exceRpt/exceRpt_smallRNA at master · rkitchen/exceRpt · GitHub

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##############################################################################################
##                                                                                          ##
##                      ____        _                                ________  _   _____    ##
##     _____  _____ ___|  _ \ _ __ | |_        _________ ___  ____ _/ / / __ \/ | / /   |   ##
##    / _ \ \/ / __/ _ \ |_) | '_ \| __|      / ___/ __ `__ \/ __ `/ / / /_/ /  |/ / /| |   ##
##   |  __/>  < (_|  __/  _ <| |_) | |_      (__  ) / / / / / /_/ / / / _, _/ /|  / ___ |   ##
##    \___/_/\_\___\___|_| \_\ .__/ \__|    /____/_/ /_/ /_/\__,_/_/_/_/ |_/_/ |_/_/  |_|   ##
##                           |_|                                                            ##
##                                                                                          ##
##                                                                                          ##
##                                                                                          ##
##   The extra-cellular RNA processing toolkit (exceRpt) optimised for smallRNA analysis    ##
##                                                                                          ##
##   This pipeline processes a single smallRNA sequence file from a single sample           ##
##                                                                                          ##
##   Author: Rob Kitchen (r.r.kitchen@gmail.com)                                            ##
##                                                                                          ##
##   Learn more at github.com/rkitchen/exceRpt                                              ##
##                                                                                          ##
##   Version 5.0.0 (2020-01-03)                                                             ##
##                                                                                          ##
##############################################################################################
EXCERPT_VERSION := 5.0.0


##
## 1) On the command line, be sure to specify the following MANDATORY parameters
##
OUTPUT_DIR      := NULL
INPUT_FILE_PATH	:= NULL
SAMPLE_NAME     := NULL

##
## 2) Choose the main organism for smallRNA / 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               := guessKnown
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	  := miRNA,tRNA,piRNA,gencode,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_outFilterScoreMinOverLread  := 0.5
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 	:= 2G
	## NB: The 'EXE_DIR' MUST be an ABSOLUTE PATH or sRNABench will fail!
	EXE_DIR   := home/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
	BOWTIE2_EXE           := $(EXE_DIR)/bowtie2/bowtie2
	SAMTOOLS_EXE          := $(EXE_DIR)/samtools/samtools
	FASTQC_EXE            := $(JAVA_EXE) -classpath $(EXE_DIR)/FastQC:$(EXE_DIR)/FastQC/sam-1.103.jar:$(EXE_DIR)/FastQC/jbzip2-0.9.jar
	SRATOOLS_EXE          := $(EXE_DIR)/sratoolkit.2.10.2-ubuntu64/bin/fastq-dump
	EXCERPT_TOOLS_EXE     := $(EXE_DIR)/exceRpt_Tools.jar
