- TCR-seq - EDA -

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Last updated: 2020-01-15

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Knit directory: Simon_et_al_2020/

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File	Version	Author	Date	Message
Rmd	d9398bc	ValentinVoillet	2020-01-15	Edits .Rmd (TCR-seq_EDA_2)
html	7145ac8	ValentinVoillet	2020-01-14	Add .html files
Rmd	49f6c61	ValentinVoillet	2020-01-14	Edits .Rmd (TCR-seq_EDA_2)
Rmd	99d00e5	ValentinVoillet	2020-01-14	Edits .Rmd (TCR-seq_EDA_2)

---

File creation: January, 12th 2020
Update: January, 14th 2020

1 Description & importing data

---

RNA was extracted from 12 patients. Alignment and quantification of TCR sequences have been performed by QIAGEN

• 12 patients: P5, P6, P7, P8, P14, P15, P16, P18, P19, P21, P22 and P23;

• Four time points: T0, M1, M2 & M6;

• One treatment: anti-PD1;

• Four fractions: PD-1+TIGIT+, PD-1+, TIGIT+ and PD-1-TIGIT-;

• Two outcomes: NR and R;

• Three batches.

QC have already been performed - please look at the TCR-QC section. Fourteen samples have been removed.

###--- Importation
#- TCR.pData
here("output", "TCR_pData.rds") %>%
  readRDS() -> TCR.pData
# pData (patient/fraction w/ at least T0 & M1)
TCR.pData %>% select(patient.id, time.point, fraction, outcome) %>% View("pData")
TCR.pData <- TCR.pData[c(1:39, 41:43, 45:114, 117:120, 123:125, 127, 130, 132, 135:146, 148:154, 157:158), ] 
TCR.pData %>% filter(time.point != "M2" & time.point != "M6") -> TCR.pData
TCR.pData %>%
  arrange(patient.id) %>%
  select(patient.id, time.point, fraction, outcome) %>% 
  View("pData")

#- TCR.exprs
here("output", "TCR_count.rds") %>%
  readRDS() %>%
  filter(QIAGEN.id %in% TCR.pData$QIAGEN.id) -> TCR.exprs

2 Summary statistics

---

The clonality score is derived from the Shannon entropy, which is calculated from the frequencies of all productive sequences divided by the logarithm of the total number of unique productive sequences. This normalized entropy value is then inverted (1 - normalized entropy) to produce the clonality metric.
The Gini coefficient is an alternative metric used to calculate repertoire diversity.
Both Gini coefficient and clonality are reported on a scale from 0 to 1 where 0 indicates all sequences have the same frequency and 1 indicates the repertoire is dominated by a single sequence.

###--- Extracting productive sequences - aggregate samples having the same cdr3aa, cdr3nt and V/J-regions
TCR.exprs %>%
  group_by(QIAGEN.id, chain, cdr3nt, cdr3aa, `V-region`, `J-region`) %>%
  summarise(frequency = sum(freq.after.filtering), 
            `UMIs with >= 1 reads` = sum(`UMIs with >= 1 reads`),
            `UMIs with >= 2 reads` = sum(`UMIs with >= 2 reads`),
            `UMIs with >= 3 reads` = sum(`UMIs with >= 3 reads`),
            `UMIs with >= 4 reads` = sum(`UMIs with >= 4 reads`),
            `UMIs with >= 5 reads` = sum(`UMIs with >= 5 reads`),
            `UMIs with >= 6 reads` = sum(`UMIs with >= 6 reads`),
            `UMIs with >= 7 reads` = sum(`UMIs with >= 7 reads`),
            total.reads = sum(`# reads`),
            total.UMIs = sum(total.UMIs)) %>%
  arrange(QIAGEN.id, chain, desc(`UMIs with >= 1 reads`)) -> productive.aa

