Onecut3 in mouse hypothalamus development
Evgenii Tretiakov
2021-01-28
Last updated: 2022-01-29
Checks: 7 0
Knit directory: Zupancic_2022/
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# Load tidyverse infrastructure packages
library(here)
# here() starts at /home/etretiakov/src/Zupancic_2022
library(tidyverse)
# ── Attaching packages ─────────────────────────────────────── tidyverse 1.3.1 ──
# ✔ ggplot2 3.3.5 ✔ purrr 0.3.4
# ✔ tibble 3.1.6 ✔ dplyr 1.0.7
# ✔ tidyr 1.1.4 ✔ stringr 1.4.0
# ✔ readr 2.1.1 ✔ forcats 0.5.1
# ── Conflicts ────────────────────────────────────────── tidyverse_conflicts() ──
# ✖ dplyr::filter() masks stats::filter()
# ✖ dplyr::lag() masks stats::lag()
library(magrittr)
#
# Attaching package: 'magrittr'
# The following object is masked from 'package:purrr':
#
# set_names
# The following object is masked from 'package:tidyr':
#
# extract
library(zeallot)
library(future)
#
# Attaching package: 'future'
# The following objects are masked from 'package:zeallot':
#
# %->%, %<-%
# Load packages for scRNA-seq analysis and visualisation
library(sctransform)
library(Seurat)
# Registered S3 method overwritten by 'spatstat.geom':
# method from
# print.boxx cli
# Attaching SeuratObject
library(SeuratWrappers)
library(SeuratDisk)
# Registered S3 method overwritten by 'SeuratDisk':
# method from
# as.sparse.H5Group Seurat
library(UpSetR)
library(patchwork)
library(Nebulosa)src_dir <- here("code")
data_dir <- here("data")
output_dir <- here("output")
plots_dir <- here(output_dir, "figures")
tables_dir <- here(output_dir, "tables")source(here(src_dir, "functions.R"))
source(here(src_dir, "genes.R"))reseed <- 42
set.seed(seed = reseed)# available cores
n_cores <- available_cores(prop2use = .5)
# Parameters for parallel execution
plan("multicore", workers = n_cores)
options(future.globals.maxSize = Inf,
future.rng.onMisuse = "ignore")
plan()
# multicore:
# - args: function (..., envir = parent.frame(), workers = 16)
# - tweaked: TRUE
# - call: plan("multicore", workers = n_cores)Read data
rar2020_ages_all <- c("E15", "E17", "P00", "P02", "P10", "P23")
rar2020_ages_postnat <- c("P02", "P10", "P23")
samples_df <- read_tsv(here("data/samples.tsv"))
