Assign Alleles to Isoloci Based on Distribution of Genotypes

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Description

Where a single locus represents two or more independent isoloci (as in an allopolyploid, or a diploidized autopolyploid), these two functions can be used in sequence to assign alleles to isoloci. alleleCorrelations uses K-means and UPGMA clustering of pairwise p-values from Fisher's exact test to make initial groupings of alleles into putative isoloci. testAlGroups is then used to check those groupings against individual genotypes, and adjust the assignments if necessary.

Usage

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alleleCorrelations(object, samples = Samples(object), locus = 1,
                   alpha = 0.05, n.subgen = 2, n.start = 50)

testAlGroups(object, fisherResults, SGploidy=2, samples=Samples(object),
             null.weight=0.5, tolerance=0.05, swap = TRUE,
             R = 100, rho = 0.95, T0 = 1, maxreps = 100)

Arguments

object

A "genambig" or "genbinary" object containing the data to analyze.

samples

An optional character or numeric vector indicating which samples to analyze.

locus

A single character string or integer indicating which locus to analyze.

alpha

The significance threshold, before multiple correction, for determining whether two alleles are significantly correlated.

n.subgen

The number of subgenomes (number of isoloci) for this locus. This would be 2 for an allotetraploid or 3] for an allohexaploid. For an allo-octoploid, the value would be 2 if there were two tetraploid subgenomes, or 4 if there were four diploid subgenomes.

n.start

Integer, passed directly to the nstart argument of the R base function kmeans. Lowering this number will speed up computation time, whereas increasing it will improve the probability of finding the correct allele assignments. The default value of 50 should work well in most cases.

fisherResults

A list output from alleleCorrelations.

SGploidy

The ploidy of each subgenome (each isolocus). This is 2 for an allotetraploid, an allohexaploid, or an allo-octoploid with four tetraploid subgenomes, or 4 for an allo-octoploid with two tetraploid genomes.

null.weight

Numeric, indicating how genotypes with potential null alleles should be counted when looking for signs of homoplasy. null.weight should be 0 if null alleles are expected to be common, and 1 if there are no null alleles in the dataset. The default of 0.5 was chosen to reflect the fact that the presence of null alleles is generally unknown.

tolerance

The proportion of genotypes that are allowed to be in disagreement with the allele assignments. This is the proportion of genotypes that are expected to have meiotic error or scoring error.

swap

Boolean indicating whether or not to use the allele swapping algorithm before checking for homoplasy. TRUE will yield more accurate results in most cases, but FALSE may be preferable for loci with null or homoplasious alleles at high frequency.

R

Simulated annealing parameter for the allele swapping algorithm. Indicates how many swaps to attempt in each rep (i.e. how many swaps to attempt before changing the temperature).

rho

Simulated annealing parameter for the allele swapping algorithm. Factor by which to reduce the temperature at the end of each rep.

T0

Simulated annealing parameter for the allele swapping algorithm. Starting temperature.

maxreps

Simulated annealing parameter for the allele swapping algorithm. Maximum number of reps if convergence is not achieved.

Details

These functions implement a novel methodology, introduced in polysat version 1.4 and updated in version 1.6, for cases where one pair of microsatellite primers amplifies alleles at two or more independently-segregating loci (referred to here as isoloci). This is not typically the case with new autopolyploids, in which all copies of a locus have equal chances of pairing with each other at meiosis. It is, however, frequently the case with allopolyploids, in which there are two homeologous subgenomes that do not pair (or infrequently pair) at meiosis, or ancient autopolyploids, in which duplicated chromosomes have diverged to the point of no longer pairing at meiosis.

