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exceRpt_longRNA
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exceRpt_longRNA
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#########################################################################################
## ##
## ____ _ __ ____ _ _____ ##
## _____ _____ ___| _ \ _ __ | |_ / /___ ____ ____ _/ __ \/ | / / | ##
## / _ \ \/ / __/ _ \ |_) | '_ \| __| / / __ \/ __ \/ __ `/ /_/ / |/ / /| | ##
## | __/> < (_| __/ _ <| |_) | |_ / / /_/ / / / / /_/ / _, _/ /| / ___ | ##
## \___/_/\_\___\___|_| \_\ .__/ \__| /_/\____/_/ /_/\__, /_/ |_/_/ |_/_/ |_| ##
## |_| /____/ ##
## ##
## ##
## ##
## The extra-cellular RNA processing toolkit (exceRpt) optimised for longRNA analysis ##
## ##
## This pipeline processes a single longRNA sequence file from a single sample ##
## ##
## Author: Rob Kitchen (r.r.kitchen@gmail.com) ##
## ##
## Learn more at github.com/rkitchen/exceRpt ##
## ##
## Version 4.6.2 (2017-01-24) ##
## ##
#########################################################################################
EXCERPT_VERSION := 4.6.2
##
## 1) On the command line, be sure to specify the following MANDATORY parameters
##
OUTPUT_DIR := NULL
INPUT_FILE_PATH_R1 := NULL
INPUT_FILE_PATH_R2 := NULL
SAMPLE_NAME := NULL
## small hack to use only the first read of each pair
INPUT_FILE_PATH := $(INPUT_FILE_PATH_R1)
##
## 2) Choose the main organism for longRNA / genome alignment (hsa + hg19, hsa + hg38, or mmu + mm10)
##
MAIN_ORGANISM_GENOME_ID := hg38
##
## 3) *OPTIONAL* parameters for calibrator library and random barcodes
##
ADAPTER_SEQ := none
MIN_ADAPTER_BASES_3p := 7
CALIBRATOR_LIBRARY := NULL
TRIM_N_BASES_5p := 0
TRIM_N_BASES_3p := 0
RANDOM_BARCODE_LENGTH := 0
RANDOM_BARCODE_LOCATION := -5p -3p
KEEP_RANDOM_BARCODE_STATS := false
ENDOGENOUS_LIB_PRIORITY := gencode,miRNA,tRNA,piRNA,circRNA
##
## 4) Select whether pipeline is run locally, should be 'true' unless this is the Genboree.org implementation!
##
LOCAL_EXECUTION := true
REMOVE_LARGE_INTERMEDIATE_FILES := false
##
## 5) READ QUALITY filtering parameters
##
QFILTER_MIN_READ_FRAC := 80
QFILTER_MIN_QUAL := 20
##
## 6) ENDOGENOUS alignment parameters
##
DOWNSAMPLE_RNA_READS := NULL
MIN_READ_LENGTH := 18
STAR_outFilterMatchNmin := $(MIN_READ_LENGTH)
STAR_outFilterMatchNminOverLread := 0.9
STAR_outFilterMismatchNmax := 1
STAR_outFilterMismatchNoverLmax := 0.3
STAR_alignEndsType := Local
##
## 7) EXOGENOUS alignment parameters
##
## Choose what kind of EXOGENOUS alignments to attempt:
## - off : none
## - miRNA : map only to exogenous miRNAs in miRbase
## - on : map to exogenous miRNAs in miRbase AND the genomes of all sequenced species in ensembl/NCBI
##
MAP_EXOGENOUS := off
## for exogenous alignments (can only be 0 or 1)
MAX_MISMATCHES_EXOGENOUS := 0
## For the taxonomic interpretation:
EXOGENOUS_RIBOSOMAL_TAXA_MINFRAC := 0.95
EXOGENOUS_RIBOSOMAL_TAXA_BATCHSIZE := 20000
EXOGENOUS_RIBOSOMAL_TAXA_MINREADPERCENT := 0.001
EXOGENOUS_RIBOSOMAL_TAXA_MINREADS := 3
EXOGENOUS_GENOMES_TAXA_MINFRAC := 0.95
EXOGENOUS_GENOMES_TAXA_BATCHSIZE := 500000
EXOGENOUS_GENOMES_TAXA_MINREADPERCENT := 0.001
EXOGENOUS_GENOMES_TAXA_MINREADS := 3
##
## 8) If this is a local installation of the pipeline, be sure to also modify the parameters in steps 4, 5, and 6 below...
##
ifeq ($(LOCAL_EXECUTION),true)
##
## 5) Modify installation-specific variables
##
N_THREADS := 4
JAVA_RAM := 10G
#MAX_RAM := 64000000000
MAX_RAM := 10000000000
BOWTIE_CHUNKMBS := 2000
SAMTOOLS_SORT_MEM := 8G
## NB: The 'EXE_DIR' MUST be an ABSOLUTE PATH or sRNABench will fail!
EXE_DIR := /gpfs/scratch/fas/gerstein/rrk24/bin/smallRNAPipeline
mkfile_path := $(abspath $(lastword $(MAKEFILE_LIST)))
#current_dir_rel := $(notdir $(patsubst %/,%,$(dir $(mkfile_path))))
EXE_DIR := $(dir $(mkfile_path))
##
## 6) Check that the paths to the required 3rd party executables work!
##
JAVA_EXE := /usr/bin/java
FASTX_CLIP_EXE := $(EXE_DIR)/fastx_0.0.14/bin/fastx_clipper
FASTX_FILTER_EXE := $(EXE_DIR)/fastx_0.0.14/bin/fastq_quality_filter
#VIENNA_PATH := $(EXE_DIR)/ViennaRNA_2.1.5/bin
BOWTIE2_EXE := $(EXE_DIR)/bowtie2-2.2.4/bowtie2
#SAMTOOLS_EXE := $(EXE_DIR)/samtools-1.1/samtools
SAMTOOLS_EXE := $(EXE_DIR)/samtools-1.3/samtools
FASTQC_EXE := $(JAVA_EXE) -classpath $(EXE_DIR)/FastQC_0.11.2:$(EXE_DIR)/FastQC_0.11.2/sam-1.103.jar:$(EXE_DIR)/FastQC_0.11.2/jbzip2-0.9.jar
SRATOOLS_EXE := $(EXE_DIR)/sratoolkit.2.5.1-centos_linux64/bin/fastq-dump
EXCERPT_TOOLS_EXE := $(EXE_DIR)/exceRpt_Tools.jar
DATABASE_PATH := $(EXE_DIR)/DATABASE
STAR_EXE := $(EXE_DIR)/STAR_2.4.2a/bin/Linux_x86_64/STAR
STAR_GENOMES_DIR := $(DATABASE_PATH)/Genomes_BacteriaFungiMammalPlantProtistVirus
STAR_PARAMS_FILE_PATH := $(STAR_GENOMES_DIR)/STAR_Parameters_Exogenous.in
EXPRESS_EXE := $(EXE_DIR)/express-1.5.1-linux_x86_64/express
##
## Use the input path to infer filetype and short name
##
INPUT_FILE_NAME := $(notdir $(INPUT_FILE_PATH))
INPUT_FILE_ID := $(basename $(INPUT_FILE_NAME))
else
##
## These parameters are for the Genboree installation only
##
EXE_DIR := $(SCRATCH_DIR)
N_THREADS := $(N_THREADS)
JAVA_RAM := 64G
MAX_RAM := 64000000000
BOWTIE_CHUNKMBS := 8000
SAMTOOLS_SORT_MEM := 2G
FASTX_CLIP_EXE := fastx_clipper
FASTX_FILTER_EXE := fastq_quality_filter
VIENNA_PATH := NULL
BOWTIE2_EXE := bowtie2
SAMTOOLS_EXE := samtools
FASTQC_EXE := $(JAVA_EXE) -classpath $(FASTQC_EXE_DIR):$(FASTQC_EXE_DIR)/sam-1.103.jar:$(FASTQC_EXE_DIR)/jbzip2-0.9.jar
SRATOOLS_EXE := fastq-dump
SRNABENCH_EXE := $(SRNABENCH_EXE)
EXCERPT_TOOLS_EXE := $(EXCERPT_TOOLS_EXE)
DATABASE_PATH := $(EXCERPT_DATABASE)
## Path to sRNABench libraries
SRNABENCH_LIBS := $(SRNABENCH_LIBS)
STAR_EXE := STAR
STAR_GENOMES_DIR := $(STAR_GENOMES_DIR)
STAR_PARAMS_FILE_PATH := $(STAR_GENOMES_DIR)/STAR_Parameters_Exogenous.in
INPUT_FILE_NAME := $(notdir $(INPUT_FILE_PATH))
INPUT_FILE_ID := $(INPUT_FILE_ID)
endif
## Define current time
ts := `/bin/date "+%Y-%m-%d--%H:%M:%S"`
ONE := 1
## Define tool name for logging
PIPELINE_NAME := exceRpt_longRNA
##
## Initialise longRNA alignment parameters
##
#BOWTIE_SEED_LENGTH := 19
BOWTIE_SEED_LENGTH := $(MIN_READ_LENGTH)
##
## For sample quality control (QC)
##
MIN_TRANSCRIPTOME_MAPPED := 100000
MIN_GENOME_TRANSCRIPTOME_RATIO := 0.5
USEAGE :=
ifeq ($(INPUT_FILE_ID),NULL)
#USEAGE := "make -f smallRNA_pipeline INPUT_FILE_PATH=[required: absolute/path/to/input/.fa|.fq|.sra] N_THREADS=[required: number of threads] OUTPUT_DIR=<required: absolute/path/to/output> INPUT_FILE_ID=[required: samplename] ADAPTER_SEQ=[optional: will guess sequence if not provided here; none, if already clipped input] MAIN_ORGANISM=[optional: defaults to 'hsa'] MAIN_ORGANISM_GENOME_ID=[optional: defaults to 'hg38'] CALIBRATOR_LIBRARY=[optional: path/to/bowtie/index/containing/calibrator/sequences] TRNA_MAPPING=[optional: TRUE|FALSE, default is TRUE] GENCODE_MAPPING=[optional: TRUE|FALSE, default is TRUE] PIRNA_MAPPING=[optional: TRUE|FALSE, default is TRUE] MAP_EXOGENOUS=[optional: off|miRNA|on, default is miRNA]"
USEAGE := "make -f longRNA_pipeline INPUT_FILE_PATH=[required: absolute/path/to/input/.fa|.fq|.sra] N_THREADS=[required: number of threads] OUTPUT_DIR=<required: absolute/path/to/output> INPUT_FILE_ID=[required: samplename] ADAPTER_SEQ=[optional: will guess sequence if not provided here; none, if already clipped input] MAIN_ORGANISM=[optional: defaults to 'hsa'] MAIN_ORGANISM_GENOME_ID=[optional: defaults to 'hg38'] CALIBRATOR_LIBRARY=[optional: path/to/bowtie/index/containing/calibrator/sequences] MAP_EXOGENOUS=[optional: off|miRNA|on, default is miRNA]"
endif
##
## STAR dynamic parameter string
##
STAR_ENDOGENOUS_DYNAMIC_PARAMS := --alignEndsType $(STAR_alignEndsType) --outFilterMatchNmin $(MIN_READ_LENGTH) --outFilterMatchNminOverLread $(STAR_outFilterMatchNminOverLread) --outFilterMismatchNmax $(STAR_outFilterMismatchNmax) --outFilterMismatchNoverLmax $(STAR_outFilterMismatchNoverLmax)
STAR_EXOGENOUS_DYNAMIC_PARAMS := --outSAMtype BAM Unsorted --outSAMattributes Standard --alignEndsType EndToEnd --outFilterMatchNmin $(MIN_READ_LENGTH) --outFilterMatchNminOverLread 1.0 --outFilterMismatchNmax $(MAX_MISMATCHES_EXOGENOUS) --outFilterMismatchNoverLmax $(STAR_outFilterMismatchNoverLmax)
##
## Map reads to plant and virus miRNAs
##
ifeq ($(MAP_EXOGENOUS),miRNA) ## ALIGNMENT TO ONLY EXOGENOUS miRNA and rRNA
PROCESS_SAMPLE_REQFILE := EXOGENOUS_rRNA/ExogenousRibosomalAlignments.result.taxaAnnotated.txt
JAVA_RAM := 90G
else ifeq ($(MAP_EXOGENOUS),on) ## COMPLETE EXOGENOUS GENOME ALIGNMENT
PROCESS_SAMPLE_REQFILE := EXOGENOUS_genomes/ExogenousGenomicAlignments.result.taxaAnnotated.txt
JAVA_RAM := 90G
else
PROCESS_SAMPLE_REQFILE := endogenousAlignments_Accepted.txt.gz
endif
##
## List of plant and virus species IDs to which to map reads that do not map to the genome of the primary organism
##
#EXOGENOUS_MIRNA_SPECIES := $(shell cat $(SRNABENCH_LIBS)/libs/mature.fa | grep ">" | awk -F '-' '{print $$1}' | sed 's/>//g'| sort | uniq | tr '\n' ':' | rev | cut -c 2- | rev)
## Parameters to use for the bowtie mapping of calibrator oligos and rRNAs
BOWTIE2_MAPPING_PARAMS_CALIBRATOR := -D 15 -R 2 -N $(STAR_outFilterMismatchNmax) -L $(BOWTIE_SEED_LENGTH) -i S,1,0
#BOWTIE2_MAPPING_PARAMS_RRNA := -D 15 -R 2 -N $(MAX_MISMATCHES_EXOGENOUS) -L $(BOWTIE_SEED_LENGTH) -i S,1,0
#################################################
##
## Generate unique ID from the input fastq filename and user's sample ID
##
SAMPLE_ID := $(INPUT_FILE_ID)
ifneq ($(SAMPLE_NAME),NULL)
SAMPLE_ID := $(SAMPLE_ID)_$(SAMPLE_NAME)
endif
##
## Detect filetype and extract from SRA format if necessary
##
COMMAND_CONVERT_SRA := cat $(INPUT_FILE_PATH)
ifeq ($(suffix $(INPUT_FILE_NAME)),.sra)
COMMAND_CONVERT_SRA := $(SRATOOLS_EXE) --stdout $(INPUT_FILE_PATH)
else ifeq ($(suffix $(INPUT_FILE_NAME)),.gz)
COMMAND_CONVERT_SRA := gunzip -c $(INPUT_FILE_PATH)
else ifeq ($(suffix $(INPUT_FILE_NAME)),.bz2)
COMMAND_CONVERT_SRA := bzip2 -dc $(INPUT_FILE_PATH)
endif
##
## Guess quality encoding
##
#COMMAND_FILTER_BY_QUALITY ?= gunzip -c $(OUTPUT_DIR)/$(SAMPLE_ID)/$(SAMPLE_ID).clipped.fastq.gz | $(FASTX_FILTER_EXE) -v -Q$(shell cat $(OUTPUT_DIR)/$(SAMPLE_ID)/$(SAMPLE_ID).qualityEncoding) -p $(QFILTER_MIN_READ_FRAC) -q $(QFILTER_MIN_QUAL) > $(OUTPUT_DIR)/$(SAMPLE_ID)/$(SAMPLE_ID).clipped.trimmed.filtered.tmp 2>>$(OUTPUT_DIR)/$(SAMPLE_ID).log