# Rows: 8 Columns: 14
# ── Column specification ────────────────────────────────────────────────────────
# Delimiter: "\t"
# chr (8): sample, age, condition, fullname, name, sex, date, date10x
# dbl (6): ncells, libbatch, seqbatch, perfussed, nt, sn
#
# ℹ Use `spec()` to retrieve the full column specification for this data.
# ℹ Specify the column types or set `show_col_types = FALSE` to quiet this message.
colours_wtree <- setNames(read_lines(here(data_dir, "colours_wtree.tsv")),
1:45)
onecut <- LoadH5Seurat(here(data_dir, "rar2020.srt.cont.oc2or3.raw.h5seurat"))
# Validating h5Seurat file
# Initializing RNA with data
# Adding counts for RNA
# Adding scale.data for RNA
# Adding variable feature information for RNA
# Adding reduction pca
# Adding cell embeddings for pca
# Adding feature loadings for pca
# Adding miscellaneous information for pca
# Adding reduction tsne
# Adding cell embeddings for tsne
# Adding miscellaneous information for tsne
# Adding reduction umap
# Adding cell embeddings for umap
# Adding miscellaneous information for umap
# Adding command information
# Adding cell-level metadata
# Adding miscellaneous information
# Adding tool-specific results
onecut3 <- subset(onecut, subset = Onecut3 > 0)Derive and filter matrix of Onecut3
mtx_oc3 <-
onecut3 %>%
GetAssayData("data", "RNA") %>%
as.data.frame() %>%
t()
rownames(mtx_oc3) <- colnames(onecut3)
# Filter features
filt_low_genes <-
colSums(mtx_oc3) %>%
.[. > quantile(., 0.4)] %>%
names()
mtx_oc3 %<>% .[, filt_low_genes]
min_filt_vector <-
mtx_oc3 %>%
as_tibble() %>%
select(all_of(filt_low_genes)) %>%
summarise(across(.fns = ~ quantile(.x, .1))) %>%
as.list %>%
map(as.double) %>%
simplify %>%
.[colnames(mtx_oc3)]
# Prepare table of intersection sets analysis
content_mtx_oc3 <-
(mtx_oc3 > min_filt_vector) %>%
as_tibble() %>%
mutate_all(as.numeric)Correlation analysis visualisation between different genes
p_corrs <- list(
ggstatsplot::ggscatterstats(as.data.frame(mtx_oc3),
x = Onecut3, y = Trh, xfill = "#ffc400", yfill = "#e22ee2"),
ggstatsplot::ggscatterstats(as.data.frame(mtx_oc3),
x = Slc32a1, y = Onecut3, xfill = "#0000da", yfill = "#ffc400"),
ggstatsplot::ggscatterstats(as.data.frame(mtx_oc3),
x = Th, y = Onecut3, xfill = "#006eff", yfill = "#ffc400"),
ggstatsplot::ggscatterstats(as.data.frame(mtx_oc3),
x = Slc32a1, y = Trh, xfill = "#0000da", yfill = "#e22ee2"),
ggstatsplot::ggscatterstats(as.data.frame(mtx_oc3),
x = Th, y = Trh, xfill = "#006eff", yfill = "#e22ee2"),
ggstatsplot::ggscatterstats(as.data.frame(mtx_oc3),
x = Slc32a1, y = Th, xfill = "#0000da", yfill = "#006eff"),
ggstatsplot::ggscatterstats(as.data.frame(mtx_oc3),
y = Slc32a1, x = Onecut3, yfill = "#0000da", xfill = "#ffc400"),
ggstatsplot::ggscatterstats(as.data.frame(mtx_oc3),
y = Th, x = Onecut3, yfill = "#006eff", xfill = "#ffc400"),
ggstatsplot::ggscatterstats(as.data.frame(mtx_oc3),
y = Slc17a6, x = Onecut3, yfill = "#ff0000", xfill = "#ffc400"),
ggstatsplot::ggscatterstats(as.data.frame(mtx_oc3),
y = Gad1, x = Onecut3, yfill = "#a50202", xfill = "#ffc400"),
ggstatsplot::ggscatterstats(as.data.frame(mtx_oc3),
y = Gad2, x = Onecut3, yfill = "#4002a5", xfill = "#ffc400"),
ggstatsplot::ggscatterstats(as.data.frame(mtx_oc3),
y = Onecut2, x = Onecut3, yfill = "#6402a5", xfill = "#ffc400"),
ggstatsplot::ggscatterstats(as.data.frame(mtx_oc3),
y = Nav1, x = Onecut3, yfill = "#2502a5", xfill = "#ffc400"),
ggstatsplot::ggscatterstats(as.data.frame(mtx_oc3),
y = Nav2, x = Onecut3, yfill = "#4002a5", xfill = "#ffc400"),
ggstatsplot::ggscatterstats(as.data.frame(mtx_oc3),
y = Trio, x = Onecut3, yfill = "#2502a5", xfill = "#ffc400")
)
# Registered S3 method overwritten by 'ggside':
# method from
# +.gg ggplot2
n_corrs <- list(
"oc3-rna-data-Onecut3-Trh",
"oc3-rna-data-Slc32a1-Onecut3",
"oc3-rna-data-Th-Onecut3",
"oc3-rna-data-Slc32a1-Trh",
"oc3-rna-data-Th-Trh",
"oc3-rna-data-Slc32a1-Th",
"oc3-rna-data-Onecut3-Slc32a1",
"oc3-rna-data-Onecut3-Th",
"oc3-rna-data-Onecut3-Slc17a6",
"oc3-rna-data-Onecut3-Gad1",
"oc3-rna-data-Onecut3-Gad2",
"oc3-rna-data-Onecut3-Onecut2",
"oc3-rna-data-Onecut3-Nav1",
"oc3-rna-data-Onecut3-Nav2",
"oc3-rna-data-Onecut3-Trio"
)
walk2(n_corrs, p_corrs, save_my_plot, type = "stat-corr-plt")
