P0 dentate gyrus
library(COTAN)
library(data.table)
library(Matrix)
library(ggrepel)
#> Loading required package: ggplot2
#library(latex2exp)mycolours <- c("A" = "#8491B4B2","B"="#E64B35FF")
my_theme = theme(axis.text.x = element_text(size = 14, angle = 0, hjust = .5, vjust = .5, face = "plain", colour ="#3C5488FF" ),
axis.text.y = element_text( size = 14, angle = 0, hjust = 0, vjust = .5, face = "plain", colour ="#3C5488FF"),
axis.title.x = element_text( size = 14, angle = 0, hjust = .5, vjust = 0, face = "plain", colour ="#3C5488FF"),
axis.title.y = element_text( size = 14, angle = 90, hjust = .5, vjust = .5, face = "plain", colour ="#3C5488FF"))data = as.data.frame(fread(paste(data_dir,"separation_age/P0.csv", sep = "/")))
data = as.data.frame(data)
rownames(data) = data$V1
data = data[,2:ncol(data)]
data[1:10,1:10]
#> X10X83_2_GCTGGAGTTAACGA. X10X83_2_TTCTGAGTAGCCGA.
#> 0610007P14Rik 1 1
#> 0610009B22Rik 1 2
#> 0610009L18Rik 0 0
#> 0610009O20Rik 1 1
#> 0610010F05Rik 0 0
#> 0610010K14Rik 2 0
#> 0610011F06Rik 0 0
#> 0610012D04Rik 0 0
#> 0610012G03Rik 0 1
#> 0610025J13Rik 0 0
#> X10X83_2_TTGAAAGTCCGGTC. X10X83_2_CAGTTAGCTAGGCA.
#> 0610007P14Rik 2 2
#> 0610009B22Rik 1 3
#> 0610009L18Rik 0 0
#> 0610009O20Rik 1 0
#> 0610010F05Rik 0 0
#> 0610010K14Rik 0 0
#> 0610011F06Rik 0 0
#> 0610012D04Rik 0 0
#> 0610012G03Rik 1 1
#> 0610025J13Rik 0 0
#> X10X83_2_CAATTAGGCCCTCA. X10X83_2_GGGATGTTACTGAC.
#> 0610007P14Rik 1 3
#> 0610009B22Rik 0 0
#> 0610009L18Rik 0 1
#> 0610009O20Rik 0 0
#> 0610010F05Rik 0 0
#> 0610010K14Rik 0 0
#> 0610011F06Rik 0 1
#> 0610012D04Rik 0 0
#> 0610012G03Rik 1 2
#> 0610025J13Rik 0 0
#> X10X83_2_CGAAGGTAAACCTC. X10X83_2_AGCTAGTATCACCA.
#> 0610007P14Rik 4 1
#> 0610009B22Rik 0 1
#> 0610009L18Rik 1 0
#> 0610009O20Rik 0 0
#> 0610010F05Rik 0 0
#> 0610010K14Rik 0 1
#> 0610011F06Rik 0 0
#> 0610012D04Rik 0 0
#> 0610012G03Rik 0 0
#> 0610025J13Rik 0 0
#> X10X83_2_GAAGGAGTCGCCGT. X10X83_2_ACCTTAGGGATACC.
#> 0610007P14Rik 2 1
#> 0610009B22Rik 0 0
#> 0610009L18Rik 0 0
#> 0610009O20Rik 0 0
#> 0610010F05Rik 0 0
#> 0610010K14Rik 0 2
#> 0610011F06Rik 0 1
#> 0610012D04Rik 0 0
#> 0610012G03Rik 2 1
#> 0610025J13Rik 0 0Define a directory where the ouput will be stored.
1 Analitical pipeline
Inizialise the COTAN object with the row count table and the metadata for the experiment.
obj = new("scCOTAN",raw = data)
obj = initRaw(obj,GEO="GSE104323" ,sc.method="10X",cond = "mouse dentate gyrus P0")
#> [1] "Initializing S4 object"Now we can start the cleaning. Analysis requires and starts from a matrix of raw UMI counts after removing possible cell doublets or multiplets and low quality or dying cells (with too high mtRNA percentage, easily done with Seurat or other tools).
If we do not want to consider the mithocondrial genes we can remove them before starting the analysis.
genes_to_rem = rownames(obj@raw[grep('^mt', rownames(obj@raw)),]) #genes to remove : mithocondrial
obj@raw = obj@raw[!rownames(obj@raw) %in% genes_to_rem,]
cells_to_rem = colnames(obj@raw[which(colSums(obj@raw) == 0)])
obj@raw = obj@raw[,!colnames(obj@raw) %in% cells_to_rem]We want also to define a prefix to identify the sample.
2 Data cleaning
First, we create the directory to store all information regarding the data cleaning.
if(!file.exists(out_dir)){
dir.create(file.path(out_dir))
}
if(!file.exists(paste(out_dir,"cleaning", sep = ""))){
dir.create(file.path(out_dir, "cleaning"))
}ttm = clean(obj)
#> [1] "Start estimation mu with linear method"
#> [1] 14162 4779
#> rowname X10X83_3_TGAGGAGCCACTAT.
#> 5860 Hba-a1 2193.613491
#> 5862 Hbb-bs 2181.620290
#> 5863 Hbb-bt 548.831701
#> 5861 Hba-a2 152.484978
#> 4626 Fth1 22.273087
#> 5670 Gpx1 21.701982
#> 12978 Tuba1a 15.990934
#> 700 Actb 14.277620
#> 4627 Ftl1 13.706515
#> 7584 Mkrn1 13.706515
#> 10874 Sec61g 12.564305
#> 9552 Prdx2 11.993201
#> 5827 H3f3a 10.850991
#> 11495 Snca 10.279886
#> 1482 Atpif1 6.853257
obj = ttm$object
ttm$pca.cell.2
Run this when B cells need to be removed.
pdf(paste(out_dir,"cleaning/",t,"_",n_it,"_plots_before_cells_exlusion.pdf", sep = ""))
ttm$pca.cell.2
ggplot(ttm$D, aes(x=n,y=means)) + geom_point() +
geom_text_repel(data=subset(ttm$D, n > (max(ttm$D$n)- 15) ), aes(n,means,label=rownames(ttm$D[ttm$D$n > (max(ttm$D$n)- 15),])),
nudge_y = 0.05,
nudge_x = 0.05,
direction = "x",
angle = 90,
vjust = 0,
segment.size = 0.2)+
ggtitle("B cell group genes mean expression")+my_theme +
theme(plot.title = element_text(color = "#3C5488FF", size = 20, face = "italic",vjust = - 5,hjust = 0.02 ))
#> Warning: ggrepel: 11 unlabeled data points (too many overlaps). Consider
#> increasing max.overlaps
dev.off()
#> png
#> 2
if (length(ttm$cl1) < length(ttm$cl2)) {
to_rem = ttm$cl1
}else{
to_rem = ttm$cl2
}
n_it = n_it+1
obj@raw = obj@raw[,!colnames(obj@raw) %in% to_rem]
#obj@raw = obj@raw[rownames(obj@raw) %in% rownames(cells),colnames(obj@raw) %in% colnames(cells)]
gc()
#> used (Mb) gc trigger (Mb) max used (Mb)
#> Ncells 3709753 198.2 6229205 332.7 6229205 332.7
#> Vcells 480834289 3668.5 702577858 5360.3 585412950 4466.4
ttm = clean(obj)
#> [1] "Start estimation mu with linear method"
#> [1] 14162 4778
#> rowname X10X83_2_GTGCGAGTGGAGTC. X10X83_2_TCTCAGTAGAGGAA.
#> 5860 Hba-a1 603.371341 1354.044610
#> 5862 Hbb-bs 579.635254 700.367902
#> 12603 Tmsb4x 148.587907 73.927723
#> 7189 Malat1 49.845784 102.136986
#> 12599 Tmsb10 58.865497 28.209263
#> 4626 Fth1 99.691567 35.018395
#> 700 Actb 9.019713 28.209263
#> 5863 Hbb-bt 8.544991 43.772994
#> 5861 Hba-a2 20.887757 52.527593
#> 2631 Cldn5 57.441331 7.781866
#> 4627 Ftl1 20.413035 26.263796
#> 10525 Rpl41 25.160253 16.536464
#> 10488 Rpl13a 34.179966 29.181996
#> 10559 Rps14 30.382192 29.181996
#> 10494 Rpl18a 34.179966 29.181996
#> X10X83_3_CTCGCTCTGTCTCG. X10X83_3_ACCAACAAATTGCC.
#> 5860 671.4304617 553.155075
#> 5862 666.8525267 524.000732
#> 12603 115.9743525 59.474861
#> 7189 74.0099486 64.917005
#> 12599 43.4903822 49.368021
#> 4626 24.4156532 15.937708
#> 700 16.0227724 89.795378
#> 5863 0.7629892 79.299814
#> 5861 16.7857615 22.546026
#> 2631 41.2014147 0.000000
#> 4627 33.5715231 22.546026
#> 10525 24.4156532 27.988170
#> 10488 20.6007073 6.997042
#> 10559 19.8377182 11.273013
#> 10494 12.9708157 5.830869
#ttm = clean.sqrt(obj, cells)
obj = ttm$object
ttm$pca.cell.2
#> Warning: ggrepel: 12 unlabeled data points (too many overlaps). Consider
#> increasing max.overlaps
#> png
#> 2
#> used (Mb) gc trigger (Mb) max used (Mb)
#> Ncells 3710705 198.2 6229205 332.7 6229205 332.7
#> Vcells 497545165 3796.0 1214345736 9264.8 1214343732 9264.8
#> [1] "Start estimation mu with linear method"
#> [1] 14147 4774
#> rowname X10X83_3_CATCTCATGAAGTA.
#> 12959 Ttr 854.13561
#> 7178 Malat1 144.24771
#> 10513 Rpl41 40.67312
#> 10560 Rps23 35.94369
#> 3758 Eef1a1 29.79543
#> 10566 Rps27a 26.01188
#> 10547 Rps14 24.12011
#> 10482 Rpl18a 23.64717
#> 10509 Rpl37a 23.64717
#> 1810 Bsg 22.22834
#> 4617 Ftl1 22.22834
#> 10554 Rps19 22.22834
#> 4616 Fth1 20.33656
#> 10476 Rpl13a 20.33656
#> 10550 Rps16 19.39068
#> Warning: ggrepel: 13 unlabeled data points (too many overlaps). Consider
#> increasing max.overlaps
#> png
#> 2
#> used (Mb) gc trigger (Mb) max used (Mb)
#> Ncells 3710869 198.2 6229205 332.7 6229205 332.7
#> Vcells 496752912 3790.0 1214345736 9264.8 1214343732 9264.8
#> [1] "Start estimation mu with linear method"
#> [1] 14143 4773
#> rowname X10X83_2_TTCTGAGTAGCCGA. X10X83_2_TTGAAAGTCCGGTC.
#> 4136 Fabp7 275.40317 134.199907
#> 7175 Malat1 68.08861 64.551854
#> 2993 Cst3 38.61742 4.246832
#> 3204 Dbi 42.68241 31.426560
#> 1150 Apoe 27.43869 22.083529
#> 12586 Tmsb4x 67.07236 34.824026
#> 9767 Ptn 30.48744 29.727827
#> 10509 Rpl41 24.38995 26.330361
#> 10550 Rps19 25.40620 41.618958
#> 10470 Rpl13 16.25997 33.974660
#> 10543 Rps14 23.37370 28.878461
#> 3756 Eef1a1 13.21122 28.029094
#> 10478 Rpl18a 11.17873 24.631628
#> 10569 Rps4x 14.22747 33.125293
#> 5816 H3f3b 13.21122 21.234162
#> X10X83_2_CAGTTAGCTAGGCA. X10X83_2_CAATTAGGCCCTCA.
#> 4136 385.62404 209.428139
#> 7175 61.69985 383.293010
#> 2993 27.54457 57.296378
#> 3204 57.29271 33.587532
#> 1150 53.98737 51.369166
#> 12586 70.51411 39.514743
#> 9767 24.23923 29.636057
#> 10509 17.62853 21.733109
#> 10550 18.73031 15.805897
#> 10470 18.73031 3.951474
#> 10543 22.03566 21.733109
#> 3756 29.74814 9.878686
#> 10478 23.13744 9.878686
#> 10569 22.03566 9.878686
#> 5816 18.73031 11.854423
#> X10X83_2_GGGATGTTACTGAC. X10X83_2_CGAAGGTAAACCTC.
#> 4136 262.26683 222.41057
#> 7175 76.38840 146.54960
#> 2993 37.34544 49.99928
#> 3204 41.58924 62.06807
#> 1150 28.85784 15.51702
#> 12586 42.43800 60.34395
#> 9767 65.35452 62.06807
#> 10509 26.31156 17.24113
#> 10550 22.91652 24.13758
#> 10470 22.91652 17.24113
#> 10543 16.97520 29.30992
#> 3756 17.82396 18.96524
#> 10478 14.42892 17.24113
#> 10569 20.37024 29.30992
#> 5816 13.58016 13.79290
#> X10X83_2_GAAGGAGTCGCCGT. X10X83_2_ACCTTAGGGATACC.
#> 4136 194.44733 353.603331
#> 7175 116.66840 94.087436
#> 2993 38.88947 19.644630
#> 3204 26.51554 41.357115
#> 1150 38.88947 17.576774
#> 12586 53.03109 61.001744
#> 9767 22.98014 20.678557
#> 10509 28.28325 24.814269
#> 10550 38.88947 26.882125
#> 10470 35.35406 25.848197
#> 10543 26.51554 14.474990
#> 3756 35.35406 18.610702
#> 10478 31.81865 16.542846
#> 10569 38.88947 22.746413
#> 5816 19.44473 9.305351
#> X10X83_2_GACCTGTCTTGTCC. X10X83_2_AACGAGTGTTCGAT.
#> 4136 120.646849 301.66254
#> 7175 151.968627 45.11668
#> 2993 17.400988 71.65591
#> 3204 32.481844 57.50166
#> 1150 6.960395 34.50099
#> 12586 18.561054 58.38630
#> 9767 100.925730 42.46276
#> 10509 42.922437 30.07779
#> 10550 42.922437 10.61569
#> 10470 44.082503 17.69282
#> 10543 31.321778 26.53923
#> 3756 25.521449 20.34674
#> 10478 29.001646 12.38497
#> 10569 18.561054 15.03889
#> 5816 16.240922 20.34674
#> X10X83_2_CCGAACACGGTGTC. X10X83_2_CAGAAGTAAGTTCC.
#> 4136 273.451720 172.61317
#> 7175 128.826144 198.23543
#> 2993 29.168184 24.27373
#> 3204 34.029547 41.80475
#> 1150 35.244888 25.62227
#> 12586 25.522161 37.75913
#> 9767 29.168184 47.19891
#> 10509 26.737502 32.36497
#> 10550 18.230115 20.22811
#> 10470 25.522161 25.62227
#> 10543 25.522161 29.66789
#> 3756 29.168184 16.18248
#> 10478 27.952843 18.87957
#> 10569 30.383524 17.53102
#> 5816 9.722728 22.92519
#> X10X83_2_TATGCAGCTAGTCT. X10X83_2_AGCCGGTCGAACAG.
#> 4136 244.675951 365.744171
#> 7175 193.753545 179.618134
#> 2993 70.794565 81.999583
#> 3204 53.406426 84.602744
#> 1150 79.488634 52.063227
#> 12586 29.808238 49.460066
#> 9767 31.050248 23.428452
#> 10509 26.082208 15.618968
#> 10550 19.872158 11.714226
#> 10470 18.630149 13.015807
#> 10543 19.872158 7.809484
#> 3756 19.872158 10.412645
#> 10478 16.146129 5.206323
#> 10569 22.356178 16.920549
#> 5816 7.452059 10.412645
#> X10X83_2_TTCCCCAGTACACT. X10X83_2_ACCTCTCGACCAGC.
#> 4136 212.813620 295.86300
#> 7175 0.000000 84.93008
#> 2993 102.465817 46.64193
#> 3204 43.350923 36.89586
#> 1150 126.111775 38.28815
#> 12586 59.114894 80.05705
#> 9767 55.173901 50.12267
#> 10509 45.321419 22.27674
#> 10550 47.291916 27.84593
#> 10470 31.527944 18.09985
#> 10543 41.380426 18.09985
#> 3756 25.616454 20.88445
#> 10478 39.409930 18.79600
#> 10569 41.380426 23.66904
#> 5816 5.911489 12.53067
#> X10X83_2_GCGTTGTTGCGTTA. X10X83_2_TAAGCTCCTAGGGC.
#> 4136 123.43190 337.80733
#> 7175 105.14569 98.65170
#> 2993 48.76322 46.33640
#> 3204 56.38247 29.89445
#> 1150 77.71638 83.70447
#> 12586 19.81006 35.87334
#> 9767 143.24196 35.87334
#> 10509 32.00086 10.46306
#> 10550 21.33391 16.44195
#> 10470 25.90546 11.95778
#> 10543 21.33391 10.46306
#> 3756 18.28621 16.44195
#> 10478 25.90546 13.45250
#> 10569 22.85776 16.44195
#> 5816 28.95316 17.93667
#> X10X83_2_TAGGCTCGGCGCAT. X10X83_2_TGGCTCACTCAGGC.
