In j-a-thia/genomalicious: A smorgasbord of R functions for population genomic analyses

knitr::opts_chunk$set(echo = TRUE)

Preamble

Genetic data obtained from population genomic analyses is rarely perfect (although we often wish it was!). Missing data can arise from a failure to recover reads from a particular SNP position, or poor read coverage at a locus which reduces our confidence in the SNP calls at that position. We may want to remove SNPs that are below a certain depth threshold or minor allele frequency, or reduce the non-independence among loci by filtering loci from the same genomic region.

In this tutorial, you will:

1. Use genomalicious to investigate the distribution of missing data.
2. Use genomalicious to filter for samples and loci for missing data and replace missing genotypes using a "most common" genotype approach.
3. Use genomalicious to filter loci for minor allele frequency, read depth, and independence within genomic regions.

Load genomalicious

library(genomalicious)

Missing data

Population genomic datasets typically come with some amount of missingness. Some programs have built in methods that account for missing data. Others require complete datasets without missing genotypes. In the second case, we want to reduce the missingness in our dataset:

1. Loci with too much missing data can be removed.
2. Individuals with too much missing ddata can be removed.
3. Missing data can be imputed.

The choice to remove loci and/or inidivduals versus impute genotypes will depend on your dataset and what seems reasonable. Ultimately, you want to aim for a dateset with as many (independent) loci as possible, with high confidence genotypes, represented in most individuals and populations. When missingness is biased to certain loci, samples, or populations, this can be problematic. Decisions on how to deal with missingness are, however, not the subject of this lesson.

Instead, this lesson will walk you through the functions available in genomalicious to help you visualise and understand the distribution of missingness in your data.

We will work with a simulated data set. This data set was simulated with no missingness, so we will need to make missingness with some simple code. Our data set comprises four populations, 30 individuals per populations, and 200 independent SNP loci.

# Import genotype data 
data(data_Genos)

# Number of samples per population
data_Genos[, length(unique(SAMPLE)), by=POP]

# Number of unique loci
data_Genos$LOCUS %>% unique %>% length

# Working genotype dataset
missGenos <- data_Genos %>% copy

# Add missing values.
missGenos <- do.call(
  'rbind', 
  # Split data table by sample, and iterate through samples, X
  split(missGenos, by='POP') %>% 
    lapply(., function(Dpop){
      pop <- Dpop$POP[1]

      if(pop=='Pop1'){
        pr <- 0.1
      } else if(pop=='Pop2'){
        pr <- 0.2
      } else if(pop %in% c('Pop3','Pop4')){
        pr <- 0.05
      }

      # Numbers and unique loci and samples
      num.loc <- Dpop$LOCUS %>% unique %>% length
      uniq.loc <- Dpop$LOCUS %>% unique
      num.samp <- Dpop$SAMPLE %>% unique %>% length
      uniq.samp <- Dpop$SAMPLE %>% unique

      # Vector of missingness
      num.miss <- rbinom(n=num.samp, size=num.loc, prob=pr)

      # Iterate through samples and add unique loci
      for(i in 1:num.samp){
        locs <- sample(uniq.loc, size=num.miss[i], replace=FALSE)
        Dpop[SAMPLE==uniq.samp[i] & LOCUS%in%locs, GT:=NA]
      }

      # Return
      return(Dpop)
      }
    )
  )

Note in the above code the use of the %>%. This is called a pipe, and is from the magrittr package. Pipes are incredibly useful for moving data from function to function without having to save intermediate objects.

Anyway, we have now created a data.table classed object called missGenos, which will be our working data set. We have simulated 10% missingness from Pop1, 20% missingness in Pop2, and 5% missingness from Pop3 and Pop4, per sample.

Note, your results may be a little different to those simulated here because of the randomisation in my code.

Let's take a look:

# Per sample missingness in each population
missGenos[, .(MISS.PROP=sum(is.na(GT))/length(GT)), by=c('POP','SAMPLE')] %>% 
  .[, .(MISS.PROP.MEAN=mean(MISS.PROP)), by=POP]

# Per locus missingness in each population
missGenos[, .(MISS.PROP=sum(is.na(GT))/length(GT)), by=c('POP','LOCUS')] %>% 
  .[, .(MISS.PROP.MEAN=mean(MISS.PROP)), by=POP]

Let's first create a histogram of the distribution of missingness using the missHist function. This function has a number of ways slice the data and visualise the distribution of missingness. It also has features that allow us to customise the output to suit our aesthetic tastes.