# `stat_bin()` using `bins = 30`. Pick better value with `binwidth`.
# `stat_bin()` using `bins = 30`. Pick better value with `binwidth`.
# `stat_bin()` using `bins = 30`. Pick better value with `binwidth`.
# `stat_bin()` using `bins = 30`. Pick better value with `binwidth`.
# `stat_bin()` using `bins = 30`. Pick better value with `binwidth`.
# `stat_bin()` using `bins = 30`. Pick better value with `binwidth`.
# `stat_bin()` using `bins = 30`. Pick better value with `binwidth`.
# `stat_bin()` using `bins = 30`. Pick better value with `binwidth`.
# `stat_bin()` using `bins = 30`. Pick better value with `binwidth`.
# `stat_bin()` using `bins = 30`. Pick better value with `binwidth`.
# `stat_bin()` using `bins = 30`. Pick better value with `binwidth`.
# `stat_bin()` using `bins = 30`. Pick better value with `binwidth`.
# `stat_bin()` using `bins = 30`. Pick better value with `binwidth`.
# `stat_bin()` using `bins = 30`. Pick better value with `binwidth`.
# `stat_bin()` using `bins = 30`. Pick better value with `binwidth`.
# `stat_bin()` using `bins = 30`. Pick better value with `binwidth`.
# `stat_bin()` using `bins = 30`. Pick better value with `binwidth`.
# `stat_bin()` using `bins = 30`. Pick better value with `binwidth`.
# `stat_bin()` using `bins = 30`. Pick better value with `binwidth`.
# `stat_bin()` using `bins = 30`. Pick better value with `binwidth`.
# `stat_bin()` using `bins = 30`. Pick better value with `binwidth`.
# `stat_bin()` using `bins = 30`. Pick better value with `binwidth`.
# `stat_bin()` using `bins = 30`. Pick better value with `binwidth`.
# `stat_bin()` using `bins = 30`. Pick better value with `binwidth`.
# `stat_bin()` using `bins = 30`. Pick better value with `binwidth`.
# `stat_bin()` using `bins = 30`. Pick better value with `binwidth`.
# `stat_bin()` using `bins = 30`. Pick better value with `binwidth`.
# `stat_bin()` using `bins = 30`. Pick better value with `binwidth`.
# `stat_bin()` using `bins = 30`. Pick better value with `binwidth`.
# `stat_bin()` using `bins = 30`. Pick better value with `binwidth`.Visualise intersections sets that we are going to use (highlighted)
upset(as.data.frame(content_mtx_oc3),
order.by = "freq",
sets.x.label = "Number of cells",
text.scale = c(2, 1.6, 2, 1.3, 2, 3),
nsets = 15,
sets = c("Gad1", "Gad2", "Slc32a1", "Slc17a6"),
queries = list(
list(
query = intersects,
params = list("Gad1", "Gad2", "Slc32a1"),
active = T
),
list(
query = intersects,
params = list("Slc17a6"),
active = T
)
),
nintersects = 60,
empty.intersections = "on"
)
Regroup factor by stages for more balanced groups
onecut3$age %>% forcats::fct_count()
# # A tibble: 6 × 2
# f n
# <fct> <int>
# 1 E15 156
# 2 E17 104
# 3 P00 77
# 4 P02 74
# 5 P10 174
# 6 P23 7
onecut3$stage <-
onecut3$age %>%
forcats::fct_collapse(`Embrionic day 15` = "E15",
`Embrionic day 17` = "E17",
Neonatal = c("P00", "P02"),
