysisProduct>(applicableProteolysisProducts ?? new List <ProteolysisProduct>()),
name : GetName(appliedSequenceVariations, protein.Name),
fullName : GetName(appliedSequenceVariations, protein.FullName),
isDecoy : protein.IsDecoy,
isContaminant : protein.IsContaminant,
databaseReferences : new List <DatabaseReference>(protein.DatabaseReferences),
sequenceVariations : new List <SequenceVariation>(protein.SequenceVariations),
disulfideBonds : new List <DisulfideBond>(protein.DisulfideBonds),
spliceSites : new List <SpliceSite>(protein.SpliceSites),
databaseFilePath : protein.DatabaseFilePath)
{
NonVariantProtein = protein.NonVariantProtein;
OriginalNonVariantModifications = NonVariantProtein.OriginalNonVariantModifications;
AppliedSequenceVariations = (appliedSequenceVariations ?? new List <SequenceVariation>()).ToList();
SampleNameForVariants = sampleNameForVariants;
}
/// <summary>
/// Applies multiple variant changes to a protein sequence
/// </summary>
/// <param name="protein"></param>
/// <param name="uniqueEffectsToApply"></param>
/// <returns></returns>
internal static List <Protein> ApplyVariants(Protein protein, IEnumerable <SequenceVariation> sequenceVariations, int maxAllowedVariantsForCombinitorics, int minAlleleDepth)
{
List <SequenceVariation> uniqueEffectsToApply = sequenceVariations
.GroupBy(v => v.SimpleString())
.Select(x => x.First())
.Where(v => v.Description.Genotypes.Count > 0) // this is a VCF line
.OrderByDescending(v => v.OneBasedBeginPosition) // apply variants at the end of the protein sequence first
.ToList();
Protein proteinCopy = new Protein(protein.BaseSequence, protein, null, protein.ProteolysisProducts, protein.OneBasedPossibleLocalizedModifications, null);
// If there aren't any variants to apply, just return the base protein
if (uniqueEffectsToApply.Count == 0)
{
return(new List <Protein> {
proteinCopy
});
}
HashSet <string> individuals = new HashSet <string>(uniqueEffectsToApply.SelectMany(v => v.Description.Genotypes.Keys));
List <Protein> variantProteins = new List <Protein>();
// loop through genotypes for each sample/individual (e.g. tumor and normal)
foreach (string individual in individuals)
{
bool tooManyHeterozygousVariants = uniqueEffectsToApply.Count(v => v.Description.Heterozygous[individual]) > maxAllowedVariantsForCombinitorics;
List <Protein> newVariantProteins = new List <Protein> {
proteinCopy
};
foreach (var variant in uniqueEffectsToApply)
{
bool variantAlleleIsInTheGenotype = variant.Description.Genotypes[individual].Contains(variant.Description.AlleleIndex.ToString()); // should catch the case where it's -1 if the INFO isn't from SnpEff
if (!variantAlleleIsInTheGenotype)
{
continue;
}
bool isHomozygousAlternate = variant.Description.Homozygous[individual] && variant.Description.Genotypes[individual].All(d => d == variant.Description.AlleleIndex.ToString()); // note this isn't a great test for homozygosity, since the genotype could be 1/2 and this would still return true. But currently, alleles 1 and 2 will be included as separate variants, so this is fine for now.
bool isDeepReferenceAllele = int.TryParse(variant.Description.AlleleDepths[individual][0], out int depthRef) && depthRef >= minAlleleDepth;
bool isDeepAlternateAllele = int.TryParse(variant.Description.AlleleDepths[individual][variant.Description.AlleleIndex], out int depthAlt) && depthAlt >= minAlleleDepth;
// homozygous alternate
if (isHomozygousAlternate && isDeepAlternateAllele)
{
newVariantProteins = newVariantProteins.Select(p => ApplySingleVariant(variant, p, individual)).ToList();
}
// heterozygous basic
// first protein with variants contains all homozygous variation, second contains all variations
else if (variant.Description.Heterozygous[individual] && tooManyHeterozygousVariants)
{
if (isDeepAlternateAllele && isDeepReferenceAllele)
{
if (newVariantProteins.Count == 1 && maxAllowedVariantsForCombinitorics > 0)
{
Protein variantProtein = ApplySingleVariant(variant, newVariantProteins[0], individual);
newVariantProteins.Add(variantProtein);
}
else if (maxAllowedVariantsForCombinitorics > 0)
{
newVariantProteins[1] = ApplySingleVariant(variant, newVariantProteins[1], individual);
}
