From 9662cb3ded3111953a9989cddd82639b9666489e Mon Sep 17 00:00:00 2001 From: Anoushka Date: Wed, 22 Sep 2021 15:40:28 -0400 Subject: [PATCH] Fixed outstanding issues. Double check and push new release in next commit --- .Rbuildignore | 1 + .gitignore | 2 + DESCRIPTION | 6 +- NAMESPACE | 1 + R/ExonQuant.R | 17 +- R/InfoPerLongRead.R | 20 +- R/sigSplitPie.R | 1 + inst/RScript/ExonCounting.R | 113 ++- inst/RScript/PieChart_Viz.R | 20 +- inst/python/Get_IsoformXClusterMat.py | 4 +- man/ExonQuant.Rd | 11 +- man/InfoPerLongRead.Rd | 22 +- man/sigSplitPie.Rd | 1 + vignettes/scisorseqr_standardWorkflow.R | 111 --- vignettes/scisorseqr_standardWorkflow.Rmd | 12 +- vignettes/scisorseqr_standardWorkflow.html | 908 ++++++++++----------- vignettes/scisorseqr_standardWorkflow.pdf | Bin 393576 -> 0 bytes 17 files changed, 547 insertions(+), 703 deletions(-) delete mode 100644 vignettes/scisorseqr_standardWorkflow.R delete mode 100644 vignettes/scisorseqr_standardWorkflow.pdf diff --git a/.Rbuildignore b/.Rbuildignore index 7a08803..e76dbf8 100644 --- a/.Rbuildignore +++ b/.Rbuildignore @@ -2,3 +2,4 @@ ^\.Rproj\.user$ ^doc$ +^Meta$ diff --git a/.gitignore b/.gitignore index 5b6a065..a535e8d 100644 --- a/.gitignore +++ b/.gitignore @@ -2,3 +2,5 @@ .Rhistory .RData .Ruserdata +doc +Meta diff --git a/DESCRIPTION b/DESCRIPTION index 5d35c3e..f0df0a6 100644 --- a/DESCRIPTION +++ b/DESCRIPTION @@ -1,11 +1,11 @@ Package: scisorseqr Type: Package Title: Processes long reads for differential isoform analysis -Version: 0.1.1 +Version: 0.1.2 Author: Anoushka Joglekar Maintainer: Anoushka Joglekar Description: For any comparative (e.g. case-control) studies of alternative splicing, - scisorseqr is the one stop shop for analyzing single-cell long read data. The linux + scisorseqr is the one stop shop for analyzing single-cell long read data. The linux based package includes functions for barcode deconvolution from fastqs, integration with long read alignment tools like STARlong and minimap2, mapping and filtering of high confidence full-length spliced reads, and some @@ -27,7 +27,7 @@ Suggests: utils, methods, spelling, - knitr, + knitr, rmarkdown, ggplot2, cowplot diff --git a/NAMESPACE b/NAMESPACE index 7b67b4a..aff5b88 100644 --- a/NAMESPACE +++ b/NAMESPACE @@ -17,5 +17,6 @@ export(triHeatmap) import(cowplot) import(dplyr) import(ggplot2) +import(parallel) import(scales) importFrom(magrittr,"%>%") diff --git a/R/ExonQuant.R b/R/ExonQuant.R index 581e1ec..4355830 100644 --- a/R/ExonQuant.R +++ b/R/ExonQuant.R @@ -6,12 +6,10 @@ #' #' For ONT data we recomment \url{https://github.com/ablab/IsoQuant.git} #' which allows for non-exact splice-site matching -#' @param allInfoFile file containing cell barcode and celltype information +#' @param allInfoFile file containing barcode, celltype, and exon information #' per read. Defaults to output of the InfoPerLongRead function -#' @param exon.gff gzipped file containing exon mapping information. -#' Defaults to output from MapAndFilter #' @param groupingFactor "Celltype" or "Barcode" to group reads by for -#' counting inclusion levels. Defaults to celltype +#' counting inclusion levels. Defaults to Celltype #' @param threshold minimum number of reads per grouping factor in order #' to consider that exon to be sufficiently expressed. Defaults to 10 #' @param threads number of threads to parallelize the function. Defaults @@ -19,18 +17,21 @@ #' @seealso \code{\link{MapAndFilter}} #' @seealso \code{\link{InfoPerLongRead}} #' @return ExonQuantOutput/InclusionExclusionCounts.tsv +#' +#' @import dplyr +#' @import parallel #' @export -#' -ExonQuant <- function(allInfoFile = 'LongReadInfo/AllInfo', - exon.gff = 'LRProcessingOutput/mapping.bestperRead.RNAdirection.withConsensIntrons.gff.gz', +ExonQuant <- function(allInfoFile = 'LongReadInfo/AllInfo_IncompleteReads', groupingFactor = "Celltype",threshold = 10, numThreads = 4) { if(!dir.exists('ExonQuantOutput')){dir.create("ExonQuantOutput/")} + if(file.exists('ExonQuantOutput/InclusionExclusionCounts.tsv')){ + file.remove('ExonQuantOutput/InclusionExclusionCounts.tsv')} R_file <- system.file("RScript", "ExonCounting.R", package = "scisorseqr") - countExons <- paste("Rscript", R_file, allInfoFile, exon.gff, threshold, groupingFactor, numThreads) + countExons <- paste("Rscript", R_file, allInfoFile, groupingFactor, numThreads, threshold) system(countExons) } diff --git a/R/InfoPerLongRead.R b/R/InfoPerLongRead.R index 515a010..6fb7ac4 100644 --- a/R/InfoPerLongRead.R +++ b/R/InfoPerLongRead.R @@ -1,9 +1,15 @@ -#' Combine all information per read to a flat file +#' Combine all available information per read to a flat file #' @aliases LongReadInfo #' @description Function to concatenate all the information per read, i.e #' gene-name, cellular barcode, UMI, cell-type information, and isoform -#' information into one file. Also outputs basic stats such as number of -#' reads/ genes/ UMIs per cellular barcode +#' information into one file. Isoform information includes a string separated list of +#' introns, Cage and PolyA peak information if available, and a +#' string separated list of exons. +#' AllInfo contains the list of complete and full-length