	DATABASE_PATH         := $(EXE_DIR)/DATABASE
	STAR_EXE              := $(EXE_DIR)/STAR/bin/Linux_x86_64/STAR
	STAR_GENOMES_DIR      := $(DATABASE_PATH)/Genomes_BacteriaFungiMammalPlantProtistVirus
	#STAR_PARAMS_FILE_PATH := $(STAR_GENOMES_DIR)/STAR_Parameters_Exogenous.in
	STAR_PARAMS_FILE_PATH := $(EXE_DIR)/
	BBDUK_EXE	      := $(EXE_DIR)/bbmap/bbduk.sh
	BBDUK_RESOURCES	      := $(EXE_DIR)/bbmap/resources

	##
	## 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_smallRNA


##
## Initialise smallRNA 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 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] 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),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

#COMMAND_CLIP_ADAPTER ?= $(COMMAND_CONVERT_SRA) > $(OUTPUT_DIR)/$(SAMPLE_ID)/$(SAMPLE_ID).preClipped.fastq.tmp; $(BBDUK_EXE) ref=$(BBDUK_RESOURCES)/adapters.fa in=$(OUTPUT_DIR)/$(SAMPLE_ID)/$(SAMPLE_ID).preClipped.fastq.tmp out=$(OUTPUT_DIR)/$(SAMPLE_ID)/$(SAMPLE_ID).clipped.fastq.tmp.gz ktrim=r k=21 mink=$(MIN_ADAPTER_BASES_3p) tbo tpe hdist=1 minlen=$(MIN_READ_LENGTH) threads=$(N_THREADS) qtrim=r trimq=10 maq=10 entropy=0.3 entropywindow=50 entropyk=5 >> $(OUTPUT_DIR)/$(SAMPLE_ID).log 2>> $(OUTPUT_DIR)/$(SAMPLE_ID).err; rm $(OUTPUT_DIR)/$(SAMPLE_ID)/$(SAMPLE_ID).preClipped.fastq.tmp

COMMAND_CLIP_ADAPTER ?= $(COMMAND_CONVERT_SRA) > $(OUTPUT_DIR)/$(SAMPLE_ID)/$(SAMPLE_ID).preClipped.fastq.tmp; $(BBDUK_EXE) ref=$(DATABASE_PATH)/adapters.fa in=$(OUTPUT_DIR)/$(SAMPLE_ID)/$(SAMPLE_ID).preClipped.fastq.tmp out=$(OUTPUT_DIR)/$(SAMPLE_ID)/$(SAMPLE_ID).clipped.fastq.tmp.gz ktrim=r k=10 mink=$(MIN_ADAPTER_BASES_3p) hdist=0 minlen=$(MIN_READ_LENGTH) trimpolya=7 threads=$(N_THREADS) >> $(OUTPUT_DIR)/$(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 "" | awk '{print "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


##
## STAR command to align both to UniVec and to the rRNAs
##
COMMAND_MAP_UNIVEC_AND_RRNA ?= $(STAR_EXE) --runThreadN $(N_THREADS) --outFileNamePrefix $(OUTPUT_DIR)/$(SAMPLE_ID)/filteringAlignments_UniVec_and_rRNA_ --genomeDir $(DATABASE_PATH)/$(MAIN_ORGANISM_GENOME_ID)/STAR_INDEX_Univec_rRNA --readFilesIn $(FILE_TO_INPUT_TO_UNIVEC_ALIGNMENT) --readFilesCommand "gunzip -c" --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_and_rRNA_Aligned.out.bam | awk '{print $$3}' | sort -k 2,2 2>> $(OUTPUT_DIR)/$(SAMPLE_ID).err | uniq -c > $(OUTPUT_DIR)/$(SAMPLE_ID)/$(SAMPLE_ID).clipped.trimmed.filtered.UniVec_and_rRNA.counts 2>> $(OUTPUT_DIR)/$(SAMPLE_ID).err; \
$(SAMTOOLS_EXE) view $(OUTPUT_DIR)/$(SAMPLE_ID)/filteringAlignments_UniVec_and_rRNA_Aligned.out.bam | awk '{print $$1}' | sort 2>> $(OUTPUT_DIR)/$(SAMPLE_ID).err | uniq -c | wc -l > $(OUTPUT_DIR)/$(SAMPLE_ID)/$(SAMPLE_ID).clipped.trimmed.filtered.UniVec_and_rRNA.readCount 2>> $(OUTPUT_DIR)/$(SAMPLE_ID).err; \
gzip -c $(OUTPUT_DIR)/$(SAMPLE_ID)/filteringAlignments_UniVec_and_rRNA_Unmapped.out.mate1 > $(OUTPUT_DIR)/$(SAMPLE_ID)/$(SAMPLE_ID).clipped.trimmed.filtered.noUniVecOrRiboRNA.fastq.gz; \
rm $(OUTPUT_DIR)/$(SAMPLE_ID)/filteringAlignments_UniVec_and_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 smallRNA pipeline (version v.$(EXCERPT_VERSION))"
	@echo -e ""
	@echo -e "Usage: make -f path/to/exceRpt_smallRNA [OPTION1=value1 OPTION2=value2 ...]"
