Kmer statistics computation

Counting kmer occurrences in immunarch is very easy. All you need to do is to run the getKmers() function on your data:

kmers <- getKmers(immdata$data[[1]], 3)
kmers

##  [38;5;246m# A tibble: 4,405 × 2 [39m
##    Kmer  Count
##     [3m [38;5;246m<chr> [39m [23m  [3m [38;5;246m<int> [39m [23m
##  [38;5;250m 1 [39m AAA       6
##  [38;5;250m 2 [39m AAD       5
##  [38;5;250m 3 [39m AAE       9
##  [38;5;250m 4 [39m AAF       2
##  [38;5;250m 5 [39m AAG      37
##  [38;5;250m 6 [39m AAI       2
##  [38;5;250m 7 [39m AAK       5
##  [38;5;250m 8 [39m AAL       4
##  [38;5;250m 9 [39m AAM       1
##  [38;5;250m10 [39m AAN      13
##  [38;5;246m# … with 4,395 more rows [39m

If is possible to compute occurrences of kmers in a batch of immune repertoires. In order to do that, you just need to provide a list of immune repertoires to the function. NA means that there is no such kmer found in a sample, specifyed by the column name.

kmers <- getKmers(immdata$data, 5)
kmers

##  [38;5;246m# A tibble: 172,926 × 13 [39m
##    Kmer  `A2-i129` `A2-i131` `A2-i133` `A2-i132` `A4-i191` `A4-i192`   MS1   MS2
##     [3m [38;5;246m<chr> [39m [23m      [3m [38;5;246m<int> [39m [23m      [3m [38;5;246m<int> [39m [23m      [3m [38;5;246m<int> [39m [23m      [3m [38;5;246m<int> [39m [23m      [3m [38;5;246m<int> [39m [23m      [3m [38;5;246m<int> [39m [23m  [3m [38;5;246m<int> [39m [23m  [3m [38;5;246m<int> [39m [23m
##  [38;5;250m 1 [39m AAAAG         [31mNA [39m         [31mNA [39m         [31mNA [39m         [31mNA [39m         [31mNA [39m         [31mNA [39m     [31mNA [39m     1
##  [38;5;250m 2 [39m AAAAL         [31mNA [39m         [31mNA [39m         [31mNA [39m         [31mNA [39m         [31mNA [39m         [31mNA [39m     [31mNA [39m     [31mNA [39m
##  [38;5;250m 3 [39m AAAAW         [31mNA [39m         [31mNA [39m         [31mNA [39m         [31mNA [39m         [31mNA [39m         1     [31mNA [39m     [31mNA [39m
##  [38;5;250m 4 [39m AAADE         [31mNA [39m         [31mNA [39m         [31mNA [39m         [31mNA [39m         [31mNA [39m         [31mNA [39m     [31mNA [39m     [31mNA [39m
##  [38;5;250m 5 [39m AAADN         [31mNA [39m         1         [31mNA [39m         [31mNA [39m         [31mNA [39m         [31mNA [39m     [31mNA [39m     [31mNA [39m
##  [38;5;250m 6 [39m AAADT         [31mNA [39m         [31mNA [39m         [31mNA [39m         [31mNA [39m         [31mNA [39m         [31mNA [39m     [31mNA [39m     [31mNA [39m
##  [38;5;250m 7 [39m AAAEA         [31mNA [39m         [31mNA [39m         [31mNA [39m         [31mNA [39m         [31mNA [39m         [31mNA [39m     [31mNA [39m     [31mNA [39m
##  [38;5;250m 8 [39m AAAEN         [31mNA [39m         [31mNA [39m         [31mNA [39m         1         [31mNA [39m         [31mNA [39m     [31mNA [39m     [31mNA [39m
##  [38;5;250m 9 [39m AAAET         [31mNA [39m         [31mNA [39m         [31mNA [39m         [31mNA [39m         [31mNA [39m         [31mNA [39m     [31mNA [39m     [31mNA [39m
##  [38;5;250m10 [39m AAAFE         [31mNA [39m         [31mNA [39m         [31mNA [39m         1         [31mNA [39m         [31mNA [39m     [31mNA [39m     [31mNA [39m
##  [38;5;246m# … with 172,916 more rows, and 4 more variables: MS3 <int>, MS4 <int>, [39m
##  [38;5;246m#   MS5 <int>, MS6 <int> [39m