mapped, spliced, and barcoded reads. +#' AllInfo_Incomplete is a superset of the above, and also contains reads that are not +#' classified as complete based on Cage/PolyA data. +#' The function also outputs basic stats +#' such as number of reads/ genes/ UMIs per cellular barcode #' @seealso \code{\link{MapAndFilter}} #' @seealso \code{\link{GetBarcodes}} #' @param barcodeOutputFile .csv file containing barcode and cell-type information @@ -13,8 +19,10 @@ #' to that output, else it uses the canonically spliced full-length reads #' @param minTimesIsoObserve minimum number of times an isoform is observed in the #' dataset. Defaults to 5 +#' @param rmTmpFolder Logical indicating whether you want to delete contents of the +#' pre-processing folder. Defaults to TRUE #' @export -InfoPerLongRead <- function(barcodeOutputFile, mapAndFilterOut, minTimesIsoObserve = 5) { +InfoPerLongRead <- function(barcodeOutputFile, mapAndFilterOut, minTimesIsoObserve = 5, rmTmpFolder = TRUE) { longReadInfo_sh <- system.file("bash", "longReadInfo.sh", package = "scisorseqr") longReadInfoFolder <- "LongReadInfo/" @@ -35,4 +43,8 @@ InfoPerLongRead <- function(barcodeOutputFile, mapAndFilterOut, minTimesIsoObser exonFile, minTimesIsoObserve, incompFile) system(longReadInfoComm) + removeDirComm <- paste("rm -r",mapAndFilterOut) + + if(rmTmpFolder == TRUE){system(removeDirComm)} + } diff --git a/R/sigSplitPie.R b/R/sigSplitPie.R index 0301463..da41291 100644 --- a/R/sigSplitPie.R +++ b/R/sigSplitPie.R @@ -6,6 +6,7 @@ #' @return Directory labelled 'Visualizations' containing a pie chart containing #' the breakup of number of isoforms needed to cross deltaPI threshold #' of pairwise comparisons containing cell groups provided in input +#' Legend indicates the number of isoforms needed to cross the deltaPI = 0.1 threshold #' @usage sigSplitPie('compDir/') #' #' @import dplyr diff --git a/inst/RScript/ExonCounting.R b/inst/RScript/ExonCounting.R index 4af8394..ee2c554 100644 --- a/inst/RScript/ExonCounting.R +++ b/inst/RScript/ExonCounting.R @@ -1,68 +1,63 @@ -#' @import dplyr #' @importFrom magrittr %>% +#' @import parallel +#' @import dplyr +#' @export `%>%` <- magrittr::`%>%` args <- commandArgs(trailingOnly=TRUE) -## arguments : AllInfo file, exonInfo.gff, threshold, grouping factor - +## arguments : AllInfo file, groupingFactor, nThreads, threshold allInfo <- data.table::fread(args[1],sep="\t") -allInfo <- allInfo[,1:6] ## Read in file with read - gene - celltype assignments -colnames(allInfo) <- c("Read","Gene","Celltype","Barcode","UMI","Stretch") -groupingFactor <- args[4] - -geneInfo <- allInfo %>% dplyr::select(Read,Gene,groupingFactor) - -print("Reading in GFF file, will take a while") - -exonGFF <- read.table(gzfile(args[2]))[,-c(2,3,6,8,9,11)] ## GFF file with exon info -colnames(exonGFF) <- c("chr","start","end","strand","readname") -exonGFF <- exonGFF %>% tidyr::separate(readname,into=c("Read","path"),sep=".path") %>% - dplyr::select(-path) %>% tidyr::unite(exon, c(chr,start,end,strand),sep="_",remove=FALSE) -exonGFF <- dplyr::right_join(exonGFF,geneInfo) ## Merge gene information with GFF in case of multi-mapping exons - -threshold <- as.integer(args[3]) -numThreads <- as.integer(args[5]) - - -uniqExons <- exonGFF %>% dplyr::select(-c(Read,groupingFactor)) %>% dplyr::distinct() ## Get unique exons to iterate over -edges <- exonGFF %>% dplyr::group_by(Read) %>% dplyr::mutate(s=min(start),e=max(end)) %>% - dplyr::select(Read,Gene,s,e,exon,groupingFactor) %>% dplyr::distinct() %>% dplyr::as_data_frame() -print(paste0("About to start processing exons per ",groupingFactor)) - -calcPSI <- function(x){ ## x for line in uniqExons - geneDF <- edges %>% dplyr::filter(Gene == uniqExons$Gene[x]) %>% dplyr::group_by(.dots = groupingFactor) %>% - dplyr::filter(s <= uniqExons$start[x] & e >= uniqExons$end[x]) %>% as.data.frame() - numReads <- geneDF %>% dplyr::group_by(.dots = groupingFactor, Read) %>% dplyr::select(Read,groupingFactor) %>% - dplyr::ungroup() %>% dplyr::group_by(.dots = groupingFactor) %>% dplyr::distinct() %>% - dplyr::add_count() %>% dplyr::filter(n>=threshold) %>% as.data.frame() - geneDF <- geneDF %>% dplyr::filter(Read %in% numReads$Read) %>% as.data.frame() - u_gf <- geneDF %>% dplyr::select(groupingFactor) %>% dplyr::distinct() ## make list of unique grouping factors - psiGF <- NULL - for(gf in u_gf[,1]){ ## If total reads spanning exon more than sampling rate, extract those reads - reads <- geneDF %>% dplyr::filter(get(groupingFactor) == gf) - psi = NULL - sr <- reads %>% dplyr::select(Read) %>% dplyr::distinct() - inc <- reads %>% dplyr::filter(Read %in% sr$Read) %>% - dplyr::filter(exon == uniqExons$exon[x]) %>% nrow() - psi <- inc/nrow(sr) ## Total reads spanning the exon = sr - psi <- as.data.frame(psi) - psi$inclusion <- inc - psi$exclusion <- nrow(sr) - inc - psi$exon <- uniqExons$exon[x] - psi$Gene <- uniqExons$Gene[x] - psi[,as.character(groupingFactor)] <- gf - psiGF <- rbind(psiGF,psi) } - return(psiGF) +if(ncol(allInfo) == 11){ + allInfo <- allInfo[,c(1:4,9)] +} else { allInfo <- allInfo[,c(1:4,7)]} ## Read in file with read - gene - celltype assignments + +colnames(allInfo) <- c("Read","Gene","Celltype","Barcode","Exons") +groupingFactor <- args[2] + +nThreads <- as.integer(args[3]) +rpg <- as.integer(args[4]) + +outDir <- 'ExonQuantOutput/' + +allInfo_SE <- allInfo %>% dplyr::group_by(Gene) %>% dplyr::add_count() %>% + dplyr::filter(n >= rpg) %>% dplyr::select(-n) %>% dplyr::rowwise() %>% + dplyr::mutate(start = unlist(strsplit(Exons,"_"))[2], + end = rev(unlist(strsplit(Exons,"_")))[2]) %>% + as.data.frame() + +internalExons = allInfo_SE %>% tidyr::separate_rows(Exons,sep = ";%;") %>% dplyr::filter(Exons != "") %>% + dplyr::group_by(Read) %>% dplyr::add_count() %>% dplyr::filter(n>=3) %>% dplyr::slice(2:(dplyr::n()-1)) + +uniqExons <- internalExons %>% dplyr::ungroup() %>% dplyr::select(Exons, Gene) %>% dplyr::distinct() + +inclusionCounts <- internalExons %>% dplyr::ungroup() %>% dplyr::select(Exons,Gene,all_of(groupingFactor)) %>% + dplyr::group_by(Exons,Gene,.dots=groupingFactor) %>% dplyr::add_count(name = "Inclusion") %>% + dplyr::distinct() %>% as.data.frame() + +readSE <- internalExons %>% dplyr::select(Read,Gene,all_of(groupingFactor),start,end) %>% dplyr::distinct() %>% as.data.frame() + +checkSpanningReads <- function(gene){ + exons <- uniqExons %>% dplyr::filter(Gene == gene) %>% + tidyr::separate(Exons,into=c("chr","s","e","strand"),sep = "_",remove=FALSE) + reads <- readSE %>% dplyr::filter(Gene == gene) + spanningReads <- dplyr::left_join(reads,exons,by = "Gene") %>% dplyr::filter(s >= start && e <= end) %>% + dplyr::select(Exons,Gene,all_of(groupingFactor)) %>% dplyr::group_by(Exons,Gene,.dots = groupingFactor) %>% + dplyr::add_count(name = "Total") %>% dplyr::distinct() %>% as.data.frame() + inclusionReads <- inclusionCounts %>% dplyr::filter(Gene == gene) + inc_tot <- dplyr::right_join(inclusionReads,spanningReads,by = c('Exons',"Gene",groupingFactor)) %>% + replace(is.na(.),0) %>% + dplyr::mutate(PSI = Inclusion/Total, Exclusion = Total-Inclusion) %>% as.data.frame() + fName <- file.path(outDir,"InclusionExclusionCounts.tsv") + if(file.exists(fName)){ + write.table(inc_tot,file.path(outDir,"InclusionExclusionCounts.tsv"),sep ="\t", + append= T, quote = F, row.names = F, col.names = FALSE) + } else{ + write.table(inc_tot,file.path(outDir,"InclusionExclusionCounts.tsv"),sep ="\t", + quote = F, row.names = F, col.names = TRUE) + } + return } - -sampledPSI <- parallel::mclapply(1:nrow(uniqExons), function(x) calcPSI(x), mc.cores=numThreads) -#sampledPSI <- parallel::mclapply(1:200, function(x) calcPSI(x), mc.cores=28) ## testing purposes - -print("Converting to dataframe") -psiDF <- dplyr::bind_rows(sampledPSI) %>% dplyr::select(exon,Gene,groupingFactor,inclusion,exclusion,psi) - -write.table(psiDF, file="ExonQuantOutput/InclusionExclusionCounts.tsv", - sep="\t",quote=FALSE,row.names=FALSE,col.names=TRUE) +byGene = parallel::mclapply(unique(uniqExons$Gene), function(g) checkSpanningReads(g), mc.cores = nThreads) diff --git a/inst/RScript/PieChart_Viz.R b/inst/RScript/PieChart_Viz.R index 4ce3fa7..c58b90f 100644 --- a/inst/RScript/PieChart_Viz.R +++ b/inst/RScript/PieChart_Viz.R @@ -3,6 +3,11 @@ #' @import scales #' @importFrom magrittr %>% +#suppressPackageStartupMessages({ +# library(dplyr) +# library(ggplot2) +#}) + `%>%` <- magrittr::`%>%` args <- commandArgs(trailingOnly=TRUE) @@ -51,19 +56,18 @@ nonSig <- length(which(resultsFile$FDR>0.05)) df <- as.data.frame(table(breakup)) df$breakup <- as.character(df$breakup) df <- rbind(c("NonSig",nonSig),df) -df$value <- as.numeric(df$Freq)/total +df$value <- as.numeric(df$Freq)*100/total df <- df %>% dplyr::mutate(ypos = cumsum(value)-0.5*value) cols <- c('NonSig'="#F9C6BA",'1'="#930077",'2'="#DD6892",'3'="#3c6f9c",'4'="#29cdb5",'5'="#048998",'6'="#553c8b") -final_pie <- ggplot2::ggplot(df, aes(x="", y=value, fill=breakup)) + - geom_bar(stat="identity", width=1) + - coord_polar("y", start=0) + - theme_void() + - theme(legend.position="none") + - geom_text(aes(y = ypos, label = percent(value)), color = "white", size=6) + - scale_fill_manual("",values=cols) +final_pie <- ggplot2::ggplot(df, ggplot2::aes(x="", y=value, fill=breakup)) + + ggplot2::geom_bar(stat="identity", width=1) + + ggplot2::coord_polar("y", start=0) + + ggplot2::theme_void() + + ggplot2::geom_text(ggplot2::aes(y = ypos, label = format(value,digits=2)), color = "white", size=6) + + ggplot2::scale_fill_manual("",values=cols) pdf(file=paste0("Visualizations/PieCharts_BreakUp_",name,".pdf"),8,6) diff --git a/inst/python/Get_IsoformXClusterMat.py b/inst/python/Get_IsoformXClusterMat.py index 55dca24..f551594 100755 --- a/inst/python/Get_IsoformXClusterMat.py +++ b/inst/python/Get_IsoformXClusterMat.py @@ -7,10 +7,12 @@ start_time = time.time() print("Creating Isoform X cell matrix") -print("Processing file with ",len(all_info)," entries") input_file = sys.argv[1] all_info = [x.strip('\n').split('\t') for x in open(input_file).readlines()][1:] + +print("Processing file with ",len(all_info)," entries") + gene_iso_names = [x[0] for x in all_info] cluster_names = [x[1] for x in all_info] num_iso = [int(x[2]) for x in all_info] diff --git a/man/ExonQuant.Rd b/man/ExonQuant.Rd index 31c72d7..76b3546 100644 --- a/man/ExonQuant.Rd +++ b/man/ExonQuant.Rd @@ -5,23 +5,18 @@ \title{Count exon inclusion and exclusion according to grouping factor} \usage{ ExonQuant( - allInfoFile = "LongReadInfo/AllInfo", - - exon.gff = "LRProcessingOutput/mapping.bestperRead.RNAdirection.withConsensIntrons.gff.gz", + allInfoFile = "LongReadInfo/AllInfo_IncompleteReads", groupingFactor = "Celltype", threshold = 10, numThreads = 4 ) } \arguments{ -\item{allInfoFile}{file containing cell barcode and celltype information +\item{allInfoFile}{file containing barcode, celltype, and exon information per read. Defaults to output of the InfoPerLongRead function} -\item{exon.gff}{gzipped file containing exon mapping information. -Defaults to output from MapAndFilter} - \item{groupingFactor}{"Celltype" or "Barcode" to group reads by for -counting inclusion levels. Defaults to celltype} +counting inclusion levels. Defaults to Celltype} \item{threshold}{minimum number of reads per grouping factor in order to consider that exon to be sufficiently expressed. Defaults to 10} diff --git a/man/InfoPerLongRead.Rd b/man/InfoPerLongRead.Rd index ac9488a..cb6855d 100644 --- a/man/InfoPerLongRead.Rd +++ b/man/InfoPerLongRead.Rd @@ -3,9 +3,14 @@ \name{InfoPerLongRead} \alias{InfoPerLongRead} \alias{LongReadInfo} -\title{Combine all information per read to a flat file} +\title{Combine all available information per read to a flat file} \usage{ -InfoPerLongRead(barcodeOutputFile, mapAndFilterOut, minTimesIsoObserve = 5) +InfoPerLongRead( + barcodeOutputFile, + mapAndFilterOut, + minTimesIsoObserve = 5, + rmTmpFolder = TRUE +) } \arguments{ \item{barcodeOutputFile}{.csv file containing barcode and cell-type information @@ -17,12 +22,21 @@ to that output, else it uses the canonically spliced full-length reads} \item{minTimesIsoObserve}{minimum number of times an isoform is observed in the dataset. Defaults to 5} + +\item{rmTmpFolder}{Logical indicating whether you want to delete contents of the +pre-processing folder. Defaults to TRUE} } \description{ Function to concatenate all the information per read, i.e gene-name, cellular barcode, UMI, cell-type information, and isoform -information into one file. Also outputs basic stats such as number of -reads/ genes/ UMIs per cellular barcode +information into one file. Isoform information includes a string separated list of +introns, Cage and PolyA peak information if available, and a +string separated list of exons. +AllInfo contains the list of complete and full-length mapped, spliced, and barcoded reads. +AllInfo_Incomplete is a superset of the above, and also contains reads that are not +classified as complete based on Cage/PolyA data. +The function also outputs basic stats +such as number of reads/ genes/ UMIs per cellular barcode } \seealso{ \code{\link{MapAndFilter}} diff --git a/man/sigSplitPie.Rd b/man/sigSplitPie.Rd index 46eb8e2..6128a27 100644 --- a/man/sigSplitPie.Rd +++ b/man/sigSplitPie.Rd @@ -14,6 +14,7 @@ specific comparison} Directory labelled 'Visualizations' containing a pie chart containing the breakup of number of isoforms needed to cross deltaPI threshold of pairwise comparisons containing cell groups provided in input +Legend indicates the number of isoforms needed to cross the deltaPI = 0.1 threshold } \description{ A quick way to summarize DIE for a single comparison diff --git a/vignettes/scisorseqr_standardWorkflow.R b/vignettes/scisorseqr_standardWorkflow.R deleted file mode 100644 index 8c22f3f..0000000 --- a/vignettes/scisorseqr_standardWorkflow.R +++ /dev/null @@ -1,111 +0,0 @@ -## ----setup, include = FALSE--------------------------------------------------- -knitr::opts_chunk$set( - collapse = TRUE, - comment = "#>" -) - -## ----makeBCfile, eval=FALSE, echo=TRUE---------------------------------------- -# -# library(Seurat) -# library(tidyr) -# library(dplyr) -# -# bc <- data.frame("Barcode"=rownames(my_object@meta.data), -# "Celltype"=my_object@meta.data$celltype) %>% -# separate(Barcode,into=c("BC","suffix"),sep="-") %>% -# select(-suffix) %>% as.data.frame() -# -# write.table(bc, file = "PATH_TO_FILE", sep="\t", row.names = FALSE, -# col.names = FALSE, quote = FALSE) - -## ---- echo=FALSE-------------------------------------------------------------- -bc_clust <- read.table(system.file("extdata/", "userInput/bc_celltype_assignments", - package = "scisorseqr"), - header = FALSE, sep = "\t") -knitr::kable(bc_clust[sample(nrow(bc_clust),8,replace = FALSE),], - row.names = FALSE) - -## ----getBarcodes, eval=FALSE, echo=TRUE--------------------------------------- -# -# bc_clust <- system.file("extdata/", "userInput/bc_celltype_assignments", -# package = "scisorseqr") -# fastqFolder <- system.file("extdata/", "userInput/FastqFiles", -# package = "scisorseqr") -# -# ## run command -# GetBarcodes(fqFolder = fastqFolder, BCClustAssignFile = bc_clust, -# chemistry = "v2", concatenate = TRUE, filterReads = FALSE) - -## ----STARalign, eval = FALSE, echo = TRUE------------------------------------- -# -# ## Load example data -# fastqFolder <- system.file("extdata/", "userInput/FastqFiles", -# package = "scisorseqr") -# -# STARalign(fqFolder = fastqFolder, starProgPath = '~/bin/Linux_x86_64/STARlong', -# refGenomeIndex = '~/starIndex_gencode10_sequins/', -# numThreads = 24) -# -# MMalign(fqFolder = fastqFolder, mmProgPath = '~/minimap2/minimap2', -# refGenome='~/genomes/mm10.fa', -# numThreads = 16) - -## ----MapAndFilter, eval = FALSE, echo = TRUE---------------------------------- -# -# MapAndFilter(annoGZ='[PATH_TO_annotation.gtf.gz]',numThreads=24, -# seqDir = '[PATH_TO_DIR/chr*.fa.gz]', -# filterFullLength=TRUE, -# cageBed = '[PATH_TO_Cage.bed.gz]', -# polyABed = '[PATH_TO_PolyA.bed.gz]', -# cp_distance=50) -# - -## ----InfoPerLongRead, eval=FALSE, echo=TRUE----------------------------------- -# -# InfoPerLongRead(barcodeOutputFile = -# 'OutputFiltered/FilteredDeconvBC_AllFiles.csv', -# mapAndFilterOut = 'LRProcessingOutput/', -# minTimesIsoObserve = 3) -# - -## ----IsoQuant, echo = TRUE, eval= FALSE--------------------------------------- -# IsoQuant(AllInfoFile = 'LongReadInfo/AllInfo', -# Iso = TRUE, TSS = TRUE, PolyA = TRUE) -# - -## ----ExonQuant, echo = TRUE, eval= FALSE-------------------------------------- -# ExonQuant(AllInfoFile = 'LongReadInfo/AllInfo', -# groupingFactor = "Celltype", threshold = 10) - -## ----DiffSplicing, eval=FALSE, echo=TRUE-------------------------------------- -# config <- system.file("extdata/", "userInput/config", -# package = "scisorseqr") -# -# DiffSplicingAnalysis(configFile = config, -# typeOfTest = 'Iso', -# minNumReads = 25, is.hier = FALSE) -# -# ## or for exons -# DiffSplicingAnalysis(configFile = config, -# typeOfTest = 'Exon', -# minNumReads = 25, -# is.hier = FALSE) - -## ----TriHeatmap, echo = TRUE, eval= FALSE------------------------------------- -# -# condensedCellTypes = system.file("extdata/", "userInput/condensedCellTypes", -# package = "scisorseqr") -# -# triHeatmap(treeDir = 'TreeTraversal_Iso/', -# comparisonList =condensedCellTypes, -# outName =condensedCellTypes) - -## ----heatmap, out.width = '60%', echo=F--------------------------------------- -knitr::include_graphics("../man/figures/condensedCellTypes.png") - -## ----PieChart, echo = TRUE, eval= FALSE--------------------------------------- -# sigSplitPie(compDir = 'Uniq_TreeTraversal_Iso/Astro_ExcitNeuron_10/') - -## ----pie, out.width = '60%', echo=F------------------------------------------- -knitr::include_graphics("../man/figures/examplePie.png") - diff --git a/vignettes/scisorseqr_standardWorkflow.Rmd b/vignettes/scisorseqr_standardWorkflow.Rmd index 53b0745..af03e75 100644 --- a/vignettes/scisorseqr_standardWorkflow.Rmd +++ b/vignettes/scisorseqr_standardWorkflow.Rmd @@ -18,7 +18,7 @@ knitr::opts_chunk$set( The *scisorseqr* package allows you to go from short-read, single cell assignments and a set of fastq files to a full-blown cell-type specific analysis of alternative splicing patterns. -## Software required +## Software required * [STARlong](https://github.com/alexdobin/STAR/) (and corresponding genome index file) * [Minimap2](https://github.com/lh3/minimap2) if not using STARlong @@ -67,7 +67,7 @@ Example files included in the package and loaded as follows: bc_clust <- system.file("extdata/", "userInput/bc_celltype_assignments", package = "scisorseqr") -fastqFolder <- system.file("extdata/", "userInput/FastqFiles", +fastqFolder <- system.file("extdata/", "userInput/FastqFiles/", package = "scisorseqr") ## run command @@ -82,7 +82,7 @@ You will see two output directories - file_summary (Stats for run) - OutputFiltered - file.CSV (One line per barcoded read with the following structure:) - + |ReadName |T9_Status |Strand|T9_position|Barcodes| |:----------|:-----|:---|:--|:----------------| |ReadX|poly_T_found|rev|47|GTCGGGTTCCAAAGTC| @@ -255,8 +255,9 @@ triHeatmap(treeDir = 'TreeTraversal_Iso/', outName =condensedCellTypes) ``` -**NOTE** Cell-type labels should correspond to the barcode-celltype list. If you input a cell-type for which pairwise DIE has not been calculated, it will output all zeros +**NOTE** Cell-type labels should correspond to the barcode-celltype list. If you input a cell-type for which pairwise DIE has not been calculated, it will output all zeros (e.g. DivOligo in this case) +**NOTE** The example data included in this package does not yield the plot below. This is just an example plot generated from the full dataset. ```{r heatmap, out.width = '60%', echo=F} knitr::include_graphics("../man/figures/condensedCellTypes.png") @@ -271,8 +272,9 @@ sigSplitPie(compDir = 'Uniq_TreeTraversal_Iso/Astro_ExcitNeuron_10/') ```{r pie, out.width = '60%', echo=F} knitr::include_graphics("../man/figures/examplePie.png") ``` - +Each number in the legend corresponds to the number of isoforms needed to cross the deltaPI = 0.1 threshold **NOTE** Percentage labels do not always arrange themselves neatly in the figure (requires editing by hand) +**NOTE** The example data included in this package does not yield the plot above. This is just an example plot generated from the full dataset. ## Done! diff --git a/vignettes/scisorseqr_standardWorkflow.html b/vignettes/scisorseqr_standardWorkflow.html index b1eaedb..8374c37 100644 --- a/vignettes/scisorseqr_standardWorkflow.html +++ b/vignettes/scisorseqr_standardWorkflow.html @@ -1,437 +1,342 @@ - - - - - - - - - - - - - -scisorseqr: Standard Workflow - - - - - - - - +