Within the two functions there are four major steps:

  1. alleleCorrelations checks to see if there are any alleles that are present in every genotype in the dataset. Such invariable alleles are assumed to be fixed at one isolocus (which is not necessarily true, but may be corrected by testAlGroups in steps 4 and 5). If present, each invariable allele is assigned to its own isolocus. If there are more invariable alleles than isoloci, the function throws an error. If only one isolocus remains, all remaining (variable) alleles are assigned to that isolocus. If there are as many invariable alleles as isoloci, all remaining (variable) alleles are assigned to all isoloci (i.e. they are considered homoplasious because they cannot be assigned).

  2. If, after step 1, two or more isoloci remain without alleles assigned to them, correlations between alleles are tested by alleleCorrelations. The dataset is converted to "genbinary" if not already in that format, and a Fisher's exact test, with negative association (odds ratio being less than one) as the alternative hypothesis, is performed between each pair of columns (alleles) in the genotype matrix. The p-value of this test between each pair of alleles is stored in a square matrix, and zeros are inserted into the diagonal of the matrix. K-means clustering and UPGMA are then performed on the square matrix of p-values, and the clusters that are produced represent initial assignments of alleles to isoloci.

  3. The output of alleleCorrelations is then passed to testAlGroups. If the results of K-means clustering and UPGMA were not identical, testAlGroups checks both sets of assignments against all genotypes in the dataset. For a genotype to be consistent with a set of assignments, it should have at least one allele and no more than SGploidy alleles belonging to each isolocus. The set of assignments that is consistent with the greatest number of genotypes is chosen, or in the case of a tie, the set of assignments produced by K-means clustering.

  4. If swap = TRUE and the assignments chosen in the previous step are inconsistent with some genotypes, testAlGroups attempts to swap the isoloci of single alleles, using a simulated annealing (Bertsimas and Tsitsiklis 1993) algorithm to search for a new set of assignments that is consistent with as many genotypes as possible. At each step, an allele is chosen at random to be moved to a different isolocus (which is also chosen at random if there are more than two isoloci). If the new set of allele assignments is consistent with an equal or greater number of genotypes than the previous set of assignments, the new set is retained. If the new set is consistent with fewer genotypes than the old set, there is a small probability of retaining the new set, dependent on how much worse the new set of assignments is and what the current “temperature” of the algorithm is. After R allele swapping attempts, the temperature is lowered, reducing the probability of retaining a set of allele assignments that is worse than the previous set. A new rep of R swapping attempts then begins. If a set of allele assignments is found that is consistent with all genotypes, the algorithm stops immediately. Otherwise it stops if no changes are made during an entire rep of R swap attempts, or if maxreps reps are performed.

  5. testAlGroups then checks through all genotypes to look for signs of homoplasy, meaning single alleles that should be assigned to more than one isolocus. For each genotype, there should be no more than SGploidy alleles assigned to each isolocus. Additionally, if there are no null alleles, each genotype should have at least one allele belonging to each isolocus. Each time a genotype is encountered that does not meet these criteria, the a score is increased for all alleles that might be homoplasious. (The second criterion is not checked if null.weight = 0.) This score starts at zero and is increased by 1 if there are too many alleles per isolocus or by null.weight if an isolocus has no alleles. Once all genotypes have been checked, the allele with the highest score is considered to be homoplasious and is added to the other isolocus. (In a hexaploid or higher, which isolocus the allele is added to depends on the genotypes that were found to be inconsistent with the allele assignments, and which isolocus or isoloci the allele could have belonged to in order to fix the assignment.) Allele scores are reset to zero and all alleles are then checked again with the new set of allele assignments. The process is repeated until the proportion of genotypes that are inconsistent with the allele assignments is at or below tolerance.

Value

Both functions return lists. For alleleCorrelations:

locus

The name of the locus that was analyzed.

clustering.method

The method that was ultimately used to produce value$Kmeans.groups and value$UPGMA.groups. Either "K-means and UPGMA" or "fixed alleles".

significant.neg

Square matrix of logical values indicating whether there was significant negative correlation between each pair of alleles, after multiple testing correction by Holm-Bonferroni.

significant.pos

Square matrix of logical values indicating whether there was significant positive correlation between each pair of alleles, after multiple testing correction by Holm-Bonferroni.

p.values.neg

Square matrix of p-values from Fisher's exact test for negative correlation between each pair of alleles.

p.values.pos

Square matrix of p-values from Fisher's exact test for positive correlation between each pair of alleles.

odds.ratio

Square matrix of the odds ratio estimate from Fisher's exact test for each pair of alleles.