Postnatal = c("P10", "P23"))
onecut3$stage %>% forcats::fct_count()
# # A tibble: 4 × 2
# f n
# <fct> <int>
# 1 Embrionic day 15 156
# 2 Embrionic day 17 104
# 3 Neonatal 151
# 4 Postnatal 181Make subset of stable neurons
onecut3$gaba_status <-
content_mtx_oc3 %>%
select(Gad1, Gad2, Slc32a1) %>%
mutate(gaba = if_all(.fns = ~ .x > 0)) %>%
.$gaba
onecut3$gaba_occurs <-
content_mtx_oc3 %>%
select(Gad1, Gad2, Slc32a1) %>%
mutate(gaba = if_any(.fns = ~ .x > 0)) %>%
.$gaba
onecut3$glut_status <-
content_mtx_oc3 %>%
select(Slc17a6) %>%
mutate(glut = Slc17a6 > 0) %>%
.$glut
oc3_fin <-
subset(onecut3,
cells = union(
WhichCells(onecut3,
expression = gaba_status == TRUE & glut_status == FALSE),
WhichCells(onecut3,
expression = glut_status == TRUE & gaba_occurs == FALSE)))Check contingency tables for neurotransmitter signature
oc3_fin@meta.data %>%
janitor::tabyl(glut_status, gaba_status)
# glut_status FALSE TRUE
# FALSE 0 102
# TRUE 92 0By age
oc3_fin@meta.data %>%
janitor::tabyl(age, gaba_status)
# age FALSE TRUE
# E15 29 9
# E17 12 4
# P00 12 6
# P02 16 2
# P10 21 81
# P23 2 0By stage
oc3_fin@meta.data %>%
janitor::tabyl(stage, gaba_status)
# stage FALSE TRUE
# Embrionic day 15 29 9
# Embrionic day 17 12 4
# Neonatal 28 8
# Postnatal 23 81Make splits of neurons by neurotransmitter signature
oc3_fin$status <- oc3_fin$gaba_status %>%
if_else(true = "GABAergic",
false = "glutamatergic")
Idents(oc3_fin) <- "status"
SaveH5Seurat(
object = oc3_fin,
filename = here(data_dir, "oc3_fin"),
overwrite = TRUE,
verbose = TRUE
)
# Warning: Overwriting previous file /home/etretiakov/src/Zupancic_2022/data/
# oc3_fin.h5seurat
# Creating h5Seurat file for version 3.1.5.9900
# Adding counts for RNA
# Adding data for RNA
# Adding scale.data for RNA
# Adding variable features for RNA
# No feature-level metadata found for RNA
# Adding cell embeddings for pca
# Adding loadings for pca
# No projected loadings for pca
# Adding standard deviations for pca
# No JackStraw data for pca
# Adding cell embeddings for tsne
# No loadings for tsne
# No projected loadings for tsne
# No standard deviations for tsne
# No JackStraw data for tsne
# Adding cell embeddings for umap
# No loadings for umap
# No projected loadings for umap
# No standard deviations for umap
# No JackStraw data for umap
## Split on basis of neurotrans and test for difference
oc3_fin_neurotrans <- SplitObject(oc3_fin, split.by = "status")
## Split on basis of age and test for difference
oc3_fin_ages <- SplitObject(oc3_fin, split.by = "age")DotPlots grouped by age
Expression of GABA receptors in GABAergic Onecut3 positive cells
DotPlot(object = oc3_fin_neurotrans$GABAergic,
features = gabar,
group.by = "age",
cols = c("#adffff", "#0084ff"),
col.min = -1, col.max = 1
) + RotatedAxis()
Expression of GABA receptors in glutamatergic Onecut3 positive cells
DotPlot(object = oc3_fin_neurotrans$glutamatergic,
