Clonotype tracking is a popular approach to monitor changes in the frequency of clonotypes of interest in vaccination and cancer immunology. For example, a researcher can track a clonotype across different time points in pre- and post-vaccination repertoires, or analyse the growth of malignant clonotypes in a tumor sample.
Various methods of clonotype tracking are integrated into the one
trackClonotypes function. Currently, there are three methods to choose from. The output of
trackClonotypes can be immediately visualised with the
The simplest approach is to choose the most abundant clonotypes from one of the input immune repertoires and track across all immune repertoires in a batch. Arguments
.col are used to choose the immune repertoire, the number of clonotypes to take from it and which columns to use.
To choose the top 10 most abundant clonotypes from the first repertoire and track them using their CDR3 nucleotide sequence use this code:
tc1 <- trackClonotypes(immdata$data, list(1, 5), .col = "nt")
list(1, 5) of the
.which argument (the second argument) means to choose 10 clonotypes from the 1st repertoire in the input list of repertoires
"nt" of the
.col argument means that the function should apply to CDR3 nucleotide sequences only.
To choose the 10 most abundant amino acid clonotype sequences and their V genes from the “MS1” repertoire to track:
tc2 <- trackClonotypes(immdata$data, list("MS1", 10), .col = "aa+v")
list("MS1", "10") of the
.which argument means to choose 10 clonotypes from the repertoire named “MS1” in the input list of repertoires
"aa+v" of the
.col argument means that the function should take both CDR3 amino acid sequences and V gene segments of the most abundant clonotypes.
Visualisation of both approaches:
In order to track specific clonotype sequences, you can provide nucleotide or amino acid sequences as the
.which argument, along with the column
.col specifying in which columns to search for sequences. For example, to track seven CDR3 amino acid sequences specified below you need to execute the following code:
target <- c("CASSLEETQYF", "CASSDSSGGANEQFF", "CASSDSSGSTDTQYF", "CASSLAGGYNEQFF", "CASSDSAGGTDTQYF", "CASSLDSYEQYF", "CASSSAGGYNEQFF") tc <- trackClonotypes(immdata$data, target, .col = "aa") vis(tc)
An improvement upon the previous approach, it is possible to track clonotypes using information about both sequences and gene segments. For support you can use a data frame of sequences with specific CDR3 sequences and gene segments. We will simulate this below by choosing the 10 most abundant clonotypes from the first repertoire in the batch:
## # A tibble: 10 × 2 ## CDR3.aa V.name ## <chr> <chr> ## 1 CASSQEGTGYSGELFF TRBV4-1 ## 2 CASSYRVGTDTQYF TRBV4-1 ## 3 CATSTNRGGTPADTQYF TRBV15 ## 4 CATSIGGGSYEQYF TRBV15 ## 5 CASSPWTGSMALHF TRBV27 ## 6 CASQGDSFNSPLHF TRBV4-1 ## 7 CASSQDMGGRNTGELFF TRBV4-1 ## 8 CASSEEPRLFGYTF TRBV2 ## 9 CASSQPGQGGGDEQFF TRBV4-1 ## 10 CASSWVARGPYEQYF TRBV6-6
Supply this data frame as an argument value to the
.which argument to track target clonotypes:
tc <- trackClonotypes(immdata$data, target) vis(tc)
Note that you can use any columns in the
target data frame, such as both CDR3 nucleotide and amino acid sequences and any gene segments.
There are three ways to visualise clonotype tracking, depending on your research and aesthetic needs. To choose the type of plot, you need to provide the
".plot" parameter to the
vis() function, specifying one of three plot types: -
.plot = "smooth" - used by default, a visualisation using smooth lines and stacked bar plots; -
.plot = "area" - visualises abundances using areas under the abundance lines; -
.plot = "line" - visualises only the lines, connecting levels of abundances of a same clonotype between time points.
target <- c("CASSLEETQYF", "CASSDSSGGANEQFF", "CASSDSSGSTDTQYF", "CASSLAGGYNEQFF", "CASSDSAGGTDTQYF", "CASSLDSYEQYF", "CASSSAGGYNEQFF") tc <- trackClonotypes(immdata$data, target, .col = "aa") vis(tc, .plot = "smooth")
vis(tc, .plot = "area")
vis(tc, .plot = "line")
.order argument of the
vis function controls the order of samples in the visualisation. You can pass either indices of samples you plan to visualise or sample names.
##  "A2-i129" "A2-i133" "A4-i191"
If your metadata contains information about time such as timepoints for vaccination or tumor samples, you can use it to re-order samples accordingly. In our examples
immdata$meta does not contain information about timepoints, so we will simulate this case.
First, we create an additional column in the metadata with randomly chosen time points:
## # A tibble: 12 × 7 ## Sample ID Sex Age Status Lane Timepoint ## <chr> <chr> <chr> <dbl> <chr> <chr> <int> ## 1 A2-i129 C1 M 11 C A 2 ## 2 A2-i131 C2 M 9 C A 11 ## 3 A2-i133 C4 M 16 C A 3 ## 4 A2-i132 C3 F 6 C A 6 ## 5 A4-i191 C8 F 22 C B 4 ## 6 A4-i192 C9 F 24 C B 1 ## 7 MS1 MS1 M 12 MS C 5 ## 8 MS2 MS2 M 30 MS C 7 ## 9 MS3 MS3 M 8 MS C 10 ## 10 MS4 MS4 F 14 MS C 8 ## 11 MS5 MS5 F 15 MS C 12 ## 12 MS6 MS6 F 15 MS C 9
Next, we create a vector with samples in the right order, according to the “Timepoint” column (from smallest to greatest):
sample_order <- order(immdata$meta$Timepoint)
Sanity check: timepoints are following the right order:
##  1 2 3 4 5 6 7 8 9 10 11 12
Samples, sorted by the timepoints:
##  "A4-i192" "A2-i129" "A2-i133" "A4-i191" "MS1" "A2-i132" "MS2" ##  "MS4" "MS6" "MS3" "A2-i131" "MS5"
And finally, we visualise the data:
vis(tc, .order = sample_order)
It is possible to create a one-liner with the full pipeline from ordering to plotting:
If you want to change the colour palette, add a ggplot2
scale_fill_* function to the plot. We recommend using
vis(tc) + scale_fill_brewer(palette = "Spectral")
vis(tc) + scale_fill_brewer(palette = "RdBu")
?scale_fill_brewer in the R console to learn more about ColorBrewer and it’s colour schemes.
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