#> 4136 364.40724 156.708971
#> 7175 109.07760 148.192179
#> 2993 50.38114 122.641803
#> 3204 52.82682 59.617543
#> 1150 81.68592 54.507468
#> 12586 33.26133 35.770526
#> 9767 57.22906 28.957092
#> 10509 26.90255 15.330225
#> 10550 28.36996 22.143659
#> 10470 28.85910 13.626867
#> 10543 28.36996 20.440301
#> 3756 30.32651 28.957092
#> 10478 30.81565 20.440301
#> 10569 25.92427 25.550376
#> 5816 15.65239 6.813434
#> X10X83_2_CTTCAGTATGAAAC. X10X83_2_TCACTGTCTAAACC.
#> 4136 177.78995 353.523542
#> 7175 198.50334 55.735694
#> 2993 75.94910 65.290384
#> 3204 18.98728 55.735694
#> 1150 120.82812 47.773452
#> 12586 29.34397 52.550797
#> 9767 44.87902 41.403658
#> 10509 20.71339 30.256519
#> 10550 20.71339 23.886726
#> 10470 24.16562 22.294277
#> 10543 13.80893 15.924484
#> 3756 15.53504 12.739587
#> 10478 17.26116 12.739587
#> 10569 20.71339 7.962242
#> 5816 22.43951 22.294277
#> X10X83_2_CACGATCAATCACG. X10X83_2_CGAAAGTTTAGCTG.
#> 4136 260.526175 195.74187
#> 7175 164.244763 122.22380
#> 2993 152.917538 26.65030
#> 3204 77.402704 41.35392
#> 1150 50.972513 17.46054
#> 12586 28.318063 105.68223
#> 9767 15.102967 53.30060
#> 10509 18.878708 14.70362
#> 10550 18.878708 21.13645
#> 10470 15.102967 26.65030
#> 10543 5.663613 18.37952
#> 3756 11.327225 17.46054
#> 10478 7.551483 13.78464
#> 10569 11.327225 10.10874
#> 5816 30.205933 23.89337
#> X10X83_2_CGGGTCAAGTCATC. X10X83_2_AGTGAAGTTAAGTG.
#> 4136 452.419536 266.55994
#> 7175 136.012202 0.00000
#> 2993 75.880492 33.00266
#> 3204 50.109759 50.77332
#> 1150 57.268296 15.23200
#> 12586 37.224392 27.92533
#> 9767 48.678051 50.77332
#> 10509 11.453659 30.46399
#> 10550 10.021952 45.69599
#> 10470 17.180489 45.69599
#> 10543 14.317074 22.84800
#> 3756 8.590244 15.23200
#> 10478 21.475611 35.54133
#> 10569 12.885367 33.00266
#> 5816 24.339026 17.77066
#> X10X83_2_CGTCGGTAGTGAAT. X10X83_2_CATCTTCTGAGTGT.
#> 4136 456.79845 251.650947
#> 7175 60.28812 60.825584
#> 2993 41.73793 8.348610
#> 3204 44.05670 32.201780
#> 1150 59.12873 9.541268
#> 12586 60.28812 54.862292
#> 9767 35.94099 26.238487
#> 10509 37.10038 29.816463
#> 10550 24.34713 29.816463
#> 10470 19.70958 16.697219
#> 10543 33.62222 19.082536
#> 3756 22.02835 20.275195
#> 10478 20.86896 15.504561
#> 10569 23.18774 28.623804
#> 5816 20.86896 15.504561
#> X10X83_2_CAGTTGTTAAGAAC. X10X83_2_TGGAACATTTCAGG.
#> 4136 311.59527 285.85287
#> 7175 189.22789 202.81942
#> 2993 65.59900 68.06021
#> 3204 42.89166 29.94649
#> 1150 108.49066 69.42141
#> 12586 37.84558 32.66890
#> 9767 54.24533 63.97660
#> 10509 16.39975 29.94649
#> 10550 15.13823 10.88963
#> 10470 12.61519 28.58529
#> 10543 17.66127 24.50167
#> 3756 13.87671 24.50167
#> 10478 20.18431 24.50167
#> 10569 11.35367 19.05686
#> 5816 21.44583 17.69565
#> X10X83_2_AGTTACATCGGGTC. X10X83_2_ACCAACACCGAATT.
#> 4136 172.10046 284.718799
#> 7175 349.75254 162.835751
#> 2993 47.18883 30.226996
#> 3204 27.75814 43.877897
#> 1150 22.20651 25.351674
#> 12586 55.51628 39.002575
#> 9767 58.29209 25.351674
#> 10509 22.20651 31.202060
#> 10550 24.98232 18.526223
#> 10470 24.98232 13.650901
#> 10543 36.08558 16.576094
#> 3756 24.98232 8.775579
#> 10478 33.30977 19.501288
#> 10569 16.65488 19.501288
#> 5816 33.30977 14.625966
#> X10X83_2_GCCAGGTAAGAGAG. X10X83_2_CCTATGTGCACTTA.
#> 4136 260.105536 276.419413
#> 7175 324.737820 196.844128
#> 2993 61.479490 60.728508
#> 3204 47.291916 50.258075
#> 1150 40.986327 14.658605
#> 12586 17.340369 33.505383
#> 9767 122.958980 20.940865
#> 10509 18.916766 23.034951
#> 10550 18.916766 29.317211
#> 10470 23.645958 27.223124
#> 10543 17.340369 18.846778
#> 3756 7.881986 8.376346
#> 10478 14.187575 12.564519
#> 10569 14.187575 10.470432
#> 5816 29.951547 4.188173
#> X10X83_2_GCCGCTCCACGTTC. X10X83_2_CTACTCATTTGCTT.
#> 4136 280.721544 169.80050
#> 7175 126.777472 318.94958
#> 2993 53.039554 114.73007
#> 3204 29.753896 32.12442
#> 1150 21.992010 66.54344
#> 12586 37.515782 29.82982
#> 9767 15.523772 29.82982
#> 10509 43.337197 11.47301
#> 10550 32.988016 20.65141
#> 10470 30.400720 20.65141
#> 10543 26.519777 27.53522
#> 3756 25.226130 16.06221
#> 10478 25.872953 11.47301
#> 10569 24.579306 20.65141
#> 5816 9.055534 25.24061
#> X10X83_2_ACATTTCTTGAGGT. X10X83_2_ATGCGAGTGTTTGC.
#> 4136 257.84960 138.209707
#> 7175 343.10632 240.752392
#> 2993 33.27092 57.958909
#> 3204 76.93899 46.812965
#> 1150 10.39716 28.979455
#> 12586 39.50921 15.604322
#> 9767 39.50921 22.291888
#> 10509 31.19148 35.667021
#> 10550 31.19148 28.979455
#> 10470 14.55603 26.750266
#> 10543 29.11205 40.125399
#> 3756 14.55603 20.062699
#> 10478 35.35035 28.979455
#> 10569 20.79432 20.062699
#> 5816 12.47659 6.687566
#> X10X83_2_ACCCATCAGGCGAA. X10X83_2_GGCAAAGCCACGTC.
#> 4136 294.369955 177.03559
#> 7175 317.431196 134.45741
#> 2993 42.052851 17.92765
#> 3204 58.331374 29.13244
#> 1150 44.765938 22.40957
#> 12586 18.991610 64.98775
#> 9767 25.774328 24.65052
#> 10509 21.704697 33.61435
#> 10550 24.417784 31.37339
#> 10470 35.270133 29.13244
#> 10543 24.417784 22.40957
#> 3756 21.704697 42.57818
#> 10478 17.635066 17.92765
#> 10569 8.139261 24.65052
#> 5816 10.852349 13.44574
#> X10X83_2_CGAGGCACCGAAAG. X10X83_2_CATAGGTATGCTTG.
#> 4136 262.485646 229.10630
#> 7175 265.338750 268.05437
#> 2993 57.062097 34.36594
#> 3204 65.621411 71.02295
#> 1150 31.384153 13.74638
#> 12586 28.531048 38.94807
#> 9767 19.971734 41.23913
#> 10509 25.677944 25.20169
#> 10550 24.251391 18.32850
#> 10470 19.971734 11.45531
#> 10543 19.971734 27.49276
#> 3756 18.545181 20.61957
#> 10478 21.398286 16.03744
#> 10569 8.559315 22.91063
#> 5816 11.412419 16.03744
#> X10X83_2_AGCCGGTCAAACTC. X10X83_2_TCTGTAGATAGTCA.
#> 4136 144.54171 137.518226
#> 7175 225.21616 284.328224
#> 2993 40.33722 76.192531
#> 3204 48.74081 83.625948
#> 1150 20.16861 27.875316
#> 12586 30.25292 31.592025
#> 9767 10.08431 14.866835
#> 10509 20.16861 35.308734
#> 10550 42.01794 20.441898
#> 10470 20.16861 22.300253
#> 10543 42.01794 39.025443
#> 3756 31.93363 9.291772
#> 10478 13.44574 22.300253
#> 10569 31.93363 22.300253
#> 5816 30.25292 26.016962
#> X10X83_2_GTTCATCAAGGTAA. X10X83_2_CAGGCGTGTTCTTT.
#> 4136 354.30603 256.79705
#> 7175 248.01422 154.89346
#> 2993 70.86121 39.40272
#> 3204 57.80783 54.34858
#> 1150 52.21352 36.68529
#> 12586 22.37722 31.25043
#> 9767 33.56583 23.09815
#> 10509 24.24199 23.09815
#> 10550 27.97153 40.76144
#> 10470 20.51245 17.66329
#> 10543 24.24199 42.12015
#> 3756 11.18861 31.25043
#> 10478 14.91815 28.53301
#> 10569 14.91815 27.17429
#> 5816 13.05338 20.38072
#> X10X83_2_AGCCAAGCGTGAGT. X10X83_2_AGAAATCATCTGCC.
#> 4136 261.68308 229.306475
#> 7175 144.89890 183.824199
#> 2993 48.66008 53.062655
#> 3204 72.44945 49.272466
#> 1150 17.30136 45.482276
#> 12586 58.39209 5.685284
#> 9767 22.70804 32.216612
#> 10509 14.05736 22.741138
#> 10550 20.54537 22.741138
#> 10470 12.97602 18.950948
#> 10543 12.97602 32.216612
#> 3756 8.65068 22.741138
#> 10478 14.05736 17.055853
#> 10569 15.13869 15.160759
#> 5816 12.97602 17.055853
#> X10X83_2_GGTAGCACGAGAGT. X10X83_2_GTGTGTCAACTCTT.
#> 4136 288.682840 285.094311
#> 7175 161.662390 262.446632
#> 2993 51.962911 85.261850
#> 3204 88.529404 59.949738
#> 1150 30.792836 55.953089
#> 12586 36.566493 30.640977
#> 9767 28.868284 31.973194
#> 10509 15.396418 21.315463
#> 10550 19.245523 14.654380
#> 10470 21.170075 9.325515
#> 10543 19.245523 21.315463
#> 3756 21.170075 9.325515
#> 10478 26.943732 13.322164
#> 10569 5.773657 13.322164
#> 5816 21.170075 14.654380
#> X10X83_2_TACTCCACTTCGAA. X10X83_2_CATACAGGTGCTTT.
#> 4136 287.18568 231.323599
#> 7175 367.22104 178.490431
#> 2993 14.12389 78.535790
#> 3204 80.03535 71.396172
#> 1150 10.98524 42.837703
#> 12586 18.83185 28.558469
#> 9767 18.83185 21.418852
#> 10509 26.67845 9.995464
#> 10550 31.38641 9.995464
#> 10470 18.83185 14.279234
#> 10543 26.67845 12.851311
#> 3756 18.83185 11.423388
#> 10478 29.81709 15.707158
#> 10569 15.69321 12.851311
#> 5816 18.83185 11.423388
#> X10X83_2_GCCACGTGTCCTCT. X10X83_2_GCAGTCAGTTCATG.
#> 4136 296.451092 184.94525
#> 7175 169.218864 246.04323
#> 2993 35.625024 36.32853
#> 3204 68.705403 59.44669
#> 1150 29.263413 13.21037
#> 12586 43.258958 11.55908
#> 9767 36.897346 24.76945
#> 10509 12.723223 24.76945
#> 10550 15.267867 51.19020
#> 10470 11.450901 28.07205
#> 10543 11.450901 29.72334
#> 3756 11.450901 13.21037
#> 10478 8.906256 16.51297
#> 10569 10.178578 19.81556
#> 5816 6.361611 42.93372
#> X10X83_2_ACGGCCACACGCTG. X10X83_2_GCGCACAACACCTA.
#> 4136 183.13921 112.204168
#> 7175 256.69512 275.756007
#> 2993 30.02282 9.508828
#> 3204 61.54678 32.330015
#> 1150 18.01369 7.607062
#> 12586 39.02967 26.624718
#> 9767 25.51940 11.410593
#> 10509 24.01826 28.526483
#> 10550 22.51712 38.035311
#> 10470 13.51027 39.937077
#> 10543 24.01826 19.017656
#> 3756 15.01141 28.526483
#> 10478 22.51712 30.428249
#> 10569 21.01597 22.821187
#> 5816 19.51483 11.410593
#> X10X83_2_GATCCGTACAGTGG. X10X83_2_TAACTCAGTGGAGT.
#> 4136 307.369263 275.217307
#> 7175 184.748546 206.762241
#> 2993 71.937487 55.881687
#> 3204 73.572430 82.425488
#> 1150 78.477259 61.469855
#> 12586 26.159086 36.323096
#> 9767 11.444600 29.337886
#> 10509 22.889200 29.337886
#> 10550 9.809657 19.558590
#> 10470 17.984372 20.955633
#> 10543 17.984372 22.352675
#> 3756 11.444600 18.161548
#> 10478 11.444600 16.764506
#> 10569 9.809657 8.382253
#> 5816 35.968743 16.764506
#> X10X83_2_CCATGAGCTGATAA. X10X83_2_TTACTAGGCAATTA.
#> 4136 297.58594 195.04111
#> 7175 252.33895 131.12007
#> 2993 111.37719 22.94601
#> 3204 62.64967 42.61402
#> 1150 95.71477 22.94601
#> 12586 29.58457 18.02901
#> 9767 20.88322 29.50202
#> 10509 17.40269 27.86302
#> 10550 15.66242 29.50202
#> 10470 12.18188 26.22401
#> 10543 29.58457 22.94601
#> 3756 22.62349 19.66801
#> 10478 10.44161 27.86302
#> 10569 17.40269 21.30701
#> 5816 26.10403 11.47301
#> X10X83_2_GACGCCATACTCTT. X10X83_2_GGCGTTCGTCTGAA.
#> 4136 119.859174 164.73583
#> 7175 271.347853 80.80199
#> 2993 13.317686 10.02195
#> 3204 41.617769 32.57134
#> 1150 4.994132 15.03293
#> 12586 9.988265 67.64817
#> 9767 18.311818 21.29665
#> 10509 24.970661 16.91204
#> 10550 28.300083 25.68125
#> 10470 31.629504 24.42851
#> 10543 29.964794 21.92302
#> 3756 33.294215 26.30762
#> 10478 38.288347 20.04390
#> 10569 21.641240 23.80214
#> 5816 19.976529 38.83506
#> X10X83_2_TGTTTAGATATGCA. X10X83_2_ACCTACATTCCTCG.
#> 4136 338.27663 193.73713
#> 7175 33.53179 117.81312
#> 2993 14.79344 13.09035
#> 3204 40.43540 23.56262
#> 1150 21.69704 1.74538
#> 12586 83.82949 46.25256
#> 9767 28.60065 19.19918
#> 10509 26.62819 32.28952
#> 10550 19.72458 41.01642
#> 10470 16.76590 31.41683
#> 10543 22.68327 25.30800
#> 3756 16.76590 34.03490
#> 10478 13.80721 22.68993
#> 10569 13.80721 23.56262
#> 5816 25.64196 22.68993
#> X10X83_2_CTCCTTCACCAGGC. X10X83_2_GCGAGCACGCATCG.
#> 4136 244.79837 131.122753
#> 7175 41.22920 111.697160
#> 2993 45.52391 27.924290
#> 3204 34.35767 40.065286
#> 1150 24.90931 9.712797
#> 12586 25.76825 52.206281
#> 9767 30.92190 37.637087
#> 10509 18.89672 48.563983
#> 10550 23.19142 26.710191
#> 10470 15.46095 46.135784
#> 10543 13.74307 24.281991
#> 3756 16.31989 15.783294
#> 10478 10.30730 19.425593
#> 10569 17.17883 14.569195
#> 5816 27.48613 26.710191
#> X10X83_2_ACTAGGTGTTAAGA. X10X83_2_AAGAGGTAAGTGTA.
