collapseClones - Constructs effective clonal sequences for all clones

Description

collapseClones creates effective input and germline sequences for each clonal group and appends columns containing the consensus sequences to the input data.frame.

Usage

collapseClones(
db,
cloneColumn = "clone_id",
sequenceColumn = "sequence_alignment",
germlineColumn = "germline_alignment_d_mask",
muFreqColumn = NULL,
regionDefinition = NULL,
method = c("mostCommon", "thresholdedFreq", "catchAll", "mostMutated", "leastMutated"),
minimumFrequency = NULL,
includeAmbiguous = FALSE,
breakTiesStochastic = FALSE,
breakTiesByColumns = NULL,
expandedDb = FALSE,
nproc = 1,
juncLengthColumn = "junction_length",
fields = NULL
)

Arguments

db
data.frame containing sequence data. Required.
cloneColumn
character name of the column containing clonal identifiers. Required.
sequenceColumn
character name of the column containing input sequences. Required. The length of each input sequence should match that of its corresponding germline sequence.
germlineColumn
character name of the column containing germline sequences. Required. The length of each germline sequence should match that of its corresponding input sequence.
muFreqColumn
character name of the column containing mutation frequency. Optional. Applicable to the "mostMutated" and "leastMutated" methods. If not supplied, mutation frequency is computed by calling observedMutations. Default is NULL. See Cautions for note on usage.
regionDefinition
RegionDefinition object defining the regions and boundaries of the Ig sequences. Optional. Default is NULL.
method
method for calculating input consensus sequence. Required. One of "thresholdedFreq", "mostCommon", "catchAll", "mostMutated", or "leastMutated". See “Methods” for details.
minimumFrequency
frequency threshold for calculating input consensus sequence. Applicable to and required for the "thresholdedFreq" method. A canonical choice is 0.6. Default is NULL.
includeAmbiguous
whether to use ambiguous characters to represent positions at which there are multiple characters with frequencies that are at least minimumFrequency or that are maximal (i.e. ties). Applicable to and required for the "thresholdedFreq" and "mostCommon" methods. Default is FALSE. See “Choosing ambiguous characters” for rules on choosing ambiguous characters.
breakTiesStochastic
In case of ties, whether to randomly pick a sequence from sequences that fulfill the criteria as consensus. Applicable to and required for all methods except for "catchAll". Default is FALSE. See “Methods” for details.
breakTiesByColumns
A list of the form list(c(col_1, col_2, ...), c(fun_1, fun_2, ...)), where col_i is a character name of a column in db, and fun_i is a function to be applied on that column. Currently, only max and min are supported. Note that the two c()’s in list() are essential (i.e. if there is only 1 column, the list should be of the form list(c(col_1), c(func_1)). Applicable to and optional for the "mostMutated" and "leastMutated" methods. If supplied, fun_i’s are applied on col_i’s to help break ties. Default is NULL. See “Methods” for details.
expandedDb
logical indicating whether or not to return the expanded db, containing all the sequences (as opposed to returning just one sequence per clone).
nproc
Number of cores to distribute the operation over. If the cluster has already been set earlier, then pass the cluster. This will ensure that it is not reset.
juncLengthColumn
character name of the column containing the junction length. Needed when regionDefinition includes CDR3 and FWR4.
fields
additional fields used for grouping. Use sample_id, to avoid combining sequences with the same clone_id that belong to different sample_id.

Value

A modified db with the following additional columns:

  • clonal_sequence: effective sequence for the clone.
  • clonal_germline: germline sequence for the clone.
  • clonal_sequence_mufreq: mutation frequency of clonal_sequence; only added for the "mostMutated" and "leastMutated" methods.

clonal_sequence is generated with the method of choice indicated by method, and clonal_germline is generated with the "mostCommon" method, along with, where applicable, user-defined parameters such as minimumFrequency, includeAmbiguous, breakTiesStochastic, and breakTiesByColumns.

Consensus lengths

For each clone, clonal_sequence and clonal_germline have the same length.

  • For the "thresholdedFreq", "mostCommon", and "catchAll" methods:

The length of the consensus sequences is determined by the longest possible consensus sequence (baesd on inputSeq and germlineSeq) and regionDefinition@seqLength (if supplied), whichever is shorter.