##
## Are we downsampling the transcriptome alignments?
##
ifeq ($(DOWNSAMPLE_RNA_READS),NULL)
COMMAND_DOWNSAMPLE :=
else
COMMAND_DOWNSAMPLE := mv $(OUTPUT_DIR)/$(SAMPLE_ID)/endogenousAlignments_Accepted.txt $(OUTPUT_DIR)/$(SAMPLE_ID)/endogenousAlignments_Accepted.original.txt; \
cat $(OUTPUT_DIR)/$(SAMPLE_ID)/endogenousAlignments_Accepted.original.txt | shuf -n $(DOWNSAMPLE_RNA_READS) --random-source=$(DATABASE_PATH)/randomBits.dat > $(OUTPUT_DIR)/$(SAMPLE_ID)/endogenousAlignments_Accepted.txt; \
gzip -c $(OUTPUT_DIR)/$(SAMPLE_ID)/endogenousAlignments_Accepted.original.txt > $(OUTPUT_DIR)/$(SAMPLE_ID)/endogenousAlignments_Accepted.original.txt.gz
endif
##
## Logic block to write the adapter sequence (whether or not one is provided by the user) to the .adapterSeq file
##
#ifeq ($(ADAPTER_SEQ),NULL)
# COMMAND_WRITE_ADAPTER_SEQ := $(COMMAND_CONVERT_SRA) 2>> $(OUTPUT_DIR)/$(SAMPLE_ID).err | head -n 40000000 2>> $(OUTPUT_DIR)/$(SAMPLE_ID).err | $(BOWTIE2_EXE) --no-head -p $(N_THREADS) --local -D 15 -R 2 -N 0 -L 20 -i S,1,0.75 -k 2 --upto 10000000 -x $(DATABASE_PATH)/$(MAIN_ORGANISM_GENOME_ID)/$(MAIN_ORGANISM_GENOME_ID) -U - 2>> $(OUTPUT_DIR)/$(SAMPLE_ID).log | awk '{if ($$5==255) print $$0}' > $(OUTPUT_DIR)/$(SAMPLE_ID)/tmp.unique.sam 2>> $(OUTPUT_DIR)/$(SAMPLE_ID).log; \
# cat $(OUTPUT_DIR)/$(SAMPLE_ID)/tmp.unique.sam 2>> $(OUTPUT_DIR)/$(SAMPLE_ID).err | awk '{print $$6}' 2>> $(OUTPUT_DIR)/$(SAMPLE_ID).err | sort 2>> $(OUTPUT_DIR)/$(SAMPLE_ID).err | uniq -c 2>> $(OUTPUT_DIR)/$(SAMPLE_ID).err | sort -rnk 1 2>> $(OUTPUT_DIR)/$(SAMPLE_ID).err | head -n 100 > $(OUTPUT_DIR)/$(SAMPLE_ID)/$(SAMPLE_ID).cigarFreqs 2>> $(OUTPUT_DIR)/$(SAMPLE_ID).err; \
# cat $(OUTPUT_DIR)/$(SAMPLE_ID)/tmp.unique.sam 2>> $(OUTPUT_DIR)/$(SAMPLE_ID).err | awk '{if ($$2==0) print $$3"\t"$$4"\t"$$6"\t"$$10}' 2>> $(OUTPUT_DIR)/$(SAMPLE_ID).err | grep "[[:space:]]2[0-9]M[0-9][0-9]S" > $(OUTPUT_DIR)/$(SAMPLE_ID)/tmp.sam 2>> $(OUTPUT_DIR)/$(SAMPLE_ID).err; \
# cat $(OUTPUT_DIR)/$(SAMPLE_ID)/tmp.sam 2>> $(OUTPUT_DIR)/$(SAMPLE_ID).err | awk '{print $$3}' 2>> $(OUTPUT_DIR)/$(SAMPLE_ID).err | sort 2>> $(OUTPUT_DIR)/$(SAMPLE_ID).err | uniq -c 2>> $(OUTPUT_DIR)/$(SAMPLE_ID).err | sort -rnk 1 2>> $(OUTPUT_DIR)/$(SAMPLE_ID).err | head -n 100 > $(OUTPUT_DIR)/$(SAMPLE_ID)/$(SAMPLE_ID).okCigarFreqs 2>> $(OUTPUT_DIR)/$(SAMPLE_ID).err; \
# cat $(OUTPUT_DIR)/$(SAMPLE_ID)/$(SAMPLE_ID).okCigarFreqs 2>> $(OUTPUT_DIR)/$(SAMPLE_ID).err | head -n 1 2>> $(OUTPUT_DIR)/$(SAMPLE_ID).err | awk '{print substr($$2,1,2)}' > $(OUTPUT_DIR)/$(SAMPLE_ID)/tmp.txt 2>> $(OUTPUT_DIR)/$(SAMPLE_ID).err; \
# cat $(OUTPUT_DIR)/$(SAMPLE_ID)/tmp.sam 2>> $(OUTPUT_DIR)/$(SAMPLE_ID).err | grep "[[:space:]]$$(<$(OUTPUT_DIR)/$(SAMPLE_ID)/tmp.txt)" 2>> $(OUTPUT_DIR)/$(SAMPLE_ID).err | awk '{getline len<"$(OUTPUT_DIR)/$(SAMPLE_ID)/tmp.txt"; print substr($$4,len+1)}' 2>> $(OUTPUT_DIR)/$(SAMPLE_ID).err | sed 's/[A]*$$//' 2>> $(OUTPUT_DIR)/$(SAMPLE_ID).err | sort 2>> $(OUTPUT_DIR)/$(SAMPLE_ID).err | uniq -c 2>> $(OUTPUT_DIR)/$(SAMPLE_ID).err | sort -rnk 1 2>> $(OUTPUT_DIR)/$(SAMPLE_ID).err | awk '{if ($$1 > 75) print $$0}' > $(OUTPUT_DIR)/$(SAMPLE_ID)/$(SAMPLE_ID).potentialAdapters.txt 2>> $(OUTPUT_DIR)/$(SAMPLE_ID).err; \
# head -n 1 $(OUTPUT_DIR)/$(SAMPLE_ID)/$(SAMPLE_ID).potentialAdapters.txt | awk '{print $$2}' > $(OUTPUT_DIR)/$(SAMPLE_ID)/$(SAMPLE_ID).adapterSeq; \
# rm $(OUTPUT_DIR)/$(SAMPLE_ID)/tmp.*
# LOGENTRY_WRITE_ADAPTER := $(ts) $(PIPELINE_NAME): Identifying unknown 3' adapter sequence. Removing 3' adapter sequence using fastX:\n
#else ifeq ($(ADAPTER_SEQ),guessKnown)
ifeq ($(ADAPTER_SEQ),guessKnown)
COMMAND_WRITE_ADAPTER_SEQ := $(COMMAND_CONVERT_SRA) 2>> $(OUTPUT_DIR)/$(SAMPLE_ID).err | $(JAVA_EXE) -Xmx$(JAVA_RAM) -jar $(EXCERPT_TOOLS_EXE) FindAdapter -n 10000 -m 1000000 -s 4 -a $(DATABASE_PATH)/adapters/adapters.fa - > $(OUTPUT_DIR)/$(SAMPLE_ID)/$(SAMPLE_ID).adapterSeq 2>> $(OUTPUT_DIR)/$(SAMPLE_ID).log
LOGENTRY_WRITE_ADAPTER := $(ts) $(PIPELINE_NAME): Identifying 3' adapter from list of known sequences. Removing 3' adapter sequence using fastX:\n
else ifeq ($(ADAPTER_SEQ),none)
COMMAND_WRITE_ADAPTER_SEQ := echo 'no adapter' > $(OUTPUT_DIR)/$(SAMPLE_ID)/$(SAMPLE_ID).adapterSeq;
COMMAND_CLIP_ADAPTER := $(COMMAND_CONVERT_SRA) | gzip -c > $(OUTPUT_DIR)/$(SAMPLE_ID)/$(SAMPLE_ID).clipped.fastq.tmp.gz 2>> $(OUTPUT_DIR)/$(SAMPLE_ID).err; gunzip -c $(OUTPUT_DIR)/$(SAMPLE_ID)/$(SAMPLE_ID).clipped.fastq.tmp.gz | wc -l | awk '{print "input\t"$$0/4"\nsuccessfully_clipped\tNA"}' >> $(OUTPUT_DIR)/$(SAMPLE_ID).stats
LOGENTRY_WRITE_ADAPTER := Provided 3' adapter clipped input sequence file. No clipping necessary.\n
else
COMMAND_WRITE_ADAPTER_SEQ := echo $(ADAPTER_SEQ) > $(OUTPUT_DIR)/$(SAMPLE_ID)/$(SAMPLE_ID).adapterSeq
LOGENTRY_WRITE_ADAPTER := $(ts) $(PIPELINE_NAME): Provided 3' adapter sequence. Removing 3' adapter sequence using fastX:\n
endif
## If no adapter clipping command has been set- use this one:
COMMAND_CLIP_ADAPTER ?= $(COMMAND_CONVERT_SRA) > $(OUTPUT_DIR)/$(SAMPLE_ID)/$(SAMPLE_ID).preClipped.fastq.tmp; $(FASTX_CLIP_EXE) $(FASTX_CLIP_COMMANDS_FOR_RANDOM_BARCODE) -Q$(shell cat $(OUTPUT_DIR)/$(SAMPLE_ID)/$(SAMPLE_ID).qualityEncoding) -a $(shell cat $(OUTPUT_DIR)/$(SAMPLE_ID)/$(SAMPLE_ID).adapterSeq) -l $(MIN_READ_LENGTH) -v -n -M $(MIN_ADAPTER_BASES_3p) -i $(OUTPUT_DIR)/$(SAMPLE_ID)/$(SAMPLE_ID).preClipped.fastq.tmp -z -o $(OUTPUT_DIR)/$(SAMPLE_ID)/$(SAMPLE_ID).clipped.fastq.tmp.gz >> $(OUTPUT_DIR)/$(SAMPLE_ID).log 2>> $(OUTPUT_DIR)/$(SAMPLE_ID).err; rm $(OUTPUT_DIR)/$(SAMPLE_ID)/$(SAMPLE_ID).preClipped.fastq.tmp
##
## Logic block to handle random adapter removal
##
ifeq ($(KEEP_RANDOM_BARCODE_STATS),true)
BARCODE_STATS_COMMAND := -stats $(OUTPUT_DIR)/$(SAMPLE_ID)/$(SAMPLE_ID).clipped.barcodeStats
else
BARCODE_STATS_COMMAND :=
endif
ifeq ($(RANDOM_BARCODE_LENGTH),0)
COMMAND_REMOVE_RANDOM_BARCODE := mv $(OUTPUT_DIR)/$(SAMPLE_ID)/$(SAMPLE_ID).clipped.fastq.tmp.gz $(OUTPUT_DIR)/$(SAMPLE_ID)/$(SAMPLE_ID).clipped.fastq.gz
ENDOGENOUS_QUANT_RANDOM_BARCODE_STATS :=
FASTX_CLIP_COMMANDS_FOR_RANDOM_BARCODE :=
else
#COMMAND_REMOVE_RANDOM_BARCODE := gunzip -c $(OUTPUT_DIR)/$(SAMPLE_ID)/$(SAMPLE_ID).clipped.fastq.tmp.gz | $(JAVA_EXE) -Xmx$(JAVA_RAM) -jar $(EXCERPT_TOOLS_EXE) ProcessFastqWithRandomBarcode -n $(RANDOM_BARCODE_LENGTH) $(RANDOM_BARCODE_LOCATION) -stats $(OUTPUT_DIR)/$(SAMPLE_ID)/$(SAMPLE_ID).clipped.barcodeStats - 2>> $(OUTPUT_DIR)/$(SAMPLE_ID).log | gzip -c > $(OUTPUT_DIR)/$(SAMPLE_ID)/$(SAMPLE_ID).clipped.fastq.gz
#ENDOGENOUS_QUANT_RANDOM_BARCODE_STATS := -randombarcode $(OUTPUT_DIR)/$(SAMPLE_ID)/$(SAMPLE_ID).clipped.barcodeStats
COMMAND_REMOVE_RANDOM_BARCODE := gunzip -c $(OUTPUT_DIR)/$(SAMPLE_ID)/$(SAMPLE_ID).clipped.fastq.tmp.gz | $(JAVA_EXE) -Xmx$(JAVA_RAM) -jar $(EXCERPT_TOOLS_EXE) ProcessFastqWithRandomBarcode -min $(MIN_READ_LENGTH) -n $(RANDOM_BARCODE_LENGTH) $(RANDOM_BARCODE_LOCATION) $(BARCODE_STATS_COMMAND) - 2>> $(OUTPUT_DIR)/$(SAMPLE_ID).log | gzip -c > $(OUTPUT_DIR)/$(SAMPLE_ID)/$(SAMPLE_ID).clipped.fastq.gz
ENDOGENOUS_QUANT_RANDOM_BARCODE_STATS :=
#
# if we are using random barcodes, reads input to the pipeline MUST HAVE A 3' ADAPTER!
FASTX_CLIP_COMMANDS_FOR_RANDOM_BARCODE := -c
endif
##
## Logic block for removing rRNAs and [optionally] calibrator sequences that may have been spiked into the sample
##
ifeq ($(CALIBRATOR_LIBRARY),NULL)
LOGENTRY_MAP_CALIBRATOR_1 := No calibrator sequences\n
LOGENTRY_MAP_CALIBRATOR_2 := Moving on to UniVec and rRNA sequences\n
COMMAND_COUNT_CALIBRATOR := echo -e "calibrator\tNA" >> $(OUTPUT_DIR)/$(SAMPLE_ID).stats
COMMAND_MAP_CALIBRATOR :=
FILE_TO_INPUT_TO_UNIVEC_ALIGNMENT := $(OUTPUT_DIR)/$(SAMPLE_ID)/$(SAMPLE_ID).clipped.trimmed.filtered.fastq.gz
else
COMMAND_COUNT_CALIBRATOR := cat $(OUTPUT_DIR)/$(SAMPLE_ID)/$(SAMPLE_ID).clipped.trimmed.filtered.calibratormapped.counts | awk 'BEGIN{sum=0} {sum+=$$1} END{print "calibrator\t"sum}' >> $(OUTPUT_DIR)/$(SAMPLE_ID).stats
COMMAND_MAP_CALIBRATOR := $(BOWTIE2_EXE) -p $(N_THREADS) $(BOWTIE2_MAPPING_PARAMS_CALIBRATOR) --un-gz $(OUTPUT_DIR)/$(SAMPLE_ID)/$(SAMPLE_ID).clipped.trimmed.filtered.noCalibrator.fastq.gz -x $(CALIBRATOR_LIBRARY) -U $(OUTPUT_DIR)/$(SAMPLE_ID)/$(SAMPLE_ID).clipped.trimmed.filtered.fastq.gz 2>> $(OUTPUT_DIR)/$(SAMPLE_ID).log | awk '$$2 != 4 {print $$0}' | $(SAMTOOLS_EXE) view -Sb - 2>> $(OUTPUT_DIR)/$(SAMPLE_ID).log | tee $(OUTPUT_DIR)/$(SAMPLE_ID)/$(SAMPLE_ID).clipped.trimmed.filtered.calibratormapped.bam | $(SAMTOOLS_EXE) view - 2>> $(OUTPUT_DIR)/$(SAMPLE_ID).log | awk '{print $$3}' | sort -k 2 2>> $(OUTPUT_DIR)/$(SAMPLE_ID).err | uniq --count > $(OUTPUT_DIR)/$(SAMPLE_ID)/$(SAMPLE_ID).clipped.trimmed.filtered.calibratormapped.counts 2>> $(OUTPUT_DIR)/$(SAMPLE_ID).err
LOGENTRY_MAP_CALIBRATOR_1 := $(ts) $(PIPELINE_NAME): Mapping reads to calibrator sequences using bowtie:\n
LOGENTRY_MAP_CALIBRATOR_2 := $(ts) $(PIPELINE_NAME): Finished mapping to the calibrators\n
FILE_TO_INPUT_TO_UNIVEC_ALIGNMENT := $(OUTPUT_DIR)/$(SAMPLE_ID)/$(SAMPLE_ID).clipped.trimmed.filtered.noCalibrator.fastq.gz
endif
##
## STAR command to align reads to the UniVec contaminant sequence database
##
COMMAND_MAP_UNIVEC ?= $(STAR_EXE) --runThreadN $(N_THREADS) --outFileNamePrefix $(OUTPUT_DIR)/$(SAMPLE_ID)/filteringAlignments_UniVec_ --genomeDir $(DATABASE_PATH)/UniVec/STAR_INDEX_UniVec --readFilesIn $(FILE_TO_INPUT_TO_UNIVEC_ALIGNMENT) --outReadsUnmapped Fastx --parametersFiles $(DATABASE_PATH)/STAR_Parameters_Endogenous_smallRNA.in $(STAR_ENDOGENOUS_DYNAMIC_PARAMS) >> $(OUTPUT_DIR)/$(SAMPLE_ID).log 2>> $(OUTPUT_DIR)/$(SAMPLE_ID).err; \
$(SAMTOOLS_EXE) view $(OUTPUT_DIR)/$(SAMPLE_ID)/filteringAlignments_UniVec_Aligned.out.bam | awk '{print $$3}' | sort -k 2,2 2>> $(OUTPUT_DIR)/$(SAMPLE_ID).err | uniq --count > $(OUTPUT_DIR)/$(SAMPLE_ID)/$(SAMPLE_ID).clipped.trimmed.filtered.uniVecContaminants.counts 2>> $(OUTPUT_DIR)/$(SAMPLE_ID).err; \
$(SAMTOOLS_EXE) view $(OUTPUT_DIR)/$(SAMPLE_ID)/filteringAlignments_UniVec_Aligned.out.bam | awk '{print $$1}' | sort 2>> $(OUTPUT_DIR)/$(SAMPLE_ID).err | uniq -c | wc -l > $(OUTPUT_DIR)/$(SAMPLE_ID)/$(SAMPLE_ID).clipped.trimmed.filtered.uniVecContaminants.readCount 2>> $(OUTPUT_DIR)/$(SAMPLE_ID).err; \
gzip -c $(OUTPUT_DIR)/$(SAMPLE_ID)/filteringAlignments_UniVec_Unmapped.out.mate1 > $(OUTPUT_DIR)/$(SAMPLE_ID)/$(SAMPLE_ID).clipped.trimmed.filtered.noUniVecContaminants.fastq.gz; \
rm $(OUTPUT_DIR)/$(SAMPLE_ID)/filteringAlignments_UniVec_Unmapped.out.mate1
##
## STAR command to align reads to the rRNA sequences
##
COMMAND_MAP_RRNAS ?= $(STAR_EXE) --runThreadN $(N_THREADS) --outFileNamePrefix $(OUTPUT_DIR)/$(SAMPLE_ID)/filteringAlignments_rRNA_ --genomeDir $(DATABASE_PATH)/$(MAIN_ORGANISM_GENOME_ID)/STAR_INDEX_rRNA --readFilesIn $(OUTPUT_DIR)/$(SAMPLE_ID)/$(SAMPLE_ID).clipped.trimmed.filtered.noUniVecContaminants.fastq.gz --outReadsUnmapped Fastx --parametersFiles $(DATABASE_PATH)/STAR_Parameters_Endogenous_smallRNA.in $(STAR_ENDOGENOUS_DYNAMIC_PARAMS) >> $(OUTPUT_DIR)/$(SAMPLE_ID).log 2>> $(OUTPUT_DIR)/$(SAMPLE_ID).err; \
$(SAMTOOLS_EXE) view $(OUTPUT_DIR)/$(SAMPLE_ID)/filteringAlignments_rRNA_Aligned.out.bam | awk '{print $$3}' | sort -k 2,2 2>> $(OUTPUT_DIR)/$(SAMPLE_ID).err | uniq -c > $(OUTPUT_DIR)/$(SAMPLE_ID)/$(SAMPLE_ID).clipped.trimmed.filtered.rRNA.counts 2>> $(OUTPUT_DIR)/$(SAMPLE_ID).err; \
$(SAMTOOLS_EXE) view $(OUTPUT_DIR)/$(SAMPLE_ID)/filteringAlignments_rRNA_Aligned.out.bam | awk '{print $$1}' | sort 2>> $(OUTPUT_DIR)/$(SAMPLE_ID).err | uniq -c | wc -l > $(OUTPUT_DIR)/$(SAMPLE_ID)/$(SAMPLE_ID).clipped.trimmed.filtered.rRNA.readCount 2>> $(OUTPUT_DIR)/$(SAMPLE_ID).err; \
gzip -c $(OUTPUT_DIR)/$(SAMPLE_ID)/filteringAlignments_rRNA_Unmapped.out.mate1 > $(OUTPUT_DIR)/$(SAMPLE_ID)/$(SAMPLE_ID).clipped.trimmed.filtered.noRiboRNA.fastq.gz; \
rm $(OUTPUT_DIR)/$(SAMPLE_ID)/filteringAlignments_rRNA_Unmapped.out.mate1
##
## Remove some potentially large intermediate pipeline output (can save as much as 50% total output size)
##
TIDYUP_COMMAND :=
ifeq ($(REMOVE_LARGE_INTERMEDIATE_FILES),true)
TIDYUP_COMMAND := rm $(OUTPUT_DIR)/$(SAMPLE_ID)/genome.parsed; rm $(OUTPUT_DIR)/$(SAMPLE_ID)/$(SAMPLE_ID).clipped*.fastq.gz