###--- Summary statistics
productive.aa %>%
  filter(chain == "TRAC" | chain == "TRBC") %>% 
  group_by(QIAGEN.id, chain) %>%
  summarise(total.reads = sum(total.reads),
            total.UMIs = sum(total.UMIs),
            unique.productive = n(),
            entropy = -sum(frequency * log2(frequency), na.rm = TRUE),
            gini.coef = ineq::Gini(frequency)) %>%
  mutate(clonality = 1 - round(entropy / log2(unique.productive), digits = 6)) -> dt
dt <- merge(dt, TCR.pData[, c("QIAGEN.id", "sample.number", "sample.id", "treatment", "batch", "patient.id", "time.point", "fraction.desc", "outcome")], by = "QIAGEN.id")

Past versions of unnamed-chunk-5-1.png

Version	Author	Date
7145ac8	ValentinVoillet	2020-01-14

3 Clustering

---

Within each fraction, let"s look at sequences (both TRAC and TRBC). We declare as emerging, expanding, emerging-contracting, expanding-contracting, contracting-expanding, contracting sequences (regarding to M1) that are declared as significant (Fisher exact test to calculate differential abundance of each TRBC (or TRAC) between two time points –total.UMIs value is used) in the following contrasts T0 vs M1. Other sequences are declared as non-expanding/contracting.

3.1 PD-1+TIGIT+

3.1.1 Patient - P5

Past versions of P5_DPOS_1-1.png

Version	Author	Date
7145ac8	ValentinVoillet	2020-01-14

3.1.2 Patient - P6

Past versions of P6_DPOS_1-1.png

Version	Author	Date
7145ac8	ValentinVoillet	2020-01-14

3.1.3 Patient - P7

Past versions of P7_DPOS_1-1.png

Version	Author	Date
7145ac8	ValentinVoillet	2020-01-14

3.1.4 Patient - P8

Past versions of P8_DPOS_1-1.png

Version	Author	Date
7145ac8	ValentinVoillet	2020-01-14

3.1.5 Patient - P14

Past versions of P14_DPOS_1-1.png

Version	Author	Date
7145ac8	ValentinVoillet	2020-01-14

3.1.6 Patient - P15

Past versions of P15_DPOS_1-1.png

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7145ac8	ValentinVoillet	2020-01-14

3.1.7 Patient - P16

Past versions of P16_DPOS_1-1.png

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7145ac8	ValentinVoillet	2020-01-14

3.1.8 Patient - P18

Past versions of P18_DPOS_1-1.png

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7145ac8	ValentinVoillet	2020-01-14

3.1.9 Patient - P19

Past versions of P19_DPOS_1-1.png

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7145ac8	ValentinVoillet	2020-01-14

3.1.10 Patient - P21

Past versions of P21_DPOS_1-1.png

Version	Author	Date
7145ac8	ValentinVoillet	2020-01-14

3.1.11 Patient - P22

No PD-1+TIGIT+ sample (T0 & M1).

3.1.12 Patient - P23

No PD-1+TIGIT+ sample (T0 & M1).

3.2 PD-1+

3.2.1 Patient - P5

Past versions of P5_PD1_1-1.png

Version	Author	Date
7145ac8	ValentinVoillet	2020-01-14

3.2.2 Patient - P6

No PD-1+ sample (T0 & M1).

3.2.3 Patient - P7

Past versions of P7_PD1_1-1.png

Version	Author	Date
7145ac8	ValentinVoillet	2020-01-14

3.2.4 Patient - P8

Past versions of P8_PD1_1-1.png

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7145ac8	ValentinVoillet	2020-01-14

3.2.5 Patient - P14

Past versions of P14_PD1_1-1.png

Version	Author	Date
7145ac8	ValentinVoillet	2020-01-14

3.2.6 Patient - P15

Past versions of P15_PD1_1-1.png

Version	Author	Date
7145ac8	ValentinVoillet	2020-01-14

3.2.7 Patient - P16

Past versions of P16_PD1_1-1.png

Version	Author	Date
7145ac8	ValentinVoillet	2020-01-14

3.2.8 Patient - P18

Past versions of P18_PD1_1-1.png

Version	Author	Date
7145ac8	ValentinVoillet	2020-01-14

3.2.9 Patient - P19

Past versions of P19_PD1_1-1.png

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7145ac8	ValentinVoillet	2020-01-14

3.2.10 Patient - P21

Past versions of P21_PD1_1-1.png

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7145ac8	ValentinVoillet	2020-01-14

3.2.11 Patient - P22

Past versions of P22_PD1_1-1.png

Version	Author	Date
7145ac8	ValentinVoillet	2020-01-14

3.2.12 Patient - P23

No PD-1+ sample (T0 & M1).