else
{
// no heterozygous variants
}
}
else if (isDeepAlternateAllele && maxAllowedVariantsForCombinitorics > 0)
{
newVariantProteins = newVariantProteins.Select(p => ApplySingleVariant(variant, p, individual)).ToList();
}
else
{
// keep reference only
}
}
// heterozygous combinitorics
else if (variant.Description.Heterozygous[individual] && isDeepAlternateAllele && !tooManyHeterozygousVariants)
{
List <Protein> combinitoricProteins = new List <Protein>();
foreach (Protein ppp in newVariantProteins)
{
if (isDeepAlternateAllele && maxAllowedVariantsForCombinitorics > 0 && isDeepReferenceAllele)
{
// keep reference allele
if (variant.Description.Genotypes[individual].Contains("0"))
{
combinitoricProteins.Add(ppp);
}
// alternate allele (replace all, since in heterozygous with two alternates, both alternates are included)
combinitoricProteins.Add(ApplySingleVariant(variant, ppp, individual));
}
else if (isDeepAlternateAllele && maxAllowedVariantsForCombinitorics > 0)
{
combinitoricProteins.Add(ApplySingleVariant(variant, ppp, individual));
}
else if (variant.Description.Genotypes[individual].Contains("0"))
{
combinitoricProteins.Add(ppp);
}
else
{
// must be two alternate alleles with not enough depth
}
}
newVariantProteins = combinitoricProteins;
}
}
variantProteins.AddRange(newVariantProteins);
}
return(variantProteins.GroupBy(x => x.BaseSequence).Select(x => x.First()).ToList());
}
/// <summary>
/// Restores modification index on a variant protein to the index on the nonvariant protein,
/// or if it falls on a variant, this restores the position on the protein with only that variant
/// </summary>
/// <param name="variantProteinIndex"></param>
/// <param name="modification"></param>
/// <returns></returns>
public static int RestoreModificationIndex(Protein protein, int variantProteinIndex)
{
return(variantProteinIndex - protein.AppliedSequenceVariations
.Where(v => v.OneBasedEndPosition < variantProteinIndex)
.Sum(v => v.VariantSequence.Length - v.OriginalSequence.Length));
}
/// <summary>
/// Adjusts modification indices.
/// </summary>
/// <param name="variant"></param>
/// <param name="modificationDictionary"></param>
/// <returns></returns>
internal static Dictionary <int, List <Modification> > AdjustModificationIndices(SequenceVariation variant, string variantAppliedProteinSequence, Protein protein)
{
IDictionary <int, List <Modification> > modificationDictionary = protein.OneBasedPossibleLocalizedModifications;
IDictionary <int, List <Modification> > variantModificationDictionary = variant.OneBasedModifications;
Dictionary <int, List <Modification> > mods = new Dictionary <int, List <Modification> >();
int sequenceLengthChange = variant.VariantSequence.Length - variant.OriginalSequence.Length;
// change modification indices for variant sequence
if (modificationDictionary != null)
{
foreach (KeyValuePair <int, List <Modification> > kv in modificationDictionary)
{
if (kv.Key > variantAppliedProteinSequence.Length)
{
continue; // it was cut out by a stop gain
}
// mod is before the variant
else if (kv.Key < variant.OneBasedBeginPosition)
{
mods.Add(kv.Key, kv.Value);
}
// mod is after the variant and not affected by a stop gain
else if (variant.OneBasedEndPosition < kv.Key && kv.Key + sequenceLengthChange <= variantAppliedProteinSequence.Length)
{
mods.Add(kv.Key + sequenceLengthChange, kv.Value);
}
else // sequence variant conflicts with modification site (modification site substitution)
{
continue;
}
}
}
// sequence variant modifications are indexed to the variant sequence
// NOTE: this code assumes variants are added from end to beginning of protein, so that previously added variant mods are adjusted above
if (variantModificationDictionary != null)
{
foreach (var kv in variantModificationDictionary)
{
if (mods.TryGetValue(kv.Key, out var modsAtPos))
{
modsAtPos.AddRange(kv.Value);
}
else
{
mods.Add(kv.Key, kv.Value);
}
}
}
return(mods);
}
/// <summary>
/// Eliminates proteolysis products that overlap sequence variations.
/// Since frameshift indels are written across the remaining sequence,
/// this eliminates proteolysis products that conflict with large deletions and other structural variations.