The scisorseqr package allows you to go from short-read, single cell assignments and a set of fastq files to a full-blown cell-type specific analysis of alternative splicing patterns.

- - - -

scisorseqr: Standard Workflow

-

Anoushka Joglekar

-

2020-11-25

- - - -

The scisorseqr package allows you to go from short-read, single cell assignments and a set of fastq files to a full-blown cell-type specific analysis of alternative splicing patterns.

-

Software required

+
    -
  • STARlong (and corresponding genome index file)
  • -
  • Minimap2 if not using STARlong
  • +
  • STARlong (and corresponding genome index file)
  • +
  • Minimap2 if not using STARlong
  • samtools
  • bedtools
  • python version >= 3.7 with the following libraries +
    • pandas
    • multiprocessing
-
-
-

Step1: Barcode deconvolution

-

The first step is to deconvolve barcodes from either the PacBio or ONT fastq output For that the user needs to specify

+ +

Step 1: Barcode deconvolution

+ +

The first step is to deconvolve barcodes from either the PacBio or ONT fastq output +For that the user needs to specify

+
  • Tab separated Barcode-Celltype file
  • Directory containing fastq.gz files
-
+

Create a barcode - celltype file assuming analysis using Seurat

- + +

+library(Seurat)
+library(tidyr)
+library(dplyr)
+
+bc <- data.frame("Barcode"=rownames(my_object@meta.data),
+                 "Celltype"=my_object@meta.data$celltype) %>% 
+  separate(Barcode,into=c("BC","suffix"),sep="-") %>% 
+  select(-suffix) %>% as.data.frame()
+
+write.table(bc, file = "PATH_TO_FILE", sep="\t", row.names = FALSE,
+            col.names = FALSE, quote = FALSE)
+
+