endif
##
## Compress only the most vital output!
##
#ls -lh $(OUTPUT_DIR)/$(SAMPLE_ID)/noGenome | awk '{print $$9}' | grep "sense.grouped\|stat" | awk '{print "$(SAMPLE_ID)/noGenome/"$$1}' >> $(OUTPUT_DIR)/$(SAMPLE_ID)_filesToCompress.txt;
COMPRESS_COMMAND := ls -lh $(OUTPUT_DIR)/$(SAMPLE_ID) | awk '{print $$9}' | grep "readCounts_\|.readLengths.txt\|_fastqc.zip\|.counts\|.knownAdapterSeq\|.adapterSeq\|.qualityEncoding\|.CIGARstats.txt\|.coverage.txt" | awk '{print "$(SAMPLE_ID)/"$$1}' > $(OUTPUT_DIR)/$(SAMPLE_ID)_filesToCompress.txt; \
echo $(SAMPLE_ID).log >> $(OUTPUT_DIR)/$(SAMPLE_ID)_filesToCompress.txt; \
echo $(SAMPLE_ID).stats >> $(OUTPUT_DIR)/$(SAMPLE_ID)_filesToCompress.txt; \
echo $(SAMPLE_ID).qcResult >> $(OUTPUT_DIR)/$(SAMPLE_ID)_filesToCompress.txt; \
ls -lh $(OUTPUT_DIR)/$(SAMPLE_ID) | awk '{print $$9}' | grep "calibratormapped.counts" | awk '{print "$(SAMPLE_ID)/"$$1}' >> $(OUTPUT_DIR)/$(SAMPLE_ID)_filesToCompress.txt; \
ls -lh $(OUTPUT_DIR)/$(SAMPLE_ID)/EXOGENOUS_miRNA | awk '{print $$9}' | grep "readCounts_" | awk '{print "$(SAMPLE_ID)/EXOGENOUS_miRNA/"$$1}' >> $(OUTPUT_DIR)/$(SAMPLE_ID)_filesToCompress.txt; \
ls -lh $(OUTPUT_DIR)/$(SAMPLE_ID)/EXOGENOUS_rRNA | awk '{print $$9}' | grep "ExogenousRibosomalAlignments.result.taxaAnnotated.txt" | awk '{print "$(SAMPLE_ID)/EXOGENOUS_rRNA/"$$1}' >> $(OUTPUT_DIR)/$(SAMPLE_ID)_filesToCompress.txt; \
ls -lh $(OUTPUT_DIR)/$(SAMPLE_ID)/EXOGENOUS_genomes | awk '{print $$9}' | grep "ExogenousGenomicAlignments.result.taxaAnnotated.txt" | awk '{print "$(SAMPLE_ID)/EXOGENOUS_genomes/"$$1}' >> $(OUTPUT_DIR)/$(SAMPLE_ID)_filesToCompress.txt
#ls -lh $(OUTPUT_DIR)/$(SAMPLE_ID)/EXOGENOUS_rRNA | awk '{print $$9}' | grep "readCounts_" | awk '{print "$(SAMPLE_ID)/EXOGENOUS_rRNA/"$$1}' >> $(OUTPUT_DIR)/$(SAMPLE_ID)_filesToCompress.txt; \
#ls -lh $(OUTPUT_DIR)/$(SAMPLE_ID)/EXOGENOUS_miRNA | awk '{print $$9}' | grep "exogenous_miRBase_mapped" | awk '{print "$(SAMPLE_ID)/EXOGENOUS_miRNA/"$$1}' >> $(OUTPUT_DIR)/$(SAMPLE_ID)_filesToCompress.txt
#ls -lh $(OUTPUT_DIR)/$(SAMPLE_ID)/EXOGENOUS_rRNA | awk '{print $$9}' | grep "exogenous_miRBase_mapped" | awk '{print "$(SAMPLE_ID)/EXOGENOUS_rRNA/"$$1}' >> $(OUTPUT_DIR)/$(SAMPLE_ID)_filesToCompress.txt
OUTDIR := $(OUTPUT_DIR)/$(SAMPLE_ID)
###########################################################
###########################################################
###########################################################
##
## Main make target
##
.PHONY: all
.DEFAULT: all
all: processSample
##
## Target to print help
##
help:
@echo -e ""
@echo -e "Help for the exceRpt longRNA pipeline (version v.$(EXCERPT_VERSION))"
@echo -e ""
@echo -e "Usage: make -f path/to/exceRpt_longRNA [OPTION1=value1 OPTION2=value2 ...]"
@echo -e ""
@echo -e "*Required* OPTIONs:"
@echo -e " INPUT_FILE_PATH_R1 | Path to the input fastq file corresponding to the FIRST read of each pair"
@echo -e " OUTPUT_DIR | Path to store the output results"
@echo -e ""
@echo -e "For paired-end RNA-seq reads:"
@echo -e " INPUT_FILE_PATH_R2 | Path to the input fastq file corresponding to the SECOND read of each pair"
@echo -e ""
@echo -e "Main analysis OPTIONs:"
@echo -e " DATABASE_PATH | <Path> | [default: '$(DATABASE_PATH)''] change the location of the exceRpt database [by default this is within the EXE_DIR specified above]"
@echo -e " ADAPTER_SEQ | 'guessKnown'/'none'/<String> | [default: '$(ADAPTER_SEQ)'] will attempt to guess the 3 adapter using known sequences. The actual adapter can be input here if known, or specify 'none' if the adapter is already removed"
@echo -e " SAMPLE_NAME | <String> | add an optional ID to the input file specified above"
@echo -e " MAIN_ORGANISM_GENOME_ID | 'hg38'/'hg19'/'mm10' | [default: '$(MAIN_ORGANISM_GENOME_ID)'] changes the organism/genome build used for alignment"
@echo -e " CALIBRATOR_LIBRARY | <Path> | path to a bowtie2 index of calibrator oligos used for QC or normalisation"
@echo -e " MAP_EXOGENOUS | 'off'/'miRNA'/'on' | [default: '$(MAP_EXOGENOUS)'] choose whether to also align to known exogenous miRNAs+rRNAs ['miRNA'] or also to the full genomes of exogenous species ['on']"
@echo -e " ENDOGENOUS_LIB_PRIORITY | <comma,separated,list,no,spaces> | [default: '$(ENDOGENOUS_LIB_PRIORITY)'] choose the priority of each library during read assignment and quantification"
@echo -e ""
@echo -e "Additional analysis OPTIONs:"
@echo -e " TRIM_N_BASES_5p | <int> | [default: '$(TRIM_N_BASES_5p)'] remove N bases from the 5' end of every read"
@echo -e " TRIM_N_BASES_3p | <int> | [default: '$(TRIM_N_BASES_3p)'] remove N bases from the 3' end of every read"
@echo -e " RANDOM_BARCODE_LENGTH | <int> | [default: $(RANDOM_BARCODE_LENGTH)] identify and remove random barcodes of this number of nucleotides. For a Bioo prep with a 4N random barcode on both the 3' and 5' adapter, this value should be '4'."
@echo -e " RANDOM_BARCODE_LOCATION | '-5p -3p'/'-5p'/'-3p' | [default: '$(RANDOM_BARCODE_LOCATION)'] specify where to look for the random barcode(s)"
@echo -e " KEEP_RANDOM_BARCODE_STATS | 'false'/'true' | [default: '$(KEEP_RANDOM_BARCODE_STATS)'] specify whether or not to calculate overrepresentation statistics using the random barcodes (this may be slow and memory intensive!)"
@echo -e " DOWNSAMPLE_RNA_READS | <int> | [default: $(DOWNSAMPLE_RNA_READS)] choose whether to downsample to this number of reads after assigning reads to the various transcriptome libraries (may be useful for normalising very different yields)"
@echo -e ""
@echo -e "Hardware-specific OPTIONs:"
@echo -e " N_THREADS | <int> | [default: $(N_THREADS)] change the number of threads used in the alignments performed by exceRpt"
@echo -e " JAVA_RAM | <String> | [default: '$(JAVA_RAM)'] change the amount of memory (RAM) available to Java. This may need to be higher if crashes occur during quantification or random barcode stats calculation"
@echo -e " REMOVE_LARGE_INTERMEDIATE_FILES | 'false'/'true' | [default: '$(REMOVE_LARGE_INTERMEDIATE_FILES)'] when exceRpt finishes, choose whether to remove the large alignment files that can take a lot of disk space"
@echo -e ""
@echo -e "Alignment/QC OPTIONs:"
@echo -e " MIN_READ_LENGTH | <int> | [default: $(MIN_READ_LENGTH)] minimum read-length to use after adapter (+ random barcode) removal"
@echo -e " QFILTER_MIN_QUAL | <int> | [default: $(QFILTER_MIN_QUAL)] minimum base-call quality of the read"
@echo -e " QFILTER_MIN_READ_FRAC | <double> | [default: $(QFILTER_MIN_READ_FRAC)] read must have base-calls higher than the value above for at least this fraction of its length"
@echo -e " STAR_alignEndsType | 'Local'/'EndToEnd' | [default: $(STAR_alignEndsType)] defines the alignment mode; local alignment is recommended to allow for isomiRs"
@echo -e " STAR_outFilterMatchNmin | <int> | [default: $(STAR_outFilterMatchNmin)] minimum number of bases to include in the alignment (should match the minimum read length defined above)"
@echo -e " STAR_outFilterMatchNminOverLread | <double> | [default: $(STAR_outFilterMatchNminOverLread)] minimum fraction of the read that *must* remain following soft-clipping in a local alignment"
@echo -e " STAR_outFilterMismatchNmax | <int> | [default: $(STAR_outFilterMismatchNmax)] maximum allowed mismatched bases in the aligned portion of the read"
@echo -e " MAX_MISMATCHES_EXOGENOUS | <int> | [default: $(MAX_MISMATCHES_EXOGENOUS)] maximum allowed mismatched bases in the *entire* read when aligning to exogenous sequences"
@echo -e ""
##
## Target to selectively compress only the most useful results for downstream processing
##
## - this will typically reduce the volume of data needing to be transferred by 100x
##
compressCoreResults:
$(COMPRESS_COMMAND)
tar -cvz -C $(OUTPUT_DIR) -T $(OUTPUT_DIR)/$(SAMPLE_ID)_filesToCompress.txt -f $(OUTPUT_DIR)/$(SAMPLE_ID)_CORE_RESULTS_v$(EXCERPT_VERSION).tgz 2> /dev/null
rm $(OUTPUT_DIR)/$(SAMPLE_ID)_filesToCompress.txt
##
## Delete sample results and logfiles
##
#clean:
# rm -r $(OUTPUT_DIR)/$(SAMPLE_ID)
####
#### Main sub-target
####
processSample: $(OUTDIR)/$(PROCESS_SAMPLE_REQFILE)
## Wrap up logging and stats files
@echo -e "$(ts) $(PIPELINE_NAME): END longRNA-seq Pipeline for sample $(SAMPLE_ID)\n======================\n" >> $(OUTPUT_DIR)/$(SAMPLE_ID).log
@echo -e "$(ts) $(PIPELINE_NAME): END\n" >> $(OUTPUT_DIR)/$(SAMPLE_ID).err
@echo -e "#END OF STATS from the exceRpt longRNA-seq pipeline. Run completed at $(ts)" >> $(OUTPUT_DIR)/$(SAMPLE_ID).stats
#
## Adapter confidence
echo -e "known: " >> $(OUTPUT_DIR)/$(SAMPLE_ID).qctmp
cat $(OUTPUT_DIR)/$(SAMPLE_ID)/$(SAMPLE_ID).knownAdapterSeq >> $(OUTPUT_DIR)/$(SAMPLE_ID).qctmp
echo -e "used: " >> $(OUTPUT_DIR)/$(SAMPLE_ID).qctmp
cat $(OUTPUT_DIR)/$(SAMPLE_ID)/$(SAMPLE_ID).adapterSeq >> $(OUTPUT_DIR)/$(SAMPLE_ID).qctmp
cat $(OUTPUT_DIR)/$(SAMPLE_ID).qctmp | tr '\n' ' ' | awk -F ' ' '{if($$2=="used:"){ if(NF==2){print "Adapter_confidence: LOW"}else{print "Adapter_confidence: WARN_unableToGuessAdapter_usingProvided("$$3")"}}else{if($$2==$$4){print "Adapter_confidence: HIGH"}else{print "Adapter_confidence: WARN_providedAdapter("$$4")DisagreesWithGuessed("$$2")"}}}' > $(OUTPUT_DIR)/$(SAMPLE_ID).qcResult
#
## Calculate QC result
cat $(OUTPUT_DIR)/$(SAMPLE_ID).stats | grep "^input" | head -n 1 | awk '{print $$2}' > $(OUTPUT_DIR)/$(SAMPLE_ID).qctmp
cat $(OUTPUT_DIR)/$(SAMPLE_ID).stats | grep "^genome" | awk '{print $$2}' >> $(OUTPUT_DIR)/$(SAMPLE_ID).qctmp
cat $(OUTPUT_DIR)/$(SAMPLE_ID).stats | grep "sense" | awk '{SUM+=$$2}END{print SUM}' >> $(OUTPUT_DIR)/$(SAMPLE_ID).qctmp
cat $(OUTPUT_DIR)/$(SAMPLE_ID).qctmp | tr '\n' '\t' | awk '{result="FAIL"; ratio=0; if($$2>0){ratio=$$3/$$2}; if(ratio>$(MIN_GENOME_TRANSCRIPTOME_RATIO) && $$3>$(MIN_TRANSCRIPTOME_MAPPED))result="PASS"}END{print "QC_result: "result"\nInputReads: "$$1"\nGenomeReads: "$$2"\nTranscriptomeReads: "$$3"\nTranscriptomeGenomeRatio: "ratio}' >> $(OUTPUT_DIR)/$(SAMPLE_ID).qcResult
gunzip -c $(OUTPUT_DIR)/$(SAMPLE_ID)/endogenousAlignments_Accepted.txt.gz | wc -l > $(OUTPUT_DIR)/$(SAMPLE_ID).qctmp
gunzip -c $(OUTPUT_DIR)/$(SAMPLE_ID)/endogenousAlignments_Accepted.txt.gz | awk '{print $$2}' | uniq | wc -l >> $(OUTPUT_DIR)/$(SAMPLE_ID).qctmp
#
cat $(OUTPUT_DIR)/$(SAMPLE_ID).qctmp | tr '\n' '\t' | awk '{if($$1>0){print "TranscriptomeComplexity: "($$2/$$1)}else{print "TranscriptomeComplexity: 0"}}' >> $(OUTPUT_DIR)/$(SAMPLE_ID).qcResult
rm $(OUTPUT_DIR)/$(SAMPLE_ID).qctmp
#
## Compress core results files automatically
$(COMPRESS_COMMAND)
#tar -cvz -C $(OUTPUT_DIR) -T $(OUTPUT_DIR)/$(SAMPLE_ID)_filesToCompress.txt -f $(OUTPUT_DIR)/$(SAMPLE_ID)_results.tgz 2> /dev/null
tar -cvz -C $(OUTPUT_DIR) -T $(OUTPUT_DIR)/$(SAMPLE_ID)_filesToCompress.txt -f $(OUTPUT_DIR)/$(SAMPLE_ID)_CORE_RESULTS_v$(EXCERPT_VERSION).tgz 2> /dev/null
rm $(OUTPUT_DIR)/$(SAMPLE_ID)_filesToCompress.txt
## END PIPELINE
##
###
#### BEGIN PIPELINE
####
#### vvv Sub-targets to do the read-preprocessing, calibrator mapping, rRNA mapping, en-exRNA mapping, and ex-exRNA mapping vvv
###
##
##
## Make results directory & Write adapter sequence
##
$(OUTDIR)/Progress_1_FoundAdapter.dat:
#$(EXPORT_CMD)
@echo -e "$(USEAGE)"
mkdir -p $(OUTPUT_DIR)/$(SAMPLE_ID)
@echo -e "$(ts) $(PIPELINE_NAME): BEGIN exceRpt longRNA-seq pipeline v.$(EXCERPT_VERSION) for sample $(SAMPLE_ID)\n======================\n" > $(OUTPUT_DIR)/$(SAMPLE_ID).log