#> 4136 147.862065 0.000000
#> 7175 99.871746 148.429915
#> 2993 3.891107 0.000000
#> 3204 15.564428 0.000000
#> 1150 23.346642 0.000000
#> 12586 101.168781 26.193514
#> 9767 16.861464 0.000000
#> 10509 32.425891 20.372733
#> 10550 31.128856 17.462343
#> 10470 22.049606 14.551952
#> 10543 15.564428 11.641562
#> 3756 33.722927 14.551952
#> 10478 24.643678 0.000000
#> 10569 22.049606 0.000000
#> 5816 10.376285 8.731171
#> X10X83_2_TTGGTAGTCTTGCA. X10X83_3_GGCAAAGTTCGCAT.
#> 4136 0.000000 135.76841
#> 7175 163.116404 174.95930
#> 2993 4.292537 40.59056
#> 3204 0.000000 25.19414
#> 1150 2.146268 50.38828
#> 12586 30.047759 34.99186
#> 9767 0.000000 88.17949
#> 10509 12.877611 34.99186
#> 10550 12.877611 39.19088
#> 10470 4.292537 33.59218
#> 10543 4.292537 27.99349
#> 3756 27.901490 46.18925
#> 10478 4.292537 27.99349
#> 10569 6.438805 11.19739
#> 5816 8.585074 26.59381
#> X10X83_3_TACAGAGTGCAAGC. X10X83_3_TGCAGGTGATAAAC.
#> 4136 274.36119 242.52795
#> 7175 91.45373 99.60969
#> 2993 79.65325 71.45913
#> 3204 41.30168 40.06042
#> 1150 134.23047 81.20355
#> 12586 13.27554 16.24071
#> 9767 30.97626 122.34669
#> 10509 17.70072 33.56414
#> 10550 26.55108 25.98514
#> 10470 39.82662 25.98514
#> 10543 25.07602 30.31599
#> 3756 28.02614 20.57157
#> 10478 35.40144 19.48885
#> 10569 22.12590 16.24071
#> 5816 14.75060 25.98514
#> X10X83_3_TCCAGAGCGCCTTG. X10X83_3_GGCAGAGTTCGCAT.
#> 4136 152.979387 284.744160
#> 7175 46.357390 168.134647
#> 2993 6.953608 112.541739
#> 3204 17.384021 47.457360
#> 1150 2.317869 113.897664
#> 12586 50.993129 42.033662
#> 9767 18.542956 24.406642
#> 10509 28.973369 20.338869
#> 10550 20.860825 5.423698
#> 10470 40.562716 12.203321
#> 10543 24.337630 10.847397
#> 3756 37.085912 5.423698
#> 10478 22.019760 8.135547
#> 10569 23.178695 8.135547
#> 5816 12.748282 16.271095
#> X10X83_3_AGGATTCCACGAAT. X10X83_3_GACGTGTATAGGGC.
#> 4136 181.24050 253.295686
#> 7175 79.69440 199.402987
#> 2993 24.42248 39.072207
#> 3204 32.13484 48.503429
#> 1150 25.70787 40.419524
#> 12586 61.05620 22.904397
#> 9767 26.35057 29.640984
#> 10509 39.20451 21.557080
#> 10550 25.70787 21.557080
#> 10470 28.92136 26.946350
#> 10543 18.63821 21.557080
#> 3756 21.85169 20.209762
#> 10478 20.56630 17.515127
#> 10569 31.49214 22.904397
#> 5816 10.28315 9.431222
#> X10X83_3_GTTCAAGACGCTTT. X10X83_3_AACGATCTCGCTTG.
#> 4136 198.66245 185.57610
#> 7175 154.11390 199.32248
#> 2993 62.60877 66.44083
#> 3204 58.99673 29.78382
#> 1150 52.97665 66.44083
#> 12586 19.26424 25.20169
#> 9767 37.32446 66.44083
#> 10509 28.89636 27.49276
#> 10550 20.46825 18.32850
#> 10470 25.28431 29.78382
#> 10543 25.28431 11.45531
#> 3756 16.85621 18.32850
#> 10478 20.46825 22.91063
#> 10569 22.87628 20.61957
#> 5816 26.48833 11.45531
#> X10X83_3_CAGTACAATCGGTT. X10X83_3_CCTGGTCACGCGGT.
#> 4136 270.28001 332.27619
#> 7175 65.99861 85.21738
#> 2993 17.59963 35.08951
#> 3204 33.31358 41.53452
#> 1150 18.22819 63.01790
#> 12586 68.51284 39.38619
#> 9767 22.62809 27.92839
#> 10509 18.22819 29.36061
#> 10550 24.51377 20.05115
#> 10470 21.99954 25.06394
#> 10543 16.34251 20.76726
#> 3756 14.45684 19.33504
#> 10478 18.85674 22.91560
#> 10569 21.99954 20.76726
#> 5816 10.05693 13.60614
#> X10X83_3_GGGTCAGTGAAGAG. X10X83_3_TCAGTAGGCTATCT.
#> 4136 260.40881 129.45988
#> 7175 65.91598 188.79565
#> 2993 78.93642 17.53102
#> 3204 52.08176 31.01643
#> 1150 46.38532 18.87957
#> 12586 33.36488 57.98724
#> 9767 54.52310 12.13686
#> 10509 24.41333 37.75913
#> 10550 22.78577 31.01643
#> 10470 23.59955 31.01643
#> 10543 23.59955 32.36497
#> 3756 21.97199 33.71351
#> 10478 21.15822 32.36497
#> 10569 17.90311 14.83394
#> 5816 17.90311 12.13686
#> X10X83_3_GAAGTGTGCACTTA. X10X83_3_AGCAAGTACAGACG.
#> 4136 222.61966 164.504575
#> 7175 78.84446 335.960047
#> 2993 25.04471 92.678634
#> 3204 44.52393 67.192009
#> 1150 23.18955 53.290214
#> 12586 43.59635 23.169658
#> 9767 47.30668 39.388419
#> 10509 29.68262 13.901795
#> 10550 44.52393 6.950898
#> 10470 33.39295 16.218761
#> 10543 24.11713 18.535727
#> 3756 36.17570 2.316966
#> 10478 28.75504 11.584829
#> 10569 25.04471 6.950898
#> 5816 18.55164 27.803590
#> X10X83_3_CCAAGAGCTGGAAC. X10X83_3_CTCAAGTGTCTGAT.
#> 4136 356.50438 320.360673
#> 7175 118.83479 199.408174
#> 2993 103.50127 91.531621
#> 3204 71.55643 35.958851
#> 1150 44.72277 124.221486
#> 12586 51.11174 27.786385
#> 9767 51.11174 24.517398
#> 10509 17.88911 26.151892
#> 10550 23.00028 13.075946
#> 10470 24.27808 26.151892
#> 10543 21.72249 9.806959
#> 3756 10.22235 9.806959
#> 10478 14.05573 14.710439
#> 10569 12.77793 14.710439
#> 5816 16.61131 27.786385
#> X10X83_3_CTTAGGTAGAGCTG. X10X83_3_GAGCTAGAGCCCAA.
#> 4136 274.729405 136.44683
#> 7175 168.382539 59.26478
#> 2993 113.436658 16.53901
#> 3204 31.904060 28.94327
#> 1150 143.568270 38.59102
#> 12586 26.586717 50.99528
#> 9767 33.676508 24.80851
#> 10509 12.407134 33.07802
#> 10550 5.317343 39.96927
#> 10470 15.952030 31.69977
#> 10543 8.862239 31.69977
#> 3756 8.862239 41.34752
#> 10478 8.862239 22.05201
#> 10569 5.317343 30.32152
#> 5816 31.904060 17.91726
#> X10X83_3_CTTGAGTCAGAAGC. X10X83_3_AGCGAAGTGTTGAA.
#> 4136 235.66810 328.013195
#> 7175 239.46920 64.633142
#> 2993 72.22087 40.395714
#> 3204 76.02197 56.553999
#> 1150 72.22087 35.548228
#> 12586 30.40879 46.859028
#> 9767 81.72362 27.469085
#> 10509 15.20439 32.316571
#> 10550 24.70714 19.389943
#> 10470 24.70714 21.005771
#> 10543 30.40879 12.926628
#> 3756 17.10494 21.005771
#> 10478 19.00549 24.237428
#> 10569 11.40330 9.694971
#> 5816 30.40879 11.310800
#> X10X83_3_CCCTACATCGGAAG. X10X83_3_CTGCCAGCGCCTTG.
#> 4136 316.047900 236.411340
#> 7175 64.526446 214.588755
#> 2993 61.892714 101.838731
#> 3204 38.189121 25.459683
#> 1150 82.962574 149.120999
#> 12586 44.773453 24.247317
#> 9767 46.090319 27.884415
#> 10509 18.436128 16.973122
#> 10550 28.971058 13.336024
#> 10470 22.386726 13.336024
#> 10543 15.802395 12.123658
#> 3756 17.119261 7.274195
#> 10478 7.901198 7.274195
#> 10569 19.752994 15.760756
#> 5816 6.584331 15.760756
#> X10X83_3_AACTTAGCTCCCAG. X10X83_3_CAGAAGTTTCGCTC.
#> 4136 140.00779 258.768481
#> 7175 83.58674 134.675046
#> 2993 21.94152 57.717877
#> 3204 32.38986 24.049115
#> 1150 21.94152 51.946089
#> 12586 54.33138 18.277328
#> 9767 21.94152 75.033240
#> 10509 44.92787 26.935009
#> 10550 48.06238 26.935009
#> 10470 41.79337 27.896974
#> 10543 40.74854 29.820903
#> 3756 38.65887 21.163222
#> 10478 28.21053 16.353398
#> 10569 38.65887 24.049115
#> 5816 22.98635 7.695717
#> X10X83_3_CCTTATCGCTTAGA. X10X83_3_TGTAAGTGTGACGA.
#> 4136 84.41786 226.029514
#> 7175 85.54343 105.220636
#> 2993 29.26486 38.970606
#> 3204 18.00914 50.661788
#> 1150 11.25571 23.382363
#> 12586 102.42700 66.250030
#> 9767 27.01372 58.455909
#> 10509 22.51143 21.433833
#> 10550 20.26029 33.125015
#> 10470 20.26029 21.433833
#> 10543 14.63243 17.536773
#> 3756 23.63700 9.742651
#> 10478 18.00914 17.536773
#> 10569 20.26029 17.536773
#> 5816 22.51143 15.588242
#> X10X83_3_AGACCGTAAACGCG. X10X83_3_ACGAATCGCCCTTA.
#> 4136 204.08097 166.45650
#> 7175 179.41185 243.18255
#> 2993 72.88606 32.51104
#> 3204 59.43017 50.71722
#> 1150 96.43387 61.12075
#> 12586 43.73164 27.30927
#> 9767 42.61031 87.12958
#> 10509 23.54780 41.61413
#> 10550 22.42648 29.91015
#> 10470 23.54780 37.71280
#> 10543 22.42648 48.11633
#> 3756 25.79045 32.51104
#> 10478 19.06251 27.30927
#> 10569 22.42648 31.21059
#> 5816 21.30516 20.80706
#> X10X83_3_TGACATCCTTTACA. X10X83_3_ATGAAGTTCACCTC.
#> 4136 143.046867 171.66900
#> 7175 54.125841 320.85754
#> 2993 9.278716 18.39311
#> 3204 23.970015 22.48046
#> 1150 6.959037 44.96093
#> 12586 51.806163 16.34943
#> 9767 17.784205 38.82989
#> 10509 30.155826 26.56782
#> 10550 24.743242 24.52414
#> 10470 38.661315 24.52414
#> 10543 26.289694 28.61150
#> 3756 41.754221 28.61150
#> 10478 20.103884 26.56782
#> 10569 27.062921 24.52414
#> 5816 15.464526 20.43679
#> X10X83_3_ACCTGCATATACCG. X10X83_3_CCAATGTAGCGCAA.
#> 4136 281.97670 167.46820
#> 7175 87.61988 95.93656
#> 2993 18.32052 21.03872
#> 3204 28.67560 42.07744
#> 1150 18.32052 14.30633
#> 12586 22.30324 171.67595
#> 9767 34.25141 53.85912
#> 10509 29.47214 32.82040
#> 10550 20.71015 41.23589
#> 10470 28.67560 36.18660
#> 10543 25.48942 27.77111
#> 3756 16.72743 25.24646
#> 10478 11.94817 24.40491
#> 10569 19.91361 26.92956
#> 5816 16.72743 16.83098
#> X10X83_3_AGCCTGTCTCTCTG. X10X83_3_ACGTACAGGCAGTA.
#> 4136 304.57241 193.926542
#> 7175 126.58389 165.114598
#> 2993 28.91510 57.623887
#> 3204 36.62580 54.299432
#> 1150 32.12789 59.840190
#> 12586 105.37948 45.434218
#> 9767 20.56185 41.001612
#> 10509 30.20022 7.757062
#> 10550 18.63418 23.271185
#> 10470 23.13208 19.946730
#> 10543 10.28093 27.703792
#> 3756 16.70650 18.838578
#> 10478 14.77883 21.054882
#> 10569 10.92348 22.163033
#> 5816 10.28093 17.730427
#> X10X83_3_GTCCGGTGTATGGG. X10X83_3_TCAGTCATGAGCGA.
#> 4136 236.61745 287.812939
#> 7175 217.74612 108.729332
#> 2993 46.45251 34.537553
#> 3204 46.45251 71.633442
#> 1150 42.09758 35.816721
#> 12586 20.32297 26.862541
#> 9767 29.03282 69.075105
#> 10509 24.67789 23.025035
#> 10550 37.74266 23.025035
#> 10470 17.41969 17.908361
#> 10543 27.58117 23.025035
#> 3756 15.96805 17.908361
#> 10478 27.58117 7.675012
#> 10569 14.51641 19.187529
#> 5816 14.51641 23.025035
#> X10X83_3_CATTTCAGATAATG. X10X83_3_ACTCCAGGGAAACA.
#> 4136 291.78316 158.145994
#> 7175 309.67552 158.145994
#> 2993 127.99922 61.077074
#> 3204 42.66641 38.173171
#> 1150 60.55877 75.255680
#> 12586 26.15038 28.357213
#> 9767 81.20380 61.077074
#> 10509 17.89236 14.178606
#> 10550 12.38702 14.178606
#> 10470 16.51603 22.903903
#> 10543 15.13969 19.631917
#> 3756 9.63435 10.906620
#> 10478 12.38702 19.631917
#> 10569 13.76336 9.815958
#> 5816 28.90305 11.997282
#> X10X83_3_TATCCGTCCGCTGA. X10X83_3_TATGGTCAGTCCCT.
#> 4136 180.71047 236.60257
#> 7175 116.28326 108.81722
#> 2993 21.99954 30.94802
#> 3204 34.57070 43.92622
#> 1150 17.28535 52.91112
#> 12586 53.42744 25.95640
#> 9767 48.71326 73.87591
#> 10509 29.85651 21.96311
#> 10550 32.99930 23.95975
#> 10470 37.71349 19.96646
#> 10543 34.57070 26.95472
#> 3756 25.14233 30.94802
#> 10478 32.99930 18.96814
#> 10569 18.85674 37.93628
#> 5816 18.85674 14.97485
#> X10X83_3_GTAGGAGATGCCTT. X10X83_3_CTTACTCTTAACCT.
#> 4136 259.20348 221.390600
#> 7175 74.65060 329.039240
#> 2993 23.84672 83.275363
#> 3204 46.65663 67.026512
#> 1150 49.76707 103.586427
#> 12586 34.21486 12.186639
#> 9767 24.88353 20.311064
#> 10509 19.69946 30.466596
#> 10550 11.40495 8.124426
#> 10470 18.66265 16.248851
#> 10543 16.58902 28.435490
#> 3756 14.51539 2.031106
#> 10478 15.55221 16.248851
#> 10569 26.95716 8.124426
#> 5816 17.62584 26.404383
#> X10X83_3_TGATGTCTCCGGTT. X10X83_3_CTCTATCAAAGACA.
#> 4136 278.464440 289.399108
#> 7175 206.405659 256.095242
#> 2993 57.402757 8.038864
#> 3204 62.288098 52.826821
#> 1150 50.074746 12.632501
#> 12586 41.525399 29.858638
#> 9767 43.968069 47.084775
#> 10509 19.541364 20.671365
#> 10550 18.320029 28.710229
#> 10470 8.549347 25.265001
#> 10543 13.434688 17.226137
#> 3756 7.328012 2.296818
#> 10478 10.992017 9.187273
#> 10569 14.656023 17.226137
#> 5816 18.320029 17.226137
#> X10X83_3_AGAAGAGGAATTAC. X10X83_3_ACTAGCAAACAACA.
#> 4136 183.38873 311.100575
#> 7175 155.88042 129.532980
#> 2993 21.39535 45.391899
#> 3204 41.56811 68.641408
#> 1150 20.78406 58.677333
#> 12586 19.56146 28.785107
#> 9767 18.95017 14.392553
#> 10509 25.67442 17.713912
#> 10550 33.62127 14.392553
#> 10470 32.39868 18.821031
#> 10543 26.89701 7.749836
#> 3756 22.00665 9.964075
#> 10478 29.95349 9.964075
#> 10569 25.67442 15.499673
#> 5816 15.89369 12.178314
#> X10X83_3_CGTAATCTGATACG. X10X83_3_CTCAGCATTCCTCG.