3.3 TIGIT+

3.3.1 Patient - P5

Past versions of P5_TIGIT_1-1.png

Version	Author	Date
7145ac8	ValentinVoillet	2020-01-14

3.3.2 Patient - P6

Past versions of P6_TIGIT_1-1.png

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7145ac8	ValentinVoillet	2020-01-14

3.3.3 Patient - P7

Past versions of P7_TIGIT_1-1.png

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7145ac8	ValentinVoillet	2020-01-14

3.3.4 Patient - P8

Past versions of P8_TIGIT_1-1.png

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7145ac8	ValentinVoillet	2020-01-14

3.3.5 Patient - P14

Past versions of P14_TIGIT_1-1.png

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7145ac8	ValentinVoillet	2020-01-14

3.3.6 Patient - P15

Past versions of P15_TIGIT_1-1.png

Version	Author	Date
7145ac8	ValentinVoillet	2020-01-14

3.3.7 Patient - P16

Past versions of P16_TIGIT_1-1.png

Version	Author	Date
7145ac8	ValentinVoillet	2020-01-14

3.3.8 Patient - P18

Past versions of P18_TIGIT_1-1.png

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7145ac8	ValentinVoillet	2020-01-14

3.3.9 Patient - P19

Past versions of P19_TIGIT_1-1.png

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7145ac8	ValentinVoillet	2020-01-14

3.3.10 Patient - P21

Past versions of P21_TIGIT_1-1.png

Version	Author	Date
7145ac8	ValentinVoillet	2020-01-14

3.3.11 Patient - P22

No TIGIT+ sample (T0 & M1).

3.3.12 Patient - P23

Past versions of P23_TIGIT_1-1.png

Version	Author	Date
7145ac8	ValentinVoillet	2020-01-14

3.4 PD-1-TIGIT-

3.4.1 Patient - P5

Past versions of P5_DNEG_1-1.png

Version	Author	Date
7145ac8	ValentinVoillet	2020-01-14

3.4.2 Patient - P6

Past versions of P6_DNEG_1-1.png

Version	Author	Date
7145ac8	ValentinVoillet	2020-01-14

3.4.3 Patient - P7

Past versions of P7_DNEG_1-1.png

Version	Author	Date
7145ac8	ValentinVoillet	2020-01-14

3.4.4 Patient - P8

Past versions of P8_DNEG_1-1.png

Version	Author	Date
7145ac8	ValentinVoillet	2020-01-14

3.4.5 Patient - P14

Past versions of P14_DNEG_1-1.png

Version	Author	Date
7145ac8	ValentinVoillet	2020-01-14

3.4.6 Patient - P15

Past versions of P15_DNEG_1-1.png

Version	Author	Date
7145ac8	ValentinVoillet	2020-01-14

3.4.7 Patient - P16

Past versions of P16_DNEG_1-1.png

Version	Author	Date
7145ac8	ValentinVoillet	2020-01-14

3.4.8 Patient - P18

Past versions of P18_DNEG_1-1.png

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7145ac8	ValentinVoillet	2020-01-14

3.4.9 Patient - P19

Past versions of P19_DNEG_1-1.png

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7145ac8	ValentinVoillet	2020-01-14

3.4.10 Patient - P21

Past versions of P21_DNEG_1-1.png

Version	Author	Date
7145ac8	ValentinVoillet	2020-01-14

3.4.11 Patient - P22

Past versions of P22_DNEG_1-1.png

Version	Author	Date
7145ac8	ValentinVoillet	2020-01-14

3.4.12 Patient - P23

No PD-1-TIGIT- sample (T0 & M1).