@echo -e "$(ts) $(PIPELINE_NAME): BEGIN \n" > $(OUTPUT_DIR)/$(SAMPLE_ID).err
@echo -e "$(ts) $(PIPELINE_NAME): Created results dir: $(OUTPUT_DIR)/$(SAMPLE_ID)\n" >> $(OUTPUT_DIR)/$(SAMPLE_ID).log
#
@echo -e "======================\n" >> $(OUTPUT_DIR)/$(SAMPLE_ID).log
@echo -e "$(ts) $(PIPELINE_NAME): Processing adapter sequence:\n" >> $(OUTPUT_DIR)/$(SAMPLE_ID).log
@echo -e "$(ts) $(PIPELINE_NAME): $(COMMAND_WRITE_ADAPTER_SEQ)\n" >> $(OUTPUT_DIR)/$(SAMPLE_ID).log 2>> $(OUTPUT_DIR)/$(SAMPLE_ID).err
$(COMMAND_WRITE_ADAPTER_SEQ)
@echo -e "$(ts) $(PIPELINE_NAME): Progress_1_FoundAdapter" > $(OUTPUT_DIR)/$(SAMPLE_ID)/Progress_1_FoundAdapter.dat
#
@echo -e "#STATS from the exceRpt longRNA-seq pipeline v.$(EXCERPT_VERSION) for sample $(SAMPLE_ID). Run started at $(ts)" > $(OUTPUT_DIR)/$(SAMPLE_ID).stats
@echo -e "Stage\tReadCount" >> $(OUTPUT_DIR)/$(SAMPLE_ID).stats
##
## Guess Fastq quality encoding
##
$(OUTDIR)/$(SAMPLE_ID).qualityEncoding: $(OUTDIR)/Progress_1_FoundAdapter.dat
@echo -e "======================\n" >> $(OUTPUT_DIR)/$(SAMPLE_ID).log
@echo -e "$(ts) $(PIPELINE_NAME): Guessing encoding of fastq read-qualities:\n" >> $(OUTPUT_DIR)/$(SAMPLE_ID).log
## ASCII 84 is equal to Q20 (p<0.01) in Phred+64, so any file with max quals greater than this can reasonably assumed to be Phred+64
@echo -e "$(ts) $(PIPELINE_NAME): $(COMMAND_CONVERT_SRA) | head -n 40000 | awk '{if(NR%4==0) printf("%s",$$0);}' | od -A n -t u1 | grep -v "^\*" | awk 'BEGIN{min=100;max=0;}{for(i=1;i<=NF;i++) {if($$i>max) max=$$i; if($$i<min) min=$$i;}}END{if(max<84) print "33"; else print "64";}' > $@\n" >> $(OUTPUT_DIR)/$(SAMPLE_ID).log
$(COMMAND_CONVERT_SRA) | head -n 40000 | awk '{if(NR%4==0) printf("%s",$$0);}' | od -A n -t u1 | grep -v "^\*" | awk 'BEGIN{min=100;max=0;}{for(i=1;i<=NF;i++) {if($$i>max) max=$$i; if($$i<min) min=$$i;}}END{if(max<84) print "33"; else print "64";}' > $@
@echo -e "$(ts) $(PIPELINE_NAME): Finished guessing encoding of fastq read-qualities:\n" >> $(OUTPUT_DIR)/$(SAMPLE_ID).log
##
## CLIP 3' adapter sequence
##
$(OUTDIR)/$(SAMPLE_ID).clipped.fastq.gz: $(OUTDIR)/$(SAMPLE_ID).qualityEncoding
## Run the SW alignment of known adapters regardless of user preference
@echo -e "======================\n" >> $(OUTPUT_DIR)/$(SAMPLE_ID).log
@echo -e "$(ts) $(PIPELINE_NAME): Checking adapter against known sequences: $(COMMAND_CONVERT_SRA) 2>> $(OUTPUT_DIR)/$(SAMPLE_ID).err | $(JAVA_EXE) -Xmx$(JAVA_RAM) -jar $(EXCERPT_TOOLS_EXE) FindAdapter -n 1000 -m 100000 -s 4 -a $(DATABASE_PATH)/adapters/adapters.fa - > $(OUTPUT_DIR)/$(SAMPLE_ID)/$(SAMPLE_ID).knownAdapterSeq 2>> $(OUTPUT_DIR)/$(SAMPLE_ID).log\n" >> $(OUTPUT_DIR)/$(SAMPLE_ID).log
$(COMMAND_CONVERT_SRA) 2>> $(OUTPUT_DIR)/$(SAMPLE_ID).err | $(JAVA_EXE) -Xmx$(JAVA_RAM) -jar $(EXCERPT_TOOLS_EXE) FindAdapter -n 1000 -m 100000 -s 4 -a $(DATABASE_PATH)/adapters/adapters.fa - > $(OUTPUT_DIR)/$(SAMPLE_ID)/$(SAMPLE_ID).knownAdapterSeq 2>> $(OUTPUT_DIR)/$(SAMPLE_ID).log
#@echo -e "$(ts) $(PIPELINE_NAME): Known adapter sequence: $(shell cat $(OUTPUT_DIR)/$(SAMPLE_ID)/$(SAMPLE_ID).knownAdapterSeq)\n" >> $(OUTPUT_DIR)/$(SAMPLE_ID).log
## Carry on with the adapter provided / guessed
@echo -e "======================\n" >> $(OUTPUT_DIR)/$(SAMPLE_ID).log
@echo -e "$(ts) $(PIPELINE_NAME): Provided/guessed adapter sequence: $(shell cat $(OUTPUT_DIR)/$(SAMPLE_ID)/$(SAMPLE_ID).adapterSeq)" >> $(OUTPUT_DIR)/$(SAMPLE_ID).log
@echo -e "$(ts) $(PIPELINE_NAME): $(LOGENTRY_WRITE_ADAPTER)" >> $(OUTPUT_DIR)/$(SAMPLE_ID).log 2>> $(OUTPUT_DIR)/$(SAMPLE_ID).err
@echo -e "$(ts) $(PIPELINE_NAME): $(COMMAND_CLIP_ADAPTER)\n" >> $(OUTPUT_DIR)/$(SAMPLE_ID).log 2>> $(OUTPUT_DIR)/$(SAMPLE_ID).err
$(COMMAND_CLIP_ADAPTER)
@echo -e "$(ts) $(PIPELINE_NAME): Finished removing adapters\n" >> $(OUTPUT_DIR)/$(SAMPLE_ID).log
## Count reads input to adapter clipping
grep "Input: " $(OUTPUT_DIR)/$(SAMPLE_ID).log | awk '{print "input\t"$$2}' >> $(OUTPUT_DIR)/$(SAMPLE_ID).stats
## Count reads output following adapter clipping
grep "Output: " $(OUTPUT_DIR)/$(SAMPLE_ID).log | awk '{print "successfully_clipped\t"$$2}' >> $(OUTPUT_DIR)/$(SAMPLE_ID).stats
## Remove random barcodes if there are any
@echo -e "$(ts) $(PIPELINE_NAME): Removing $(RANDOM_BARCODE_LENGTH)N random barcode:\n" >> $(OUTPUT_DIR)/$(SAMPLE_ID).log
@echo -e "$(ts) $(PIPELINE_NAME): $(COMMAND_REMOVE_RANDOM_BARCODE)\n" >> $(OUTPUT_DIR)/$(SAMPLE_ID).log 2>> $(OUTPUT_DIR)/$(SAMPLE_ID).err
$(COMMAND_REMOVE_RANDOM_BARCODE)
@echo -e "$(ts) $(PIPELINE_NAME): Finished removing random barcode\n" >> $(OUTPUT_DIR)/$(SAMPLE_ID).log
##
## Trim reads at the 5' and/or 3' end?
##
$(OUTDIR)/$(SAMPLE_ID).clipped.trimmed.fastq.gz: $(OUTDIR)/$(SAMPLE_ID).clipped.fastq.gz
@echo -e "======================\n" >> $(OUTPUT_DIR)/$(SAMPLE_ID).log
@echo -e "$(ts) $(PIPELINE_NAME): Trimming $(TRIM_N_BASES_5p) bases from the 5' and $(TRIM_N_BASES_3p) bases from the 3' of all reads:\n" >> $(OUTPUT_DIR)/$(SAMPLE_ID).log
#@echo -e "$(ts) $(PIPELINE_NAME): gunzip -c $(OUTPUT_DIR)/$(SAMPLE_ID)/$(SAMPLE_ID).clipped.fastq.gz | $(FASTX_TRIMMER_EXE) -z -Q$(shell cat $(OUTPUT_DIR)/$(SAMPLE_ID)/$(SAMPLE_ID).qualityEncoding) -f $(shell expr $(TRIM_N_BASES_5p) + $(ONE) ) > $(OUTPUT_DIR)/$(SAMPLE_ID)/$(SAMPLE_ID).clipped.trimmed.fastq.gz 2>>$(OUTPUT_DIR)/$(SAMPLE_ID).log\n" >> $(OUTPUT_DIR)/$(SAMPLE_ID).log
#gunzip -c $(OUTPUT_DIR)/$(SAMPLE_ID)/$(SAMPLE_ID).clipped.fastq.gz | $(FASTX_TRIMMER_EXE) -z -Q$(shell cat $(OUTPUT_DIR)/$(SAMPLE_ID)/$(SAMPLE_ID).qualityEncoding) -f $(shell expr $(TRIM_N_BASES_5p) + $(ONE) ) > $(OUTPUT_DIR)/$(SAMPLE_ID)/$(SAMPLE_ID).clipped.trimmed.fastq.gz 2>>$(OUTPUT_DIR)/$(SAMPLE_ID).log
@echo -e "$(ts) $(PIPELINE_NAME): gunzip -c $(OUTPUT_DIR)/$(SAMPLE_ID)/$(SAMPLE_ID).clipped.fastq.gz | $(JAVA_EXE) -Xmx$(JAVA_RAM) -jar $(EXCERPT_TOOLS_EXE) TrimFastq -5p $(TRIM_N_BASES_5p) -3p $(TRIM_N_BASES_3p) | gzip -c > $(OUTPUT_DIR)/$(SAMPLE_ID)/$(SAMPLE_ID).clipped.trimmed.fastq.gz 2>>$(OUTPUT_DIR)/$(SAMPLE_ID).log\n" >> $(OUTPUT_DIR)/$(SAMPLE_ID).log
gunzip -c $(OUTPUT_DIR)/$(SAMPLE_ID)/$(SAMPLE_ID).clipped.fastq.gz | $(JAVA_EXE) -Xmx$(JAVA_RAM) -jar $(EXCERPT_TOOLS_EXE) TrimFastq -5p $(TRIM_N_BASES_5p) -3p $(TRIM_N_BASES_3p) | gzip -c > $(OUTPUT_DIR)/$(SAMPLE_ID)/$(SAMPLE_ID).clipped.trimmed.fastq.gz 2>>$(OUTPUT_DIR)/$(SAMPLE_ID).log
@echo -e "$(ts) $(PIPELINE_NAME): Finished trimming bases from the 5' and 3' end of all reads\n" >> $(OUTPUT_DIR)/$(SAMPLE_ID).log
##
## FILTER clipped reads that have poor overall base quality & Remove homopolymer repeats
##
$(OUTDIR)/$(SAMPLE_ID).clipped.trimmed.filtered.fastq.gz: $(OUTDIR)/$(SAMPLE_ID).clipped.trimmed.fastq.gz $(OUTDIR)/$(SAMPLE_ID).qualityEncoding
@echo -e "======================\n" >> $(OUTPUT_DIR)/$(SAMPLE_ID).log
@echo -e "$(ts) $(PIPELINE_NAME): Filtering reads by base quality:\n" >> $(OUTPUT_DIR)/$(SAMPLE_ID).log
#@echo -e "$(ts) $(PIPELINE_NAME): $(COMMAND_FILTER_BY_QUALITY)\n" >> $(OUTPUT_DIR)/$(SAMPLE_ID).log 2>> $(OUTPUT_DIR)/$(SAMPLE_ID).err
#$(COMMAND_FILTER_BY_QUALITY)
@echo -e "$(ts) $(PIPELINE_NAME): gunzip -c $(OUTPUT_DIR)/$(SAMPLE_ID)/$(SAMPLE_ID).clipped.trimmed.fastq.gz | $(FASTX_FILTER_EXE) -v -Q$(shell cat $(OUTPUT_DIR)/$(SAMPLE_ID)/$(SAMPLE_ID).qualityEncoding) -p $(QFILTER_MIN_READ_FRAC) -q $(QFILTER_MIN_QUAL) > $(OUTPUT_DIR)/$(SAMPLE_ID)/$(SAMPLE_ID).clipped.trimmed.filtered.tmp\n" >> $(OUTPUT_DIR)/$(SAMPLE_ID).log 2>> $(OUTPUT_DIR)/$(SAMPLE_ID).err
gunzip -c $(OUTPUT_DIR)/$(SAMPLE_ID)/$(SAMPLE_ID).clipped.trimmed.fastq.gz | $(FASTX_FILTER_EXE) -v -Q$(shell cat $(OUTPUT_DIR)/$(SAMPLE_ID)/$(SAMPLE_ID).qualityEncoding) -p $(QFILTER_MIN_READ_FRAC) -q $(QFILTER_MIN_QUAL) > $(OUTPUT_DIR)/$(SAMPLE_ID)/$(SAMPLE_ID).clipped.trimmed.filtered.tmp 2>>$(OUTPUT_DIR)/$(SAMPLE_ID).log
@echo -e "$(ts) $(PIPELINE_NAME): Finished filtering reads by base quality\n" >> $(OUTPUT_DIR)/$(SAMPLE_ID).log
## Count reads that failed the quality filter
grep "low-quality reads" $(OUTPUT_DIR)/$(SAMPLE_ID).log | awk '{print "failed_quality_filter\t"$$2}' >> $(OUTPUT_DIR)/$(SAMPLE_ID).stats
#
# Filter homopolymer reads (those that have too many single nt repeats)
@echo -e "======================\n" >> $(OUTPUT_DIR)/$(SAMPLE_ID).log
@echo -e "$(ts) $(PIPELINE_NAME): Filtering homopolymer repeat reads:\n" >> $(OUTPUT_DIR)/$(SAMPLE_ID).log
@echo -e "$(ts) $(PIPELINE_NAME): $(JAVA_EXE) -Xmx$(JAVA_RAM) -jar $(EXCERPT_TOOLS_EXE) RemoveHomopolymerRepeats -m 0.66 -i $(OUTPUT_DIR)/$(SAMPLE_ID)/$(SAMPLE_ID).clipped.trimmed.filtered.tmp -o $(OUTPUT_DIR)/$(SAMPLE_ID)/$(SAMPLE_ID).clipped.trimmed.filtered.fastq\n" >> $(OUTPUT_DIR)/$(SAMPLE_ID).log 2>> $(OUTPUT_DIR)/$(SAMPLE_ID).err
$(JAVA_EXE) -Xmx$(JAVA_RAM) -jar $(EXCERPT_TOOLS_EXE) RemoveHomopolymerRepeats --verbose -m 0.66 -i $(OUTPUT_DIR)/$(SAMPLE_ID)/$(SAMPLE_ID).clipped.trimmed.filtered.tmp -o $(OUTPUT_DIR)/$(SAMPLE_ID)/$(SAMPLE_ID).clipped.trimmed.filtered.fastq >> $(OUTPUT_DIR)/$(SAMPLE_ID).log 2>> $(OUTPUT_DIR)/$(SAMPLE_ID)/$(SAMPLE_ID).clipped.REMOVEDRepeatReads.fastq
gzip $(OUTPUT_DIR)/$(SAMPLE_ID)/$(SAMPLE_ID).clipped.REMOVEDRepeatReads.fastq
gzip $(OUTPUT_DIR)/$(SAMPLE_ID)/$(SAMPLE_ID).clipped.trimmed.filtered.fastq
rm $(OUTPUT_DIR)/$(SAMPLE_ID)/$(SAMPLE_ID).clipped.trimmed.filtered.tmp
@echo -e "$(ts) $(PIPELINE_NAME): Finished filtering homopolymer repeat reads\n" >> $(OUTPUT_DIR)/$(SAMPLE_ID).log
## Count homopolymer repeat reads that failed the quality filter
grep "Done. Sequences removed" $(OUTPUT_DIR)/$(SAMPLE_ID).log | awk -F "=" '{print "failed_homopolymer_filter\t"$$2}' >> $(OUTPUT_DIR)/$(SAMPLE_ID).stats
##
## Assess Read-lengths after clipping
##
$(OUTDIR)/$(SAMPLE_ID).clipped.trimmed.filtered.readLengths.txt: $(OUTDIR)/$(SAMPLE_ID).clipped.trimmed.filtered.fastq.gz
@echo -e "======================" >> $(OUTPUT_DIR)/$(SAMPLE_ID).log
@echo -e "$(ts) $(PIPELINE_NAME): Calculating length distribution of clipped reads:\n" >> $(OUTPUT_DIR)/$(SAMPLE_ID).log
@echo -e "$(ts) $(PIPELINE_NAME): $(JAVA_EXE) -Xmx$(JAVA_RAM) -jar $(EXCERPT_TOOLS_EXE) GetSequenceLengths $(OUTPUT_DIR)/$(SAMPLE_ID)/$(SAMPLE_ID).clipped.trimmed.filtered.fastq > $@ 2>> $(OUTPUT_DIR)/$(SAMPLE_ID).err\n" >> $(OUTPUT_DIR)/$(SAMPLE_ID).log
gunzip -c $< > $(OUTPUT_DIR)/$(SAMPLE_ID)/$(SAMPLE_ID).clipped.trimmed.filtered.fastq
$(JAVA_EXE) -Xmx$(JAVA_RAM) -jar $(EXCERPT_TOOLS_EXE) GetSequenceLengths $(OUTPUT_DIR)/$(SAMPLE_ID)/$(SAMPLE_ID).clipped.trimmed.filtered.fastq > $@ 2>> $(OUTPUT_DIR)/$(SAMPLE_ID).err
rm $(OUTPUT_DIR)/$(SAMPLE_ID)/$(SAMPLE_ID).clipped.trimmed.filtered.fastq
@echo -e "$(ts) $(PIPELINE_NAME): Finished calculating read-lengths\n" >> $(OUTPUT_DIR)/$(SAMPLE_ID).log
##
## Perform FastQC after adapter removal
##
$(OUTDIR)/$(SAMPLE_ID).clipped.trimmed.filtered_fastqc.zip: $(OUTDIR)/$(SAMPLE_ID).clipped.trimmed.filtered.fastq.gz
@echo -e "======================" >> $(OUTPUT_DIR)/$(SAMPLE_ID).log
@echo -e "$(ts) $(PIPELINE_NAME): Running FastQC on clipped reads:\n" >> $(OUTPUT_DIR)/$(SAMPLE_ID).log
@echo -e "$(ts) $(PIPELINE_NAME): $(FASTQC_EXE) -Xmx$(JAVA_RAM) -Dfastqc.threads=$(N_THREADS) -Dfastqc.unzip=false -Dfastqc.output_dir=$(OUTPUT_DIR)/$(SAMPLE_ID)/ uk/ac/bbsrc/babraham/FastQC/FastQCApplication $< >> $(OUTPUT_DIR)/$(SAMPLE_ID).log 2>> $(OUTPUT_DIR)/$(SAMPLE_ID).err\n" >> $(OUTPUT_DIR)/$(SAMPLE_ID).log