The table should look something like this

- - - + +
+ - - - - - + + + + - - - + + + + + + + - - + + - - + + - - - + + + - - - + + + - - - + + + - - - - - -
V1 V2
CAGTAACAGTGATCGGHipp_Oligo
GTACGTAAGAGCCTAGHipp_Astro
ACTTGTTGTAGCGTCCHipp_InhibNeuron
CAAGAAACATGGGAACHipp_GranuleNB
GTGTTAGTCCCTTGTGHipp_NIPCs
TGTGGTAAGTGCAAGC
TGTTCCGTCAGGATCT Hipp_Astro
TCAGGATAGGATGCGT
AACTGGTAGCCAGTAG PFC_Astro
GGAATAACAGGTGGATPFC_ExciteNeuron
CGGTTAATCACCGTAAHipp_NIPCs
CTAGAGTCAGCTGTTAPFC_InhibNeuron
TGGACGCAGCACCGTCPFC_Astro
GGCGACTCACAACTGTHipp_Oligo
TGTCCCACAGGAATCGHipp_RGL
GCTGCAGGTTCGTCTCHipp_ExciteNeuron
-
-
+ +

Use that file to locate barcodes from the fastq files

-

Example files included in the package and loaded as follows:

- + +

Example files included in the package and loaded as follows:

+ +

+bc_clust <- system.file("extdata/", "userInput/bc_celltype_assignments", 
+                        package = "scisorseqr")
+fastqFolder <- system.file("extdata/", "userInput/FastqFiles/", 
+                           package = "scisorseqr")
+
+## run command
+GetBarcodes(fqFolder = fastqFolder, BCClustAssignFile = bc_clust, 
+            chemistry = "v2", concatenate = TRUE, filterReads = FALSE)
+
+

You will see two output directories

+
  • OutputRaw +
    • file.CSV (One line per read)
    • file_summary (Stats for run)
  • OutputFiltered +
    • file.CSV (One line per barcoded read with the following structure:)
- - - + +
+ - - - + + - -
ReadName T9_Status Strand T9_position Barcodes
ReadX poly_T_found rev 47 GTCGGGTTCCAAAGTC
- - - +
+ + + @@ -439,9 +344,8 @@

Use that file to locate barcodes from the fastq files

- - - + + @@ -449,54 +353,67 @@

Use that file to locate barcodes from the fastq files

- -
Barcode_position Cluster UMI TSO_position Length
22 Hipp_GranuleNB ATGGCGGGAT 26 583
-
-
-
-

Step2: Align your fastq files to the reference

+ + +

Step 2: Align your fastq files to the reference

+

We have made two aligners available and compatible with our package

+
  • STARlong
  • Minimap2
- + +

+## Load example data
+fastqFolder <- system.file("extdata/", "userInput/FastqFiles", 
+                           package = "scisorseqr")
+
+STARalign(fqFolder = fastqFolder, starProgPath = '~/bin/Linux_x86_64/STARlong',
+          refGenomeIndex = '~/starIndex_gencode10_sequins/',
+          numThreads = 24)
+
+MMalign(fqFolder = fastqFolder, mmProgPath = '~/minimap2/minimap2',
+        refGenome='~/genomes/mm10.fa',
+        numThreads = 16)
+
+

This process will convert all the aligned sam files to bam files, and dump the output into STARoutput

-
-
-

Step3: Map and filter for full-length, spliced reads

- -

NOTE the parameter seqDir takes in a reference genome broken down by chromosome so as to parallelize the process. This function has not yet been made generalizable to loading in the full reference

+ +

Step 3: Map and filter for full-length, spliced reads

+ +

We used annotated CAGE peaks and PolyA sites for our study

+ +

+MapAndFilter(annoGZ='[PATH_TO_annotation.gtf.gz]',numThreads=24, 
+             seqDir = '[PATH_TO_DIR/chr*.fa.gz]',
+             filterFullLength=TRUE,
+             cageBed = '[PATH_TO_Cage.bed.gz]',
+             polyABed = '[PATH_TO_PolyA.bed.gz]',
+             cp_distance=50)
+
+
+ +

NOTE the parameter seqDir takes in a reference genome broken down by chromosome so +as to parallelize the process. +This function has not yet been made generalizable to loading in the full reference

+

This will result in multiple files being stored in yet another output directory: LRProcessingOutput

-
-
-

Step4: Concatenate all the information collected so far

- + +

Step 4: Concatenate all the information collected so far

+ +

+InfoPerLongRead(barcodeOutputFile = 
+                  'OutputFiltered/FilteredDeconvBC_AllFiles.csv',
+                mapAndFilterOut = 'LRProcessingOutput/',
+                minTimesIsoObserve = 3)
+
+
+

This will yield a directory LongReadInfo with file AllInfo which has the following structure

- - - + +
+ @@ -506,96 +423,106 @@

Step4: Concatenate all the information collected so far

- - - -
ReadName Gene Celltype Status # Introns
-
-
-

Step5a: Quantify the various isoforms observed in your data

-

If you provided CAGE and PolyA peaks at the MapAndFilter() stage, you can choose to quantify unique cage and polyA sites per gene. Otherwise the default option is False

- -

Depending on which modality you chose as TRUE, you will see the following files in the IsoQuantOutput folder:

+ + + +

Step 5a: Quantify the various isoforms observed in your data

+ +

If you provided CAGE and PolyA peaks at the MapAndFilter() stage, you can choose to quantify unique +cage and polyA sites per gene. Otherwise the default option is False

+ +
IsoQuant(AllInfoFile = 'LongReadInfo/AllInfo',
+          Iso = TRUE, TSS = TRUE, PolyA = TRUE)
+
+
+ +

Depending on which modality you chose as TRUE, you will see the following files in the IsoQuantOutput folder:

+
    -
  • Modality-ModalityID.csv
  • -
  • NumModalityPerCluster
  • +
  • Modality-*Modality*ID.csv
  • +
  • Num*Modality*PerCluster
+

These will be used for the differential splicing analysis step

-
-
-

Step5b: [OPTIONAL] Quantify the various exons observed in your data

-

For ONT reads we don’t have too much confidence in this method and recommend using our sister package IsoQuant.