3.5 Summary

TRAC

Past versions of unnamed-chunk-24-1.png

Version	Author	Date
7145ac8	ValentinVoillet	2020-01-14

Past versions of unnamed-chunk-26-1.png

Version	Author	Date
7145ac8	ValentinVoillet	2020-01-14

Table - # of patients per fraction

Table - NR - # of patients per cluster

Table - R - # of patients per cluster

TRBC

Past versions of unnamed-chunk-31-1.png

Version	Author	Date
7145ac8	ValentinVoillet	2020-01-14

Past versions of unnamed-chunk-33-1.png

Version	Author	Date
7145ac8	ValentinVoillet	2020-01-14

Table - # of patients per fraction

Table - NR - # of patients per cluster

Table - R - # of patients per cluster

Session information

sessionInfo()

R version 3.6.2 (2019-12-12)
Platform: x86_64-apple-darwin15.6.0 (64-bit)
Running under: macOS Mojave 10.14.6

Matrix products: default
BLAS:   /Library/Frameworks/R.framework/Versions/3.6/Resources/lib/libRblas.0.dylib
LAPACK: /Library/Frameworks/R.framework/Versions/3.6/Resources/lib/libRlapack.dylib

Random number generation:
 RNG:     Mersenne-Twister 
 Normal:  Inversion 
 Sample:  Rounding 
 
locale:
[1] fr_FR.UTF-8/fr_FR.UTF-8/fr_FR.UTF-8/C/fr_FR.UTF-8/fr_FR.UTF-8

attached base packages:
[1] parallel  grid      stats     graphics  grDevices utils     datasets 
[8] methods   base     

other attached packages:
 [1] broom_0.5.3          doMC_1.3.6           iterators_1.0.12    
 [4] foreach_1.4.7        ComplexHeatmap_2.2.0 gridExtra_2.3       
 [7] VennDiagram_1.6.20   futile.logger_1.4.3  here_0.1            
[10] data.table_1.12.8    janitor_1.2.0        forcats_0.4.0       
[13] stringr_1.4.0        dplyr_0.8.3          purrr_0.3.3         
[16] readr_1.3.1          tidyr_1.0.0          tibble_2.1.3        
[19] ggplot2_3.2.1        tidyverse_1.3.0     

loaded via a namespace (and not attached):
 [1] nlme_3.1-143         fs_1.3.1             lubridate_1.7.4     
 [4] RColorBrewer_1.1-2   httr_1.4.1           rprojroot_1.3-2     
 [7] tools_3.6.2          backports_1.1.5      R6_2.4.1            
[10] DBI_1.1.0            lazyeval_0.2.2       colorspace_1.4-1    
[13] GetoptLong_0.1.8     withr_2.1.2          tidyselect_0.2.5    
[16] compiler_3.6.2       git2r_0.26.1         cli_2.0.1           
[19] rvest_0.3.5          formatR_1.7          xml2_1.2.2          
[22] labeling_0.3         scales_1.1.0         digest_0.6.23       
[25] rmarkdown_2.0        pkgconfig_2.0.3      htmltools_0.4.0     
[28] dbplyr_1.4.2         rlang_0.4.2          GlobalOptions_0.1.1 
[31] readxl_1.3.1         rstudioapi_0.10      farver_2.0.2        
[34] shape_1.4.4          generics_0.0.2       jsonlite_1.6        
[37] magrittr_1.5         Rcpp_1.0.3           munsell_0.5.0       
[40] fansi_0.4.1          lifecycle_0.1.0      stringi_1.4.5       
[43] whisker_0.4          yaml_2.2.0           plyr_1.8.5          
[46] promises_1.1.0       crayon_1.3.4         lattice_0.20-38     
[49] haven_2.2.0          cowplot_1.0.0        circlize_0.4.8      
[52] hms_0.5.3            zeallot_0.1.0        knitr_1.26          
[55] pillar_1.4.3         rjson_0.2.20         reshape2_1.4.3      
[58] codetools_0.2-16     futile.options_1.0.1 reprex_0.3.0        
[61] glue_1.3.1           evaluate_0.14        lambda.r_1.2.4      
[64] modelr_0.1.5         png_0.1-7            vctrs_0.2.1         
[67] httpuv_1.5.2         cellranger_1.1.0     gtable_0.3.0        
[70] clue_0.3-57          assertthat_0.2.1     xfun_0.12           
[73] later_1.0.0          viridisLite_0.3.0    ineq_0.2-13         
[76] workflowr_1.6.0      cluster_2.1.0