	@echo -e ""
	@echo -e "*Required* OPTIONs:"
	@echo -e "  INPUT_FILE_PATH                  | Path to the input fastq/fasta/sra file"
	@echo -e "  OUTPUT_DIR                       | Path to store the output results"
	@echo -e ""
	@echo -e "Main analysis OPTIONs:"
	@echo -e "  DATABASE_PATH                    | <Path>                           | [default: '$(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 "$(ts) $(PIPELINE_NAME): END smallRNA-seq Pipeline for sample $(SAMPLE_ID)\n======================\n" >> $(OUTPUT_DIR)/$(SAMPLE_ID).log
	@echo "$(ts) $(PIPELINE_NAME): END\n" >> $(OUTPUT_DIR)/$(SAMPLE_ID).err
	@echo "#END OF STATS from the exceRpt smallRNA-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)_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) $(PIPELINE_NAME): BEGIN exceRpt smallRNA-seq pipeline v.$(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


##
## CLIP 3' adapter sequence with bbduk
##
$(OUTDIR)/$(SAMPLE_ID).clipped.fastq.gz: $(OUTDIR)/Progress_1_FoundAdapter.dat
	@echo -e "======================\n" >> $(OUTPUT_DIR)/$(SAMPLE_ID).log
	@echo -e "$(ts) $(PIPELINE_NAME): Trimming adapter sequences:\n" >> $(OUTPUT_DIR)/$(SAMPLE_ID).log
	@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).err | awk '{print "input\t"$$2}' >> $(OUTPUT_DIR)/$(SAMPLE_ID).stats
	## Count reads output following adapter clipping
	grep "Result: " $(OUTPUT_DIR)/$(SAMPLE_ID).err | awk '{print "successfully_clipped\t"$$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 | $(JAVA_EXE) -Xmx$(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): $(BBDUK_EXE) in=$(OUTPUT_DIR)/$(SAMPLE_ID)/$(SAMPLE_ID).clipped.fastq.gz out=$(OUTPUT_DIR)/$(SAMPLE_ID)/$(SAMPLE_ID).clipped.trimmed.fastq.gz forcetrimleft=$(TRIM_N_BASES_5p) forcetrimright2=$(TRIM_N_BASES_3p) minlen=$(MIN_READ_LENGTH) threads=$(N_THREADS) \n">> $(OUTPUT_DIR)/$(SAMPLE_ID).log
	$(BBDUK_EXE) in=$(OUTPUT_DIR)/$(SAMPLE_ID)/$(SAMPLE_ID).clipped.fastq.gz out=$(OUTPUT_DIR)/$(SAMPLE_ID)/$(SAMPLE_ID).clipped.trimmed.fastq.gz forcetrimleft=$(TRIM_N_BASES_5p) forcetrimright2=$(TRIM_N_BASES_3p) minlen=$(MIN_READ_LENGTH) threads=$(N_THREADS) >>$(OUTPUT_DIR)/$(SAMPLE_ID).log 2>>$(OUTPUT_DIR)/$(SAMPLE_ID).err
	@echo -e "$(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
	@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): $(BBDUK_EXE) in=$(OUTPUT_DIR)/$(SAMPLE_ID)/$(SAMPLE_ID).clipped.trimmed.fastq.gz out=$(OUTPUT_DIR)/$(SAMPLE_ID)/$(SAMPLE_ID).clipped.trimmed.filtered.tmp minlen=$(MIN_READ_LENGTH) threads=$(N_THREADS) qtrim=r trimq=10 maq=10 \n" >> $(OUTPUT_DIR)/$(SAMPLE_ID).log 2>> $(OUTPUT_DIR)/$(SAMPLE_ID).err
	$(BBDUK_EXE) in=$(OUTPUT_DIR)/$(SAMPLE_ID)/$(SAMPLE_ID).clipped.trimmed.fastq.gz out=$(OUTPUT_DIR)/$(SAMPLE_ID)/$(SAMPLE_ID).clipped.trimmed.filtered.tmp minlen=$(MIN_READ_LENGTH) threads=$(N_THREADS) qtrim=r trimq=10 maq=10 2>>$(OUTPUT_DIR)/$(SAMPLE_ID)/$(SAMPLE_ID).clipped.trimmed.filtered.tmp.log
	## Count reads that PASS the quality filter