Note that by default getKmers() filter out all non-coding sequences before counting the kmer statistics. You can use both coding and non-coding sequences by setting the .coding argument to FALSE:

kmers <- getKmers(immdata$data[[1]], 3, .coding = F)
kmers

##  [38;5;246m# A tibble: 4,645 × 2 [39m
##    Kmer  Count
##     [3m [38;5;246m<chr> [39m [23m  [3m [38;5;246m<int> [39m [23m
##  [38;5;250m 1 [39m **G       1
##  [38;5;250m 2 [39m *~G       4
##  [38;5;250m 3 [39m *~L       1
##  [38;5;250m 4 [39m *AA       1
##  [38;5;250m 5 [39m *D~       1
##  [38;5;250m 6 [39m *EE       1
##  [38;5;250m 7 [39m *G~       1
##  [38;5;250m 8 [39m *GG       1
##  [38;5;250m 9 [39m *GP       1
##  [38;5;250m10 [39m *HL       1
##  [38;5;246m# … with 4,635 more rows [39m

Kmer statistics visualisation

To visualise your kmer statistics, the vis() function comes to help:

kmers <- getKmers(immdata$data, 5)
vis(kmers)

The vis() function for kmers has a number of arguments to manipulate the plot. First, the .head argument specifies the number of the most abundant kmers to visualise.

p1 <- vis(kmers, .head = 5)
p2 <- vis(kmers, .head = 10)
p3 <- vis(kmers, .head = 30)

(p1 + p2) / p3

Second, there are three options to choose from for positions of bars: “stack”, “dodge” and “fill”, adjusted by providing the correposnding option to the .position argument:

p1 <- vis(kmers, .head = 10, .position = "stack")
p2 <- vis(kmers, .head = 10, .position = "fill")
p3 <- vis(kmers, .head = 10, .position = "dodge")

(p1 + p2) / p3

Option “stack” stacks all bars on top of each other so you can see the full distribution of kmers. Option “fill” stack all bars on top of each other as well, but normalises it in a such way so you see distribution of counts per-kmer, i.e., you can clearly see which repertoire has more kmer counts than others for a specific kmer. Option “dodge” groups kmer bars of different samples so you can clearly see, which samples has more kmer occurrences overall.

Additional argument is .log needed if your distribution of kmer counts is vastly imbalanced for some of repertoires. It permits to use the log-transformation of y-axis so you can see differences in orders of magnitude in kmer counts rather.

p1 <- vis(kmers, .head = 10, .position = "stack")
p2 <- vis(kmers, .head = 10, .position = "stack", .log = T)

p1 + p2

Sequence motifs analysis

immunarch utilises common approaches to sequence motif analysis and uses different types of matrices to represent sequence motifs:

position frequency matrix (PFM) - a matrix with occurences of each amino acid in each position;
position probability matrix (PPM) - a matrix with probabilities of occurences of each amino acid in each position;
position weight matrix (PWM) - a matrix with log likelihoods of PPM elements;
a matrix with self-information of elements in PWM.

To compute and visualise sequence motifs, first you need to compute kmer statistics for one of the input immune repertoires, and then apply the kmer_profile() function to compute sequence motif matrices:

kmers <- getKmers(immdata$data[[1]], 5)
kmer_profile(kmers)

##    [,1]  [,2]  [,3] [,4] [,5]
## A  8955  8976  3791 3246 3159
## C  6469    26    27   18   10
## D  2129  2131  2131 2130 2085
## E  3315  5868  5872 5870 5720
## F   688   691   691 2600 9029
## G  9194  9603  9603 9566 9326
## H   405   405   405  680  659
## I   750  1020  1107  896  852
## K   721  1099  1100 1100 1044
## L  3073  3078  4108 4097 4033
## M   241   245   246  246  226
## N  2421  2423  2424 2419 2355
## P  2285  2564  2564 2559 2509
## Q  2459  2466  7087 7086 7051
## R  3487  3502  3504 3496 2917
## S 14021 14029 13082 8128 3462
## T  3408  5669  5671 6024 5797
## V  1703  1927  1927 1561 1508
## W   485   486   487  450  439
## Y  2060  2061  2442 6097 6088
## attr(,"class")
## [1] "immunr_kmer_profile_pfm" "matrix"                 
## [3] "array"

Currently we are not supporting sequence motifs analysis for more than one sample, but we are working on it. In order to compute and visualise sequence motif matrices for all of your samples you need to process them one-by-one, which can be easily done in for-loops or via the lapply() function.