#> 4136 272.04859 185.17678
#> 7175 151.90095 228.57759
#> 2993 59.21562 46.29420
#> 3204 72.08859 47.74089
#> 1150 45.48447 36.16734
#> 12586 22.31313 26.04049
#> 9767 36.04429 39.06073
#> 10509 23.17133 18.80702
#> 10550 22.31313 27.48718
#> 10470 18.02215 28.93387
#> 10543 29.17871 15.91363
#> 3756 17.16395 15.91363
#> 10478 14.58936 17.36032
#> 10569 24.02953 21.70040
#> 5816 15.44755 10.12686
#> X10X83_3_TCTGTGTCCGCTGA. X10X83_3_CGTGCGTACATGTC.
#> 4136 339.17965 191.97376
#> 7175 108.49535 206.26968
#> 2993 76.89477 14.29592
#> 3204 58.98777 59.22595
#> 1150 81.10818 61.26822
#> 12586 33.70729 59.22595
#> 9767 16.85365 51.05685
#> 10509 25.28047 34.71866
#> 10550 21.06706 30.63411
#> 10470 16.85365 28.59184
#> 10543 15.80029 46.97230
#> 3756 20.01371 20.42274
#> 10478 10.53353 36.76093
#> 10569 12.64024 24.50729
#> 5816 14.74694 24.50729
#> X10X83_3_ATGAAGTAACGACG. X10X83_3_CCTCAGTGGAAAGA.
#> 4136 139.01076 148.493845
#> 7175 269.41023 148.493845
#> 2993 24.60367 32.636010
#> 3204 71.35065 48.954015
#> 1150 41.82625 6.527202
#> 12586 24.60367 24.477007
#> 9767 46.74698 31.004209
#> 10509 36.90551 39.163212
#> 10550 18.45276 47.322214
#> 10470 30.75459 35.899611
#> 10543 38.13569 37.531411
#> 3756 12.30184 26.108808
#> 10478 22.14331 24.477007
#> 10569 20.91312 35.899611
#> 5816 27.06404 16.318005
#> X10X83_3_AACTTGTCTAGTGT. X10X83_3_ACCTACATCACCCT.
#> 4136 227.781154 190.704662
#> 7175 294.681913 295.225486
#> 2993 76.458010 44.008768
#> 3204 47.786256 62.345755
#> 1150 60.529258 16.503288
#> 12586 17.521627 44.008768
#> 9767 9.557251 20.170685
#> 10509 23.893128 11.002192
#> 10550 17.521627 20.170685
#> 10470 27.078878 36.673973
#> 10543 25.486003 14.669589
#> 3756 11.150126 9.168493
#> 10478 15.928752 12.835891
#> 10569 15.928752 11.002192
#> 5816 6.371501 27.505480
#> X10X83_3_TTTCCTCACCATAG. X10X83_3_TATTGAGTTCGCGC.
#> 4136 159.317282 188.64364
#> 7175 239.780555 258.45664
#> 2993 111.039318 51.98841
#> 3204 59.542822 81.69607
#> 1150 37.013106 34.16381
#> 12586 35.403840 20.79536
#> 9767 22.529717 22.28075
#> 10509 22.529717 16.33921
#> 10550 12.874124 17.82460
#> 10470 27.357513 13.36845
#> 10543 16.092655 17.82460
#> 3756 25.748248 16.33921
#> 10478 22.529717 11.88306
#> 10569 8.046327 23.76613
#> 5816 49.887230 16.33921
#> X10X83_3_TCAGTCACTCGACG. X10X83_3_AGAGAAGTCATCCA.
#> 4136 124.225447 229.869759
#> 7175 268.254950 159.140603
#> 2993 102.621021 91.947904
#> 3204 27.005532 74.265615
#> 1150 43.208851 47.742181
#> 12586 3.600738 22.986976
#> 9767 73.815121 31.828121
#> 10509 12.602582 7.072916
#> 10550 16.203319 15.914060
#> 10470 25.205163 12.377602
#> 10543 14.402950 8.841145
#> 3756 14.402950 7.072916
#> 10478 10.802213 17.682289
#> 10569 16.203319 7.072916
#> 5816 30.606270 19.450518
#> X10X83_3_ACTAGCATTTGCTT. X10X83_3_GCGGTCATCACGTA.
#> 4136 179.18645 137.54655
#> 7175 293.04451 358.96295
#> 2993 41.06356 40.25753
#> 3204 55.99577 46.96711
#> 1150 44.79661 26.83835
#> 12586 16.79873 48.64451
#> 9767 16.79873 67.09588
#> 10509 24.26483 23.48356
#> 10550 20.53178 16.77397
#> 10470 13.06568 23.48356
#> 10543 14.93220 30.19315
#> 3756 27.99788 30.19315
#> 10478 14.93220 30.19315
#> 10569 13.06568 18.45137
#> 5816 39.19704 23.48356
#> X10X83_3_ATTCTGTTATCGGT. X10X83_3_GTTAGTCAGTACGT.
#> 4136 276.243406 204.53980
#> 7175 232.725609 240.90243
#> 2993 34.057406 43.18062
#> 3204 68.114812 49.99862
#> 1150 41.625719 34.08997
#> 12586 22.704937 22.72664
#> 9767 7.568312 29.54464
#> 10509 9.460391 22.72664
#> 10550 28.381172 20.45398
#> 10470 24.597016 24.99931
#> 10543 7.568312 20.45398
#> 3756 24.597016 15.90865
#> 10478 18.920781 29.54464
#> 10569 13.244547 11.36332
#> 5816 20.812859 15.90865
#> X10X83_3_GGTGACAAAGGTGC. X10X83_3_GCGATTCACCCGAG.
#> 4136 243.456941 220.896268
#> 7175 165.083816 405.480820
#> 2993 130.066037 77.162395
#> 3204 31.682753 46.902632
#> 1150 33.350266 42.363668
#> 12586 35.017779 25.720798
#> 9767 70.035558 27.233786
#> 10509 8.337566 19.668846
#> 10550 26.680213 15.129881
#> 10470 15.007620 15.129881
#> 10543 3.335027 21.181834
#> 3756 23.345186 22.694822
#> 10478 11.672593 15.129881
#> 10569 16.675133 6.051953
#> 5816 20.010160 13.616893
#> X10X83_3_GCTGGCAACACCCG. X10X83_3_TATGCCAGGAATCG.
#> 4136 119.227201 132.14433
#> 7175 311.824986 246.90125
#> 2993 6.114215 27.81986
#> 3204 47.385170 41.72979
#> 1150 24.456862 27.81986
#> 12586 15.285539 12.17119
#> 9767 22.928308 34.77482
#> 10509 24.456862 34.77482
#> 10550 9.171323 34.77482
#> 10470 22.928308 26.08112
#> 10543 12.228431 27.81986
#> 3756 4.585662 10.43245
#> 10478 6.114215 22.60364
#> 10569 10.699877 20.86489
#> 5816 22.928308 13.90993
#> X10X83_3_CACGAGTTAGAACA. X10X83_3_CGTACCATTGTGCA.
#> 4136 185.93058 163.38192
#> 7175 253.54170 317.68707
#> 2993 25.35417 31.76871
#> 3204 35.49584 65.80661
#> 1150 23.66389 52.19145
#> 12586 13.52222 18.15355
#> 9767 35.49584 24.96113
#> 10509 13.52222 20.42274
#> 10550 37.18612 15.88435
#> 10470 27.04445 22.69193
#> 10543 37.18612 36.30709
#> 3756 13.52222 15.88435
#> 10478 27.04445 24.96113
#> 10569 13.52222 18.15355
#> 5816 10.14167 24.96113
#> X10X83_3_ACAGAAGACAAAGG. X10X83_3_GGGTTGTGAGCGAT.
#> 4136 182.437800 243.86670
#> 7175 138.949719 208.62585
#> 2993 31.820546 50.74683
#> 3204 40.306025 46.51793
#> 1150 14.849588 43.69866
#> 12586 20.153013 21.14451
#> 9767 39.245341 22.55415
#> 10509 27.577807 25.37341
#> 10550 30.759862 18.32524
#> 10470 27.577807 21.14451
#> 10543 24.395752 28.19268
#> 3756 19.092328 16.91561
#> 10478 33.941916 18.32524
#> 10569 22.274383 25.37341
#> 5816 7.424794 25.37341
#> X10X83_3_CTACGTCCTCATTA. X10X83_3_AACATAGGCTACGA.
#> 4136 157.255964 196.46338
#> 7175 332.124596 224.79945
#> 2993 16.354620 109.56612
#> 3204 62.902386 64.22841
#> 1150 35.225336 100.12076
#> 12586 50.321908 17.00164
#> 9767 11.322429 35.89235
#> 10509 30.193145 11.33443
#> 10550 23.902907 11.33443
#> 10470 26.419002 15.11257
#> 10543 21.386811 26.44699
#> 3756 15.096573 13.22350
#> 10478 7.548286 15.11257
#> 10569 15.096573 17.00164
#> 5816 18.870716 20.77978
#> X10X83_3_CGTGGTCCCGGATG. X10X83_3_GCGATCAAATTGCC.
#> 4136 180.446506 213.023306
#> 7175 171.072661 297.899780
#> 2993 39.838839 106.511653
#> 3204 30.464995 56.584316
#> 1150 37.495378 54.920071
#> 12586 30.464995 26.627913
#> 9767 67.960372 19.970935
#> 10509 28.121533 9.985467
#> 10550 21.091150 13.313957
#> 10470 21.091150 14.978201
#> 10543 16.404228 13.313957
#> 3756 11.717306 11.649712
#> 10478 14.060767 14.978201
#> 10569 7.030383 8.321223
#> 5816 18.747689 19.970935
#> X10X83_3_ATCTTGTAAGAGGA. X10X83_3_GTGGTGTAGCACGA.
#> 4136 165.59491 294.679365
#> 7175 138.33649 279.470108
#> 2993 42.25055 89.354388
#> 3204 33.39157 64.639345
#> 1150 16.35505 70.342816
#> 12586 49.74662 15.209258
#> 9767 22.48820 38.023144
#> 10509 23.16966 17.110415
#> 10550 21.80674 13.308100
#> 10470 13.62921 15.209258
#> 10543 24.53258 15.209258
#> 3756 26.57696 13.308100
#> 10478 27.25842 5.703472
#> 10569 19.76236 7.604629
#> 5816 23.85112 24.715044
#> X10X83_3_ATTCGAGACTAGAT. X10X83_3_CTTACTCCGCGGTA.
#> 4136 149.77647 167.408941
#> 7175 60.75838 55.146475
#> 2993 32.49867 26.588479
#> 3204 24.02076 23.634203
#> 1150 33.91165 4.923792
#> 12586 56.51942 64.501680
#> 9767 16.95583 35.451305
#> 10509 40.97658 35.943684
#> 10550 32.49867 21.172307
#> 10470 55.10644 27.080858
#> 10543 38.15061 16.740894
#> 3756 28.25971 24.126583
#> 10478 33.91165 16.740894
#> 10569 26.84673 25.603720
#> 5816 28.25971 19.202790
#> X10X83_3_ATACGGTGTCGCTG. X10X83_3_CGAGTTCAACACGT.
#> 4136 204.35359 187.06350
#> 7175 39.91281 72.52462
#> 2993 19.95640 67.52292
#> 3204 24.74594 41.01392
#> 1150 20.75466 34.01155
#> 12586 66.25526 40.51375
#> 9767 15.16687 25.50866
#> 10509 39.91281 26.50900
#> 10550 26.34245 28.00951
#> 10470 19.95640 33.51138
#> 10543 22.35117 19.00645
#> 3756 26.34245 26.50900
#> 10478 19.95640 18.00611
#> 10569 21.55292 25.50866
#> 5816 10.37733 15.50526
#> X10X83_3_CACGACAGGTGCCT. X10X83_3_TTCAGAGCGCTCCA.
#> 4136 0.000000 0.000000
#> 7175 290.289482 308.657851
#> 2993 5.277991 0.000000
#> 3204 0.000000 0.000000
#> 1150 0.000000 0.000000
#> 12586 8.796651 21.660200
#> 9767 0.000000 0.000000
#> 10509 8.796651 13.537625
#> 10550 3.518660 10.830100
#> 10470 5.277991 5.415050
#> 10543 5.277991 8.122575
#> 3756 21.111962 27.075250
#> 10478 5.277991 2.707525
#> 10569 3.518660 2.707525
#> 5816 12.315311 10.830100
Run this only in the last iteration, instead the previous code, when B cells group has not to be removed.
pdf(paste(out_dir,"cleaning/",t,"_",n_it,"_plots_before_cells_exlusion.pdf", sep = ""))
ttm$pca.cell.2
ggplot(ttm$D, aes(x=n,y=means)) + geom_point() +
geom_text_repel(data=subset(ttm$D, n > (max(ttm$D$n)- 15) ), aes(n,means,label=rownames(ttm$D[ttm$D$n > (max(ttm$D$n)- 15),])),
nudge_y = 0.05,
nudge_x = 0.05,
direction = "x",
angle = 90,
vjust = 0,
segment.size = 0.2)+
ggtitle(label = "B cell group genes mean expression", subtitle = " - B group NOT removed -")+my_theme +
theme(plot.title = element_text(color = "#3C5488FF", size = 20, face = "italic",vjust = - 10,hjust = 0.02 ),
plot.subtitle = element_text(color = "darkred",vjust = - 15,hjust = 0.01 ))
#> Warning: ggrepel: 11 unlabeled data points (too many overlaps). Consider
#> increasing max.overlaps
dev.off()
#> png
#> 2To colour the pca based on nu_j (so the cells’ efficiency).
nu_est = round(obj@nu, digits = 7)
plot.nu <-ggplot(ttm$pca_cells,aes(x=PC1,y=PC2, colour = log(nu_est)))
plot.nu = plot.nu + geom_point(size = 1,alpha= 0.8)+
scale_color_gradient2(low = "#E64B35B2",mid = "#4DBBD5B2", high = "#3C5488B2" ,
midpoint = log(mean(nu_est)),name = "ln (nu)")+
ggtitle("Cells PCA coloured by cells efficiency") +
my_theme + theme(plot.title = element_text(color = "#3C5488FF", size = 20),
legend.title=element_text(color = "#3C5488FF", size = 14,face = "italic"),
legend.text = element_text(color = "#3C5488FF", size = 11),
legend.key.width = unit(2, "mm"),
legend.position="right")
pdf(paste(out_dir,"cleaning/",t,"_plots_PCA_efficiency_colored.pdf", sep = ""))
plot.nu
dev.off()
#> png
#> 2
plot.nu
The next part is used to remove the cells with efficiency too low.
nu_df = data.frame("nu"= sort(obj@nu), "n"=c(1:length(obj@nu)))
ggplot(nu_df, aes(x = n, y=nu)) +
geom_point(colour = "#8491B4B2", size=1)+
my_theme #+ ylim(0,1) + xlim(0,70)
We can zoom on the smallest values and, if we detect a clear elbow, we can decide to remove the cells.
yset = 0.4#threshold to remove low UDE cells
plot.ude <- ggplot(nu_df, aes(x = n, y=nu)) +
geom_point(colour = "#8491B4B2", size=1) +
my_theme + ylim(0.,0.7) + xlim(0,2000) +
geom_hline(yintercept=yset, linetype="dashed", color = "darkred") +
annotate(geom="text", x=500, y=0.5,
label=paste("to remove cells with nu < ",yset,sep = " "),
color="darkred", size=4.5)
pdf(paste(out_dir,"cleaning/",t,"_plots_efficiency.pdf", sep = ""))
plot.ude
#> Warning: Removed 2840 rows containing missing values (geom_point).
dev.off()
#> png
#> 2
plot.ude
#> Warning: Removed 2840 rows containing missing values (geom_point).
We also save the defined threshold in the metadata and re-run the estimation.
obj@meta[(nrow(obj@meta)+1),1:2] = c("Threshold low UDE cells:",yset)
to_rem = rownames(nu_df[which(nu_df$nu < yset),])
obj@raw = obj@raw[, !colnames(obj@raw) %in% to_rem]Repeat the estimation after the cells are removed
ttm = clean(obj)
#> [1] "Start estimation mu with linear method"
#> [1] 14121 4690
#> rowname X10X83_2_TTCTGAGTAGCCGA. X10X83_2_TTGAAAGTCCGGTC.
#> 4130 Fabp7 278.40788 135.68345
#> 7161 Malat1 68.83147 65.26546
#> 2989 Cst3 39.03874 4.29378
#> 3200 Dbi 43.14808 31.77397
#> 1147 Apoe 27.73805 22.32766
#> 12565 Tmsb4x 67.80413 35.20900
#> 9749 Ptn 30.82006 30.05646
#> 10491 Rpl41 24.65605 26.62144
#> 10452 Rpl13 16.43737 34.35024
#> 10532 Rps19 25.68338 42.07905
#> 10525 Rps14 23.62871 29.19770
#> 10460 Rpl18a 11.30069 24.90392
#> 3750 Eef1a1 13.35536 28.33895
#> 5804 H3f3b 13.35536 21.46890
#> 10551 Rps4x 14.38269 33.49148
#> X10X83_2_CAGTTAGCTAGGCA. X10X83_2_CAATTAGGCCCTCA.