$(FASTQC_EXE) -Xmx$(JAVA_RAM) -Dfastqc.threads=$(N_THREADS) -Dfastqc.unzip=false -Dfastqc.output_dir=$(OUTPUT_DIR)/$(SAMPLE_ID)/ uk/ac/babraham/FastQC/FastQCApplication $< >> $(OUTPUT_DIR)/$(SAMPLE_ID).log 2>> $(OUTPUT_DIR)/$(SAMPLE_ID).err
@echo -e "$(ts) $(PIPELINE_NAME): Finished running FastQC\n" >> $(OUTPUT_DIR)/$(SAMPLE_ID).log
##
## MAP to external bowtie (calibrator?) library and to UniVec sequences
##
$(OUTDIR)/$(SAMPLE_ID).clipped.trimmed.filtered.noUniVecContaminants.fastq.gz: $(OUTDIR)/$(SAMPLE_ID).clipped.trimmed.filtered.fastq.gz $(OUTDIR)/$(SAMPLE_ID).clipped.trimmed.filtered.readLengths.txt $(OUTDIR)/$(SAMPLE_ID).clipped.trimmed.filtered_fastqc.zip
@echo -e "======================\n" >> $(OUTPUT_DIR)/$(SAMPLE_ID).log
@echo -e "$(ts) $(PIPELINE_NAME): $(LOGENTRY_MAP_CALIBRATOR_1)" >> $(OUTPUT_DIR)/$(SAMPLE_ID).log
@echo -e "$(ts) $(PIPELINE_NAME): $(COMMAND_MAP_CALIBRATOR)" >> $(OUTPUT_DIR)/$(SAMPLE_ID).log
$(COMMAND_MAP_CALIBRATOR)
@echo -e "$(ts) $(PIPELINE_NAME): $(LOGENTRY_MAP_CALIBRATOR_2)" >> $(OUTPUT_DIR)/$(SAMPLE_ID).log
## Count calibrator oligo reads
$(COMMAND_COUNT_CALIBRATOR)
#
@echo -e "======================\n" >> $(OUTPUT_DIR)/$(SAMPLE_ID).log
@echo -e "$(ts) $(PIPELINE_NAME): Mapping reads to contaminant sequences in UniVec using STAR:\n" >> $(OUTPUT_DIR)/$(SAMPLE_ID).log
@echo -e "$(ts) $(PIPELINE_NAME): $(COMMAND_MAP_UNIVEC)\n" >> $(OUTPUT_DIR)/$(SAMPLE_ID).log
$(COMMAND_MAP_UNIVEC)
@echo -e "$(ts) $(PIPELINE_NAME): Finished mapping to the UniVec contaminant DB\n" >> $(OUTPUT_DIR)/$(SAMPLE_ID).log
## Count UniVec contaminant reads
cat $(OUTPUT_DIR)/$(SAMPLE_ID)/$(SAMPLE_ID).clipped.trimmed.filtered.uniVecContaminants.readCount | awk '{print "UniVec_contaminants\t"$$1}' >> $(OUTPUT_DIR)/$(SAMPLE_ID).stats
##
## MAP to rRNA sequences
##
$(OUTDIR)/$(SAMPLE_ID).clipped.trimmed.filtered.noRiboRNA.fastq.gz: $(OUTDIR)/$(SAMPLE_ID).clipped.trimmed.filtered.noUniVecContaminants.fastq.gz
@echo -e "======================\n" >> $(OUTPUT_DIR)/$(SAMPLE_ID).log
@echo -e "$(ts) $(PIPELINE_NAME): Mapping reads to ribosomal RNA sequences using STAR:\n" >> $(OUTPUT_DIR)/$(SAMPLE_ID).log
@echo -e "$(ts) $(PIPELINE_NAME): $(COMMAND_MAP_RRNAS)\n" >> $(OUTPUT_DIR)/$(SAMPLE_ID).log
$(COMMAND_MAP_RRNAS)
## Count rRNA reads
cat $(OUTPUT_DIR)/$(SAMPLE_ID)/$(SAMPLE_ID).clipped.trimmed.filtered.rRNA.readCount | awk ' {print "rRNA\t"$$1}' >> $(OUTPUT_DIR)/$(SAMPLE_ID).stats
#
#$(SAMTOOLS_EXE) sort $(OUTPUT_DIR)/$(SAMPLE_ID)/$(SAMPLE_ID).clipped.trimmed.filtered.rRNAmapped.bam $(OUTPUT_DIR)/$(SAMPLE_ID)/$(SAMPLE_ID).clipped.trimmed.filtered.rRNAmapped.sorted
$(SAMTOOLS_EXE) sort -@ $(N_THREADS) -m 8G -O bam -T $(OUTPUT_DIR)/$(SAMPLE_ID)/tmp $(OUTPUT_DIR)/$(SAMPLE_ID)/filteringAlignments_rRNA_Aligned.out.bam > $(OUTPUT_DIR)/$(SAMPLE_ID)/filteringAlignments_rRNA_Aligned.out.sorted.bam
$(SAMTOOLS_EXE) index $(OUTPUT_DIR)/$(SAMPLE_ID)/filteringAlignments_rRNA_Aligned.out.sorted.bam
rm $(OUTPUT_DIR)/$(SAMPLE_ID)/filteringAlignments_rRNA_Aligned.out.bam
@echo -e "$(ts) $(PIPELINE_NAME): Finished mapping to the rRNAs\n" >> $(OUTPUT_DIR)/$(SAMPLE_ID).log
##
## Perform FastQC again after rRNA / UniVec removal
##
$(OUTDIR)/$(SAMPLE_ID).clipped.trimmed.filtered.noRiboRNA_fastqc.zip: $(OUTDIR)/$(SAMPLE_ID).clipped.trimmed.filtered.noRiboRNA.fastq.gz
@echo -e "======================" >> $(OUTPUT_DIR)/$(SAMPLE_ID).log
@echo -e "$(ts) $(PIPELINE_NAME): Running FastQC on cleaned reads:\n" >> $(OUTPUT_DIR)/$(SAMPLE_ID).log
@echo -e "$(ts) $(PIPELINE_NAME): $(FASTQC_EXE) -Xmx$(JAVA_RAM) -Dfastqc.threads=$(N_THREADS) -Dfastqc.unzip=false -Dfastqc.output_dir=$(OUTPUT_DIR)/$(SAMPLE_ID)/ uk/ac/bbsrc/babraham/FastQC/FastQCApplication $< >> $(OUTPUT_DIR)/$(SAMPLE_ID).log 2>> $(OUTPUT_DIR)/$(SAMPLE_ID).err\n" >> $(OUTPUT_DIR)/$(SAMPLE_ID).log
$(FASTQC_EXE) -Xmx$(JAVA_RAM) -Dfastqc.threads=$(N_THREADS) -Dfastqc.unzip=false -Dfastqc.output_dir=$(OUTPUT_DIR)/$(SAMPLE_ID)/ uk/ac/babraham/FastQC/FastQCApplication $< >> $(OUTPUT_DIR)/$(SAMPLE_ID).log 2>> $(OUTPUT_DIR)/$(SAMPLE_ID).err
@echo -e "$(ts) $(PIPELINE_NAME): Finished running FastQC\n" >> $(OUTPUT_DIR)/$(SAMPLE_ID).log
##
## Map reads to the endogenous genome and transcriptome
##
## map ALL READS to the GENOME (STAR ungapped)
$(OUTDIR)/endogenousAlignments_genomeUnmapped_transcriptome_Unmapped.R1.fastq.gz: $(OUTDIR)/$(SAMPLE_ID).clipped.trimmed.filtered.noRiboRNA.fastq.gz $(OUTDIR)/$(SAMPLE_ID).clipped.trimmed.filtered.noRiboRNA_fastqc.zip
@echo -e "======================\n" >> $(OUTPUT_DIR)/$(SAMPLE_ID).log
@echo -e "$(ts) $(PIPELINE_NAME): Mapping reads to the genome of the primary organism:\n" >> $(OUTPUT_DIR)/$(SAMPLE_ID).log
@echo -e "$(ts) $(PIPELINE_NAME): $(STAR_EXE) --runThreadN $(N_THREADS) --outFileNamePrefix $(OUTPUT_DIR)/$(SAMPLE_ID)/endogenousAlignments_genome_ --genomeDir $(DATABASE_PATH)/$(MAIN_ORGANISM_GENOME_ID)/STAR_INDEX_genome --readFilesIn $(OUTPUT_DIR)/$(SAMPLE_ID)/$(SAMPLE_ID).clipped.trimmed.filtered.noRiboRNA.fastq.gz --outReadsUnmapped Fastx --parametersFiles $(DATABASE_PATH)/STAR_Parameters_Endogenous_smallRNA.in $(STAR_ENDOGENOUS_DYNAMIC_PARAMS) >> $(OUTPUT_DIR)/$(SAMPLE_ID).log 2>> $(OUTPUT_DIR)/$(SAMPLE_ID).err\n" >> $(OUTPUT_DIR)/$(SAMPLE_ID).log
$(STAR_EXE) --runThreadN $(N_THREADS) --outFileNamePrefix $(OUTPUT_DIR)/$(SAMPLE_ID)/endogenousAlignments_genome_ --genomeDir $(DATABASE_PATH)/$(MAIN_ORGANISM_GENOME_ID)/STAR_INDEX_genome --readFilesIn $(OUTPUT_DIR)/$(SAMPLE_ID)/$(SAMPLE_ID).clipped.trimmed.filtered.noRiboRNA.fastq.gz --outReadsUnmapped Fastx --parametersFiles $(DATABASE_PATH)/STAR_Parameters_Endogenous_smallRNA.in $(STAR_ENDOGENOUS_DYNAMIC_PARAMS) >> $(OUTPUT_DIR)/$(SAMPLE_ID).log 2>> $(OUTPUT_DIR)/$(SAMPLE_ID).err
@echo -e "$(ts) $(PIPELINE_NAME): Finished mapping to the genome of the primary organism\n" >> $(OUTPUT_DIR)/$(SAMPLE_ID).log
#
## sort the alignments by ReadID just in case these are paired end reads in a single file? -- no, better to flag that this is an invalid file (ToDo)
#
## v use this line when we start dealing with paired-end reads
#$(SAMTOOLS_EXE) fastq -1 $(OUTPUT_DIR)/$(SAMPLE_ID)/endogenousAlignments_genome_Mapped.out.mate1 -2 $(OUTPUT_DIR)/$(SAMPLE_ID)/endogenousAlignments_genome_Mapped.out.mate2 $(OUTPUT_DIR)/$(SAMPLE_ID)/endogenousAlignments_genome_Aligned.out.bam
$(SAMTOOLS_EXE) fastq $(OUTPUT_DIR)/$(SAMPLE_ID)/endogenousAlignments_genome_Aligned.out.bam > $(OUTPUT_DIR)/$(SAMPLE_ID)/endogenousAlignments_genome_Mapped.out.mate1
#
## map ALL READS to the TRANSCRIPTOME (STAR ungapped)
@echo -e "======================\n" >> $(OUTPUT_DIR)/$(SAMPLE_ID).log
@echo -e "$(ts) $(PIPELINE_NAME): Mapping genome-aligned reads to the transcriptome of the primary organism:\n" >> $(OUTPUT_DIR)/$(SAMPLE_ID).log
@echo -e "$(ts) $(PIPELINE_NAME): $(STAR_EXE) --runThreadN $(N_THREADS) --outFileNamePrefix $(OUTPUT_DIR)/$(SAMPLE_ID)/endogenousAlignments_genomeMapped_transcriptome_ --readFilesIn $(OUTPUT_DIR)/$(SAMPLE_ID)/endogenousAlignments_genome_Mapped.out.mate1 --genomeDir $(DATABASE_PATH)/$(MAIN_ORGANISM_GENOME_ID)/STAR_INDEX_transcriptome --parametersFiles $(DATABASE_PATH)/STAR_Parameters_Endogenous_smallRNA.in $(STAR_ENDOGENOUS_DYNAMIC_PARAMS) --readFilesCommand - >> $(OUTPUT_DIR)/$(SAMPLE_ID).log 2>> $(OUTPUT_DIR)/$(SAMPLE_ID).err\n" >> $(OUTPUT_DIR)/$(SAMPLE_ID).log
$(STAR_EXE) --runThreadN $(N_THREADS) --outFileNamePrefix $(OUTPUT_DIR)/$(SAMPLE_ID)/endogenousAlignments_genomeMapped_transcriptome_ --readFilesIn $(OUTPUT_DIR)/$(SAMPLE_ID)/endogenousAlignments_genome_Mapped.out.mate1 --genomeDir $(DATABASE_PATH)/$(MAIN_ORGANISM_GENOME_ID)/STAR_INDEX_transcriptome --parametersFiles $(DATABASE_PATH)/STAR_Parameters_Endogenous_smallRNA.in $(STAR_ENDOGENOUS_DYNAMIC_PARAMS) --readFilesCommand - >> $(OUTPUT_DIR)/$(SAMPLE_ID).log 2>> $(OUTPUT_DIR)/$(SAMPLE_ID).err
@echo -e "$(ts) $(PIPELINE_NAME): Finished mapping genome-aligned reads to the transcriptome of the primary organism\n" >> $(OUTPUT_DIR)/$(SAMPLE_ID).log
gzip -c $(OUTPUT_DIR)/$(SAMPLE_ID)/endogenousAlignments_genomeMapped_transcriptome_Unmapped.out.mate1 > $(OUTPUT_DIR)/$(SAMPLE_ID)/endogenousAlignments_genomeMapped_transcriptome_Unmapped.R1.fastq.gz
#
@echo -e "$(ts) $(PIPELINE_NAME): Mapping genome-unaligned reads to the transcriptome of the primary organism:\n" >> $(OUTPUT_DIR)/$(SAMPLE_ID).log
@echo -e "$(ts) $(PIPELINE_NAME): $(STAR_EXE) --runThreadN $(N_THREADS) --outFileNamePrefix $(OUTPUT_DIR)/$(SAMPLE_ID)/endogenousAlignments_genomeUnmapped_transcriptome_ --readFilesIn $(OUTPUT_DIR)/$(SAMPLE_ID)/endogenousAlignments_genome_Unmapped.out.mate1 --outReadsUnmapped Fastx --genomeDir $(DATABASE_PATH)/$(MAIN_ORGANISM_GENOME_ID)/STAR_INDEX_transcriptome --parametersFiles $(DATABASE_PATH)/STAR_Parameters_Endogenous_smallRNA.in $(STAR_ENDOGENOUS_DYNAMIC_PARAMS) --readFilesCommand - >> $(OUTPUT_DIR)/$(SAMPLE_ID).log 2>> $(OUTPUT_DIR)/$(SAMPLE_ID).err\n" >> $(OUTPUT_DIR)/$(SAMPLE_ID).log
$(STAR_EXE) --runThreadN $(N_THREADS) --outFileNamePrefix $(OUTPUT_DIR)/$(SAMPLE_ID)/endogenousAlignments_genomeUnmapped_transcriptome_ --readFilesIn $(OUTPUT_DIR)/$(SAMPLE_ID)/endogenousAlignments_genome_Unmapped.out.mate1 --outReadsUnmapped Fastx --genomeDir $(DATABASE_PATH)/$(MAIN_ORGANISM_GENOME_ID)/STAR_INDEX_transcriptome --parametersFiles $(DATABASE_PATH)/STAR_Parameters_Endogenous_smallRNA.in $(STAR_ENDOGENOUS_DYNAMIC_PARAMS) --readFilesCommand - >> $(OUTPUT_DIR)/$(SAMPLE_ID).log 2>> $(OUTPUT_DIR)/$(SAMPLE_ID).err
@echo -e "$(ts) $(PIPELINE_NAME): Finished mapping genome-unaligned reads to the transcriptome of the primary organism\n" >> $(OUTPUT_DIR)/$(SAMPLE_ID).log
gzip -c $(OUTPUT_DIR)/$(SAMPLE_ID)/endogenousAlignments_genomeUnmapped_transcriptome_Unmapped.out.mate1 > $(OUTPUT_DIR)/$(SAMPLE_ID)/endogenousAlignments_genomeUnmapped_transcriptome_Unmapped.R1.fastq.gz
#
## Count # mapped reads
cat $(OUTPUT_DIR)/$(SAMPLE_ID)/endogenousAlignments_genome_Log.final.out | grep "Number of input reads" | awk -F "|\t" '{print "reads_used_for_alignment\t"$$2}' >> $(OUTPUT_DIR)/$(SAMPLE_ID).stats
#cat $(OUTPUT_DIR)/$(SAMPLE_ID)/endogenousAlignments_genome_Log.final.out | grep "Uniquely mapped reads number\|Number of reads mapped to multiple loci" | awk -F "|\t" '{SUM+=$$2}END{print "reads_used_for_alignment\t"SUM}' >> $(OUTPUT_DIR)/$(SAMPLE_ID).stats
cat $(OUTPUT_DIR)/$(SAMPLE_ID)/endogenousAlignments_genome*apped_transcriptome_Log.final.out | grep "Number of input reads\|Uniquely mapped reads number\|Number of reads mapped to multiple loci" | sed '2,4d' | awk -F "|\t" '{SUM+=$$2}END{print "genome\t"SUM}' >> $(OUTPUT_DIR)/$(SAMPLE_ID).stats
#
## Compress STAR logs
gzip $(OUTPUT_DIR)/$(SAMPLE_ID)/endogenousAlignments_genomeMapped_transcriptome_Log.out
gzip $(OUTPUT_DIR)/$(SAMPLE_ID)/endogenousAlignments_genomeUnmapped_transcriptome_Log.out
#
## Tidy up
rm $(OUTPUT_DIR)/$(SAMPLE_ID)/endogenousAlignments_genome_SJ.out.tab
rm $(OUTPUT_DIR)/$(SAMPLE_ID)/endogenousAlignments_genomeUnmapped_transcriptome_SJ.out.tab
rm $(OUTPUT_DIR)/$(SAMPLE_ID)/endogenousAlignments_genome_Mapped.out.mate1
rm $(OUTPUT_DIR)/$(SAMPLE_ID)/endogenousAlignments_genome_Unmapped.out.mate1
rm $(OUTPUT_DIR)/$(SAMPLE_ID)/endogenousAlignments_genomeMapped_transcriptome_Unmapped.out.mate1
rm $(OUTPUT_DIR)/$(SAMPLE_ID)/endogenousAlignments_genomeUnmapped_transcriptome_Unmapped.out.mate1
