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annotate scpipe.R @ 0:32e1bfc6b7b2 draft

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planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/scpipe commit 8908da9cdd112ae0943dbf1eccb221e84cd99ca7
author iuc
date Wed, 15 Aug 2018 13:54:40 -0400
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children 5c4bca9dd4a2
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32e1bfc6b7b2 planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/scpipe commit 8908da9cdd112ae0943dbf1eccb221e84cd99ca7
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1 options( show.error.messages=F, error = function () { cat( geterrmessage(), file=stderr() ); q( "no", 1, F ) } )
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32e1bfc6b7b2 planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/scpipe commit 8908da9cdd112ae0943dbf1eccb221e84cd99ca7
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3 # we need that to not crash galaxy with an UTF8 error on German LC settings.
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4 loc <- Sys.setlocale("LC_MESSAGES", "en_US.UTF-8")
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5
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6 suppressPackageStartupMessages({
32e1bfc6b7b2 planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/scpipe commit 8908da9cdd112ae0943dbf1eccb221e84cd99ca7
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7 library(scPipe)
32e1bfc6b7b2 planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/scpipe commit 8908da9cdd112ae0943dbf1eccb221e84cd99ca7
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8 library(SingleCellExperiment)
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9 library(optparse)
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10 library(readr)
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11 library(ggplot2)
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12 library(plotly)
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13 library(DT)
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14 library(scater)
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15 library(scran)
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16 library(scales)
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17 library(Rtsne)
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18 })
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20 option_list <- list(
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21 make_option(c("-fasta","--fasta"), type="character", help="Genome fasta file"),
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22 make_option(c("-exons","--exons"), type="character", help="Exon annotation gff3 file"),
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23 make_option(c("-barcodes","--barcodes"), type="character", help="Cell barcodes csv file"),
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24 make_option(c("-read1","--read1"), type="character", help="Read 1 fastq.gz"),
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25 make_option(c("-read2","--read2"), type="character", help="Read 2 fastq.gz"),
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26 make_option(c("-samplename","--samplename"), type="character", help="Name to use for sample"),
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27 make_option(c("-bs1","--bs1"), type="integer", help="Barcode start in Read 1"),
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28 make_option(c("-bl1","--bl1"), type="integer", help="Barcode length in Read 1"),
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29 make_option(c("-bs2","--bs2"), type="integer", help="Barcode start in Read 2"),
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30 make_option(c("-bl2","--bl2"), type="integer", help="Barcode length in Read 2"),
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31 make_option(c("-us","--us"), type="integer", help="UMI start in Read 2"),
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32 make_option(c("-ul","--ul"), type="integer", help="UMI length in Read 2"),
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33 make_option(c("-rmlow","--rmlow"), type="logical", help="Remove reads with N in barcode or UMI"),
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34 make_option(c("-rmN","--rmN"), type="logical", help="Remove reads with low quality"),
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35 make_option(c("-minq","--minq"), type="integer", help="Minimum read quality"),
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36 make_option(c("-numbq","--numbq"), type="integer", help="Maximum number of bases below minq"),
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37 make_option(c("-stnd","--stnd"), type="logical", help="Perform strand-specific mapping"),
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38 make_option(c("-max_mis","--max_mis"), type="integer", help="Maximum mismatch allowed in barcode"),
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39 make_option(c("-UMI_cor","--UMI_cor"), type="integer", help="Correct UMI sequence error"),
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40 make_option(c("-gene_fl","--gene_fl"), type="logical", help="Remove low abundant genes"),
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41 make_option(c("-max_reads","--max_reads"), type="integer", help="Maximum reads processed"),
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42 make_option(c("-min_count","--min_count"), type="integer", help="Minimum count to keep"),
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43 make_option(c("-report","--report"), type="logical", help="HTML report of plots"),
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44 make_option(c("-rdata","--rdata"), type="logical", help="Output RData file"),
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45 make_option(c("-nthreads","--nthreads"), type="integer", help="Number of threads")
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46 )
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47
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48 parser <- OptionParser(usage = "%prog [options] file", option_list=option_list)
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49 args = parse_args(parser)
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50
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51 fa_fn = args$fasta
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52 anno_fn = args$exons
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53 fq_R1 = args$read1
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54 fq_R2 = args$read2
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55 read_structure = list(
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56 bs1 = args$bs1, # barcode start position in fq_R1, -1 indicates no barcode
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57 bl1 = args$bl1, # barcode length in fq_R1, 0 since no barcode present
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58 bs2 = args$bs2, # barcode start position in fq_R2
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59 bl2 = args$bl2, # barcode length in fq_R2
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60 us = args$us, # UMI start position in fq_R2
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61 ul = args$ul # UMI length
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62 )
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63
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64 if (args$us == -1) {
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65 has_umi = FALSE
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66 } else {
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67 has_umi = TRUE
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68 }
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69
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70 filter_settings=list(rmlow=args$rmlow, rmN=args$rmN, minq=args$minq, numbq=args$numbq)
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71
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72 # Outputs
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73 out_dir = "."