#> 4130 389.83129 211.713051
#> 7161 62.37301 387.474829
#> 2989 27.84509 57.921495
#> 3200 57.91779 33.953980
#> 1147 54.57638 51.929616
#> 12565 71.28344 39.945859
#> 9749 24.50368 29.959394
#> 10491 17.82086 21.970222
#> 10452 18.93466 3.994586
#> 10532 18.93466 15.978343
#> 10525 22.27607 21.970222
#> 10460 23.38988 9.986465
#> 3750 30.07270 9.986465
#> 5804 18.93466 11.983758
#> 10551 22.27607 9.986465
#> X10X83_2_GGGATGTTACTGAC. X10X83_2_CGAAGGTAAACCTC.
#> 4130 265.12823 224.90237
#> 7161 77.22181 148.19148
#> 2989 37.75289 50.55945
#> 3200 42.04299 62.76345
#> 1147 29.17269 15.69086
#> 12565 42.90101 61.02002
#> 9749 66.06755 62.76345
#> 10491 26.59862 17.43429
#> 10452 23.16654 17.43429
#> 10532 23.16654 24.40801
#> 10525 17.16040 29.63830
#> 10460 14.58634 17.43429
#> 3750 18.01842 19.17772
#> 5804 13.72832 13.94743
#> 10551 20.59248 29.63830
#> X10X83_2_GAAGGAGTCGCCGT. X10X83_2_ACCTTAGGGATACC.
#> 4130 196.56879 357.523428
#> 7161 117.94128 95.130503
#> 2989 39.31376 19.862413
#> 3200 26.80484 41.815606
#> 1147 39.31376 17.771632
#> 12565 53.60967 61.678018
#> 9749 23.23086 20.907803
#> 10491 28.59182 25.089363
#> 10452 35.73978 26.134754
#> 10532 39.31376 27.180144
#> 10525 26.80484 14.635462
#> 10460 32.16580 16.726242
#> 3750 35.73978 18.817023
#> 5804 19.65688 9.408511
#> 10551 39.31376 22.998583
#> X10X83_2_AACGAGTGTTCGAT. X10X83_2_CCGAACACGGTGTC.
#> 4130 304.95375 276.435145
#> 7161 45.60892 130.231668
#> 2989 72.43769 29.486415
#> 3200 58.12901 34.400818
#> 1147 34.87741 35.629419
#> 12565 59.02331 25.800613
#> 9749 42.92604 29.486415
#> 10491 30.40595 27.029214
#> 10452 17.88585 25.800613
#> 10532 10.73151 18.429010
#> 10525 26.82878 25.800613
#> 10460 12.52010 28.257815
#> 3750 20.56873 29.486415
#> 5804 20.56873 9.828805
#> 10551 15.20297 30.715016
#> X10X83_2_CAGAAGTAAGTTCC. X10X83_2_GGAGAGTTTACTCT.
#> 4130 174.49642 148.59319
#> 7161 200.39823 132.95180
#> 2989 24.53856 53.18072
#> 3200 42.26085 32.84691
#> 1147 25.90181 23.46208
#> 12565 38.17109 40.66761
#> 9749 47.71386 26.59036
#> 10491 32.71808 23.46208
#> 10452 25.90181 31.28278
#> 10532 20.44880 26.59036
#> 10525 29.99157 39.10347
#> 10460 19.08555 32.84691
#> 3750 16.35904 29.71864
#> 5804 23.17531 18.76967
#> 10551 17.72229 35.97519
#> X10X83_2_TATGCAGCTAGTCT. X10X83_2_AGCCGGTCGAACAG.
#> 4130 247.345425 369.734529
#> 7161 195.867443 181.577811
#> 2989 71.566950 82.894218
#> 3200 53.989103 85.525781
#> 1147 80.355874 52.631250
#> 12565 30.133453 49.999687
#> 9749 31.389013 23.684062
#> 10491 26.366771 15.789375
#> 10452 18.833408 13.157812
#> 10532 20.088969 11.842031
#> 10525 20.088969 7.894687
#> 10460 16.322287 5.263125
#> 3750 20.088969 10.526250
#> 5804 7.533363 10.526250
#> 10551 22.600090 17.105156
#> X10X83_2_TTCCCCAGTACACT. X10X83_2_ACCTCTCGACCAGC.
#> 4130 215.135468 299.09093
#> 7161 0.000000 85.85669
#> 2989 103.583744 47.15081
#> 3200 43.823892 37.29840
#> 1147 127.487685 38.70589
#> 12565 59.759852 80.93049
#> 9749 55.775862 50.66952
#> 10491 45.815887 22.51979
#> 10452 31.871921 18.29733
#> 10532 47.807882 28.14973
#> 10525 41.831897 18.29733
#> 10460 39.839901 19.00107
#> 3750 25.895936 21.11230
#> 5804 5.975985 12.66738
#> 10551 41.831897 23.92727
#> X10X83_2_TAAGCTCCTAGGGC. X10X83_2_TAGGCTCGGCGCAT.
#> 4130 341.49289 368.38301
#> 7161 99.72801 110.26767
#> 2989 46.84194 50.93081
#> 3200 30.22061 53.40317
#> 1147 84.61771 82.57713
#> 12565 36.26473 33.62422
#> 9749 36.26473 57.85344
#> 10491 10.57721 27.19606
#> 10452 12.08824 29.17396
#> 10532 16.62134 28.67948
#> 10525 10.57721 28.67948
#> 10460 13.59927 31.15185
#> 3750 16.62134 30.65738
#> 5804 18.13237 15.82316
#> 10551 16.62134 26.20711
#> X10X83_2_TGGCTCACTCAGGC. X10X83_2_CTTCAGTATGAAAC.
#> 4130 158.50957 179.72968
#> 7161 149.89492 200.66906
#> 2989 124.05096 76.77773
#> 3200 60.30255 19.19443
#> 1147 55.13376 122.14638
#> 12565 36.18153 29.66412
#> 9749 29.28981 45.36866
#> 10491 15.50637 20.93938
#> 10452 13.78344 24.42928
#> 10532 22.39809 20.93938
#> 10525 20.67516 13.95959
#> 10460 20.67516 17.44948
#> 3750 29.28981 15.70454
#> 5804 6.89172 22.68433
#> 10551 25.84395 20.93938
#> X10X83_2_TCACTGTCTAAACC. X10X83_2_CACGATCAATCACG.
#> 4130 357.380570 263.368579
#> 7161 56.343783 166.036713
#> 2989 66.002718 154.585905
#> 3200 56.343783 78.247186
#> 1147 48.294672 51.528635
#> 12565 53.124139 28.627019
#> 9749 41.855382 15.267744
#> 10491 30.586625 19.084680
#> 10452 22.537513 15.267744
#> 10532 24.147336 19.084680
#> 10525 16.098224 5.725404
#> 10460 12.878579 7.633872
#> 3750 12.878579 11.450808
#> 5804 22.537513 30.535487
#> 10551 8.049112 11.450808
#> X10X83_2_CGAAAGTTTAGCTG. X10X83_2_CGGGTCAAGTCATC.
#> 4130 197.87747 457.355543
#> 7161 123.55729 137.496128
#> 2989 26.94106 76.708366
#> 3200 41.80510 50.656468
#> 1147 17.65104 57.893107
#> 12565 106.83525 37.630519
#> 9749 53.88213 49.209141
#> 10491 14.86403 11.578621
#> 10452 26.94106 17.367932
#> 10532 21.36705 10.131294
#> 10525 18.58004 14.473277
#> 10460 13.93503 21.709915
#> 3750 17.65104 8.683966
#> 5804 24.15406 24.604570
#> 10551 10.21902 13.025949
#> X10X83_2_GTCTGTCGAGAACG. X10X83_2_CGTCGGTAGTGAAT.
#> 4130 161.89321 461.78223
#> 7161 135.99030 60.94588
#> 2989 43.71117 42.19330
#> 3200 77.70874 44.53737
#> 1147 11.33252 59.77384
#> 12565 11.33252 60.94588
#> 9749 22.66505 36.33312
#> 10491 27.52185 37.50516
#> 10452 25.90291 19.92461
#> 10532 25.90291 24.61276
#> 10525 21.04612 33.98905
#> 10460 21.04612 21.09665
#> 3750 29.14078 22.26869
#> 5804 33.99757 21.09665
#> 10551 19.42719 23.44072
#> X10X83_2_CATCTTCTGAGTGT. X10X83_2_CAGTTGTTAAGAAC.
#> 4130 254.396520 314.99484
#> 7161 61.489206 191.29242
#> 2989 8.439695 66.31470
#> 3200 32.553109 43.35961
#> 1147 9.645366 109.67432
#> 12565 55.460853 38.25848
#> 9749 26.524756 54.83716
#> 10491 30.141768 16.57868
#> 10452 16.879390 12.75283
#> 10532 30.141768 15.30339
#> 10525 19.290731 17.85396
#> 10460 15.673719 20.40452
#> 3750 20.496402 14.02811
#> 5804 15.673719 21.67981
#> 10551 28.936097 11.47754
#> X10X83_2_TGGAACATTTCAGG. X10X83_2_ACCAACACCGAATT.
#> 4130 288.97160 287.825149
#> 7161 205.03223 164.612329
#> 2989 68.80276 30.556780
#> 3200 30.27322 44.356615
#> 1147 70.17882 25.628267
#> 12565 33.02533 39.428103
#> 9749 64.67460 25.628267
#> 10491 30.27322 31.542482
#> 10452 28.89716 13.799836
#> 10532 11.00844 18.728349
#> 10525 24.76899 16.756944
#> 10460 24.76899 19.714051
#> 3750 24.76899 8.871323
#> 5804 17.88872 14.785538
#> 10551 19.26477 19.714051
#> X10X83_2_GCCAGGTAAGAGAG. X10X83_2_CCTATGTGCACTTA.
#> 4130 262.94335 279.435216
#> 7161 328.28079 198.991745
#> 2989 62.15025 61.391070
#> 3200 47.80788 50.806403
#> 1147 41.43350 14.818534
#> 12565 17.52956 33.870935
#> 9749 124.30049 21.169335
#> 10491 19.12315 23.286268
#> 10452 23.90394 27.520135
#> 10532 19.12315 29.637068
#> 10525 17.52956 19.052401
#> 10460 14.34236 12.701601
#> 3750 7.96798 8.467734
#> 5804 30.27833 4.233867
#> 10551 14.34236 10.584667
#> X10X83_2_GCCGCTCCACGTTC. X10X83_2_GCTAAGTACAGTTC.
#> 4130 283.784284 176.01144
#> 7161 128.160644 55.31788
#> 2989 53.618229 43.58379
#> 3200 30.078519 65.37568
#> 1147 22.231949 25.14449
#> 12565 37.925089 18.43929
#> 9749 15.693140 25.14449
#> 10491 43.810016 20.11559
#> 10452 30.732399 26.82079
#> 10532 33.347923 26.82079
#> 10525 26.809114 11.73410
#> 10460 26.155234 20.11559
#> 3750 25.501353 16.76299
#> 5804 9.154332 33.52599
#> 10551 24.847472 21.79189
#> X10X83_2_CTACTCATTTGCTT. X10X83_2_ACATTTCTTGAGGT.
#> 4130 171.65306 260.66280
#> 7161 322.42940 346.84970
#> 2989 115.98180 33.63391
#> 3200 32.47490 77.77842
#> 1147 67.26944 10.51060
#> 12565 30.15527 39.94027
#> 9749 30.15527 39.94027
#> 10491 11.59818 31.53179
#> 10452 20.87672 14.71484
#> 10532 20.87672 31.53179
#> 10525 27.83563 29.42967
#> 10460 11.59818 35.73603
#> 3750 16.23745 14.71484
#> 5804 25.51600 12.61272
#> 10551 20.87672 21.02119
#> X10X83_2_ATGCGAGTGTTTGC. X10X83_2_ACCCATCAGGCGAA.
#> 4130 139.717608 297.581602
#> 7161 243.379059 320.894447
#> 2989 58.591255 42.511657
#> 3200 47.323706 58.967783
#> 1147 29.295627 45.254345
#> 12565 15.774569 19.198813
#> 9749 22.535098 26.055532
#> 10491 36.056157 21.941501
#> 10452 27.042118 35.654939
#> 10532 29.295627 24.684188
#> 10525 40.563177 24.684188
#> 10460 29.295627 17.827469
#> 3750 20.281588 21.941501
#> 5804 6.760529 10.970750
#> 10551 20.281588 8.228063
#> X10X83_2_GTCATAGGCATGGT. X10X83_2_GGCAAAGCCACGTC.
#> 4130 186.509206 178.96709
#> 7161 99.848363 135.92437
#> 2989 16.955382 18.12325
#> 3200 39.562559 29.45028
#> 1147 22.607176 22.65406
#> 12565 9.419657 65.69678
#> 9749 56.517941 24.91947
#> 10491 15.071451 33.98109
#> 10452 28.258971 29.45028
#> 10532 24.491108 31.71569
#> 10525 16.955382 22.65406
#> 10460 11.303588 18.12325
#> 3750 20.723245 43.04272
#> 5804 22.607176 13.59244
#> 10551 1.883931 24.91947
#> X10X83_2_CGAGGCACCGAAAG. X10X83_2_CATAGGTATGCTTG.
#> 4130 265.349427 231.60590
#> 7161 268.233660 270.97891
#> 2989 57.684658 34.74089
#> 3200 66.337357 71.79783
#> 1147 31.726562 13.89635
#> 12565 28.842329 39.37300
#> 9749 20.189630 41.68906
#> 10491 25.958096 25.47665
#> 10452 20.189630 11.58030
#> 10532 24.515980 18.52847
#> 10525 20.189630 27.79271
#> 10460 21.631747 16.21241
#> 3750 18.747514 20.84453
#> 5804 11.536932 16.21241
#> 10551 8.652699 23.16059
#> X10X83_2_AGCCGGTCAAACTC. X10X83_2_TCTGTAGATAGTCA.
#> 4130 146.11870 139.018583
#> 7161 227.67332 287.430314
#> 2989 40.77731 77.023810
#> 3200 49.27258 84.538328
#> 1147 20.38866 28.179443
#> 12565 30.58298 31.936702
#> 9749 10.19433 15.029036
#> 10491 20.38866 35.693961
#> 10452 20.38866 22.543554
#> 10532 42.47637 20.664925
#> 10525 42.47637 39.451220
#> 10460 13.59244 22.543554
#> 3750 32.28204 9.393148
#> 5804 30.58298 26.300813
#> 10551 32.28204 22.543554
#> X10X83_2_CACGGTCCCTAACC. X10X83_2_GTTCATCAAGGTAA.
#> 4130 95.87006 358.17159
#> 7161 246.93804 250.72011
#> 2989 17.43092 71.63432
#> 3200 66.81853 58.43852
#> 1147 15.97834 52.78318
#> 12565 36.31442 22.62136
#> 9749 13.07319 33.93205
#> 10491 36.31442 24.50648
#> 10452 31.95669 20.73625
#> 10532 29.05153 28.27670
#> 10525 31.95669 24.50648
#> 10460 37.76699 15.08091
#> 3750 18.88350 11.31068
#> 5804 13.07319 13.19580
#> 10551 27.59896 15.08091
#> X10X83_2_AACATGTACGACCC. X10X83_2_GTCCGAGGCAGTCA.
#> 4130 100.043195 149.821874
#> 7161 210.617253 141.260624
#> 2989 13.163578 21.403125
#> 3200 46.072524 29.964375
#> 1147 7.898147 14.982187
#> 12565 27.643514 2.140312
#> 9749 18.429010 19.262812
#> 10491 35.541661 25.683750
#> 10452 22.378083 23.543437
#> 10532 34.225304 12.841875
#> 10525 32.908946 23.543437
#> 10460 31.592588 17.122500
#> 3750 22.378083 23.543437
#> 5804 17.112652 34.245000
#> 10551 15.796294 19.262812
#> X10X83_2_TGCAGGTTCCCGAG. X10X83_2_CAGGCGTGTTCTTT.
#> 4130 190.10217 259.71752
#> 7161 69.12806 156.65501
#> 2989 48.70386 39.85084
#> 3200 42.41949 54.96667
#> 1147 31.42185 37.10250
#> 12565 18.85311 31.60584
#> 9749 15.71092 23.36084
#> 10491 20.42420 23.36084
#> 10452 25.13748 17.86417
#> 10532 17.28202 41.22500
#> 10525 28.27966 42.59917
#> 10460 15.71092 28.85750
#> 3750 10.99765 31.60584
#> 5804 18.85311 20.61250
#> 10551 21.99529 27.48334
#> X10X83_2_CTATCCAGTTAACC. X10X83_2_AGCCAAGCGTGAGT.
#> 4130 145.052969 264.538106
#> 7161 210.178791 146.479778
#> 2989 8.880794 49.190970
#> 3200 45.884102 73.239889
#> 1147 22.201985 17.490123
#> 12565 34.043044 59.029164
#> 9749 20.721853 22.955786
#> 10491 22.201985 14.210725
#> 10452 26.642382 13.117592
#> 10532 17.761588 20.769521
#> 10525 14.801323 13.117592
#> 10460 16.281456 14.210725
#> 3750 5.920529 8.745061
#> 5804 19.241720 13.117592
#> 10551 10.360926 15.303857
#> X10X83_2_AGAAATCATCTGCC. X10X83_2_GGTAGCACGAGAGT.