# We need to specify whether we want the histogram to summarise missingness
# across samples or across loci.
plot_miss_hist_samps <- missHist(missGenos, plotBy='samples')
plot_miss_hist_samps

plot_miss_hist_loci <- missHist(missGenos, plotBy='loci')
plot_miss_hist_loci

# By default, the arguments `sampCol`=='SAMPLE', `locusCol`=='LOCUS' and
# `genoCol`=='GT'. The argument `popCol` is NA by default. 
# But if we specify a population # column, then we can get our results plotted 
# with respect to population.
plot_miss_hist_pop <- missHist(missGenos, plotBy='samples', popCol='POP')
plot_miss_hist_pop

# By default, missHist produces a ggplot style plot, but we can produce a classic
# R style plot by specifying the `look` argument.
plot_miss_hist_pop_classic <- missHist(
  missGenos, plotBy='samples', popCol='POP', look='classic'
  )
plot_miss_hist_pop_classic

# We also have options to manipulate the colours of bars (`plotColours`) in 
# the histogram and the grid number of columns in the grid (`plotNCol`).
plot_miss_hist_pop_colour <- missHist(
  missGenos, plotBy='samples', popCol='POP', look='classic', 
  plotColours='#D886CB', plotNCol=1
  )
plot_miss_hist_pop_colour

We may be interested in visualising the patterns of missingness among loci, samples, and populations. We can do this using the missHeatmap function. This function also has several features to customise the style, colours and grid structure to suit your aesthetic.

# The default output, with default arguments sampCol`=='SAMPLE', `locusCol`=='LOCUS' 
# and `genoCol`=='GT'.
plot_heat <- missHeatmap(missGenos)
plot_heat

# Now, split by population. Make missing values black and non-missing values
# pink. Use 4 columns in the grid strucutre.
plot_heat_colours <- missHeatmap(
  missGenos, popCol='POP', plotColours=c('black','#D886CB'), 
  plotNCol=4
  )
plot_heat_colours

Filtering missing data

There are two ways to filter missing data using genomalicious. The filtering functions return lists of "good loci", those that pass the function's missingness threshold crtieria.

Filtering by loci is achieved using the filter_missing_loci function. This function calculates missingness for each locus and removes loci based on a missingness threshold. This threshold is set with the missingness argument, which takes a value betwee 0 and 1.

filter_missing_loci can filter either a long-format data.table object of individual genotypes or population allele frequencies. We have to specify the type of data using the type argument, e.g., type=="genos" or type=="freqs", respectively.

For individually sequenced genotypes, we have two ways of filtering loci using the method argument. When method=="samples", missingness at each locus is calculated across all samples, and if this value is above the threshold, the locus will be removed. When method=="pop", missingness at each locus is calculated for each population, and if any population has missingness above the threshold, the locus will be removed.

Let's start with filtering loci for individually genotypes samples.

# Filter across all samples, irrespective of population
good_loci_genos_samps <- filter_missing_loci(
  missGenos, missing=0.1, type="genos", method="samples",
  sampCol='SAMPLE', locusCol='LOCUS', genoCol='GT'
  )

# Filter if any population does not meet the threshold
good_loci_genos_pops <- filter_missing_loci(
  missGenos, missing=0.1, type="genos", method="pops",
  sampCol='SAMPLE', locusCol='LOCUS', genoCol='GT', popCol='POP'
  )

# Take a look at the number of loci and the overlapping (intersecting) loci
length(good_loci_genos_samps)
length(good_loci_genos_pops)
intersect(good_loci_genos_samps, good_loci_genos_pops)

# Subset the genotypes based on the vector of good loci
missGenos[LOCUS %in% good_loci_genos_samps]
missGenos[LOCUS %in% good_loci_genos_pops]

Filtering populations allele frequency data is a bit different. In this case, the samples are populations (not individuals), and missingness is calculated as the proportion of populations for which allele frequencies are available. Let's take a look.

# Make some missing population frequency data from population pool-seq data.
data("data_PoolFreqs")

missFreqs <- data_PoolFreqs %>% copy
missFreqs$FREQ[sample(1:nrow(missFreqs), size=0.1*nrow(missFreqs), replace=FALSE)] <- NA 

# Take a look at the missing data (note, this may look different on your system)
missFreqs.props <- missFreqs[, .(MISS.PROP=sum(is.na(FREQ))/length(FREQ)), by=c('LOCUS')]

missFreqs.props$MISS.PROP %>% 
  hist(
    ., 
    main='Missing data for population frequencies', 
    xlab='Proportion of populations missing',
    ylab='Number of loci'
    )

# Filter
good_loci_freqs <- filter_missing_loci(
  missFreqs, missing=0.1, type='freqs', 
  locusCol='LOCUS', freqCol='FREQ', popCol='POOL'
  )

length(good_loci_freqs)

# Subset for the good loci
missFreqs[LOCUS %in% good_loci_freqs]

We can also filter missing data by sample, removing samples with too many loci missing genotypes or population allele frequencies. The necessary function is filter_missing_units. In this case, the "units" refers to the sampled unit, individuals or populations. The parameterisation of filter_missing_units is basically identical to that of filter_missing_loci that we explored above. Except this time, missing data is calculated across loci (instead of samples).