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74 fasta_index = file.path(out_dir, paste0(fa_fn, ".fasta_index"))
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75 combined_fastq = file.path(out_dir, "combined.fastq")
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76 aligned_bam = file.path(out_dir, "aligned.bam")
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77 mapped_bam = file.path(out_dir, "aligned.mapped.bam")
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78
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79
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80 print("Trimming barcodes")
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81 sc_trim_barcode(combined_fastq,
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82 fq_R1,
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83 fq_R2,
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84 read_structure=read_structure,
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85 filter_settings=filter_settings)
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86
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87 print("Building genome index")
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88 Rsubread::buildindex(basename=fasta_index, reference=fa_fn)
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89
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90 print("Aligning reads to genome")
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91 Rsubread::align(index=fasta_index,
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92 readfile1=combined_fastq,
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93 output_file=aligned_bam,
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94 nthreads=args$nthreads)
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95
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96 if (!is.null(args$barcodes)) {
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97 barcode_anno=args$barcodes
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98 } else {
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99 print("Detecting barcodes")
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100 # detect 10X barcodes and generate sample_index.csv file
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101 barcode_anno = "sample_index.csv"
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102 sc_detect_bc(
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103 infq=combined_fastq,
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104 outcsv=barcode_anno,
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105 bc_len=read_structure$bl2,
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106 max_reads=args$max_reads,
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107 min_count=args$min_count,
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108 max_mismatch=args$max_mis
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109 )
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110 }
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111
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112 print("Assigning reads to exons")
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113 sc_exon_mapping(aligned_bam, mapped_bam, anno_fn, bc_len=read_structure$bl2, UMI_len=read_structure$ul, stnd=args$stnd)
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114
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115 print("De-multiplexing data")
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116 sc_demultiplex(mapped_bam, out_dir, barcode_anno, has_UMI=has_umi)
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117
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118 print("Counting genes")
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119 sc_gene_counting(out_dir, barcode_anno, UMI_cor=args$UMI_cor, gene_fl=args$gene_fl)
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120
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121 print("Creating SingleCellExperiment object")
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122 sce <- create_sce_by_dir(out_dir)
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123
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124 if (!is.null(args$report)) {
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125 print("Creating report")
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126 create_report(sample_name=args$samplename,
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127 outdir=out_dir,
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128 r1=fq_R1,
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129 r2=fq_R2,
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130 outfq=combined_fastq,
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131 read_structure=read_structure,
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132 filter_settings=filter_settings,
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133 align_bam=aligned_bam,
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134 genome_index=fasta_index,
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135 map_bam=mapped_bam,
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136 exon_anno=anno_fn,
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137 stnd=args$stnd,
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138 fix_chr=FALSE,
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139 barcode_anno=barcode_anno,
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140 max_mis=args$max_mis,
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141 UMI_cor=args$UMI_cor,
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142 gene_fl=args$gene_fl)
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143 }
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144
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145 if (!is.null(args$rdata) ) {
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146 save(sce, file = file.path(out_dir,"scPipe_analysis.RData"))
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147 }
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148
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149 sessionInfo()