/// </summary>
/// <param name="variants"></param>
/// <param name="proteolysisProducts"></param>
/// <returns></returns>
internal static List <ProteolysisProduct> AdjustProteolysisProductIndices(SequenceVariation variant, string variantAppliedProteinSequence, Protein protein, IEnumerable <ProteolysisProduct> proteolysisProducts)
{
List <ProteolysisProduct> products = new List <ProteolysisProduct>();
if (proteolysisProducts == null)
{
return(products);
}
int sequenceLengthChange = variant.VariantSequence.Length - variant.OriginalSequence.Length;
foreach (ProteolysisProduct p in proteolysisProducts.Where(p => p.OneBasedEndPosition.HasValue && p.OneBasedBeginPosition.HasValue))
{
// proteolysis product is entirely before the variant
if (variant.OneBasedBeginPosition > p.OneBasedEndPosition)
{
products.Add(p);
}
// proteolysis product straddles the variant, but the cleavage site(s) are still intact; the ends aren't considered cleavage sites
else if ((p.OneBasedBeginPosition < variant.OneBasedBeginPosition || p.OneBasedBeginPosition == 1 || p.OneBasedBeginPosition == 2) &&
(p.OneBasedEndPosition > variant.OneBasedEndPosition || p.OneBasedEndPosition == protein.NonVariantProtein.BaseSequence.Length))
{
if (variant.VariantSequence.EndsWith("*"))
{
products.Add(new ProteolysisProduct(p.OneBasedBeginPosition, variantAppliedProteinSequence.Length, p.Type));
}
else if (p.OneBasedEndPosition + sequenceLengthChange <= variantAppliedProteinSequence.Length)
{
products.Add(new ProteolysisProduct(p.OneBasedBeginPosition, p.OneBasedEndPosition + sequenceLengthChange, p.Type));
}
else
{
// cleavage site is not intact
}
}
// proteolysis product is after the variant and there is no stop gain
else if (p.OneBasedBeginPosition > variant.OneBasedEndPosition &&
p.OneBasedBeginPosition + sequenceLengthChange <= variantAppliedProteinSequence.Length &&
p.OneBasedEndPosition + sequenceLengthChange <= variantAppliedProteinSequence.Length &&
!variant.VariantSequence.EndsWith("*"))
{
products.Add(new ProteolysisProduct(p.OneBasedBeginPosition + sequenceLengthChange, p.OneBasedEndPosition + sequenceLengthChange, p.Type));
}
else // sequence variant conflicts with proteolysis cleavage site (cleavage site was lost)
{
continue;
}
}
return(products);
}
/// <summary>
/// Applies a single variant to a protein sequence
/// </summary>
/// <param name="variantGettingApplied"></param>
/// <returns></returns>
internal static Protein ApplySingleVariant(SequenceVariation variantGettingApplied, Protein protein, string individual)
{
string seqBefore = protein.BaseSequence.Substring(0, variantGettingApplied.OneBasedBeginPosition - 1);
string seqVariant = variantGettingApplied.VariantSequence;
int afterIdx = variantGettingApplied.OneBasedBeginPosition + variantGettingApplied.OriginalSequence.Length - 1;
SequenceVariation variantAfterApplication = new SequenceVariation(
variantGettingApplied.OneBasedBeginPosition,
variantGettingApplied.OneBasedBeginPosition + variantGettingApplied.VariantSequence.Length - 1,
variantGettingApplied.OriginalSequence,
variantGettingApplied.VariantSequence,
variantGettingApplied.Description.Description,
variantGettingApplied.OneBasedModifications.ToDictionary(kv => kv.Key, kv => kv.Value));
// check to see if there is incomplete indel overlap, which would lead to weird variant sequences
// complete overlap is okay, since it will be overwritten; this can happen if there are two alternate alleles,
// e.g. reference sequence is wrong at that point
bool intersectsAppliedRegionIncompletely = protein.AppliedSequenceVariations.Any(x => variantGettingApplied.Intersects(x) && !variantGettingApplied.Includes(x));
IEnumerable <SequenceVariation> appliedVariations = new[] { variantAfterApplication };
string seqAfter = null;
if (intersectsAppliedRegionIncompletely)
{
// use original protein sequence for the remaining sequence
seqAfter = protein.BaseSequence.Length - afterIdx <= 0 ? "" : protein.NonVariantProtein.BaseSequence.Substring(afterIdx);
}
else
{
// use this variant protein sequence for the remaining sequence
seqAfter = protein.BaseSequence.Length - afterIdx <= 0 ? "" : protein.BaseSequence.Substring(afterIdx);
appliedVariations = appliedVariations
.Concat(protein.AppliedSequenceVariations.Where(x => !variantGettingApplied.Includes(x)))
.ToList();
}
string variantSequence = (seqBefore + seqVariant + seqAfter).Split('*')[0]; // there may be a stop gained
// adjust indices
List <ProteolysisProduct> adjustedProteolysisProducts = AdjustProteolysisProductIndices(variantGettingApplied, variantSequence, protein, protein.ProteolysisProducts);
Dictionary <int, List <Modification> > adjustedModifications = AdjustModificationIndices(variantGettingApplied, variantSequence, protein);
List <SequenceVariation> adjustedAppliedVariations = AdjustSequenceVariationIndices(variantGettingApplied, variantSequence, appliedVariations);
return(new Protein(variantSequence, protein, adjustedAppliedVariations, adjustedProteolysisProducts, adjustedModifications, individual));
}
/// <summary>
/// Gets the accession for a protein with applied variations
/// </summary>
/// <param name="protein"></param>
/// <param name="sequenceVariation"></param>
public static string GetAccession(Protein protein, IEnumerable <SequenceVariation> appliedSequenceVariations)
{
return(protein.NonVariantProtein.Accession +
(appliedSequenceVariations == null || appliedSequenceVariations.Count() == 0 ? "" : $"_{CombineSimpleStrings(appliedSequenceVariations)}"));
}