features = gabar,
group.by = "age",
cols = c("#ffc2c2", "#ff3c00"),
col.min = -1, col.max = 1
) + RotatedAxis()
Expression of glutamate receptors in GABAergic Onecut3 positive cells
DotPlot(object = oc3_fin_neurotrans$GABAergic,
features = glutr,
group.by = "age",
cols = c("#adffff", "#0084ff"),
col.min = -1, col.max = 1
) + RotatedAxis()
Expression of glutamate receptors in glutamatergic Onecut3 positive cells
DotPlot(object = oc3_fin_neurotrans$glutamatergic,
features = glutr,
group.by = "age",
cols = c("#ffc2c2", "#ff3c00"),
col.min = -1, col.max = 1
) + RotatedAxis()
DotPlots grouped by stage
Expression of GABA receptors in GABAergic Onecut3 positive cells
DotPlot(object = oc3_fin_neurotrans$GABAergic,
features = gabar,
group.by = "stage",
cols = c("#adffff", "#0084ff"),
col.min = -1, col.max = 1
) + RotatedAxis()
Expression of GABA receptors in glutamatergic Onecut3 positive cells
DotPlot(object = oc3_fin_neurotrans$glutamatergic,
features = gabar,
group.by = "stage",
cols = c("#ffc2c2", "#ff3c00"),
col.min = -1, col.max = 1
) + RotatedAxis()
Expression of glutamate receptors in GABAergic Onecut3 positive cells
DotPlot(object = oc3_fin_neurotrans$GABAergic,
features = glutr,
group.by = "stage",
cols = c("#adffff", "#0084ff"),
col.min = -1, col.max = 1
) + RotatedAxis()
Expression of glutamate receptors in glutamatergic Onecut3 positive cells
DotPlot(object = oc3_fin_neurotrans$glutamatergic,
features = glutr,
group.by = "stage",
cols = c("#ffc2c2", "#ff3c00"),
col.min = -1, col.max = 1
) + RotatedAxis()
sessionInfo()
# R version 4.1.2 (2021-11-01)
# Platform: x86_64-redhat-linux-gnu (64-bit)
# Running under: Rocky Linux 8.5 (Green Obsidian)
#
# Matrix products: default
# BLAS/LAPACK: /usr/lib64/libopenblas-r0.3.12.so
#
# locale:
# [1] LC_CTYPE=C.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] Nebulosa_1.4.0 patchwork_1.1.1 UpSetR_1.4.0
# [4] SeuratDisk_0.0.0.9019 SeuratWrappers_0.3.0 SeuratObject_4.0.4
# [7] Seurat_4.0.6 sctransform_0.3.2 future_1.23.0
# [10] zeallot_0.1.0 magrittr_2.0.1 forcats_0.5.1
# [13] stringr_1.4.0 dplyr_1.0.7 purrr_0.3.4
# [16] readr_2.1.1 tidyr_1.1.4 tibble_3.1.6
# [19] ggplot2_3.3.5 tidyverse_1.3.1 here_1.0.1
# [22] workflowr_1.7.0
#
# loaded via a namespace (and not attached):
# [1] scattermore_0.7 R.methodsS3_1.8.1
# [3] coda_0.19-4 ragg_1.2.1
# [5] bit64_4.0.5 knitr_1.37
# [7] irlba_2.3.5 DelayedArray_0.20.0
# [9] R.utils_2.11.0 data.table_1.14.2
# [11] rpart_4.1-15 RCurl_1.98-1.5
# [13] generics_0.1.1 BiocGenerics_0.40.0
# [15] callr_3.7.0 cowplot_1.1.1
# [17] RANN_2.6.1 correlation_0.7.1
# [19] bit_4.0.4 tzdb_0.2.0
# [21] spatstat.data_2.1-2 xml2_1.3.3
# [23] lubridate_1.8.0 httpuv_1.6.5
# [25] SummarizedExperiment_1.24.0 assertthat_0.2.1
# [27] WRS2_1.1-3 xfun_0.29
# [29] hms_1.1.1 jquerylib_0.1.4
# [31] evaluate_0.14 promises_1.2.0.1
# [33] fansi_1.0.0 