Argument .method specifies which matrix to compute:

.method = "freq" - position frequency matrix (PFM);
.method = "prob" - position probability matrix (PPM);
.method = "wei" - position weight matrix (PWM);
.method = "self" - self-information matrix.

kmer_profile(kmers, "freq")

##    [,1]  [,2]  [,3] [,4] [,5]
## A  8955  8976  3791 3246 3159
## C  6469    26    27   18   10
## D  2129  2131  2131 2130 2085
## E  3315  5868  5872 5870 5720
## F   688   691   691 2600 9029
## G  9194  9603  9603 9566 9326
## H   405   405   405  680  659
## I   750  1020  1107  896  852
## K   721  1099  1100 1100 1044
## L  3073  3078  4108 4097 4033
## M   241   245   246  246  226
## N  2421  2423  2424 2419 2355
## P  2285  2564  2564 2559 2509
## Q  2459  2466  7087 7086 7051
## R  3487  3502  3504 3496 2917
## S 14021 14029 13082 8128 3462
## T  3408  5669  5671 6024 5797
## V  1703  1927  1927 1561 1508
## W   485   486   487  450  439
## Y  2060  2061  2442 6097 6088
## attr(,"class")
## [1] "immunr_kmer_profile_pfm" "matrix"                 
## [3] "array"

kmer_profile(kmers, "prob")

##          [,1]         [,2]         [,3]         [,4]         [,5]
## A 0.131172274 0.1314798811 0.0555303286 0.0475472030 0.0462728325
## C 0.094757503 0.0003808464 0.0003954943 0.0002636629 0.0001464794
## D 0.031185458 0.0312147534 0.0312147534 0.0312001055 0.0305409483
## E 0.048557911 0.0859540934 0.0860126851 0.0859833892 0.0837861987
## F 0.010077781 0.0101217244 0.0101217244 0.0380846358 0.1322562217
## G 0.134673131 0.1406641375 0.1406641375 0.1401221638 0.1366066590
## H 0.005932414 0.0059324144 0.0059324144 0.0099605970 0.0096529904
## I 0.010985953 0.0149408956 0.0162152661 0.0131245514 0.0124800422
## K 0.010561162 0.0160980826 0.0161127305 0.0161127305 0.0152924461
## L 0.045013110 0.0450863496 0.0601737245 0.0600125972 0.0590751293
## M 0.003530153 0.0035887445 0.0036033925 0.0036033925 0.0033104337
## N 0.035462655 0.0354919510 0.0355065989 0.0354333592 0.0344958913
## P 0.033470536 0.0375573101 0.0375573101 0.0374840704 0.0367516735
## Q 0.036019277 0.0361218122 0.1038099284 0.1037952804 0.1032826026
## R 0.051077356 0.0512970748 0.0513263707 0.0512091872 0.0427280318
## S 0.205378722 0.2054959059 0.1916243097 0.1190584306 0.0507111573
## T 0.049920169 0.0830391539 0.0830684498 0.0882391715 0.0849140899
## V 0.024945436 0.0282265743 0.0282265743 0.0228654294 0.0220890888
## W 0.007104249 0.0071188973 0.0071335452 0.0065915716 0.0064304443
## Y 0.030174750 0.0301893978 0.0357702618 0.0893084709 0.0891766395
## attr(,"class")
## [1] "immunr_kmer_profile_ppm" "matrix"                 
## [3] "array"

kmer_profile(kmers, "wei")