## convert genomic alignments to wiggle file for display - CURRENTLY DISABLED - REQUIRES BAM TO BE SORTED!
$(OUTDIR)/endogenousAlignments_unspliced.wig: $(OUTDIR)/endogenousAlignments_genomeUnmapped_transcriptome_Unmapped.R1.fastq.gz
@echo -e "======================\n" >> $(OUTPUT_DIR)/$(SAMPLE_ID).log
@echo -e "$(ts) $(PIPELINE_NAME): Converting genomic alignments to .wig format:\n" >> $(OUTPUT_DIR)/$(SAMPLE_ID).log
@echo -e "$(ts) $(PIPELINE_NAME): $(SAMTOOLS_EXE) sort -@ $(N_THREADS) -m 8G -O bam -T $(OUTPUT_DIR)/$(SAMPLE_ID)/tmp $(OUTPUT_DIR)/$(SAMPLE_ID)/endogenousAlignments_genome_Aligned.out.bam | $(SAMTOOLS_EXE) mpileup -t SP - 2>> $(OUTPUT_DIR)/$(SAMPLE_ID).log | perl -ne 'BEGIN{print "track type=wiggle_0 name=$(SAMPLE_ID) description=$(SAMPLE_ID)\n"};($$c, $$start, undef, $$depth) = split; if ($$c ne $$lastC) { print "variableStep chrom=$$c\n"; };$$lastC=$$c;next unless $$. % 10 ==0;print "$$start\t$$depth\n" unless $$depth<3;' > $(OUTPUT_DIR)/$(SAMPLE_ID)/endogenousAlignments_genome_Aligned.wig 2>> $(OUTPUT_DIR)/$(SAMPLE_ID).log\n" >> $(OUTPUT_DIR)/$(SAMPLE_ID).log
$(SAMTOOLS_EXE) sort -@ $(N_THREADS) -m 8G -O bam -T $(OUTPUT_DIR)/$(SAMPLE_ID)/tmp $(OUTPUT_DIR)/$(SAMPLE_ID)/endogenousAlignments_genome_Aligned.out.bam | $(SAMTOOLS_EXE) mpileup -t SP - 2>> $(OUTPUT_DIR)/$(SAMPLE_ID).log | perl -ne 'BEGIN{print "track type=wiggle_0 name=$(SAMPLE_ID) description=$(SAMPLE_ID)\n"};($$c, $$start, undef, $$depth) = split; if ($$c ne $$lastC) { print "variableStep chrom=$$c\n"; };$$lastC=$$c;next unless $$. % 10 ==0;print "$$start\t$$depth\n" unless $$depth<3;' > $(OUTPUT_DIR)/$(SAMPLE_ID)/endogenousAlignments_genome_Aligned.wig 2>> $(OUTPUT_DIR)/$(SAMPLE_ID).log
@echo -e "$(ts) $(PIPELINE_NAME): Finished converting genomic alignments to .wig format\n" >> $(OUTPUT_DIR)/$(SAMPLE_ID).log
##
## Do QC on alignments
##
$(OUTDIR)/endogenousAlignments_genomeMapped_transcriptome_Aligned.out.sorted.bam.coverage.txt: $(OUTDIR)/endogenousAlignments_genomeUnmapped_transcriptome_Unmapped.R1.fastq.gz
@echo -e "======================\n" >> $(OUTPUT_DIR)/$(SAMPLE_ID).log
#
@echo -e "$(ts) $(PIPELINE_NAME): Performing CIGAR QC on endogenous genome alignments:\n" >> $(OUTPUT_DIR)/$(SAMPLE_ID).log
@echo -e "$(ts) $(PIPELINE_NAME): $(JAVA_EXE) -Xmx$(JAVA_RAM) -jar $(EXCERPT_TOOLS_EXE) CIGAR_2_PWM -f $(OUTPUT_DIR)/$(SAMPLE_ID)/endogenousAlignments_genome_Aligned.out.bam > $(OUTPUT_DIR)/$(SAMPLE_ID)/endogenousAlignments_genome_Aligned.out.bam.CIGARstats.txt 2>> $(OUTPUT_DIR)/$(SAMPLE_ID).log" >> $(OUTPUT_DIR)/$(SAMPLE_ID).log
$(JAVA_EXE) -Xmx$(JAVA_RAM) -jar $(EXCERPT_TOOLS_EXE) CIGAR_2_PWM -f $(OUTPUT_DIR)/$(SAMPLE_ID)/endogenousAlignments_genome_Aligned.out.bam > $(OUTPUT_DIR)/$(SAMPLE_ID)/endogenousAlignments_genome_Aligned.out.bam.CIGARstats.txt 2>> $(OUTPUT_DIR)/$(SAMPLE_ID).log
@echo -e "$(ts) $(PIPELINE_NAME): Finished performing CIGAR QC on endogenous genome alignments\n" >> $(OUTPUT_DIR)/$(SAMPLE_ID).log
#
@echo -e "$(ts) $(PIPELINE_NAME): Sorting endogenous transcriptome alignments:\n" >> $(OUTPUT_DIR)/$(SAMPLE_ID).log
@echo -e "$(ts) $(PIPELINE_NAME): $(SAMTOOLS_EXE) sort -n -@ $(N_THREADS) -m $(SAMTOOLS_SORT_MEM) -O bam -T $(OUTPUT_DIR)/$(SAMPLE_ID)/tmp $(OUTPUT_DIR)/$(SAMPLE_ID)/endogenousAlignments_genomeMapped_transcriptome_Aligned.out.bam > $(OUTPUT_DIR)/$(SAMPLE_ID)/endogenousAlignments_genomeMapped_transcriptome_Aligned.out.sorted.bam 2>> $(OUTPUT_DIR)/$(SAMPLE_ID).log" >> $(OUTPUT_DIR)/$(SAMPLE_ID).log
$(SAMTOOLS_EXE) sort -n -@ $(N_THREADS) -m $(SAMTOOLS_SORT_MEM) -O bam -T $(OUTPUT_DIR)/$(SAMPLE_ID)/tmp $(OUTPUT_DIR)/$(SAMPLE_ID)/endogenousAlignments_genomeMapped_transcriptome_Aligned.out.bam > $(OUTPUT_DIR)/$(SAMPLE_ID)/endogenousAlignments_genomeMapped_transcriptome_Aligned.out.sorted.bam 2>> $(OUTPUT_DIR)/$(SAMPLE_ID).log
@echo -e "$(ts) $(PIPELINE_NAME): Finished sorting endogenous transcriptome alignments\n" >> $(OUTPUT_DIR)/$(SAMPLE_ID).log
#
@echo -e "$(ts) $(PIPELINE_NAME): Performing read-coverage QC on endogenous transcriptome alignments:\n" >> $(OUTPUT_DIR)/$(SAMPLE_ID).log
@echo -e "$(ts) $(PIPELINE_NAME): $(JAVA_EXE) -Xmx$(JAVA_RAM) -jar $(EXCERPT_TOOLS_EXE) ReadCoverage -exceRpt -a $(DATABASE_PATH)/$(MAIN_ORGANISM_GENOME_ID)/gencodeAnnotation.gtf -f $(OUTPUT_DIR)/$(SAMPLE_ID)/endogenousAlignments_genomeMapped_transcriptome_Aligned.out.sorted.bam 2>> $(OUTPUT_DIR)/$(SAMPLE_ID).log" >> $(OUTPUT_DIR)/$(SAMPLE_ID).log
$(JAVA_EXE) -Xmx$(JAVA_RAM) -jar $(EXCERPT_TOOLS_EXE) ReadCoverage -exceRpt -a $(DATABASE_PATH)/$(MAIN_ORGANISM_GENOME_ID)/gencodeAnnotation.gtf -f $(OUTPUT_DIR)/$(SAMPLE_ID)/endogenousAlignments_genomeMapped_transcriptome_Aligned.out.sorted.bam 2>> $(OUTPUT_DIR)/$(SAMPLE_ID).log
@echo -e "$(ts) $(PIPELINE_NAME): Finished performing read-coverage QC on endogenous transcriptome alignments\n" >> $(OUTPUT_DIR)/$(SAMPLE_ID).log
#
rm $(OUTPUT_DIR)/$(SAMPLE_ID)/endogenousAlignments_genomeMapped_transcriptome_Aligned.out.sorted.bam
## process alignments
$(OUTDIR)/endogenousAlignments_Accepted.txt.gz: $(OUTDIR)/endogenousAlignments_genomeUnmapped_transcriptome_Unmapped.R1.fastq.gz $(OUTDIR)/endogenousAlignments_genomeMapped_transcriptome_Aligned.out.sorted.bam.coverage.txt
#$(OUTPUT_DIR)/$(SAMPLE_ID)/endogenousAlignments_Accepted.txt.gz: $(OUTPUT_DIR)/$(SAMPLE_ID)/endogenousAlignments_genomeUnmapped_transcriptome_Unmapped.R1.fastq.gz $(OUTPUT_DIR)/$(SAMPLE_ID)/endogenousAlignments_unspliced.wig
@echo -e "======================\n" >> $(OUTPUT_DIR)/$(SAMPLE_ID).log
#
## Assign reads
@echo -e "$(ts) $(PIPELINE_NAME): Assigning and sorting reads:\n" >> $(OUTPUT_DIR)/$(SAMPLE_ID).log
@echo -e "$(ts) $(PIPELINE_NAME): $(JAVA_EXE) -Xmx$(JAVA_RAM) -jar $(EXCERPT_TOOLS_EXE) ProcessEndogenousAlignments --libPriority $(ENDOGENOUS_LIB_PRIORITY) --genomeMappedReads $(OUTPUT_DIR)/$(SAMPLE_ID)/endogenousAlignments_genomeMapped_transcriptome_Aligned.out.bam --transcriptomeMappedReads $(OUTPUT_DIR)/$(SAMPLE_ID)/endogenousAlignments_genomeUnmapped_transcriptome_Aligned.out.bam --hairpin2genome $(DATABASE_PATH)/$(MAIN_ORGANISM_GENOME_ID)/miRNA_precursor2genome.sam --mature2hairpin $(DATABASE_PATH)/$(MAIN_ORGANISM_GENOME_ID)/miRNA_mature2precursor.sam --dict $(OUTPUT_DIR)/$(SAMPLE_ID)/endogenousAlignments_Accepted.dict 2>> $(OUTPUT_DIR)/$(SAMPLE_ID).log | sort -k 2,2 -k 1,1 > $(OUTPUT_DIR)/$(SAMPLE_ID)/endogenousAlignments_Accepted.txt\n" >> $(OUTPUT_DIR)/$(SAMPLE_ID).log
$(JAVA_EXE) -Xmx$(JAVA_RAM) -jar $(EXCERPT_TOOLS_EXE) ProcessEndogenousAlignments --libPriority $(ENDOGENOUS_LIB_PRIORITY) --genomeMappedReads $(OUTPUT_DIR)/$(SAMPLE_ID)/endogenousAlignments_genomeMapped_transcriptome_Aligned.out.bam --transcriptomeMappedReads $(OUTPUT_DIR)/$(SAMPLE_ID)/endogenousAlignments_genomeUnmapped_transcriptome_Aligned.out.bam --hairpin2genome $(DATABASE_PATH)/$(MAIN_ORGANISM_GENOME_ID)/miRNA_precursor2genome.sam --mature2hairpin $(DATABASE_PATH)/$(MAIN_ORGANISM_GENOME_ID)/miRNA_mature2precursor.sam --dict $(OUTPUT_DIR)/$(SAMPLE_ID)/endogenousAlignments_Accepted.dict 2>> $(OUTPUT_DIR)/$(SAMPLE_ID).log | sort -k 2,2 -k 1,1 > $(OUTPUT_DIR)/$(SAMPLE_ID)/endogenousAlignments_Accepted.txt
@echo -e "$(ts) $(PIPELINE_NAME): Finished assigning and sorting reads\n" >> $(OUTPUT_DIR)/$(SAMPLE_ID).log