Given a set of sequences of potentially varying lengths, the longest possible length of their consensus sequence is taken to be the longest length along which there is information contained at every nucleotide position across majority of the sequences. Majority is defined to be greater than floor(n/2), where n is the number of sequences. If the longest possible consensus length is 0, there will be a warning and an empty string ("") will be returned.

If a length limit is defined by supplying a regionDefinition via regionDefinition@seqLength, the consensus length will be further restricted to the shorter of the longest possible length and regionDefinition@seqLength.

  • For the "mostMutated" and "leastMutated" methods:

The length of the consensus sequences depends on that of the most/least mutated input sequence, and, if supplied, the length limit defined by regionDefinition@seqLength, whichever is shorter. If the germline consensus computed using the "mostCommon" method is longer than the most/least mutated input sequence, the germline consensus is trimmed to be of the same length as the input consensus.

Methods

The descriptions below use “sequences” as a generalization of input sequences and germline sequences.

  • method="thresholdedFreq"

A threshold must be supplied to the argument minimumFrequency. At each position along the length of the consensus sequence, the frequency of each nucleotide/character across sequences is tabulated. The nucleotide/character whose frequency is at least (i.e. >=) minimumFrequency becomes the consensus; if there is none, the consensus nucleotide will be "N".

When there are ties (frequencies of multiple nucleotides/characters are at least minimumFrequency), this method can be deterministic or stochastic, depending on additional parameters.

  • With includeAmbiguous=TRUE, ties are resolved deterministically by representing ties using ambiguous characters. See “Choosing ambiguous characters” for how ambiguous characters are chosen.
  • With breakTiesStochastic=TRUE, ties are resolved stochastically by randomly picking a character amongst the ties.
  • When both TRUE, includeAmbiguous takes precedence over breakTiesStochastic.
  • When both FALSE, the first character from the ties is taken to be the consensus following the order of "A", "T", "G", "C", "N", ".", and "-".

Below are some examples looking at a single position based on 5 sequences with minimumFrequency=0.6, includeAmbiguous=FALSE, and breakTiesStochastic=FALSE:

  • If the sequences have "A", "A", "A", "T", "C", the consensus will be "A", because "A" has frequency 0.6, which is at least minimumFrequency.

  • If the sequences have "A", "A", "T", "T", "C", the consensus will be "N", because none of "A", "T", or "C" has frequency that is at least minimumFrequency.

  • method="mostCommon"

The most frequent nucleotide/character across sequences at each position along the length of the consensus sequence makes up the consensus.

When there are ties (multiple nucleotides/characters with equally maximal frequencies), this method can be deterministic or stochastic, depending on additional parameters. The same rules for breaking ties for method="thresholdedFreq" apply.

Below are some examples looking at a single position based on 5 sequences with includeAmbiguous=FALSE, and breakTiesStochastic=FALSE:

  • If the sequences have "A", "A", "T", "A", "C", the consensus will be "A".

  • If the sequences have "T", "T", "C", "C", "G", the consensus will be "T", because "T" is before "C" in the order of "A", "T", "G", "C", "N", ".", and "-".

  • method="catchAll"

This method returns a consensus sequence capturing most of the information contained in the sequences. Ambiguous characters are used where applicable. See “Choosing ambiguous characters” for how ambiguous characters are chosen. This method is deterministic and does not involve breaking ties.

Below are some examples for method="catchAll" looking at a single position based on 5 sequences:

  • If the sequences have "N", "N", "N", "N", "N", the consensus will be "N".
  • If the sequences have "N", "A", "A", "A", "A", the consensus will be "A".
  • If the sequences have "N", "A", "G", "A", "A", the consensus will be "R".
  • If the sequences have "-", "-", ".", ".", ".", the consensus will be "-".
  • If the sequences have "-", "-", "-", "-", "-", the consensus will be "-".

  • If the sequences have ".", ".", ".", ".", ".", the consensus will be ".".

  • method="mostMutated" and method="leastMutated"

These methods return the most/least mutated sequence as the consensus sequence.

When there are ties (multple sequences have the maximal/minimal mutation frequency), this method can be deterministic or stochastic, depending on additional parameters.