+ +

Step 5b: [OPTIONAL] Quantify the various exons observed in your data

+ +

For ONT reads we don't have too much confidence in this method and recommend using +our sister package IsoQuant.

+

This function however, works for annotated as well as novel exons

- + +
ExonQuant(AllInfoFile = 'LongReadInfo/AllInfo', 
+          groupingFactor = "Celltype", threshold = 10)
+
+

This will output the quantifications to a separate folder ExonQuantOutput

-
-
-

Step6: Differential isoform analysis

+ +

Step 6: Differential isoform analysis

+

This step can be done using various settings for various modalities. Options include

+
  • Full-length isoform (Iso)
  • -
  • TSS
  • +
  • TSS
  • PolyA
  • Exon (Refer to step 6b)
+

Depending on the modality chosen, this function automatically detects the input

+

It also requires a tab-separated config file in the following format to be provided by the user

+

NOTE: Header here just for demonstration

- ------ - - + +
+ - - - + + - + - + - -
Comparison1 Celltypes Comparison2 Celltypes
HippNeuron Hipp_ExciteNeuron,Hipp_InhibNeuron PFCNeuron PFC_ExciteNeuron,PFC_InhibNeuron
HippInhib Hipp_InhibNeuron PFCInhib PFC_InhibNeuron
HippExcite Hipp_ExciteNeuron HippInhib Hipp_InhibNeuron
- + + +
config <- system.file("extdata/", "userInput/config", 
+                      package = "scisorseqr")
+
+DiffSplicingAnalysis(configFile = config, 
+                     typeOfTest = 'Iso', 
+                     minNumReads = 25, is.hier = FALSE)
+
+## or for exons
+DiffSplicingAnalysis(configFile = config, 
+                     typeOfTest = 'Exon', 
+                     minNumReads = 25, 
+                     is.hier = FALSE)
+
+

Assuming you run this command, each comparison (line in the config file) yields a separate sub-directory in the folder TreeTraveral_Iso

+

The most important file in that output directory is the results file, which has the following structure:

- - - + +
+ @@ -603,9 +530,8 @@

Step6: Differential isoform analysis

- - - + + @@ -613,7 +539,7 @@

Step6: Differential isoform analysis

- + @@ -621,7 +547,7 @@

Step6: Differential isoform analysis

- + @@ -629,7 +555,7 @@

Step6: Differential isoform analysis

- + @@ -637,73 +563,71 @@

Step6: Differential isoform analysis

- -
Gene pvals dPI maxDeltaPI_ix2 FDR
ENSMUSG00000000001.4 0.563702861650773 0 NA 1
ENSMUSG00000000078.7 1 0 NA 1
ENSMUSG00000000088.7 1 0.0714285714285714 NA 1
ENSMUSG00000000326.13 0.343030146138244 0.666666666666667 3 1
+ +

NOTE Deeper data will yield significant values. These ^ are just a product of the example data subset we have provided with the package

+

You can use the above output to plot your significant genes

-
-
-

Step7: Visualizations

-
+ +

Step 7: Visualizations

+

Triangular Heatmap

-

This function plots a triangular heatmap of percentage DIE genes of pairwise comparisons in a directory It also takes in an untitled file list of cell types to be plotted as follows:

- - - + +

This function plots a triangular heatmap of percentage DIE genes of pairwise comparisons in a directory +It also takes in an untitled file list of cell types to be plotted as follows:

+ +
+ + + + + - + - + - + - + - + - -
Hipp_Astro
Hipp_ChorPlex
Hipp_DivOligo
Hipp_Ependymal
.
.
- -

NOTE Cell-type labels should correspond to the barcode-celltype list. If you input a cell-type for which pairwise DIE has not been calculated, it will output all zeros (e.g. DivOligo in this case)

-

-
-
-

Pie Chart breaking down the significant genes

- -

-

NOTE Percentage labels do not always arrange themselves neatly in the figure (requires editing by hand)

-
-
-
-

Done!

-
+ +

+condensedCellTypes = system.file("extdata/", "userInput/condensedCellTypes", 
+                      package = "scisorseqr")
 
+triHeatmap(treeDir = 'TreeTraversal_Iso/',
+           comparisonList =condensedCellTypes, 
+           outName =condensedCellTypes)
+
- +

NOTE Cell-type labels should correspond to the barcode-celltype list. If you input a cell-type for which pairwise DIE has not been calculated, it will output all zeros +(e.g. DivOligo in this case) +NOTE The example data included in this package does not yield the plot below. This is just an example plot generated from the full dataset.

+

plot of chunk heatmap

- - +

Pie Chart breaking down the significant genes

+ +
sigSplitPie(compDir = 'Uniq_TreeTraversal_Iso/Astro_ExcitNeuron_10/')
+
+ +

plot of chunk pie +Each number in the legend corresponds to the number of isoforms needed to cross the deltaPI = 0.1 threshold +NOTE Percentage labels do not always arrange themselves neatly in the figure (requires editing by hand) +NOTE The example data included in this package does not yield the plot above. This is just an example plot generated from the full dataset.

+ +

Done!

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