#
## Do we want to downsample?
@echo -e "$(ts) $(PIPELINE_NAME): If requested, downsampling to $(DOWNSAMPLE_RNA_READS) transcriptome alignments:\n" >> $(OUTPUT_DIR)/$(SAMPLE_ID).log
@echo -e "$(ts) $(PIPELINE_NAME): $(COMMAND_DOWNSAMPLE)\n" >> $(OUTPUT_DIR)/$(SAMPLE_ID).log
$(COMMAND_DOWNSAMPLE)
@echo -e "$(ts) $(PIPELINE_NAME): Finished downsampling transcriptome alignments\n" >> $(OUTPUT_DIR)/$(SAMPLE_ID).log
#
## Quantify all annotated RNAs
@echo -e "$(ts) $(PIPELINE_NAME): Quantifying:\n" >> $(OUTPUT_DIR)/$(SAMPLE_ID).log
@echo -e "$(ts) $(PIPELINE_NAME): $(JAVA_EXE) -Xmx$(JAVA_RAM) -jar $(EXCERPT_TOOLS_EXE) QuantifyEndogenousAlignments --dict $(OUTPUT_DIR)/$(SAMPLE_ID)/endogenousAlignments_Accepted.dict --acceptedAlignments $(OUTPUT_DIR)/$(SAMPLE_ID)/endogenousAlignments_Accepted.txt --outputPath $(OUTPUT_DIR)/$(SAMPLE_ID)\n" >> $(OUTPUT_DIR)/$(SAMPLE_ID).log
$(JAVA_EXE) -Xmx$(JAVA_RAM) -jar $(EXCERPT_TOOLS_EXE) QuantifyEndogenousAlignments --dict $(OUTPUT_DIR)/$(SAMPLE_ID)/endogenousAlignments_Accepted.dict --acceptedAlignments $(OUTPUT_DIR)/$(SAMPLE_ID)/endogenousAlignments_Accepted.txt --outputPath $(OUTPUT_DIR)/$(SAMPLE_ID) 2>> $(OUTPUT_DIR)/$(SAMPLE_ID).log
#
## Summarise alignment statistics
cat $(OUTPUT_DIR)/$(SAMPLE_ID)/readCounts_miRNAmature_sense.txt | awk '{SUM+=$$4}END{printf "miRNA_sense\t%.0f\n",SUM}' >> $(OUTPUT_DIR)/$(SAMPLE_ID).stats
cat $(OUTPUT_DIR)/$(SAMPLE_ID)/readCounts_miRNAmature_antisense.txt | awk '{SUM+=$$4}END{printf "miRNA_antisense\t%.0f\n",SUM}' >> $(OUTPUT_DIR)/$(SAMPLE_ID).stats
cat $(OUTPUT_DIR)/$(SAMPLE_ID)/readCounts_miRNAprecursor_sense.txt | awk '{SUM+=$$4}END{printf "miRNAprecursor_sense\t%.0f\n",SUM}' >> $(OUTPUT_DIR)/$(SAMPLE_ID).stats
cat $(OUTPUT_DIR)/$(SAMPLE_ID)/readCounts_miRNAprecursor_antisense.txt | awk '{SUM+=$$4}END{printf "miRNAprecursor_antisense\t%.0f\n",SUM}' >> $(OUTPUT_DIR)/$(SAMPLE_ID).stats
cat $(OUTPUT_DIR)/$(SAMPLE_ID)/readCounts_tRNA_sense.txt | awk '{SUM+=$$4}END{printf "tRNA_sense\t%.0f\n",SUM}' >> $(OUTPUT_DIR)/$(SAMPLE_ID).stats
cat $(OUTPUT_DIR)/$(SAMPLE_ID)/readCounts_tRNA_antisense.txt | awk '{SUM+=$$4}END{printf "tRNA_antisense\t%.0f\n",SUM}' >> $(OUTPUT_DIR)/$(SAMPLE_ID).stats
cat $(OUTPUT_DIR)/$(SAMPLE_ID)/readCounts_piRNA_sense.txt | awk '{SUM+=$$4}END{printf "piRNA_sense\t%.0f\n",SUM}' >> $(OUTPUT_DIR)/$(SAMPLE_ID).stats
cat $(OUTPUT_DIR)/$(SAMPLE_ID)/readCounts_piRNA_antisense.txt | awk '{SUM+=$$4}END{printf "piRNA_antisense\t%.0f\n",SUM}' >> $(OUTPUT_DIR)/$(SAMPLE_ID).stats
cat $(OUTPUT_DIR)/$(SAMPLE_ID)/readCounts_gencode_sense.txt | awk '{SUM+=$$4}END{printf "gencode_sense\t%.0f\n",SUM}' >> $(OUTPUT_DIR)/$(SAMPLE_ID).stats
cat $(OUTPUT_DIR)/$(SAMPLE_ID)/readCounts_gencode_antisense.txt | awk '{SUM+=$$4}END{printf "gencode_antisense\t%.0f\n",SUM}' >> $(OUTPUT_DIR)/$(SAMPLE_ID).stats
cat $(OUTPUT_DIR)/$(SAMPLE_ID)/readCounts_circRNA_sense.txt | awk '{SUM+=$$4}END{printf "circularRNA_sense\t%.0f\n",SUM}' >> $(OUTPUT_DIR)/$(SAMPLE_ID).stats
cat $(OUTPUT_DIR)/$(SAMPLE_ID)/readCounts_circRNA_antisense.txt | awk '{SUM+=$$4}END{printf "circularRNA_antisense\t%.0f\n",SUM}' >> $(OUTPUT_DIR)/$(SAMPLE_ID).stats
## Count reads not mapping to the genome or to the libraries
gunzip -c $(OUTPUT_DIR)/$(SAMPLE_ID)/endogenousAlignments_genomeUnmapped_transcriptome_Unmapped.R1.fastq.gz | wc -l | awk '{print "not_mapped_to_genome_or_libs\t"($$1/4)}' >> $(OUTPUT_DIR)/$(SAMPLE_ID).stats
#
## Tidy up
gzip -c $(OUTPUT_DIR)/$(SAMPLE_ID)/endogenousAlignments_Accepted.txt > $(OUTPUT_DIR)/$(SAMPLE_ID)/endogenousAlignments_Accepted.txt.gz
rm $(OUTPUT_DIR)/$(SAMPLE_ID)/endogenousAlignments_Accepted.txt
##
## Align reads to repetitive element sequences, just in case repetitive reads have not been mapped to the genome
##
$(OUTDIR)/endogenousAlignments_repetitiveElements_Unmapped.R1.fastq.gz: $(OUTDIR)/endogenousAlignments_Accepted.txt.gz $(OUTDIR)/endogenousAlignments_genomeUnmapped_transcriptome_Unmapped.R1.fastq.gz
@echo -e "======================\n" >> $(OUTPUT_DIR)/$(SAMPLE_ID).log
@echo -e "$(ts) $(PIPELINE_NAME): Mapping reads to repetitive elements in the host genome:\n" >> $(OUTPUT_DIR)/$(SAMPLE_ID).log
@echo -e "$(ts) $(PIPELINE_NAME): $(STAR_EXE) --runThreadN $(N_THREADS) --outFileNamePrefix $(OUTPUT_DIR)/$(SAMPLE_ID)/endogenousAlignments_repetitiveElements_ --genomeDir $(DATABASE_PATH)/$(MAIN_ORGANISM_GENOME_ID)/STAR_INDEX_repetitiveElements --readFilesIn $(OUTPUT_DIR)/$(SAMPLE_ID)/endogenousAlignments_genomeUnmapped_transcriptome_Unmapped.R1.fastq.gz --outReadsUnmapped Fastx --parametersFiles $(DATABASE_PATH)/STAR_Parameters_Endogenous_smallRNA.in $(STAR_ENDOGENOUS_DYNAMIC_PARAMS) >> $(OUTPUT_DIR)/$(SAMPLE_ID).log 2>> $(OUTPUT_DIR)/$(SAMPLE_ID).err\n" >> $(OUTPUT_DIR)/$(SAMPLE_ID).log
$(STAR_EXE) --runThreadN $(N_THREADS) --outFileNamePrefix $(OUTPUT_DIR)/$(SAMPLE_ID)/endogenousAlignments_repetitiveElements_ --genomeDir $(DATABASE_PATH)/$(MAIN_ORGANISM_GENOME_ID)/STAR_INDEX_repetitiveElements --readFilesIn $(OUTPUT_DIR)/$(SAMPLE_ID)/endogenousAlignments_genomeUnmapped_transcriptome_Unmapped.R1.fastq.gz --outReadsUnmapped Fastx --parametersFiles $(DATABASE_PATH)/STAR_Parameters_Endogenous_smallRNA.in $(STAR_ENDOGENOUS_DYNAMIC_PARAMS) >> $(OUTPUT_DIR)/$(SAMPLE_ID).log 2>> $(OUTPUT_DIR)/$(SAMPLE_ID).err
@echo -e "$(ts) $(PIPELINE_NAME): Finished mapping to repetitive elements in the host genome\n" >> $(OUTPUT_DIR)/$(SAMPLE_ID).log
## Assigned non-redundantly to annotated REs
$(SAMTOOLS_EXE) view $(OUTPUT_DIR)/$(SAMPLE_ID)/endogenousAlignments_repetitiveElements_Aligned.out.bam | grep -v "^@" | awk '{print $$1}' | sort | uniq | wc -l | awk '{print "repetitiveElements\t"$$0}' >> $(OUTPUT_DIR)/$(SAMPLE_ID).stats
gzip -c $(OUTPUT_DIR)/$(SAMPLE_ID)/endogenousAlignments_repetitiveElements_Unmapped.out.mate1 > $(OUTPUT_DIR)/$(SAMPLE_ID)/endogenousAlignments_repetitiveElements_Unmapped.R1.fastq.gz
##
## map REMAINING reads to the genome allowing gaps / novel splices
##
$(OUTDIR)/endogenousAlignments_genomeGapped_Unmapped.R1.fastq.gz: $(OUTDIR)/endogenousAlignments_repetitiveElements_Unmapped.R1.fastq.gz
@echo -e "======================\n" >> $(OUTPUT_DIR)/$(SAMPLE_ID).log
@echo -e "$(ts) $(PIPELINE_NAME): Aligning remaining reads to the genome allowing gaps \n" >> $(OUTPUT_DIR)/$(SAMPLE_ID).log
@echo -e "$(ts) $(PIPELINE_NAME): $(STAR_EXE) --runThreadN $(N_THREADS) --outFileNamePrefix $(OUTPUT_DIR)/$(SAMPLE_ID)/endogenousAlignments_genomeGapped_ --alignIntronMax 0 --alignIntronMin 21 --genomeDir $(DATABASE_PATH)/$(MAIN_ORGANISM_GENOME_ID)/STAR_INDEX_genome --readFilesIn $(OUTPUT_DIR)/$(SAMPLE_ID)/endogenousAlignments_repetitiveElements_Unmapped.R1.fastq.gz --outReadsUnmapped Fastx --parametersFiles $(DATABASE_PATH)/STAR_Parameters_Endogenous_smallRNA.in $(STAR_ENDOGENOUS_DYNAMIC_PARAMS) >> $(OUTPUT_DIR)/$(SAMPLE_ID).log 2>> $(OUTPUT_DIR)/$(SAMPLE_ID).err\n" >> $(OUTPUT_DIR)/$(SAMPLE_ID).log
$(STAR_EXE) --runThreadN $(N_THREADS) --outFileNamePrefix $(OUTPUT_DIR)/$(SAMPLE_ID)/endogenousAlignments_genomeGapped_ --alignIntronMax 0 --alignIntronMin 21 --genomeDir $(DATABASE_PATH)/$(MAIN_ORGANISM_GENOME_ID)/STAR_INDEX_genome --readFilesIn $(OUTPUT_DIR)/$(SAMPLE_ID)/endogenousAlignments_repetitiveElements_Unmapped.R1.fastq.gz --outReadsUnmapped Fastx --parametersFiles $(DATABASE_PATH)/STAR_Parameters_Endogenous_smallRNA.in $(STAR_ENDOGENOUS_DYNAMIC_PARAMS) >> $(OUTPUT_DIR)/$(SAMPLE_ID).log 2>> $(OUTPUT_DIR)/$(SAMPLE_ID).err
@echo -e "$(ts) $(PIPELINE_NAME): Finished aligning remaining reads to the genome allowing gaps\n" >> $(OUTPUT_DIR)/$(SAMPLE_ID).log
## mapped to the genome with gaps
$(SAMTOOLS_EXE) view $(OUTPUT_DIR)/$(SAMPLE_ID)/endogenousAlignments_genomeGapped_Aligned.out.bam | grep -v "^@" | awk '{print $$1}' | sort | uniq | wc -l | awk '{print "endogenous_gapped\t"$$0}' >> $(OUTPUT_DIR)/$(SAMPLE_ID).stats
gzip -c $(OUTPUT_DIR)/$(SAMPLE_ID)/endogenousAlignments_genomeGapped_Unmapped.out.mate1 > $(OUTPUT_DIR)/$(SAMPLE_ID)/endogenousAlignments_genomeGapped_Unmapped.R1.fastq.gz
##
## Use the unmapped reads and search against all miRNAs in miRBase
##
$(OUTDIR)/EXOGENOUS_miRNA/exogenous_miRBase_Unmapped.R1.fastq.gz: $(OUTDIR)/endogenousAlignments_genomeGapped_Unmapped.R1.fastq.gz
mkdir -p $(OUTPUT_DIR)/$(SAMPLE_ID)/EXOGENOUS_miRNA
@echo -e "======================\n" >> $(OUTPUT_DIR)/$(SAMPLE_ID).log
@echo -e "$(ts) $(PIPELINE_NAME): Mapping reads to all miRNAs in miRBase:\n" >> $(OUTPUT_DIR)/$(SAMPLE_ID).log
@echo -e "$(ts) $(PIPELINE_NAME): $(STAR_EXE) --runThreadN $(N_THREADS) --outFileNamePrefix $(OUTPUT_DIR)/$(SAMPLE_ID)/EXOGENOUS_miRNA/exogenous_miRBase_ --genomeDir $(DATABASE_PATH)/miRBase/STAR_INDEX_miRBaseAll --readFilesIn $(OUTPUT_DIR)/$(SAMPLE_ID)/endogenousAlignments_genomeGapped_Unmapped.R1.fastq.gz --outReadsUnmapped Fastx --parametersFiles $(DATABASE_PATH)/STAR_Parameters_Exogenous.in $(STAR_EXOGENOUS_DYNAMIC_PARAMS) >> $(OUTPUT_DIR)/$(SAMPLE_ID).log 2>> $(OUTPUT_DIR)/$(SAMPLE_ID).err\n" >> $(OUTPUT_DIR)/$(SAMPLE_ID).log
$(STAR_EXE) --runThreadN $(N_THREADS) --outFileNamePrefix $(OUTPUT_DIR)/$(SAMPLE_ID)/EXOGENOUS_miRNA/exogenous_miRBase_ --genomeDir $(DATABASE_PATH)/miRBase/STAR_INDEX_miRBaseAll --readFilesIn $(OUTPUT_DIR)/$(SAMPLE_ID)/endogenousAlignments_genomeGapped_Unmapped.R1.fastq.gz --outReadsUnmapped Fastx --parametersFiles $(DATABASE_PATH)/STAR_Parameters_Exogenous.in $(STAR_EXOGENOUS_DYNAMIC_PARAMS) >> $(OUTPUT_DIR)/$(SAMPLE_ID).log 2>> $(OUTPUT_DIR)/$(SAMPLE_ID).err
@echo -e "$(ts) $(PIPELINE_NAME): Finished mapping to all miRNAs in miRBase\n" >> $(OUTPUT_DIR)/$(SAMPLE_ID).log
gzip -c $(OUTPUT_DIR)/$(SAMPLE_ID)/EXOGENOUS_miRNA/exogenous_miRBase_Unmapped.out.mate1 > $(OUTPUT_DIR)/$(SAMPLE_ID)/EXOGENOUS_miRNA/exogenous_miRBase_Unmapped.R1.fastq.gz
rm $(OUTPUT_DIR)/$(SAMPLE_ID)/EXOGENOUS_miRNA/exogenous_miRBase_Unmapped.out.mate1
#
@echo -e "$(ts) $(PIPELINE_NAME): Assigning reads:\n" >> $(OUTPUT_DIR)/$(SAMPLE_ID).log
## quantify read alignments using a slight hack of the endogenous alignment engine
$(JAVA_EXE) -Xmx$(JAVA_RAM) -jar $(EXCERPT_TOOLS_EXE) ProcessEndogenousAlignments --forceLib miRNA --transcriptomeMappedReads $(OUTPUT_DIR)/$(SAMPLE_ID)/EXOGENOUS_miRNA/exogenous_miRBase_Aligned.out.bam --hairpin2genome $(DATABASE_PATH)/miRBase/miRNA_precursor2genome.sam --mature2hairpin $(DATABASE_PATH)/miRBase/miRNA_mature2precursor.sam --dict $(OUTPUT_DIR)/$(SAMPLE_ID)/EXOGENOUS_miRNA/exogenousAlignments_Accepted.dict 2>> $(OUTPUT_DIR)/$(SAMPLE_ID).log | sort -k 2,2 -k 1,1 > $(OUTPUT_DIR)/$(SAMPLE_ID)/EXOGENOUS_miRNA/exogenousAlignments_Accepted.txt
#
$(JAVA_EXE) -Xmx$(JAVA_RAM) -jar $(EXCERPT_TOOLS_EXE) QuantifyEndogenousAlignments --dict $(OUTPUT_DIR)/$(SAMPLE_ID)/EXOGENOUS_miRNA/exogenousAlignments_Accepted.dict --acceptedAlignments $(OUTPUT_DIR)/$(SAMPLE_ID)/EXOGENOUS_miRNA/exogenousAlignments_Accepted.txt --outputPath $(OUTPUT_DIR)/$(SAMPLE_ID)/EXOGENOUS_miRNA 2>> $(OUTPUT_DIR)/$(SAMPLE_ID).log
@echo -e "$(ts) $(PIPELINE_NAME): Finished assigning reads\n" >> $(OUTPUT_DIR)/$(SAMPLE_ID).log
#
## Tidy up:
gzip -c $(OUTPUT_DIR)/$(SAMPLE_ID)/EXOGENOUS_miRNA/exogenousAlignments_Accepted.txt > $(OUTPUT_DIR)/$(SAMPLE_ID)/EXOGENOUS_miRNA/exogenousMiRNAAlignments_Accepted.txt.gz