##       [,1]      [,2]      [,3]       [,4]      [,5]
## A 8.806550 8.8099289 7.5664346  7.3425192  7.303324
## C 8.337399 0.3785116 0.4329594 -0.1520031 -1.000000
## D 6.734032 6.7353868 6.7353868  6.7347096  6.703904
## E 7.372865 8.1967251 8.1977082  8.1972167  8.159871
## F 5.104337 5.1106138 5.1106138  7.0223678  8.818422
## G 8.844549 8.9073414 8.9073414  8.9017720  8.865115
## H 4.339850 4.3398500 4.3398500  5.0874628  5.042207
## I 5.228819 5.6724253 5.7905114  5.4854268  5.412782
## K 5.171927 5.7800476 5.7813597  5.7813597  5.705978
## L 7.263504 7.2658494 7.6822924  7.6784241  7.655710
## M 3.590961 3.6147098 3.6205864  3.6205864  3.498251
## N 6.919459 6.9206506 6.9212459  6.9182670  6.879583
## P 6.836050 7.0022525 7.0022525  6.9994363  6.970969
## Q 6.941928 6.9460290 8.4690312  8.4688277  8.461684
## R 7.445843 7.4520353 7.4528590  7.4495614  7.188342
## S 9.453374 9.4541965 9.3533674  8.6667566  7.435462
## T 7.412782 8.1469505 8.1474593  8.2345780  8.179163
## V 6.411935 6.5902128 6.5902128  6.2863267  6.236493
## W 4.599913 4.6028844 4.6058499  4.4918531  4.456149
## Y 6.686501 6.6872007 6.9319194  8.2519557  8.249825
## attr(,"class")
## [1] "immunr_kmer_profile_pwm" "matrix"                 
## [3] "array"

kmer_profile(kmers, "self")

##         [,1]        [,2]        [,3]        [,4]       [,5]
## A 0.38439580 0.384852924 0.231593692 0.208945980 0.20515943
## C 0.32213912 0.004325845 0.004470689 0.003134693 0.00186571
## D 0.15602031 0.156124588 0.156124588 0.156072452 0.15371599
## E 0.21191400 0.304302404 0.304425277 0.304363847 0.29971526
## F 0.06684268 0.067070605 0.067070605 0.179555617 0.38600202
## G 0.38953746 0.398033587 0.398033587 0.397280373 0.39232070
## H 0.04388305 0.043883048 0.043883048 0.066233509 0.06462492
## I 0.07149874 0.090610399 0.096424136 0.082049289 0.07892670
## K 0.06933496 0.095895752 0.095961867 0.095961867 0.09222931
## L 0.20136664 0.201588530 0.243987757 0.243566576 0.24110364
## M 0.02875681 0.029148878 0.029246677 0.029246677 0.02727388
## N 0.17084331 0.170942166 0.170991579 0.170744427 0.16756143
## P 0.16403791 0.177824941 0.177824941 0.177583728 0.17516018
## Q 0.17271556 0.173059094 0.339249150 0.339222412 0.33828469
## R 0.21918174 0.219806921 0.219890176 0.219557010 0.19435586
## S 0.46901135 0.469109844 0.456764807 0.365540136 0.21813673
## T 0.21586646 0.298115914 0.298178816 0.309052134 0.30211178
## V 0.13283645 0.145276593 0.145276593 0.124632326 0.12150152
## W 0.05070375 0.050787142 0.050870488 0.047757003 0.04681920
## Y 0.15239801 0.152450850 0.171879524 0.311245305 0.31097592
## attr(,"class")
## [1] "immunr_kmer_profile_self" "matrix"                  
## [3] "array"

Sequence motif visualisation

Visualisation of sequence motif matrices is done by vis(). There are two types of plots to choose from - sequence logo and “text logo”. The argument .plot regulates the type of plot: .plot = "seq" for sequence logo plots and .plot = "text" (by default) for “text logo” plots.

kp <- kmer_profile(kmers, "self")
p1 <- vis(kp)
p2 <- vis(kp, .plot = "seq")

p1 + p2

## Warning: ggrepel: 13 unlabeled data points (too many overlaps). Consider
## increasing max.overlaps

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Kmer and sequence motif analysis and visualisation

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Kmer statistics computation

Kmer statistics visualisation

Sequence motifs analysis

Sequence motif visualisation

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