	grep "Result: " $(OUTPUT_DIR)/$(SAMPLE_ID)/$(SAMPLE_ID).clipped.trimmed.filtered.tmp.log | awk '{print "passed_quality_filter\t"$$2}' >> $(OUTPUT_DIR)/$(SAMPLE_ID).stats
	#
	# Filter homopolymer reads (those that have too many single nt repeats)
	@echo -e "======================\n" >> $(OUTPUT_DIR)/$(SAMPLE_ID).log
	@echo -e "$(ts) $(PIPELINE_NAME): Filtering homopolymer repeat reads:\n" >> $(OUTPUT_DIR)/$(SAMPLE_ID).log
	@echo -e "$(ts) $(PIPELINE_NAME): $(JAVA_EXE) -Xmx$(JAVA_RAM) -jar $(EXCERPT_TOOLS_EXE) RemoveHomopolymerRepeats -m 0.66 -i $(OUTPUT_DIR)/$(SAMPLE_ID)/$(SAMPLE_ID).clipped.trimmed.filtered.tmp -o $(OUTPUT_DIR)/$(SAMPLE_ID)/$(SAMPLE_ID).clipped.trimmed.filtered.fastq\n" >> $(OUTPUT_DIR)/$(SAMPLE_ID).log 2>> $(OUTPUT_DIR)/$(SAMPLE_ID).err
	$(JAVA_EXE) -Xmx$(JAVA_RAM) -jar $(EXCERPT_TOOLS_EXE) RemoveHomopolymerRepeats --verbose -m 0.66 -i $(OUTPUT_DIR)/$(SAMPLE_ID)/$(SAMPLE_ID).clipped.trimmed.filtered.tmp -o $(OUTPUT_DIR)/$(SAMPLE_ID)/$(SAMPLE_ID).clipped.trimmed.filtered.tmp2 >> $(OUTPUT_DIR)/$(SAMPLE_ID).log 2>> $(OUTPUT_DIR)/$(SAMPLE_ID)/$(SAMPLE_ID).clipped.REMOVEDRepeatReads.fastq
	gzip --force $(OUTPUT_DIR)/$(SAMPLE_ID)/$(SAMPLE_ID).clipped.REMOVEDRepeatReads.fastq
	#gzip $(OUTPUT_DIR)/$(SAMPLE_ID)/$(SAMPLE_ID).clipped.trimmed.filtered.fastq
	rm $(OUTPUT_DIR)/$(SAMPLE_ID)/$(SAMPLE_ID).clipped.trimmed.filtered.tmp
	@echo -e "$(ts) $(PIPELINE_NAME): Finished filtering homopolymer repeat reads\n" >> $(OUTPUT_DIR)/$(SAMPLE_ID).log
	## Count homopolymer repeat reads that failed the quality filter
	grep "Done.  Sequences removed" $(OUTPUT_DIR)/$(SAMPLE_ID).log | awk -F "=" '{print "failed_homopolymer_filter\t"$$2}' >> $(OUTPUT_DIR)/$(SAMPLE_ID).stats
	#
	@echo -e "======================\n" >> $(OUTPUT_DIR)/$(SAMPLE_ID).log
	@echo -e "$(ts) $(PIPELINE_NAME): Filtering reads to remove PhiX contamination:\n" >> $(OUTPUT_DIR)/$(SAMPLE_ID).log
	@echo -e "$(ts) $(PIPELINE_NAME): $(BBDUK_EXE) in=$(OUTPUT_DIR)/$(SAMPLE_ID)/$(SAMPLE_ID).clipped.trimmed.filtered.tmp2 out=$(OUTPUT_DIR)/$(SAMPLE_ID)/$(SAMPLE_ID).clipped.trimmed.filtered.fastq.gz minlen=$(MIN_READ_LENGTH) threads=$(N_THREADS) qtrim=r trimq=10 maq=10 \n" >> $(OUTPUT_DIR)/$(SAMPLE_ID).log 2>> $(OUTPUT_DIR)/$(SAMPLE_ID).err
	$(BBDUK_EXE) in=$(OUTPUT_DIR)/$(SAMPLE_ID)/$(SAMPLE_ID).clipped.trimmed.filtered.tmp2 out=$(OUTPUT_DIR)/$(SAMPLE_ID)/$(SAMPLE_ID).clipped.trimmed.filtered.fastq minlen=$(MIN_READ_LENGTH) threads=$(N_THREADS) ref=$(DATABASE_PATH)/phiX.fa k=31 hdist=1 2>>$(OUTPUT_DIR)/$(SAMPLE_ID)/$(SAMPLE_ID).clipped.trimmed.filtered.tmp.PhiX.log
	gzip --force $(OUTPUT_DIR)/$(SAMPLE_ID)/$(SAMPLE_ID).clipped.trimmed.filtered.fastq
	## Count reads that PASS the PhiX filter
	grep "Result: " $(OUTPUT_DIR)/$(SAMPLE_ID)/$(SAMPLE_ID).clipped.trimmed.filtered.tmp.PhiX.log | awk '{print "passed_phiX_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
$(OUTPUT_DIR)/$(SAMPLE_ID)/$(SAMPLE_ID).clipped.trimmed.filtered.noUniVecOrRiboRNA.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 and ribosomal RNAs using STAR:\n" >> $(OUTPUT_DIR)/$(SAMPLE_ID).log