  • With breakTiesStochastic=TRUE, ties are resolved stochastically by randomly picking a sequence out of sequences with the maximal/minimal mutation frequency.
  • When breakTiesByColumns is supplied, ties are resolved deterministically. Column by column, a function is applied on the column and sequences with column value matching the functional value are retained, until ties are resolved or columns run out. In the latter case, the first remaining sequence is taken as the consensus.
  • When breakTiesStochastic=TRUE and breakTiesByColumns is also supplied, breakTiesStochastic takes precedence over breakTiesByColumns.
  • When breakTiesStochastic=FALSE and breakTiesByColumns is not supplied (i.e. NULL), the sequence that appears first amongst the ties is taken as the consensus.

Choosing ambiguous characters

Ambiguous characters may be present in the returned consensuses when using the "catchAll" method and when using the "thresholdedFreq" or "mostCommon" methods with includeAmbiguous=TRUE.

The rules on choosing ambiguous characters are as follows:

  • If a position contains only "N" across sequences, the consensus at that position is "N".
  • If a position contains one or more of "A", "T", "G", or "C", the consensus will be an IUPAC character representing all of the characters present, regardless of whether "N", "-", or "." is present.
  • If a position contains only "-" and "." across sequences, the consensus at that position is taken to be "-".
  • If a position contains only one of "-" or "." across sequences, the consensus at that position is taken to be the character present.

Cautions

  • Note that this function does not perform multiple sequence alignment. As a prerequisite, it is assumed that the sequences in sequenceColumn and germlineColumn have been aligned somehow. In the case of immunoglobulin repertoire analysis, this usually means that the sequences are IMGT-gapped.
  • When using the "mostMutated" and "leastMutated" methods, if you supply both muFreqColumn and regionDefinition, it is your responsibility to ensure that the mutation frequency in muFreqColumn was calculated with sequence lengths restricted to the same regionDefinition you are supplying. Otherwise, the “most/least mutated” sequence you obtain might not be the most/least mutated given the regionDefinition supplied, because your mutation frequency was based on a regionDefinition different from the one supplied.
  • If you intend to run collapseClones before building a 5-mer targeting model, you must choose parameters such that your collapsed clonal consensuses do not include ambiguous characters. This is because the targeting model functions do NOT support ambiguous characters in their inputs.

Examples

# Subset example data
data(ExampleDb, package="alakazam")
db <- subset(ExampleDb, c_call %in% c("IGHA", "IGHG") & sample_id == "+7d" &
clone_id %in% c("3100", "3141", "3184"))

# thresholdedFreq method, resolving ties deterministically without using ambiguous characters
clones <- collapseClones(db, cloneColumn="clone_id", sequenceColumn="sequence_alignment", 
germlineColumn="germline_alignment_d_mask",
method="thresholdedFreq", minimumFrequency=0.6,
includeAmbiguous=FALSE, breakTiesStochastic=FALSE)

# mostCommon method, resolving ties deterministically using ambiguous characters
clones <- collapseClones(db, cloneColumn="clone_id", sequenceColumn="sequence_alignment", 
germlineColumn="germline_alignment_d_mask",
method="mostCommon", 
includeAmbiguous=TRUE, breakTiesStochastic=FALSE)

# Make a copy of db that has a mutation frequency column
db2 <- observedMutations(db, frequency=TRUE, combine=TRUE)

# mostMutated method, resolving ties stochastically
clones <- collapseClones(db2, cloneColumn="clone_id", sequenceColumn="sequence_alignment", 
germlineColumn="germline_alignment_d_mask",
method="mostMutated", muFreqColumn="mu_freq", 
breakTiesStochastic=TRUE, breakTiesByColumns=NULL)

# mostMutated method, resolving ties deterministically using additional columns
clones <- collapseClones(db2, cloneColumn="clone_id", sequenceColumn="sequence_alignment", 
germlineColumn="germline_alignment_d_mask",
method="mostMutated", muFreqColumn="mu_freq", 
breakTiesStochastic=FALSE, 
breakTiesByColumns=list(c("duplicate_count"), c(max)))

# Build consensus for V segment only
# Capture all nucleotide variations using ambiguous characters 
clones <- collapseClones(db, cloneColumn="clone_id", sequenceColumn="sequence_alignment", 
germlineColumn="germline_alignment_d_mask",
method="catchAll", regionDefinition=IMGT_V)

# Return the same number of rows as the input
clones <- collapseClones(db, cloneColumn="clone_id", sequenceColumn="sequence_alignment", 
germlineColumn="germline_alignment_d_mask",
method="mostCommon", expandedDb=TRUE)

See also

See IMGT_SCHEMES for a set of predefined RegionDefinition objects.