dbplyr_2.1.1
# [35] readxl_1.3.1 igraph_1.2.11
# [37] DBI_1.1.2 htmlwidgets_1.5.4
# [39] reshape_0.8.8 spatstat.geom_2.3-1
# [41] kSamples_1.2-9 stats4_4.1.2
# [43] Rmpfr_0.8-7 paletteer_1.4.0
# [45] ellipsis_0.3.2 ks_1.13.3
# [47] backports_1.4.1 insight_0.15.0
# [49] deldir_1.0-6 MatrixGenerics_1.6.0
# [51] vctrs_0.3.8 SingleCellExperiment_1.16.0
# [53] Biobase_2.54.0 remotes_2.4.2
# [55] ROCR_1.0-11 abind_1.4-5
# [57] cachem_1.0.6 withr_2.4.3
# [59] vroom_1.5.7 mclust_5.4.9
# [61] goftest_1.2-3 cluster_2.1.2
# [63] lazyeval_0.2.2 crayon_1.4.2
# [65] hdf5r_1.3.5 labeling_0.4.2
# [67] pkgconfig_2.0.3 SuppDists_1.1-9.7
# [69] GenomeInfoDb_1.30.0 nlme_3.1-153
# [71] statsExpressions_1.2.0 rlang_0.4.12
# [73] globals_0.14.0 lifecycle_1.0.1
# [75] miniUI_0.1.1.1 MatrixModels_0.5-0
# [77] modelr_0.1.8 rsvd_1.0.5
# [79] cellranger_1.1.0 rprojroot_2.0.2
# [81] polyclip_1.10-0 matrixStats_0.61.0
# [83] lmtest_0.9-39 datawizard_0.2.2
# [85] Matrix_1.3-4 mc2d_0.1-21
# [87] zoo_1.8-9 reprex_2.0.1
# [89] whisker_0.4 ggridges_0.5.3
# [91] processx_3.5.2 png_0.1-7
# [93] viridisLite_0.4.0 PMCMRplus_1.9.3
# [95] parameters_0.16.0 bitops_1.0-7
# [97] getPass_0.2-2 R.oo_1.24.0
# [99] KernSmooth_2.23-20 multcompView_0.1-8
# [101] parallelly_1.30.0 S4Vectors_0.32.3
# [103] scales_1.1.1 memoise_2.0.1
# [105] plyr_1.8.6 ica_1.0-2
# [107] zlibbioc_1.40.0 compiler_4.1.2
# [109] RColorBrewer_1.1-2 fitdistrplus_1.1-6
# [111] snakecase_0.11.0 cli_3.1.0
# [113] XVector_0.34.0 listenv_0.8.0
# [115] pbapply_1.5-0 ps_1.6.0
# [117] ggside_0.2.0 MASS_7.3-54
# [119] mgcv_1.8-38 tidyselect_1.1.1
# [121] stringi_1.7.6 textshaping_0.3.6
# [123] highr_0.9 yaml_2.2.1
# [125] ggrepel_0.9.1 grid_4.1.2
# [127] sass_0.4.0 tools_4.1.2
# [129] future.apply_1.8.1 parallel_4.1.2
# [131] rstudioapi_0.13 git2r_0.29.0
# [133] janitor_2.1.0 gridExtra_2.3
# [135] farver_2.1.0 Rtsne_0.15
# [137] digest_0.6.29 BiocManager_1.30.16
# [139] shiny_1.7.1 pracma_2.3.6
# [141] BWStest_0.2.2 Rcpp_1.0.8
# [143] GenomicRanges_1.46.1 broom_0.7.11
# [145] BayesFactor_0.9.12-4.3 performance_0.8.0
# [147] later_1.3.0 RcppAnnoy_0.0.19
# [149] httr_1.4.2 effectsize_0.5
# [151] colorspace_2.0-2 rvest_1.0.2
# [153] fs_1.5.2 tensor_1.5
# [155] reticulate_1.22 IRanges_2.28.0
# [157] splines_4.1.2 uwot_0.1.11
# [159] rematch2_2.1.2 spatstat.utils_2.3-0
# [161] systemfonts_1.0.3 plotly_4.10.0
# [163] xtable_1.8-4 gmp_0.6-2.1
# [165] jsonlite_1.7.3 R6_2.5.1
# [167] pillar_1.6.4 htmltools_0.5.2
# [169] mime_0.12 glue_1.6.0
# [171] fastmap_1.1.0 codetools_0.2-18
# [173] mvtnorm_1.1-3 utf8_1.2.2
# [175] lattice_0.20-45 bslib_0.3.1
# [177] spatstat.sparse_2.1-0 leiden_0.3.9
# [179] gtools_3.9.2 survival_3.2-13
# [181] rmarkdown_2.11 munsell_0.5.0
# [183] GenomeInfoDbData_1.2.7 ggstatsplot_0.9.0
# [185] haven_2.4.3 reshape2_1.4.4
# [187] gtable_0.3.0 bayestestR_0.11.5
# [189] spatstat.core_2.3-2