rm $(OUTPUT_DIR)/$(SAMPLE_ID)/EXOGENOUS_miRNA/exogenousAlignments_Accepted.txt
#
## Stats
cat $(OUTPUT_DIR)/$(SAMPLE_ID)/EXOGENOUS_miRNA/exogenous_miRBase_Log.final.out | grep "Number of input reads" | awk -F "|\t" '{print "input_to_exogenous_miRNA\t"$$2}' >> $(OUTPUT_DIR)/$(SAMPLE_ID).stats
cat $(OUTPUT_DIR)/$(SAMPLE_ID)/EXOGENOUS_miRNA/exogenous_miRBase_Log.final.out | grep "Uniquely mapped reads number\|Number of reads mapped to multiple loci" | awk -F "|\t" '{SUM+=$$2}END{print "exogenous_miRNA\t"SUM}' >> $(OUTPUT_DIR)/$(SAMPLE_ID).stats
##
## Use the unmapped reads and search against all rRNAs in RDP (ribosome DB)
##
$(OUTDIR)/EXOGENOUS_rRNA/unaligned.fq.gz: $(OUTDIR)/EXOGENOUS_miRNA/exogenous_miRBase_Unmapped.R1.fastq.gz
mkdir -p $(OUTPUT_DIR)/$(SAMPLE_ID)/EXOGENOUS_rRNA
@echo -e "======================\n" >> $(OUTPUT_DIR)/$(SAMPLE_ID).log
@echo -e "$(ts) $(PIPELINE_NAME): Mapping reads to rRNA sequences in RDP (a.k.a. ribosome DB):\n" >> $(OUTPUT_DIR)/$(SAMPLE_ID).log
@echo -e "$(ts) $(PIPELINE_NAME): $(STAR_EXE) --runThreadN $(N_THREADS) --outFileNamePrefix $(OUTDIR)/EXOGENOUS_rRNA/exogenous_rRNA_ --genomeDir $(DATABASE_PATH)/ribosomeDatabase/exogenous_rRNAs --readFilesIn $(OUTDIR)/EXOGENOUS_miRNA/exogenous_miRBase_Unmapped.R1.fastq.gz --outReadsUnmapped Fastx --parametersFiles $(STAR_PARAMS_FILE_PATH) $(STAR_EXOGENOUS_DYNAMIC_PARAMS) >> $(OUTPUT_DIR)/$(SAMPLE_ID).log\n" >> $(OUTPUT_DIR)/$(SAMPLE_ID).log
$(STAR_EXE) --runThreadN $(N_THREADS) --outFileNamePrefix $(OUTDIR)/EXOGENOUS_rRNA/exogenous_rRNA_ --genomeDir $(DATABASE_PATH)/ribosomeDatabase/exogenous_rRNAs --readFilesIn $(OUTDIR)/EXOGENOUS_miRNA/exogenous_miRBase_Unmapped.R1.fastq.gz --outReadsUnmapped Fastx --parametersFiles $(STAR_PARAMS_FILE_PATH) $(STAR_EXOGENOUS_DYNAMIC_PARAMS) >> $(OUTPUT_DIR)/$(SAMPLE_ID).log
## Input to exogenous rRNA alignment
grep "Number of input reads" $(OUTDIR)/EXOGENOUS_rRNA/exogenous_rRNA_Log.final.out | tr '[:blank:]' ' ' | awk -F " \\\| " '{print "input_to_exogenous_rRNA\t"$$2}' >> $(OUTPUT_DIR)/$(SAMPLE_ID).stats
## Assigned non-redundantly to annotated exogenous rRNAs
cat $(OUTDIR)/EXOGENOUS_rRNA/exogenous_rRNA_Log.final.out | grep "Uniquely mapped reads number\|Number of reads mapped to multiple loci" | awk -F "|\t" '{SUM+=$$2}END{print "exogenous_rRNA\t"SUM}' >> $(OUTPUT_DIR)/$(SAMPLE_ID).stats
#$(SAMTOOLS_EXE) view $(OUTDIR)/EXOGENOUS_rRNA/exogenous_rRNA_Aligned.out.bam | awk '{print $$1}' | sort | uniq | wc -l | awk '{print "exogenous_rRNA\t"$$0}' >> $(OUTPUT_DIR)/$(SAMPLE_ID).stats
## compress and tidy up
gzip -c $(OUTDIR)/EXOGENOUS_rRNA/exogenous_rRNA_Unmapped.out.mate1 > $(OUTDIR)/EXOGENOUS_rRNA/unaligned.fq.gz
#$(SAMTOOLS_EXE) view -@ $(N_THREADS) -b $(OUTDIR)/EXOGENOUS_rRNA/exogenous_rRNA_Aligned.out.sam > $(OUTDIR)/EXOGENOUS_rRNA/exogenous_rRNA_Aligned.bam
rm $(OUTDIR)/EXOGENOUS_rRNA/exogenous_rRNA_Unmapped.out.mate1
#rm $(OUTDIR)/EXOGENOUS_rRNA/exogenous_rRNA_Aligned.out.sam
rm $(OUTDIR)/EXOGENOUS_rRNA/exogenous_rRNA_Log.out
@echo -e "$(ts) $(PIPELINE_NAME): Finished mapping to rRNA sequences in RDP\n" >> $(OUTPUT_DIR)/$(SAMPLE_ID).log
##
## Resolve the RDP rRNA alignments against the NCBI taxonomy
##
$(OUTDIR)/EXOGENOUS_rRNA/ExogenousRibosomalAlignments.result.taxaAnnotated.txt: $(OUTDIR)/EXOGENOUS_rRNA/unaligned.fq.gz
@echo -e "======================\n" >> $(OUTPUT_DIR)/$(SAMPLE_ID).log
@echo -e "$(ts) $(PIPELINE_NAME): Resolving and quantifying rRNA alignments\n" >> $(OUTPUT_DIR)/$(SAMPLE_ID).log
@echo -e "\n$(ts) $(PIPELINE_NAME): $(SAMTOOLS_EXE) view $(OUTDIR)/EXOGENOUS_rRNA/exogenous_rRNA_Aligned.out.bam | awk '{print $$1,$$3,$$4,$$6,$$10}' | sort -k 1,1 -k 2,2 | uniq > $(OUTDIR)/EXOGENOUS_rRNA/ExogenousRibosomalAlignments.txt 2>> $(OUTPUT_DIR)/$(SAMPLE_ID).log\n" >> $(OUTPUT_DIR)/$(SAMPLE_ID).log
$(SAMTOOLS_EXE) view $(OUTDIR)/EXOGENOUS_rRNA/exogenous_rRNA_Aligned.out.bam | awk '{print $$1,$$3,$$4,$$6,$$10}' | sort -k 1,1 -k 2,2 | uniq > $(OUTDIR)/EXOGENOUS_rRNA/ExogenousRibosomalAlignments.txt 2>> $(OUTPUT_DIR)/$(SAMPLE_ID).log
#
@echo -e "\n$(ts) $(PIPELINE_NAME): $(JAVA_EXE) -Xmx$(JAVA_RAM) -jar $(EXCERPT_TOOLS_EXE) ProcessExogenousAlignments -taxonomyPath $(DATABASE_PATH)/NCBI_taxonomy_taxdump -min $(EXOGENOUS_RIBOSOMAL_TAXA_MINREADPERCENT) -frac $(EXOGENOUS_RIBOSOMAL_TAXA_MINFRAC) --minReads $(EXOGENOUS_RIBOSOMAL_TAXA_MINREADS) -batchSize $(EXOGENOUS_RIBOSOMAL_TAXA_BATCHSIZE) -alignments $(OUTDIR)/EXOGENOUS_rRNA/ExogenousRibosomalAlignments.txt --rdp > $(OUTDIR)/EXOGENOUS_rRNA/ExogenousRibosomalAlignments.tmp 2>> $(OUTPUT_DIR)/$(SAMPLE_ID).log\n" >> $(OUTPUT_DIR)/$(SAMPLE_ID).log
$(JAVA_EXE) -Xmx$(JAVA_RAM) -jar $(EXCERPT_TOOLS_EXE) ProcessExogenousAlignments -taxonomyPath $(DATABASE_PATH)/NCBI_taxonomy_taxdump -min $(EXOGENOUS_RIBOSOMAL_TAXA_MINREADPERCENT) -frac $(EXOGENOUS_RIBOSOMAL_TAXA_MINFRAC) --minReads $(EXOGENOUS_RIBOSOMAL_TAXA_MINREADS) -batchSize $(EXOGENOUS_RIBOSOMAL_TAXA_BATCHSIZE) -alignments $(OUTDIR)/EXOGENOUS_rRNA/ExogenousRibosomalAlignments.txt --rdp > $(OUTDIR)/EXOGENOUS_rRNA/ExogenousRibosomalAlignments.tmp 2>> $(OUTPUT_DIR)/$(SAMPLE_ID).log
#
# Tidy up
mv $(OUTDIR)/EXOGENOUS_rRNA/ExogenousRibosomalAlignments.tmp $(OUTDIR)/EXOGENOUS_rRNA/ExogenousRibosomalAlignments.result.taxaAnnotated.txt
rm $(OUTDIR)/EXOGENOUS_rRNA/ExogenousRibosomalAlignments.txt
@echo -e "$(ts) $(PIPELINE_NAME): Finished resolving and quantifying rRNA alignments\n" >> $(OUTPUT_DIR)/$(SAMPLE_ID).log
##
## Routines for aligning unmapped reads to exogenous sequences
##
## Bacteria
$(OUTDIR)/EXOGENOUS_genomes/Bacteria10_Aligned.out.bam: $(OUTDIR)/EXOGENOUS_rRNA/ExogenousRibosomalAlignments.result.taxaAnnotated.txt
mkdir -p $(OUTPUT_DIR)/$(SAMPLE_ID)/EXOGENOUS_genomes
@echo -e "======================\n" >> $(OUTPUT_DIR)/$(SAMPLE_ID).log
@echo -e "$(ts) $(PIPELINE_NAME): Mapping reads to exogenous GENOMES of BACTERIA:\n" >> $(OUTPUT_DIR)/$(SAMPLE_ID).log
@echo -e "$(ts) $(PIPELINE_NAME): Bacteria1:" >> $(OUTPUT_DIR)/$(SAMPLE_ID).log
$(STAR_EXE) --runThreadN $(N_THREADS) --outFileNamePrefix $(OUTDIR)/EXOGENOUS_genomes/Bacteria1_ --genomeDir $(STAR_GENOMES_DIR)/STAR_GENOME_BACTERIA1 --readFilesIn $(OUTDIR)/EXOGENOUS_rRNA/unaligned.fq.gz --parametersFiles $(STAR_PARAMS_FILE_PATH) $(STAR_EXOGENOUS_DYNAMIC_PARAMS) >> $(OUTDIR).log
@echo -e "\n$(ts) $(PIPELINE_NAME): Bacteria2:" >> $(OUTDIR).log
$(STAR_EXE) --runThreadN $(N_THREADS) --outFileNamePrefix $(OUTDIR)/EXOGENOUS_genomes/Bacteria2_ --genomeDir $(STAR_GENOMES_DIR)/STAR_GENOME_BACTERIA2 --readFilesIn $(OUTDIR)/EXOGENOUS_rRNA/unaligned.fq.gz --parametersFiles $(STAR_PARAMS_FILE_PATH) $(STAR_EXOGENOUS_DYNAMIC_PARAMS) >> $(OUTDIR).log
@echo -e "\n$(ts) $(PIPELINE_NAME): Bacteria3:" >> $(OUTDIR).log
$(STAR_EXE) --runThreadN $(N_THREADS) --outFileNamePrefix $(OUTDIR)/EXOGENOUS_genomes/Bacteria3_ --genomeDir $(STAR_GENOMES_DIR)/STAR_GENOME_BACTERIA3 --readFilesIn $(OUTDIR)/EXOGENOUS_rRNA/unaligned.fq.gz --parametersFiles $(STAR_PARAMS_FILE_PATH) $(STAR_EXOGENOUS_DYNAMIC_PARAMS) >> $(OUTDIR).log
@echo -e "\n$(ts) $(PIPELINE_NAME): Bacteria4:" >> $(OUTDIR).log
$(STAR_EXE) --runThreadN $(N_THREADS) --outFileNamePrefix $(OUTDIR)/EXOGENOUS_genomes/Bacteria4_ --genomeDir $(STAR_GENOMES_DIR)/STAR_GENOME_BACTERIA4 --readFilesIn $(OUTDIR)/EXOGENOUS_rRNA/unaligned.fq.gz --parametersFiles $(STAR_PARAMS_FILE_PATH) $(STAR_EXOGENOUS_DYNAMIC_PARAMS) >> $(OUTDIR).log
@echo -e "\n$(ts) $(PIPELINE_NAME): Bacteria5:" >> $(OUTDIR).log
$(STAR_EXE) --runThreadN $(N_THREADS) --outFileNamePrefix $(OUTDIR)/EXOGENOUS_genomes/Bacteria5_ --genomeDir $(STAR_GENOMES_DIR)/STAR_GENOME_BACTERIA5 --readFilesIn $(OUTDIR)/EXOGENOUS_rRNA/unaligned.fq.gz --parametersFiles $(STAR_PARAMS_FILE_PATH) $(STAR_EXOGENOUS_DYNAMIC_PARAMS) >> $(OUTDIR).log
@echo -e "\n$(ts) $(PIPELINE_NAME): Bacteria6:" >> $(OUTDIR).log
$(STAR_EXE) --runThreadN $(N_THREADS) --outFileNamePrefix $(OUTDIR)/EXOGENOUS_genomes/Bacteria6_ --genomeDir $(STAR_GENOMES_DIR)/STAR_GENOME_BACTERIA6 --readFilesIn $(OUTDIR)/EXOGENOUS_rRNA/unaligned.fq.gz --parametersFiles $(STAR_PARAMS_FILE_PATH) $(STAR_EXOGENOUS_DYNAMIC_PARAMS) >> $(OUTDIR).log
@echo -e "\n$(ts) $(PIPELINE_NAME): Bacteria7:" >> $(OUTDIR).log
$(STAR_EXE) --runThreadN $(N_THREADS) --outFileNamePrefix $(OUTDIR)/EXOGENOUS_genomes/Bacteria7_ --genomeDir $(STAR_GENOMES_DIR)/STAR_GENOME_BACTERIA7 --readFilesIn $(OUTDIR)/EXOGENOUS_rRNA/unaligned.fq.gz --parametersFiles $(STAR_PARAMS_FILE_PATH) $(STAR_EXOGENOUS_DYNAMIC_PARAMS) >> $(OUTDIR).log
@echo -e "\n$(ts) $(PIPELINE_NAME): Bacteria8:" >> $(OUTDIR).log
$(STAR_EXE) --runThreadN $(N_THREADS) --outFileNamePrefix $(OUTDIR)/EXOGENOUS_genomes/Bacteria8_ --genomeDir $(STAR_GENOMES_DIR)/STAR_GENOME_BACTERIA8 --readFilesIn $(OUTDIR)/EXOGENOUS_rRNA/unaligned.fq.gz --parametersFiles $(STAR_PARAMS_FILE_PATH) $(STAR_EXOGENOUS_DYNAMIC_PARAMS) >> $(OUTDIR).log
@echo -e "\n$(ts) $(PIPELINE_NAME): Bacteria9:" >> $(OUTDIR).log
$(STAR_EXE) --runThreadN $(N_THREADS) --outFileNamePrefix $(OUTDIR)/EXOGENOUS_genomes/Bacteria9_ --genomeDir $(STAR_GENOMES_DIR)/STAR_GENOME_BACTERIA9 --readFilesIn $(OUTDIR)/EXOGENOUS_rRNA/unaligned.fq.gz --parametersFiles $(STAR_PARAMS_FILE_PATH) $(STAR_EXOGENOUS_DYNAMIC_PARAMS) >> $(OUTDIR).log
@echo -e "\n$(ts) $(PIPELINE_NAME): Bacteria10:" >> $(OUTDIR).log
$(STAR_EXE) --runThreadN $(N_THREADS) --outFileNamePrefix $(OUTDIR)/EXOGENOUS_genomes/Bacteria10_ --genomeDir $(STAR_GENOMES_DIR)/STAR_GENOME_BACTERIA10 --readFilesIn $(OUTDIR)/EXOGENOUS_rRNA/unaligned.fq.gz --parametersFiles $(STAR_PARAMS_FILE_PATH) $(STAR_EXOGENOUS_DYNAMIC_PARAMS) >> $(OUTDIR).log
@echo -e "\n$(ts) $(PIPELINE_NAME): Finished mapping to exogenous GENOMES of BACTERIA\n" >> $(OUTDIR).log
## Plants
$(OUTDIR)/EXOGENOUS_genomes/Plants5_Aligned.out.bam: $(OUTDIR)/EXOGENOUS_rRNA/ExogenousRibosomalAlignments.result.taxaAnnotated.txt
mkdir -p $(OUTDIR)/EXOGENOUS_genomes
@echo -e "======================\n" >> $(OUTDIR).log
@echo -e "$(ts) $(PIPELINE_NAME): Mapping reads to exogenous GENOMES of PLANTS:\n" >> $(OUTDIR).log
@echo -e "$(ts) $(PIPELINE_NAME): Plants1:" >> $(OUTDIR).log
$(STAR_EXE) --runThreadN $(N_THREADS) --outFileNamePrefix $(OUTDIR)/EXOGENOUS_genomes/Plants1_ --genomeDir $(STAR_GENOMES_DIR)/STAR_GENOME_PLANTS1 --readFilesIn $(OUTDIR)/EXOGENOUS_rRNA/unaligned.fq.gz --parametersFiles $(STAR_PARAMS_FILE_PATH) $(STAR_EXOGENOUS_DYNAMIC_PARAMS) >> $(OUTDIR).log
@echo -e "\n$(ts) $(PIPELINE_NAME): Plants2:" >> $(OUTDIR).log
$(STAR_EXE) --runThreadN $(N_THREADS) --outFileNamePrefix $(OUTDIR)/EXOGENOUS_genomes/Plants2_ --genomeDir $(STAR_GENOMES_DIR)/STAR_GENOME_PLANTS2 --readFilesIn $(OUTDIR)/EXOGENOUS_rRNA/unaligned.fq.gz --parametersFiles $(STAR_PARAMS_FILE_PATH) $(STAR_EXOGENOUS_DYNAMIC_PARAMS) >> $(OUTDIR).log
@echo -e "\n$(ts) $(PIPELINE_NAME): Plants3:" >> $(OUTDIR).log
$(STAR_EXE) --runThreadN $(N_THREADS) --outFileNamePrefix $(OUTDIR)/EXOGENOUS_genomes/Plants3_ --genomeDir $(STAR_GENOMES_DIR)/STAR_GENOME_PLANTS3 --readFilesIn $(OUTDIR)/EXOGENOUS_rRNA/unaligned.fq.gz --parametersFiles $(STAR_PARAMS_FILE_PATH) $(STAR_EXOGENOUS_DYNAMIC_PARAMS) >> $(OUTDIR).log
@echo -e "\n$(ts) $(PIPELINE_NAME): Plants4:" >> $(OUTDIR).log