Kmeans.groups

Matrix with n.subgen rows, and as many columns as there are alleles in the dataset. 1 indicates that a given allele belongs to a given isolocus, and 0 indicates that it does not. These are the groupings determined by K-means clustering.

UPGMA.groups

Matrix in the same format as value$Kmeans.groups, showing groupings determined by UPGMA.

heatmap.dist

Square matrix like value$p.values.neg but with zeros inserted on the diagonal. This is the matrix that was used for K-means clustering and UPGMA. This matrix can be passed to the heatmap function in R to visualize the clusters.

totss

Total sums of squares output from K-means clustering.

betweenss

Sums of squares between clusters output from K-means clustering. value$betweenss/value$totss can be used as an indication of clustering quality.

gentable

The table indicating presence/absence of each allele in each genotype.

For testAlGroups:

locus

Name of the locus that was tested.

SGploidy

The ploidy of each subgenome, taken from the SGploidy argument that was passed to testAlGroups.

assignments

Matrix with as many rows as there are isoloci, and as many columns as there are alleles in the dataset. 1 indicates that a given allele belongs to a given isolocus, and 0 indicates that it does not.

proportion.inconsistent.genotypes

A number ranging from zero to one indicating the proportion of genotypes from the dataset that are inconsistent with assignments.

Note

alleleCorrelations will print a warning to the console or to the standard output stream if a significant positive correlation is found between any pair of alleles. (This is not a “warning” in the technical sense usually used in R, because it can occur by random chance and I did not want it to cause polysat to fail package checks.) You can see which allele pair(s) caused this warning by looking at value$significant.pos. If you receive this warning for many loci, consider that there may be population structure in your dataset, and that you might split the dataset into multiple populations to test seperately. If it happens at just a few loci, check to make sure there are not scoring problems such as stutter peaks being miscalled as alleles. If it only happens at one locus and you can't find any evidence of scoring problems, two alleles may have been positively correlated simply from random chance, and the warning can be ignored.

alleleCorrelations can also produce an actual warning stating “Quick-TRANSfer stage steps exceeded maximum”. This warning is produced internally by kmeans and may occur if many genotypes are similar, as in mapping populations. It can be safely ignored.

Author(s)

Lindsay V. Clark

References

Clark, L. V. and Drauch Schreier, A. (2015) Resolving microsatellite genotype ambiguity in populations of allopolyploid and diploidized autopolyploid organisms using negative correlations between allelic variables. bioRxiv, DOI: 10.1101/020610.

Bertsimas, D. and Tsitsiklis, J.(1993) Simulated annealing. Statistical Science 8, 10–15.

See Also

recodeAllopoly, mergeAlleleAssignments, catalanAlleles, processDatasetAllo

Examples

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# randomly generate example data for an allotetraploid
mydata <- simAllopoly(n.alleles=c(5,5), n.homoplasy=1)
viewGenotypes(mydata)

# test allele correlations
# n.start is lowered in this example to speed up computation time
myCorr <- alleleCorrelations(mydata, n.subgen=2, n.start=10)
myCorr$Kmeans.groups
myCorr$clustering.method
if(!is.null(myCorr$heatmap.dist)) heatmap(myCorr$heatmap.dist)

# check individual genotypes 
# (low maxreps used in order to speed processing time for this example)
myRes <- testAlGroups(mydata, myCorr, SGploidy=2, maxreps = 5)
myRes$assignments
myRes2 <- testAlGroups(mydata, myCorr, SGploidy=2, swap = FALSE)
myRes2$assignments

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