#> 4130 231.882206 291.832439
#> 7161 185.889041 163.426166
#> 2989 53.658692 52.529839
#> 3200 49.825928 89.495281
#> 1147 45.993165 31.128793
#> 12565 5.749146 36.965442
#> 9749 32.578492 29.183244
#> 10491 22.996582 15.564397
#> 10452 19.163819 21.401046
#> 10532 22.996582 19.455496
#> 10525 32.578492 19.455496
#> 10460 17.247437 27.237694
#> 3750 22.996582 21.401046
#> 5804 17.247437 21.401046
#> 10551 15.331055 5.836649
#> X10X83_2_GTGTGTCAACTCTT. X10X83_2_TACTCCACTTCGAA.
#> 4130 288.204759 290.39563
#> 7161 265.309988 371.32556
#> 2989 86.192077 14.28175
#> 3200 60.603804 80.92993
#> 1147 56.563551 11.10803
#> 12565 30.975278 19.04234
#> 9749 32.322029 19.04234
#> 10491 21.548019 26.97664
#> 10452 9.427258 19.04234
#> 10532 14.814263 31.73723
#> 10525 21.548019 26.97664
#> 10460 13.467512 30.15037
#> 3750 9.427258 19.04234
#> 5804 14.814263 19.04234
#> 10551 13.467512 15.86861
#> X10X83_2_CATACAGGTGCTTT. X10X83_2_GCCACGTGTCCTCT.
#> 4130 233.84739 299.685445
#> 7161 180.43780 171.065082
#> 2989 79.39263 36.013702
#> 3200 72.17512 69.454996
#> 1147 43.30507 29.582683
#> 12565 28.87005 43.730923
#> 9749 21.65254 37.299905
#> 10491 10.10452 12.862036
#> 10452 14.43502 11.575833
#> 10532 10.10452 15.434444
#> 10525 12.99152 11.575833
#> 10460 15.87853 9.003425
#> 3750 11.54802 11.575833
#> 5804 11.54802 6.431018
#> 10551 12.99152 10.289629
#> X10X83_2_GCAGTCAGTTCATG. X10X83_2_ACGGCCACACGCTG.
#> 4130 186.96305 185.13730
#> 7161 248.72762 259.49572
#> 2989 36.72488 30.35038
#> 3200 60.09526 62.21827
#> 1147 13.35450 18.21023
#> 12565 11.68519 39.45549
#> 9749 25.03969 25.79782
#> 10491 25.03969 24.28030
#> 10452 28.37832 13.65767
#> 10532 51.74870 22.76278
#> 10525 30.04763 24.28030
#> 10460 16.69313 22.76278
#> 3750 13.35450 15.17519
#> 5804 43.40214 19.72775
#> 10551 20.03175 21.24526
#> X10X83_2_GCGCACAACACCTA. X10X83_2_GATCCGTACAGTGG.
#> 4130 113.428343 310.722735
#> 7161 278.764571 186.764197
#> 2989 9.612571 72.722342
#> 3200 32.682743 74.375123
#> 1147 7.690057 79.333464
#> 12565 26.915200 26.444488
#> 9749 11.535086 11.569464
#> 10491 28.837714 23.138927
#> 10452 40.372800 18.180586
#> 10532 38.450286 9.916683
#> 10525 19.225143 18.180586
#> 10460 30.760229 11.569464
#> 3750 28.837714 11.569464
#> 5804 11.535086 36.361171
#> 10551 23.070171 9.916683
#> X10X83_2_TAACTCAGTGGAGT. X10X83_2_CCATGAGCTGATAA.
#> 4130 278.219995 300.83267
#> 7161 209.018067 255.09203
#> 2989 56.491369 112.59234
#> 3200 83.324770 63.33319
#> 1147 62.140506 96.75905
#> 12565 36.719390 29.90734
#> 9749 29.657969 21.11106
#> 10491 29.657969 17.59255
#> 10452 21.184264 12.31479
#> 10532 19.771979 15.83330
#> 10525 22.596548 29.90734
#> 10460 16.947411 10.55553
#> 3750 18.359695 22.87032
#> 5804 16.947411 26.38883
#> 10551 8.473705 17.59255
#> X10X83_2_TTACTAGGCAATTA. X10X83_2_GACGCCATACTCTT.
#> 4130 197.16906 121.166867
#> 7161 132.55063 274.308323
#> 2989 23.19636 13.462985
#> 3200 43.07895 42.071829
#> 1147 23.19636 5.048619
#> 12565 18.22571 10.097239
#> 9749 29.82389 18.511605
#> 10491 28.16701 25.243097
#> 10452 26.51013 31.974590
#> 10532 29.82389 28.608844
#> 10525 23.19636 30.291717
#> 10460 28.16701 38.706082
#> 3750 19.88259 33.657463
#> 5804 11.59818 20.194478
#> 10551 21.53948 21.877351
#> X10X83_2_CTGCTCATTCGACA. X10X83_2_GGCGTTCGTCTGAA.
#> 4130 152.23441 166.55069
#> 7161 172.85971 81.69217
#> 2989 12.76805 10.13236
#> 3200 35.35767 32.93018
#> 1147 17.67883 15.19854
#> 12565 24.55394 68.39344
#> 9749 45.17924 21.53127
#> 10491 29.46472 17.09836
#> 10452 18.66099 24.69763
#> 10532 23.57178 25.96418
#> 10525 13.75020 22.16454
#> 10460 13.75020 20.26472
#> 3750 16.69668 26.59745
#> 5804 29.46472 39.26290
#> 10551 19.64315 24.06436
#> X10X83_2_TGTTTAGATATGCA. X10X83_2_ACCTACATTCCTCG.
#> 4130 341.96731 195.850850
#> 7161 33.89763 119.098490
#> 2989 14.95484 13.233166
#> 3200 40.87656 23.819698
#> 1147 21.93376 1.764422
#> 12565 84.74409 46.757185
#> 9749 28.91269 19.408643
#> 10491 26.91871 32.641808
#> 10452 16.94882 31.759597
#> 10532 19.93978 41.463919
#> 10525 22.93075 25.584120
#> 10460 13.95785 22.937487
#> 3750 16.94882 34.406230
#> 5804 25.92172 22.937487
#> 10551 13.95785 23.819698
#> X10X83_2_CTCCTTCACCAGGC. X10X83_3_GGCAAAGTTCGCAT.
#> 4130 247.46918 137.24968
#> 7161 41.67902 176.86814
#> 2989 46.02059 41.03341
#> 3200 34.73252 25.46901
#> 1147 25.18107 50.93803
#> 12565 26.04939 35.37363
#> 9749 31.25927 89.14154
#> 10491 19.10288 35.37363
#> 10452 15.62963 33.95868
#> 10532 23.44445 39.61846
#> 10525 13.89301 28.29890
#> 10460 10.41976 28.29890
#> 3750 16.49795 46.69319
#> 5804 27.78601 26.88396
#> 10551 17.36626 11.31956
#> X10X83_3_TACAGAGTGCAAGC. X10X83_3_TGCAGGTGATAAAC.
#> 4130 277.35454 245.21866
#> 7161 92.45151 100.71481
#> 2989 80.52228 72.25193
#> 3200 41.75230 40.50487
#> 1147 135.69496 82.10446
#> 12565 13.42038 16.42089
#> 9749 31.31422 123.70406
#> 10491 17.89384 33.93651
#> 10452 40.26114 26.27343
#> 10532 26.84076 26.27343
#> 10525 25.34961 30.65233
#> 10460 35.78768 19.70507
#> 3750 28.33192 20.79980
#> 5804 14.91153 26.27343
#> 10551 22.36730 16.42089
#> X10X83_3_TCCAGAGCGCCTTG. X10X83_3_GGCAGAGTTCGCAT.
#> 4130 154.648429 287.850787
#> 7161 46.863160 169.969036
#> 2989 7.029474 113.769597
#> 3200 17.573685 47.975131
#> 1147 2.343158 115.140315
#> 12565 51.549476 42.492259
#> 9749 18.745264 24.672925
#> 10491 29.289475 20.560771
#> 10452 41.005265 12.336462
#> 10532 21.088422 5.482872
#> 10525 24.603159 10.965744
#> 10460 22.260001 8.224308
#> 3750 37.490528 5.482872
#> 5804 12.887369 16.448616
#> 10551 23.431580 8.224308
#> X10X83_3_AGGATTCCACGAAT. X10X83_3_GACGTGTATAGGGC.
#> 4130 183.23769 256.059203
#> 7161 80.57260 201.578521
#> 2989 24.69160 39.498494
#> 3200 32.48895 49.032613
#> 1147 25.99116 40.860511
#> 12565 61.72901 23.154290
#> 9749 26.64094 29.964375
#> 10491 39.63652 21.792273
#> 10452 29.24006 27.240341
#> 10532 25.99116 21.792273
#> 10525 18.84359 21.792273
#> 10460 20.79293 17.706221
#> 3750 22.09249 20.430256
#> 5804 10.39646 9.534119
#> 10551 31.83917 23.154290
#> X10X83_3_GTTCAAGACGCTTT. X10X83_3_AACGATCTCGCTTG.
#> 4130 200.82991 187.60078
#> 7161 155.79532 201.49714
#> 2989 63.29185 67.16571
#> 3200 59.64040 30.10877
#> 1147 53.55464 67.16571
#> 12565 19.47442 25.47665
#> 9749 37.73168 67.16571
#> 10491 29.21162 27.79271
#> 10452 25.56017 30.10877
#> 10532 20.69157 18.52847
#> 10525 25.56017 11.58030
#> 10460 20.69157 23.16059
#> 3750 17.04011 18.52847
#> 5804 26.77732 11.58030
#> 10551 23.12587 20.84453
#> X10X83_3_CAGTACAATCGGTT. X10X83_3_CCTGGTCACGCGGT.
#> 4130 273.22883 335.90140
#> 7161 66.71867 86.14713
#> 2989 17.79164 35.47235
#> 3200 33.67704 41.98767
#> 1147 18.42706 63.70544
#> 12565 69.26033 39.81590
#> 9749 22.87497 28.23309
#> 10491 18.42706 29.68094
#> 10452 22.23956 25.33739
#> 10532 24.78122 20.26991
#> 10525 16.52081 20.99384
#> 10460 19.06248 23.16561
#> 3750 14.61457 19.54599
#> 5804 10.16665 13.75458
#> 10551 22.23956 20.99384
#> X10X83_3_GGGTCAGTGAAGAG. X10X83_3_TCAGTAGGCTATCT.
#> 4130 263.24994 130.87231
#> 7161 66.63514 190.85546
#> 2989 79.79764 17.72229
#> 3200 52.64999 31.35483
#> 1147 46.89139 19.08555
#> 12565 33.72890 58.61989
#> 9749 55.11796 12.26928
#> 10491 24.67968 38.17109
#> 10452 23.85703 31.35483
#> 10532 23.03437 31.35483
#> 10525 23.85703 32.71808
#> 10460 21.38906 32.71808
#> 3750 22.21171 34.08133
#> 5804 18.09843 12.26928
#> 10551 18.09843 14.99579
#> X10X83_3_GAAGTGTGCACTTA. X10X83_3_AGCAAGTACAGACG.
#> 4130 225.04850 166.299360
#> 7161 79.70468 339.625453
#> 2989 25.31796 93.689780
#> 3200 45.00970 67.925091
#> 1147 23.44255 53.871624
#> 12565 44.07200 23.422445
#> 9749 47.82281 39.818157
#> 10491 30.00647 14.053467
#> 10452 33.75727 16.395712
#> 10532 45.00970 7.026734
#> 10525 24.38025 18.737956
#> 10460 29.06876 11.711223
#> 3750 36.57038 2.342245
#> 5804 18.75404 28.106934
#> 10551 25.31796 7.026734
#> X10X83_3_CCAAGAGCTGGAAC. X10X83_3_CTCAAGTGTCTGAT.
#> 4130 360.39392 323.855886
#> 7161 120.13131 201.583766
#> 2989 104.63049 92.530253
#> 3200 72.33713 36.351171
#> 1147 45.21071 125.576772
#> 12565 51.66938 28.089541
#> 9749 51.66938 24.784889
#> 10491 18.08428 26.437215
#> 10452 24.54296 26.437215
#> 10532 23.25122 13.218608
#> 10525 21.95949 9.913956
#> 10460 14.20908 14.870934
#> 3750 10.33388 9.913956
#> 5804 16.79255 28.089541
#> 10551 12.91734 14.870934
#> X10X83_3_CTTAGGTAGAGCTG. X10X83_3_GAGCTAGAGCCCAA.
#> 4130 277.726769 137.93550
#> 7161 170.219633 59.91138
#> 2989 114.674279 16.71945
#> 3200 32.252141 29.25904
#> 1147 145.134634 39.01206
#> 12565 26.876784 51.55165
#> 9749 34.043927 25.07918
#> 10491 12.542499 33.43891
#> 10452 16.126070 32.04562
#> 10532 5.375357 40.40535
#> 10525 8.958928 32.04562
#> 10460 8.958928 22.29261
#> 3750 8.958928 41.79864
#> 5804 32.252141 18.11274
#> 10551 5.375357 30.65233
#> X10X83_3_CTTGAGTCAGAAGC. X10X83_3_AGCGAAGTGTTGAA.
#> 4130 238.23930 331.591898
#> 7161 242.08187 65.338305
#> 2989 73.00882 40.836441
#> 3200 76.85139 57.171017
#> 1147 73.00882 35.936068
#> 12565 30.74055 47.370271
#> 9749 82.61524 27.768780
#> 10491 15.37028 32.669152
#> 10452 24.97670 21.234949
#> 10532 24.97670 19.601491
#> 10525 30.74055 13.067661
#> 10460 19.21285 24.501864
#> 3750 17.29156 21.234949
#> 5804 30.74055 11.434203
#> 10551 11.52771 9.800746
#> X10X83_3_CCCTACATCGGAAG. X10X83_3_CTGCCAGCGCCTTG.
#> 4130 319.496059 238.990645
#> 7161 65.230445 216.929970
#> 2989 62.567978 102.949816
#> 3200 38.605774 25.737454
#> 1147 83.867715 150.747945
#> 12565 45.261942 24.511861
#> 9749 46.593175 28.188640
#> 10491 18.637270 17.158303
#> 10452 22.630971 13.481524
#> 10532 29.287139 13.481524
#> 10525 15.974803 12.255931
#> 10460 7.987401 7.353558
#> 3750 17.306037 7.353558
#> 5804 6.656168 15.932710
#> 10551 19.968504 15.932710
#> X10X83_3_AACTTAGCTCCCAG. X10X83_3_CAGAAGTTTCGCTC.
#> 4130 141.53531 261.634057
#> 7161 84.49869 136.166424
#> 2989 22.18091 58.357039
#> 3200 32.74324 24.315433
#> 1147 22.18091 52.521335
#> 12565 54.92415 18.479729
#> 9749 22.18091 75.864150
#> 10491 45.41805 27.233285
#> 10452 42.24935 28.205902
#> 10532 48.58675 27.233285
#> 10525 41.19311 30.151137
#> 10460 28.51831 16.534494
#> 3750 39.08065 21.397581
#> 5804 23.23714 7.780939
#> 10551 39.08065 24.315433
#> X10X83_3_TGTAAGTGTGACGA. X10X83_3_AGACCGTAAACGCG.
#> 4130 228.495550 206.30755
#> 7161 106.368618 181.36927
#> 2989 39.395785 73.68127
#> 3200 51.214520 60.07857
#> 1147 23.637471 97.48598
#> 12565 66.972834 44.20876
#> 9749 59.093677 43.07520
#> 10491 21.667681 23.80472
#> 10452 21.667681 23.80472
#> 10532 33.486417 22.67116
#> 10525 17.728103 22.67116
#> 10460 17.728103 19.27049
#> 3750 9.848946 26.07183
#> 5804 15.758314 21.53760
#> 10551 17.728103 22.67116
#> X10X83_3_ACGAATCGCCCTTA. X10X83_3_TGACATCCTTTACA.
#> 4130 168.27259 144.607542
#> 7161 245.83573 54.716367
#> 2989 32.86574 9.379949
#> 3200 51.27055 24.231534
#> 1147 61.78759 7.034962
#> 12565 27.60722 52.371380
#> 9749 88.08018 17.978235
#> 10491 42.06815 30.484833
#> 10452 38.12426 39.083120
#> 10532 30.23648 25.013197
#> 10525 48.64129 26.576521
#> 10460 27.60722 20.323222
#> 3750 32.86574 42.209769
#> 5804 21.03407 15.633248
#> 10551 31.55111 27.358184
#> X10X83_3_ATGAAGTTCACCTC. X10X83_3_ACCTGCATATACCG.