	@echo -e "$(ts) $(PIPELINE_NAME): $(COMMAND_MAP_UNIVEC_AND_RRNA)\n" >> $(OUTPUT_DIR)/$(SAMPLE_ID).log
	$(COMMAND_MAP_UNIVEC_AND_RRNA)
	@echo -e "$(ts) $(PIPELINE_NAME): Finished mapping reads to contaminant sequences in UniVec and ribosomal RNAs using STAR:\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
$(OUTDIR)/$(SAMPLE_ID).clipped.trimmed.filtered.noRiboRNA_fastqc.zip: $(OUTDIR)/$(SAMPLE_ID).clipped.trimmed.filtered.noUniVecOrRiboRNA.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.noUniVecOrRiboRNA.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.noUniVecOrRiboRNA.fastq.gz --readFilesCommand "gunzip -c" --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.noUniVecOrRiboRNA.fastq.gz --readFilesCommand "gunzip -c" --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"$$2}' >> $(OUTPUT_DIR)/$(SAMPLE_ID).stats
	cat $(OUTPUT_DIR)/$(SAMPLE_ID)/endogenousAlignments_genome*apped_transcriptome_Log.final.out | grep "Number of input reads\|Uniquely mapped reads number\|Number of reads mapped to multiple loci" | sed '2,4d' | awk -F "\t" '{SUM+=$$2}END{print "genome\t"SUM}' >> $(OUTPUT_DIR)/$(SAMPLE_ID).stats
	#
	## Compress STAR logs
	gzip --force $(OUTPUT_DIR)/$(SAMPLE_ID)/endogenousAlignments_genomeMapped_transcriptome_Log.out
	gzip --force $(OUTPUT_DIR)/$(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 $(SAMTOOLS_SORT_MEM) -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 $(SAMTOOLS_SORT_MEM) -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): gunzip -c $(DATABASE_PATH)/$(MAIN_ORGANISM_GENOME_ID)/gencodeAnnotation.gtf.gz > $(DATABASE_PATH)/$(MAIN_ORGANISM_GENOME_ID)/gencodeAnnotation.gtf" >> $(OUTPUT_DIR)/$(SAMPLE_ID).log
	gunzip -c $(DATABASE_PATH)/$(MAIN_ORGANISM_GENOME_ID)/gencodeAnnotation.gtf.gz > $(DATABASE_PATH)/$(MAIN_ORGANISM_GENOME_ID)/gencodeAnnotation.gtf 2>> $(OUTPUT_DIR)/$(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
	rm $(DATABASE_PATH)/$(MAIN_ORGANISM_GENOME_ID)/gencodeAnnotation.gtf
	@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 --readFilesCommand "gunzip -c" --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 --readFilesCommand "gunzip -c" --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 --readFilesCommand "gunzip -c" --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 --readFilesCommand "gunzip -c" --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 --readFilesCommand "gunzip -c" --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 --readFilesCommand "gunzip -c" --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  --readFilesCommand "gunzip -c" --outReadsUnmapped Fastx --parametersFiles $(DATABASE_PATH)/STAR_Parameters_Exogenous.in $(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 --readFilesCommand "gunzip -c" --outReadsUnmapped Fastx --parametersFiles $(DATABASE_PATH)/STAR_Parameters_Exogenous.in $(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
	rm $(OUTDIR)/EXOGENOUS_rRNA/exogenous_rRNA_Unmapped.out.mate1