For filter_missing_units, if type=="genos", there are again two ways to parameterise the argument method. If method=="samples", then missingness is calculated across loci for each sample, irrespective of its populations, and any sample with missingness above the threshold is removed. If method=="pops", then the mean missingness is calculated per population, and any population with missingness above the threshold is removed.

# Filter by samples, irrespective of populations. Returns vector of individuals.
good_units_samps <- filter_missing_units(
  missGenos, missing=0.1, type='genos', method='samples',
  sampCol='SAMPLE', locusCol='LOCUS', genoCol='GT'
)

# Filter by populations means. Returns vector of populations.
good_units_pops <- filter_missing_units(
  missGenos, missing=0.1, type='genos', method='pops',
  sampCol='SAMPLE', locusCol='LOCUS', genoCol='GT', popCol='POP'
)

# Subset samples
missGenos[SAMPLE %in% good_units_samps]

# Subset populations
missGenos[POP %in% good_units_pops]

And similarly, we can use type=="freqs" to remove populations from a data table of allele frequencies.

# Filter populations with too many missing loci
good_units_pops_freqs <- filter_missing_units(
  missFreqs, missing=0.1, type='freqs', 
  locusCol='LOCUS', freqCol='FREQ', popCol='POOL'
)

good_units_pops_freqs

# Subset good populations
missFreqs[POOL %in% good_units_pops_freqs]

Replacing missing genotypes

Sometimes you cannot filter missing data completely without compromising your dataset. In which case, missing genotypes need to be replaced with estimated values. Ideally, this would be done via imputation methods. However, if you are in a hurry and need to run some quick analyses, it might be easier to replace missing values using the most common genotype

The genomalicious function replace_miss_genos can be used to replace missing genotypes. How this function operate depends on how the argument popCol is parameterised. By default, popCol is NULL, so the function will identify the most common genotypes across all individuals for each locus. If, however, popCol is given a value, a population ID column, then the most common genotype for each locus will be calculated per population.

D <- data_Genos %>% copy

# Sites with missing data
D[sample(1:nrow(D), round(0.1*nrow(D)), FALSE), GT:=NA] %>%
 setnames(., 'GT', 'GT.MISS')

# Replace across individuals
D.rep.inds <- replace_miss_genos(
   dat=D, sampCol='SAMPLE', locusCol='LOCUS', genoCol='GT.MISS'
) %>%
   setnames(., 'GT', 'GT.INDS')

# Replace within populations
D.rep.pops <- replace_miss_genos(
   dat=D, sampCol='SAMPLE', locusCol='LOCUS', genoCol='GT.MISS', popCol='POP'
) %>%
   setnames(., 'GT', 'GT.POPS')

# Tabulate comparisons between methods
compReplace <- left_join(
   data_Genos[, c('LOCUS','SAMPLE','POP','GT')],
   D[, c('LOCUS','SAMPLE','POP','GT.MISS')]
) %>%
.[is.na(GT.MISS), !'GT.MISS'] %>%
   left_join(., D.rep.inds[,c('LOCUS','SAMPLE','POP','GT.INDS')]) %>%
   left_join(., D.rep.pops[,c('LOCUS','SAMPLE','POP','GT.POPS')])

# Number of correct matches is slightly higher when using the most
# common genotype within populations
compReplace[GT==GT.INDS] %>% nrow
compReplace[GT==GT.POPS] %>% nrow

Filtering for minor allele frequency, read depth, and "unlinked" loci

There are additional filtering functions available in genomalicious. It is important to note that the functions in this section cannot have handle missing data. Missing data therefore need to be removed or imputed before applying these functions.

Minor allele frequency (MAF) filtering can be performed with the function, filter_maf. The MAF threshold is set using the argument maf. This function filer a long-format data.table object of individual genotypes or population allele frequencies, specified with type=="genos" and type=="freqs", respectively.

filter_maf filters loci via one of two methods specified with the argument method. When method=="mean", the MAF is calculated as the mean across populations, and any locus with the mean MAF below the threshold is removed. When method=="any_pop", the MAF is calcualted for each population and if any population has a MAF below the threshold, the locus will be removed.