#> 4130 173.54195 285.05313
#> 7161 324.35817 88.57583
#> 2989 18.59378 18.52040
#> 3200 22.72573 28.98845
#> 1147 45.45146 18.52040
#> 12565 16.52781 22.54658
#> 9749 39.25354 34.62510
#> 10491 26.85768 29.79369
#> 10452 24.79171 28.98845
#> 10532 24.79171 20.93611
#> 10525 28.92366 25.76751
#> 10460 26.85768 12.07852
#> 3750 28.92366 16.90993
#> 5804 20.65976 16.90993
#> 10551 24.79171 20.13087
#> X10X83_3_CCAATGTAGCGCAA. X10X83_3_AGCCTGTCTCTCTG.
#> 4130 169.29532 307.89537
#> 7161 96.98325 127.96495
#> 2989 21.26826 29.23057
#> 3200 42.53651 37.02539
#> 1147 14.46241 32.47841
#> 12565 173.54897 106.52920
#> 9749 54.44674 20.78619
#> 10491 33.17848 30.52971
#> 10452 36.58140 23.38446
#> 10532 41.68578 18.83748
#> 10525 28.07410 10.39309
#> 10460 24.67118 14.94007
#> 3750 25.52191 16.88878
#> 5804 17.01461 10.39309
#> 10551 27.22337 11.04266
#> X10X83_3_ACGTACAGGCAGTA. X10X83_3_GTCCGGTGTATGGG.
#> 4130 196.042327 239.19900
#> 7161 166.916038 220.12178
#> 2989 58.252577 46.95931
#> 3200 54.891852 46.95931
#> 1147 60.493061 42.55688
#> 12565 45.929917 20.54470
#> 9749 41.448949 29.34957
#> 10491 7.841693 24.94714
#> 10452 20.164354 17.60974
#> 10532 23.525079 38.15444
#> 10525 28.006047 27.88209
#> 10460 21.284596 27.88209
#> 3750 19.044112 16.14226
#> 5804 17.923870 14.67479
#> 10551 22.404837 14.67479
#> X10X83_3_TCAGTCATGAGCGA. X10X83_3_CATTTCAGATAATG.
#> 4130 290.953047 294.966585
#> 7161 109.915596 313.054159
#> 2989 34.914366 129.395719
#> 3200 72.414981 43.131906
#> 1147 36.207490 61.219480
#> 12565 27.155618 26.435685
#> 9749 69.828731 82.089757
#> 10491 23.276244 18.087574
#> 10452 18.103745 16.696222
#> 10532 23.276244 12.522166
#> 10525 23.276244 15.304870
#> 10460 7.758748 12.522166
#> 3750 18.103745 9.739463
#> 5804 23.276244 29.218388
#> 10551 19.396870 13.913518
#> X10X83_3_ACTCCAGGGAAACA. X10X83_3_TATCCGTCCGCTGA.
#> 4130 159.871405 182.68207
#> 7161 159.871405 117.55194
#> 2989 61.743439 22.23956
#> 3200 38.589649 34.94787
#> 1147 76.076738 17.47394
#> 12565 28.666597 54.01035
#> 9749 61.743439 49.24473
#> 10491 14.333298 30.18225
#> 10452 23.153790 38.12495
#> 10532 14.333298 33.35933
#> 10525 19.846105 34.94787
#> 10460 19.846105 33.35933
#> 3750 11.025614 25.41664
#> 5804 12.128176 19.06248
#> 10551 9.923053 19.06248
#> X10X83_3_TATGGTCAGTCCCT. X10X83_3_GTAGGAGATGCCTT.
#> 4130 239.18396 262.03145
#> 7161 110.00444 75.46506
#> 2989 31.28567 24.10689
#> 3200 44.40546 47.16566
#> 1147 53.48840 50.31004
#> 12565 26.23959 34.58815
#> 9749 74.68191 25.15502
#> 10491 22.20273 19.91439
#> 10452 20.18430 18.86626
#> 10532 24.22116 11.52938
#> 10525 27.24881 16.77001
#> 10460 19.17509 15.72189
#> 3750 31.28567 14.67376
#> 5804 15.13823 17.81814
#> 10551 38.35017 27.25127
#> X10X83_3_CTTACTCTTAACCT. X10X83_3_TGATGTCTCCGGTT.
#> 4130 223.806025 281.502554
#> 7161 332.629138 208.657595
#> 2989 84.183918 58.029035
#> 3200 67.757787 62.967677
#> 1147 104.716580 50.621073
#> 12565 12.319598 41.978451
#> 9749 20.532663 44.447772
#> 10491 30.798994 19.754565
#> 10452 16.426130 8.642622
#> 10532 8.213065 18.519905
#> 10525 28.745728 13.581264
#> 10460 16.426130 11.111943
#> 3750 2.053266 7.407962
#> 5804 26.692462 18.519905
#> 10551 8.213065 14.815924
#> X10X83_3_CTCTATCAAAGACA. X10X83_3_AGAAGAGGAATTAC.
#> 4130 292.556522 185.38954
#> 7161 258.889303 157.58111
#> 2989 8.126570 21.62878
#> 3200 53.403175 42.02163
#> 1147 12.770324 21.01081
#> 12565 30.184403 19.77488
#> 9749 47.598482 19.15692
#> 10491 20.896894 25.95454
#> 10452 25.540649 32.75215
#> 10532 29.023464 33.98808
#> 10525 17.414079 27.19047
#> 10460 9.287509 30.28029
#> 3750 2.321877 22.24675
#> 5804 17.414079 16.06709
#> 10551 17.414079 25.95454
#> X10X83_3_ACTAGCAAACAACA. X10X83_3_CGTAATCTGATACG.
#> 4130 314.494757 275.01671
#> 7161 130.946216 153.55823
#> 2989 45.887135 59.86168
#> 3200 69.390302 72.87509
#> 1147 59.317516 45.98071
#> 12565 29.099159 22.55658
#> 9749 14.549580 36.43755
#> 10491 17.907175 23.42414
#> 10452 19.026373 18.21877
#> 10532 14.549580 22.55658
#> 10525 7.834389 29.49706
#> 10460 10.072786 14.74853
#> 3750 10.072786 17.35121
#> 5804 12.311183 15.61609
#> 10551 15.668778 24.29170
#> X10X83_3_CTCAGCATTCCTCG. X10X83_3_TCTGTGTCCGCTGA.
#> 4130 187.19711 342.94097
#> 7161 231.07143 109.69851
#> 2989 46.79928 77.74749
#> 3200 48.26175 59.64191
#> 1147 36.56193 82.00762
#> 12565 26.32459 34.08109
#> 9749 39.48689 17.04054
#> 10491 19.01221 25.56082
#> 10452 29.24955 17.04054
#> 10532 27.78707 21.30068
#> 10525 16.08725 15.97551
#> 10460 17.54973 10.65034
#> 3750 16.08725 20.23565
#> 5804 10.23734 14.91048
#> 10551 21.93716 12.78041
#> X10X83_3_CGTGCGTACATGTC. X10X83_3_ATGAAGTAACGACG.
#> 4130 194.06824 140.52740
#> 7161 208.52013 272.34956
#> 2989 14.45189 24.87211
#> 3200 59.87212 72.12911
#> 1147 61.93667 42.28258
#> 12565 59.87212 24.87211
#> 9749 51.61389 47.25700
#> 10491 35.09745 37.30816
#> 10452 28.90378 31.09013
#> 10532 30.96834 18.65408
#> 10525 47.48478 38.55176
#> 10460 37.16200 22.38490
#> 3750 20.64556 12.43605
#> 5804 24.77467 27.35932
#> 10551 24.77467 21.14129
#> X10X83_3_CCTCAGTGGAAAGA. X10X83_3_AACTTGTCTAGTGT.
#> 4130 150.113948 230.266302
#> 7161 150.113948 297.896964
#> 2989 32.992077 77.292185
#> 3200 49.488115 48.307616
#> 1147 6.598415 61.189647
#> 12565 24.744057 17.712792
#> 9749 31.342473 9.661523
#> 10491 39.590492 24.153808
#> 10452 36.291284 27.374316
#> 10532 47.838511 17.712792
#> 10525 37.940888 25.764062
#> 10460 24.744057 16.102539
#> 3750 26.393661 11.271777
#> 5804 16.496038 6.441015
#> 10551 36.291284 16.102539
#> X10X83_3_ACCTACATCACCCT. X10X83_3_TTTCCTCACCATAG.
#> 4130 192.785296 161.055472
#> 7161 298.446467 242.396619
#> 2989 44.488914 112.250783
#> 3200 63.025962 60.192449
#> 1147 16.683343 37.416928
#> 12565 44.488914 35.790105
#> 9749 20.390752 22.775521
#> 10491 11.122229 22.775521
#> 10452 37.074095 27.655990
#> 10532 20.390752 13.014584
#> 10525 14.829638 16.268229
#> 10460 12.975933 22.775521
#> 3750 9.268524 26.029167
#> 5804 27.805571 50.431511
#> 10551 11.122229 8.134115
#> X10X83_3_TATTGAGTTCGCGC. X10X83_3_GTCTGCAAGGACAC.
#> 4130 190.70179 108.906859
#> 7161 261.27647 36.473523
#> 2989 52.55561 3.595981
#> 3200 82.58739 19.521041
#> 1147 34.53654 4.623404
#> 12565 21.02224 56.508276
#> 9749 22.52383 28.767850
#> 10491 16.51748 52.398583
#> 10452 13.51430 35.446100
#> 10532 18.01907 40.069505
#> 10525 18.01907 25.685580
#> 10460 12.01271 26.713003
#> 3750 16.51748 30.822696
#> 5804 16.51748 10.787944
#> 10551 24.02542 34.932389
#> X10X83_3_TCAGTCACTCGACG. X10X83_3_AGAGAAGTCATCCA.
#> 4130 125.580776 232.377694
#> 7161 271.181677 160.876865
#> 2989 103.740641 92.951077
#> 3200 27.300169 75.075870
#> 1147 43.680270 48.263059
#> 12565 3.640023 23.237769
#> 9749 74.620461 32.175373
#> 10491 12.740079 7.150083
#> 10452 25.480158 12.512645
#> 10532 16.380101 16.087686
#> 10525 14.560090 8.937604
#> 10460 10.920068 17.875207
#> 3750 14.560090 7.150083
#> 5804 30.940191 19.662728
#> 10551 16.380101 7.150083
#> X10X83_3_ACTAGCATTTGCTT. X10X83_3_GAAGTGTACAGTTC.
#> 4130 181.14142 208.968944
#> 7161 296.24170 52.242236
#> 2989 41.51158 58.574628
#> 3200 56.60669 49.076040
#> 1147 45.28536 6.332392
#> 12565 16.98201 44.326746
#> 9749 16.98201 37.994353
#> 10491 24.52957 26.912667
#> 10452 13.20823 23.746471
#> 10532 20.75579 23.746471
#> 10525 15.09512 18.997177
#> 10460 15.09512 23.746471
#> 3750 28.30335 12.664784
#> 5804 39.62469 37.994353
#> 10551 13.20823 17.414079
#> X10X83_3_GCGGTCATCACGTA. X10X83_3_ATTCTGTTATCGGT.
#> 4130 139.04722 279.257288
#> 7161 362.87932 235.264702
#> 2989 40.69675 34.428981
#> 3200 47.47954 68.857962
#> 1147 27.13116 42.079865
#> 12565 49.17523 22.952654
#> 9749 67.82791 7.650885
#> 10491 23.73977 9.563606
#> 10452 23.73977 24.865375
#> 10532 16.95698 28.690817
#> 10525 30.52256 7.650885
#> 10460 30.52256 19.127212
#> 3750 30.52256 24.865375
#> 5804 23.73977 21.039933
#> 10551 18.65268 13.389048
#> X10X83_3_GTTAGTCAGTACGT. X10X83_3_GGTGACAAAGGTGC.
#> 4130 206.77137 246.113115
#> 7161 243.53073 166.884921
#> 2989 43.65173 131.485089
#> 3200 50.54411 32.028419
#> 1147 34.46190 33.714125
#> 12565 22.97460 35.399832
#> 9749 29.86698 70.799663
#> 10491 22.97460 8.428531
#> 10452 25.27206 15.171356
#> 10532 20.67714 26.971300
#> 10525 20.67714 3.371413
#> 10460 29.86698 11.799944
#> 3750 16.08222 23.599888
#> 5804 16.08222 20.228475
#> 10551 11.48730 16.857063
#> X10X83_3_GCGATTCACCCGAG. X10X83_3_GCTGGCAACACCCG.
#> 4130 223.306299 120.527998
#> 7161 409.904713 315.227072
#> 2989 78.004255 6.180923
#> 3200 47.414351 47.902153
#> 1147 42.825866 24.723692
#> 12565 26.001418 15.452307
#> 9749 27.530914 23.178461
#> 10491 19.883438 24.723692
#> 10452 15.294952 23.178461
#> 10532 15.294952 9.271384
#> 10525 21.412933 12.361846
#> 10460 15.294952 6.180923
#> 3750 22.942428 4.635692
#> 5804 13.765457 23.178461
#> 10551 6.117981 10.816615
#> X10X83_3_TATGCCAGGAATCG. X10X83_3_CACGAGTTAGAACA.
#> 4130 133.58606 187.95913
#> 7161 249.59500 256.30790
#> 2989 28.12338 25.63079
#> 3200 42.18507 35.88311
#> 1147 28.12338 23.92207
#> 12565 12.30398 13.66975
#> 9749 35.15423 35.88311
#> 10491 35.15423 13.66975
#> 10452 26.36567 27.33951
#> 10532 35.15423 37.59183
#> 10525 28.12338 37.59183
#> 10460 22.85025 27.33951
#> 3750 10.54627 13.66975
#> 5804 14.06169 10.25232
#> 10551 21.09254 13.66975
#> X10X83_3_CGTACCATTGTGCA. X10X83_3_ACAGAAGACAAAGG.
#> 4130 165.16446 184.42824
#> 7161 321.15311 140.46569
#> 2989 32.11531 32.16772
#> 3200 66.52457 40.74577
#> 1147 52.76087 15.01160
#> 12565 18.35161 20.37289
#> 9749 25.23346 39.67352
#> 10491 20.64556 27.87869
#> 10452 22.93951 27.87869
#> 10532 16.05766 31.09546
#> 10525 36.70321 24.66192
#> 10460 25.23346 34.31223
#> 3750 16.05766 19.30063
#> 5804 25.23346 7.50580
#> 10551 18.35161 22.51740
#> X10X83_3_GGGTTGTGAGCGAT. X10X83_3_CTACGTCCTCATTA.
#> 4130 246.52734 158.97166
#> 7161 210.90201 335.74816
#> 2989 51.30049 16.53305
#> 3200 47.02545 63.58867
#> 1147 44.17542 35.60965
#> 12565 21.37520 50.87093
#> 9749 22.80022 11.44596
#> 10491 25.65024 30.52256
#> 10452 21.37520 26.70724
#> 10532 18.52518 24.16369
#> 10525 28.50027 21.62015
#> 10460 18.52518 7.63064
#> 3750 17.10016 15.26128
#> 5804 25.65024 19.07660
#> 10551 25.65024 15.26128
#> X10X83_3_AACATAGGCTACGA. X10X83_3_CAGCCAGTGGGATC.
#> 4130 198.60685 187.48514
#> 7161 227.25207 0.00000
#> 2989 110.76151 14.58218
#> 3200 64.92916 39.58020
#> 1147 101.21311 20.83168
#> 12565 17.18713 29.16436
#> 9749 36.28394 45.82970
#> 10491 11.45809 49.99604
#> 10452 15.27745 66.66138
#> 10532 11.45809 56.24554
#> 10525 26.73554 31.24752
#> 10460 15.27745 39.58020
#> 3750 13.36777 33.33069
#> 5804 21.00649 12.49901
#> 10551 17.18713 24.99802
#> X10X83_3_TGAAGAGACAAAGG. X10X83_3_CGTGGTCCCGGATG.
#> 4130 181.62962 182.415221
#> 7161 56.31926 172.939105
#> 2989 40.83146 40.273490
#> 3200 42.23945 30.797375
#> 1147 19.71174 37.904461
#> 12565 21.11972 30.797375
#> 9749 21.11972 68.701836
#> 10491 29.56761 28.428346
#> 10452 21.11972 21.321260
#> 10532 12.67183 21.321260
#> 10525 18.30376 16.583202
#> 10460 25.34367 14.214173
#> 3750 21.11972 11.845144
#> 5804 25.34367 18.952231
#> 10551 12.67183 7.107087
#> X10X83_3_CAGAATCTCATTCA. X10X83_3_GCGATCAAATTGCC.
#> 4130 172.400413 215.347442
#> 7161 131.975488 301.149938
#> 2989 13.078652 107.673721
#> 3200 24.968336 57.201664
#> 1147 17.834525 55.519262
#> 12565 30.913177 26.918430
#> 9749 27.346272 20.188823
#> 10491 15.456589 10.094411
#> 10452 24.968336 15.141617
#> 10532 23.779367 13.459215
#> 10525 7.133810 13.459215
#> 10460 10.700715 15.141617
#> 3750 3.566905 11.776813
#> 5804 16.645557 20.188823
#> 10551 10.700715 8.412009
#> X10X83_3_GACCTGTTACCAGT. X10X83_3_ATCTTGTAAGAGGA.