	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 --readFilesCommand "gunzip -c" --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 --readFilesCommand "gunzip -c" --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 --readFilesCommand "gunzip -c" --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 --readFilesCommand "gunzip -c" --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 --readFilesCommand "gunzip -c" --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 --readFilesCommand "gunzip -c" --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 --readFilesCommand "gunzip -c" --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 --readFilesCommand "gunzip -c" --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 --readFilesCommand "gunzip -c" --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 --readFilesCommand "gunzip -c" --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 --readFilesCommand "gunzip -c" --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 --readFilesCommand "gunzip -c" --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 --readFilesCommand "gunzip -c" --parametersFiles $(STAR_PARAMS_FILE_PATH) $(STAR_EXOGENOUS_DYNAMIC_PARAMS) >> $(OUTDIR).log
	@echo -e "\n$(ts) $(PIPELINE_NAME): Plants4:" >> $(OUTDIR).log
	$(STAR_EXE) --runThreadN $(N_THREADS) --outFileNamePrefix $(OUTDIR)/EXOGENOUS_genomes/Plants4_ --genomeDir $(STAR_GENOMES_DIR)/STAR_GENOME_PLANTS4 --readFilesIn $(OUTDIR)/EXOGENOUS_rRNA/unaligned.fq.gz --readFilesCommand "gunzip -c" --parametersFiles $(STAR_PARAMS_FILE_PATH) $(STAR_EXOGENOUS_DYNAMIC_PARAMS) >> $(OUTDIR).log
	@echo -e "\n$(ts) $(PIPELINE_NAME): Plants5:" >> $(OUTDIR).log
	$(STAR_EXE) --runThreadN $(N_THREADS) --outFileNamePrefix $(OUTDIR)/EXOGENOUS_genomes/Plants5_ --genomeDir $(STAR_GENOMES_DIR)/STAR_GENOME_PLANTS5 --readFilesIn $(OUTDIR)/EXOGENOUS_rRNA/unaligned.fq.gz --readFilesCommand "gunzip -c" --parametersFiles $(STAR_PARAMS_FILE_PATH) $(STAR_EXOGENOUS_DYNAMIC_PARAMS) >> $(OUTDIR).log
	@echo -e "\n$(ts) $(PIPELINE_NAME): Finished mapping to exogenous GENOMES of PLANTS\n" >> $(OUTDIR).log

## Metazoa
$(OUTDIR)/EXOGENOUS_genomes/Metazoa5_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 METAZOA:\n" >> $(OUTDIR).log
	@echo -e "$(ts) $(PIPELINE_NAME): Metazoa1:" >> $(OUTDIR).log
	$(STAR_EXE) --runThreadN $(N_THREADS) --outFileNamePrefix $(OUTDIR)/EXOGENOUS_genomes/Metazoa1_ --genomeDir $(STAR_GENOMES_DIR)/STAR_GENOME_METAZOA1 --readFilesIn $(OUTDIR)/EXOGENOUS_rRNA/unaligned.fq.gz --readFilesCommand "gunzip -c" --parametersFiles $(STAR_PARAMS_FILE_PATH) $(STAR_EXOGENOUS_DYNAMIC_PARAMS) >> $(OUTDIR).log
	@echo -e "\n$(ts) $(PIPELINE_NAME): Metazoa2:" >> $(OUTDIR).log
	$(STAR_EXE) --runThreadN $(N_THREADS) --outFileNamePrefix $(OUTDIR)/EXOGENOUS_genomes/Metazoa2_ --genomeDir $(STAR_GENOMES_DIR)/STAR_GENOME_METAZOA2 --readFilesIn $(OUTDIR)/EXOGENOUS_rRNA/unaligned.fq.gz --readFilesCommand "gunzip -c" --parametersFiles $(STAR_PARAMS_FILE_PATH) $(STAR_EXOGENOUS_DYNAMIC_PARAMS) >> $(OUTDIR).log