# Loci with good MAF, taking the mean across populations
good_maf_genos_mean <- filter_maf(
  dat=data_Genos, maf=0.1, type='genos', method='mean',
  sampCol='SAMPLE', locusCol='LOCUS', genoCol='GT'
  )

length(good_maf_genos_mean)

# Loci with good MAF, where all populations meet the threshold
good_maf_genos_pops <- filter_maf(
  dat=data_Genos, maf=0.1, type='genos', method='any_pop',
  sampCol='SAMPLE', locusCol='LOCUS', genoCol='GT'
  )

length(good_maf_genos_pops)

# Overlap
intersect(good_maf_genos_mean, good_maf_genos_pops)

# Filter a data table of population allele frequencies
good_maf_freqs <- filter_maf(
  dat=data_PoolFreqs, maf=0.1, type='freqs',
  popCol='POOL', locusCol='LOCUS', freqCol='FREQ'
)

length(good_maf_freqs)

Read depth filtering can be performed using the function filter_depth. This function is relatively straight forward in that it simply requires a data.table object with depth values for different loci in different samples. The key parameterisations are the minimum depth, minDP, and maximum depth, maxDP. You can set either, or both of these arguments to filter read depth.

# Take a look at the depth distribution before filtering
data_Genos$DP %>% summary

data_Genos$DP  %>% 
  hist(., main='Initial depth distribution', xlab='Depth', ylab='Samples')

# Filtering both minimum and maximum
good_loci_dp_both <- filter_depth(
  data_Genos, minDP=10, maxDP=60, 
  locusCol='LOCUS', dpCol='DP'
  )

length(good_loci_dp_both)

data_Genos[LOCUS %in% good_loci_dp_both]$DP %>% 
  hist(., main='Min and max depth', xlab='Depth', ylab='Samples')

# Filtering minimum only
good_loci_dp_min <- filter_depth(
  data_Genos, minDP=10, maxDP=NULL, 
  locusCol='LOCUS', dpCol='DP'
  )

length(good_loci_dp_min)

data_Genos[LOCUS %in% good_loci_dp_min]$DP %>% 
  hist(., main='Min depth only', xlab='Depth', ylab='Samples')

# Filtering maximum only
good_loci_dp_max <- filter_depth(
  data_Genos, minDP=NULL, maxDP=60, 
  locusCol='LOCUS', dpCol='DP'
  )

length(good_loci_dp_max)

data_Genos[LOCUS %in% good_loci_dp_max]$DP %>% 
  hist(., main='Max depth only', xlab='Depth', ylab='Samples')

# Filtering a data table of allele frequencies is the same.
data_PoolFreqs$DP %>% 
  hist(., main='Depth distribution for frequency data', xlab='Depth', ylab='Samples')

good_loci_dp_freqs <- filter_depth(
  data_PoolFreqs, minDP=30, maxDP=100, 
  locusCol='LOCUS', dpCol='DP'
  )

data_PoolFreqs[LOCUS %in% good_loci_dp_freqs]$DP %>% 
  hist(., main='Max and min depth frequency data', xlab='Depth', ylab='Samples')

Finally, there is a function to "unlink" loci in reduced representation datasets (herein, "RADseq"). In these types of datasets, genotypes or allele frequencies are derived from short genomic fragments (herein, "RAD contigs"). To reduce the non-independence of loci within RAD contigs, it is standard practise to filter loci to keep just one locus per RAD contig. This function is called filter_unlink.

The key argument is method. When method=="random", then the locus in the RAD contig is randomly selected. When method=="first", then the first locus in the RAD contig is selected.

unlink_loci <- filter_unlink(
  data_Genos, chromCol='CHROM', locusCol='LOCUS', posCol='POS',
  method='random'
  )

length(unlink_loci)

Postamble

You have now completed the 'Quality control and filtering' tutorial! You should now be familiar with the functions in genomalicious that help visualise patterns of missingness in your population genomic data. You should also now be able to use genomalicious to filter you data for missingness, minor allele frequency, read depth, and independent loci on different genomic fragments (e.g., RAD contigs in RADseq datasets). You now also know how genomalicious can help you replace missing genotypes with a "most common genotype" approach, although it is recommended that for real analyses you should use proper genotype imputation algorithms.

j-a-thia/genomalicious documentation built on Oct. 19, 2024, 7:51 p.m.