#> 4130 111.52433 167.40159
#> 7161 228.21183 139.84577
#> 2989 11.35896 42.71152
#> 3200 54.72953 33.75588
#> 1147 17.55476 16.53349
#> 12565 27.88108 50.28937
#> 9749 26.84845 22.73355
#> 10491 43.37057 23.42245
#> 10452 35.10951 13.77791
#> 10532 36.14215 22.04465
#> 10525 27.88108 24.80024
#> 10460 35.10951 27.55582
#> 3750 29.94635 26.86692
#> 5804 15.48949 24.11134
#> 10551 28.91372 19.97797
#> X10X83_3_GTGGTGTAGCACGA. X10X83_3_ATTCGAGACTAGAT.
#> 4130 297.894388 151.41057
#> 7161 282.519194 61.42127
#> 2989 90.329266 32.85324
#> 3200 65.344575 24.28283
#> 1147 71.110273 34.28164
#> 12565 15.375194 57.13606
#> 9749 38.437986 17.14082
#> 10491 17.297094 41.42365
#> 10452 15.375194 55.70766
#> 10532 13.453295 32.85324
#> 10525 15.375194 38.56684
#> 10460 5.765698 34.28164
#> 3750 13.453295 28.56803
#> 5804 24.984691 28.56803
#> 10551 7.687597 27.13963
#> X10X83_3_GAATGAGAACTCGG. X10X83_3_AGAGAGTCCGAGTC.
#> 4130 121.607073 200.28729
#> 7161 0.000000 124.13243
#> 2989 0.000000 15.99252
#> 3200 7.015793 44.16982
#> 1147 0.000000 15.99252
#> 12565 46.771951 24.36956
#> 9749 11.692988 21.32336
#> 10491 35.078963 21.32336
#> 10452 114.591281 11.42323
#> 10532 25.724573 15.99252
#> 10525 44.433354 21.32336
#> 10460 28.063171 12.18478
#> 3750 18.708781 12.18478
#> 5804 35.078963 35.03124
#> 10551 25.724573 11.42323
#> X10X83_3_CTTACTCCGCGGTA. X10X83_3_ATACGGTGTCGCTG.
#> 4130 169.235412 206.58313
#> 7161 55.748136 40.34827
#> 2989 26.878566 20.17413
#> 3200 23.892058 25.01593
#> 1147 4.977512 20.98110
#> 12565 65.205409 66.97813
#> 9749 35.838087 15.33234
#> 10491 36.335839 40.34827
#> 10452 27.376317 20.17413
#> 10532 21.403302 26.62986
#> 10525 16.923541 22.59503
#> 10460 16.923541 20.17413
#> 3750 24.389809 26.62986
#> 5804 19.412297 10.49055
#> 10551 25.883063 21.78806
#> X10X83_3_CGAGTTCAACACGT.
#> 4130 189.10441
#> 7161 73.31588
#> 2989 68.25961
#> 3200 41.46139
#> 1147 34.38262
#> 12565 40.95577
#> 9749 25.78696
#> 10491 26.79822
#> 10452 33.87699
#> 10532 28.31510
#> 10525 19.21382
#> 10460 18.20256
#> 3750 26.79822
#> 5804 15.67443
#> 10551 25.78696
obj = ttm$object
ttm$pca.cell.2
Just to check again, we plot the final efficiency coloured PCA.
nu_est = round(obj@nu, digits = 7)
plot.nu <-ggplot(ttm$pca_cells,aes(x=PC1,y=PC2, colour = log(nu_est)))
plot.nu = plot.nu + geom_point(size = 2,alpha= 0.8)+
scale_color_gradient2(low = "#E64B35B2",mid = "#4DBBD5B2", high = "#3C5488B2" ,
midpoint = log(mean(nu_est)),name = "ln(nu)")+
ggtitle("Cells PCA coloured by cells efficiency: last") +
my_theme + theme(plot.title = element_text(color = "#3C5488FF", size = 20),
legend.title=element_text(color = "#3C5488FF", size = 14,face = "italic"),
legend.text = element_text(color = "#3C5488FF", size = 11),
legend.key.width = unit(2, "mm"),
legend.position="right")
pdf(paste(out_dir,"cleaning/",t,"_plots_PCA_efficiency_colored_FINAL.pdf", sep = ""))
plot.nu
dev.off()
#> png
#> 2
plot.nu
COTAN analysis: in this part all the contingency tables are computed and used to get the statistics (S) To storage efficiency of all the observed tables only the yes_yes is stored. Note that if will be necessary re-computing the yes-yes table, the yes-yes table should be cancelled before running cotan_analysis.
COEX evaluation and storing
saveRDS(obj,file = paste(out_dir,t,".cotan.RDS", sep = ""))
obj = get.coex(obj)
# saving the structure
saveRDS(obj,file = paste(out_dir,t,".cotan.RDS", sep = ""))Automatic data elaboration (without cleaning)
3 Analysis of the elaborated data
In this case the test possible macroscopic differences between cleaned and not cleaned data.
The initial cell number was:
obj@meta
#> V1 V2
#> 1 GEO: GSE104323
#> 2 scRNAseq method: 10X
#> 3 starting n. of cells: 4779
#> 4 Condition sample: mouse dentate gyrus P0
#> 5 Threshold low UDE cells: 0.4While the final number of cells was:
So were removed:
cells
3.1 GDI with cleaning
quant.p = get.GDI(obj)
#> [1] "function to generate GDI dataframe"
#> [1] "Using S"
#> [1] "function to generate S "
head(quant.p)
#> sum.raw.norm GDI exp.cells
#> 0610007P14Rik 8.538538 2.909430 58.635394
#> 0610009B22Rik 7.788376 2.044579 37.270789
#> 0610009L18Rik 6.464229 1.809001 12.366738
#> 0610009O20Rik 6.279491 1.787471 9.616205
#> 0610010F05Rik 7.952439 3.657138 41.300640
#> 0610010K14Rik 7.650414 2.042570 33.731343In the third column of this dataframe is reported the percentage of cells expressing the gene.
NPGs=c("Nes","Vim","Sox2","Sox1","Notch1", "Hes1","Hes5","Pax6") #,"Hes3"
PNGs=c("Map2","Tubb3","Neurod1","Nefm","Nefl","Dcx","Tbr1")
hk=c("Calm1","Cox6b1","Ppia","Rpl18","Cox7c","Erh","H3f3a","Taf1","Taf2","Gapdh","Actb","Golph3", "Mtmr12", "Zfr", "Sub1", "Tars", "Amacr")
text.size = 12
quant.p$highlight = with(quant.p, ifelse(rownames(quant.p) %in% NPGs, "NPGs",
ifelse(rownames(quant.p) %in% hk,"Constitutive" ,
ifelse(rownames(quant.p) %in% PNGs,"PNGs" , "normal"))))
textdf <- quant.p[rownames(quant.p) %in% c(NPGs,hk,PNGs), ]
mycolours <- c("Constitutive" = "#00A087FF","NPGs"="#E64B35FF","PNGs"="#F39B7FFF","normal" = "#8491B4B2")
f1 = ggplot(subset(quant.p,highlight == "normal" ), aes(x=sum.raw.norm, y=GDI)) + geom_point(alpha = 0.1, color = "#8491B4B2", size=2.5)
GDI_plot = f1 + geom_point(data = subset(quant.p,highlight != "normal" ), aes(x=sum.raw.norm, y=GDI, colour=highlight),size=2.5,alpha = 0.8) +
geom_hline(yintercept=quantile(quant.p$GDI)[4], linetype="dashed", color = "darkblue") +
geom_hline(yintercept=quantile(quant.p$GDI)[3], linetype="dashed", color = "darkblue") +
geom_hline(yintercept=1.5, linetype="dotted", color = "red", size= 0.5) +
scale_color_manual("Status", values = mycolours) +
scale_fill_manual("Status", values = mycolours) +
xlab("log normalized counts")+ylab("GDI")+
geom_label_repel(data =textdf , aes(x=sum.raw.norm, y=GDI, label = rownames(textdf),fill=highlight),
label.size = NA,
alpha = 0.5,
direction ="both",
na.rm=TRUE,
seed = 1234) +
geom_label_repel(data =textdf , aes(x=sum.raw.norm, y=GDI, label = rownames(textdf)),
label.size = NA,
segment.color = 'black',
segment.size = 0.5,
direction = "both",
alpha = 0.8,
na.rm=TRUE,
fill = NA,
seed = 1234) +
theme(axis.text.x = element_text(size = text.size, angle = 0, hjust = .5, vjust = .5, face = "plain", colour ="#3C5488FF" ),
axis.text.y = element_text( size = text.size, angle = 0, hjust = 0, vjust = .5, face = "plain", colour ="#3C5488FF"),
axis.title.x = element_text( size = text.size, angle = 0, hjust = .5, vjust = 0, face = "plain", colour ="#3C5488FF"),
axis.title.y = element_text( size = text.size, angle = 90, hjust = .5, vjust = .5, face = "plain", colour ="#3C5488FF"),
legend.title = element_blank(),
legend.text = element_text(color = "#3C5488FF",face ="italic" ),
legend.position = "bottom") # titl)
legend <- cowplot::get_legend(GDI_plot)
GDI_plot =GDI_plot + theme(
legend.position = "none")
GDI_plot
3.2 GDI without cleaning
quant.p = get.GDI(obj2)
#> [1] "function to generate GDI dataframe"
#> [1] "Using S"
#> [1] "function to generate S "
head(quant.p)
#> sum.raw.norm GDI exp.cells
#> 0610007P14Rik 8.545662 2.930231 58.108391
#> 0610009B22Rik 7.803461 2.049402 37.057962
#> 0610009L18Rik 6.474749 1.819495 12.220130
#> 0610009O20Rik 6.299620 1.809639 9.583595
#> 0610010F05Rik 7.965502 3.652991 40.929065
#> 0610010K14Rik 7.656849 2.033830 33.417033In the third column of this dataframe is reported the percentage of cells expressing the gene.
NPGs=c("Nes","Vim","Sox2","Sox1","Notch1", "Hes1","Hes5","Pax6") #,"Hes3"
PNGs=c("Map2","Tubb3","Neurod1","Nefm","Nefl","Dcx","Tbr1")
hk=c("Calm1","Cox6b1","Ppia","Rpl18","Cox7c","Erh","H3f3a","Taf1","Taf2","Gapdh","Actb","Golph3", "Mtmr12", "Zfr", "Sub1", "Tars", "Amacr")
text.size = 12
quant.p$highlight = with(quant.p, ifelse(rownames(quant.p) %in% NPGs, "NPGs",
ifelse(rownames(quant.p) %in% hk,"Constitutive" ,
ifelse(rownames(quant.p) %in% PNGs,"PNGs" , "normal"))))
textdf <- quant.p[rownames(quant.p) %in% c(NPGs,hk,PNGs), ]
mycolours <- c("Constitutive" = "#00A087FF","NPGs"="#E64B35FF","PNGs"="#F39B7FFF","normal" = "#8491B4B2")
f1 = ggplot(subset(quant.p,highlight == "normal" ), aes(x=sum.raw.norm, y=GDI)) + geom_point(alpha = 0.1, color = "#8491B4B2", size=2.5)
GDI_plot = f1 + geom_point(data = subset(quant.p,highlight != "normal" ), aes(x=sum.raw.norm, y=GDI, colour=highlight),size=2.5,alpha = 0.8) +
geom_hline(yintercept=quantile(quant.p$GDI)[4], linetype="dashed", color = "darkblue") +
geom_hline(yintercept=quantile(quant.p$GDI)[3], linetype="dashed", color = "darkblue") +
geom_hline(yintercept=1.5, linetype="dotted", color = "red", size= 0.5) +
scale_color_manual("Status", values = mycolours) +
scale_fill_manual("Status", values = mycolours) +
xlab("log normalized counts")+ylab("GDI")+
geom_label_repel(data =textdf , aes(x=sum.raw.norm, y=GDI, label = rownames(textdf),fill=highlight),
label.size = NA,
alpha = 0.5,
direction ="both",
na.rm=TRUE,
seed = 1234) +
geom_label_repel(data =textdf , aes(x=sum.raw.norm, y=GDI, label = rownames(textdf)),
label.size = NA,
segment.color = 'black',
segment.size = 0.5,
direction = "both",
alpha = 0.8,
na.rm=TRUE,
fill = NA,
seed = 1234) +
theme(axis.text.x = element_text(size = text.size, angle = 0, hjust = .5, vjust = .5, face = "plain", colour ="#3C5488FF" ),
axis.text.y = element_text( size = text.size, angle = 0, hjust = 0, vjust = .5, face = "plain", colour ="#3C5488FF"),
axis.title.x = element_text( size = text.size, angle = 0, hjust = .5, vjust = 0, face = "plain", colour ="#3C5488FF"),
axis.title.y = element_text( size = text.size, angle = 90, hjust = .5, vjust = .5, face = "plain", colour ="#3C5488FF"),
legend.title = element_blank(),
legend.text = element_text(color = "#3C5488FF",face ="italic" ),
legend.position = "bottom") # titl)
legend <- cowplot::get_legend(GDI_plot)
GDI_plot =GDI_plot + theme(
legend.position = "none")
GDI_plot
3.3 Heatmaps with cleaning
For the Gene Pair Analysis, we can plot an heatmap with the coex values between two genes sets. To do so we need to define, in a list, the different gene sets (list.genes). Then in the function parameter sets we can decide which sets need to be considered (in the example from 1 to 3). In the condition parameter we should insert an array with each file name prefix to be considered (in the example, the file names is “E17.5_cortex”).
list.genes = list("Ref.col"= PNGs, "NPGs"=NPGs, "Const."=hk )
plot_heatmap(df_genes = list.genes,sets = c(1:3),conditions = "P0_dent_cleaned",dir = "Data/")
#> [1] "plot heatmap"
#> [1] "Loading condition P0_dent_cleaned"
#> [1] "Map2" "Tubb3" "Neurod1" "Nefm" "Nefl" "Dcx" "Tbr1"
#> [1] "Get p-values on a set of genes on columns on a set of genes on rows"
#> [1] "Using function S"
#> [1] "function to generate S "
#> [1] "Ref.col"
#> [1] "NPGs"
#> [1] "Const."
#> [1] "min coex: -0.00753159172756036 max coex 0.00674563579291443"
## Heatmaps without cleaning
list.genes = list("Ref.col"= PNGs, "NPGs"=NPGs, "Const."=hk )
plot_heatmap(df_genes = list.genes,sets = c(1:3),conditions = "P0_dent_gyrus",dir = "Data/")
#> [1] "plot heatmap"
#> [1] "Loading condition P0_dent_gyrus"
#> [1] "Map2" "Tubb3" "Neurod1" "Nefm" "Nefl" "Dcx" "Tbr1"
#> [1] "Get p-values on a set of genes on columns on a set of genes on rows"
#> [1] "Using function S"
#> [1] "function to generate S "
#> [1] "Ref.col"
#> [1] "NPGs"
#> [1] "Const."
#> [1] "min coex: -0.00741399939021704 max coex 0.00658383694600149"
print(sessionInfo())
#> R version 4.0.4 (2021-02-15)
#> Platform: x86_64-pc-linux-gnu (64-bit)
#> Running under: Ubuntu 18.04.5 LTS
#>
#> Matrix products: default
#> BLAS: /usr/lib/x86_64-linux-gnu/openblas/libblas.so.3
#> LAPACK: /usr/lib/x86_64-linux-gnu/libopenblasp-r0.2.20.so
#>
#> locale:
#> [1] LC_CTYPE=en_US.UTF-8 LC_NUMERIC=C
#> [3] LC_TIME=en_US.UTF-8 LC_COLLATE=en_US.UTF-8
#> [5] LC_MONETARY=en_US.UTF-8 LC_MESSAGES=en_US.UTF-8
#> [7] LC_PAPER=en_US.UTF-8 LC_NAME=C
#> [9] LC_ADDRESS=C LC_TELEPHONE=C
#> [11] LC_MEASUREMENT=en_US.UTF-8 LC_IDENTIFICATION=C
#>
#> attached base packages:
#> [1] stats graphics grDevices utils datasets methods base
#>
#> other attached packages:
#> [1] ggrepel_0.9.1 ggplot2_3.3.3 Matrix_1.3-2 data.table_1.14.0
#> [5] COTAN_0.1.0
#>
#> loaded via a namespace (and not attached):
#> [1] Rcpp_1.0.6 lattice_0.20-41 circlize_0.4.12
#> [4] tidyr_1.1.2 png_0.1-7 assertthat_0.2.1
#> [7] digest_0.6.27 utf8_1.2.1 R6_2.5.0
#> [10] stats4_4.0.4 evaluate_0.14 highr_0.8
#> [13] pillar_1.5.1 basilisk_1.2.1 GlobalOptions_0.1.2
#> [16] rlang_0.4.10 jquerylib_0.1.3 S4Vectors_0.28.1
#> [19] GetoptLong_1.0.5 reticulate_1.18 rmarkdown_2.7
#> [22] labeling_0.4.2 stringr_1.4.0 munsell_0.5.0
#> [25] compiler_4.0.4 xfun_0.22 pkgconfig_2.0.3
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