	@echo -e "\n$(ts) $(PIPELINE_NAME): Metazoa3:" >> $(OUTDIR).log
	$(STAR_EXE) --runThreadN $(N_THREADS) --outFileNamePrefix $(OUTDIR)/EXOGENOUS_genomes/Metazoa3_ --genomeDir $(STAR_GENOMES_DIR)/STAR_GENOME_METAZOA3 --readFilesIn $(OUTDIR)/EXOGENOUS_rRNA/unaligned.fq.gz --readFilesCommand "gunzip -c" --parametersFiles $(STAR_PARAMS_FILE_PATH) $(STAR_EXOGENOUS_DYNAMIC_PARAMS) >> $(OUTDIR).log
	@echo -e "\n$(ts) $(PIPELINE_NAME): Metazoa4:" >> $(OUTDIR).log
	$(STAR_EXE) --runThreadN $(N_THREADS) --outFileNamePrefix $(OUTDIR)/EXOGENOUS_genomes/Metazoa4_ --genomeDir $(STAR_GENOMES_DIR)/STAR_GENOME_METAZOA4 --readFilesIn $(OUTDIR)/EXOGENOUS_rRNA/unaligned.fq.gz --readFilesCommand "gunzip -c" --parametersFiles $(STAR_PARAMS_FILE_PATH) $(STAR_EXOGENOUS_DYNAMIC_PARAMS) >> $(OUTDIR).log
	@echo -e "\n$(ts) $(PIPELINE_NAME): Metazoa5:" >> $(OUTDIR).log
	$(STAR_EXE) --runThreadN $(N_THREADS) --outFileNamePrefix $(OUTDIR)/EXOGENOUS_genomes/Metazoa5_ --genomeDir $(STAR_GENOMES_DIR)/STAR_GENOME_METAZOA5 --readFilesIn $(OUTDIR)/EXOGENOUS_rRNA/unaligned.fq.gz --readFilesCommand "gunzip -c" --parametersFiles $(STAR_PARAMS_FILE_PATH) $(STAR_EXOGENOUS_DYNAMIC_PARAMS) >> $(OUTDIR).log
	@echo -e "\n$(ts) $(PIPELINE_NAME): Finished mapping to exogenous GENOMES of METAZOA\n" >> $(OUTDIR).log

## Fungi, Protist, and Virus
$(OUTDIR)/EXOGENOUS_genomes/FungiProtistVirus_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 FUNGI, PROTISTS, and VIRUSES:\n" >> $(OUTDIR).log
	$(STAR_EXE) --runThreadN $(N_THREADS) --outFileNamePrefix $(OUTDIR)/EXOGENOUS_genomes/FungiProtistVirus_ --genomeDir $(STAR_GENOMES_DIR)/STAR_GENOME_FUNGI_PROTIST_VIRUS --readFilesIn $(OUTDIR)/EXOGENOUS_rRNA/unaligned.fq.gz --readFilesCommand "gunzip -c" --parametersFiles $(STAR_PARAMS_FILE_PATH) $(STAR_EXOGENOUS_DYNAMIC_PARAMS) >> $(OUTDIR).log
	@echo -e "\n$(ts) $(PIPELINE_NAME): Finished mapping to exogenous GENOMES of FUNGI, PROTISTS, and VIRUSES\n" >> $(OUTDIR).log

## Vertebrates
$(OUTDIR)/EXOGENOUS_genomes/Vertebrate4_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 VERTEBRATES:\n" >> $(OUTDIR).log
	@echo -e "$(ts) $(PIPELINE_NAME): Vertebrate1:" >> $(OUTDIR).log
	$(STAR_EXE) --runThreadN $(N_THREADS) --outFileNamePrefix $(OUTDIR)/EXOGENOUS_genomes/Vertebrate1_ --genomeDir $(STAR_GENOMES_DIR)/STAR_GENOME_VERTEBRATE1 --readFilesIn $(OUTDIR)/EXOGENOUS_rRNA/unaligned.fq.gz --readFilesCommand "gunzip -c" --parametersFiles $(STAR_PARAMS_FILE_PATH) $(STAR_EXOGENOUS_DYNAMIC_PARAMS) >> $(OUTDIR).log
	@echo -e "\n$(ts) $(PIPELINE_NAME): Vertebrate2:" >> $(OUTDIR).log
	$(STAR_EXE) --runThreadN $(N_THREADS) --outFileNamePrefix $(OUTDIR)/EXOGENOUS_genomes/Vertebrate2_ --genomeDir $(STAR_GENOMES_DIR)/STAR_GENOME_VERTEBRATE2 --readFilesIn $(OUTDIR)/EXOGENOUS_rRNA/unaligned.fq.gz --readFilesCommand "gunzip -c" --parametersFiles $(STAR_PARAMS_FILE_PATH) $(STAR_EXOGENOUS_DYNAMIC_PARAMS) >> $(OUTDIR).log
	@echo -e "\n$(ts) $(PIPELINE_NAME): Vertebrate3:" >> $(OUTDIR).log