separate barcode whitelists and adapter trimmming for 2-level or 3-level SCI-Seq (37aa5aab) · Commits · github_fork / Universc

launch_universc.sh

+73 −18

Original line number	Diff line number	Diff line
		@@ -203,10 +203,13 @@ Mandatory arguments to long options are mandatory for short options too.
		ICELL8 version 3 (11bp barcode, 14bp UMI): icell8 or custom
		inDrops version 1 (19bp barcode, 6bp UMI): indrops-v1, 1cellbio-v1
		inDrops version 2 (19bp barcode, 6bp UMI): indrops-v2, 1cellbio-v2
		inDrops version 3 (16bp barcode, 6bp UMI): indrops-v3, 1cellbio-v3
		MARS-Seq (6bp barcode, 10bp UMI): marsseq, marsseq-v1
		MARS-Seq2 (7bp barcode, 8bp UMI): marsseq2, marsseq-v2
		Quartz-Seq2 (14bp barcode, 8bp UMI): quartzseq2-384
		Quartz-Seq2 (15bp barcode, 8bp UMI): quartzseq2-1536
		SCI-Seq 2-level indexing (30 bp barcode, 8 bp UMI): sciseq2
		SCI-Seq 3-level indexing (40 bp barcode, 8 bp UMI): sciseq3
		SCRB-Seq (6bp barcode, 10bp UMI): scrbseq, mcscrbseq
		SeqWell (12bp barcode, 8bp UMI): seqwell
		Smart-seq, Smart-seq2 (16bp barcode, No UMI): smartseq2
		@@ -216,10 +219,6 @@ Mandatory arguments to long options are mandatory for short options too.
		Custom inputs are also supported by giving the name "custom" and length of barcode and UMI separated by "_"
		e.g. Custom (16bp barcode, 10bp UMI): custom_16_10

		Experimental technologies (not yet supported):
		inDrops version 3 (16bp barcode, 6bp UMI): indrops-v3, 1cellbio-v3
		Sci-Seq (8bp UMI, 30bp barcode): sciseq

		-b, --barcodefile FILE Custom barcode list in plain text (with each line containing a barcode)

		-c, --chemistry CHEM Assay configuration, autodetection is not possible for converted files: 'SC3Pv2' (default), 'SC5P-PE', or 'SC5P-R2'
		@@ -614,7 +613,11 @@ elif [[ "$technology" == "quartz-seq2-384" ]] \|\| [[ "$technology" == "quartzseq2
		elif [[ "$technology" == "quartz-seq2-1536" ]] \|\| [[ "$technology" == "quartzseq2-1536" ]] \|\| [[ "$technology" == "quartz-seq2-v3.2" ]] \|\| [[ "$technology" == "quartzseq2-v3.2" ]] \|\| [[ "$technology" == "quartzseq2v3.2" ]]; then
		technology="quartz-seq2-1536"
		elif [[ "$technology" == "sciseq" ]] \|\| [[ "$technology" == "sci-seq" ]]; then
		technology="sciseq"
		technology="sciseq3"
		elif [[ "$technology" == "sciseq2" ]] \|\| [[ "$technology" == "sci-seq2" ]]; then
		technology="sciseq2"
		elif [[ "$technology" == "sciseq3" ]] \|\| [[ "$technology" == "sci-seq3" ]]; then
		technology="sciseq3"
		elif [[ "$technology" == "scrbseq" ]] \|\| [[ "$technology" == "scrb-seq" ]] \|\| [[ "$technology" == "mcscrbseq" ]] \|\| [[ "$technology" == "mcscrb-seq" ]]; then
		technology="scrbseq"
		elif [[ "$technology" == "seqwell" ]] \|\| [[ "$technology" == "seq-well" ]]; then
		@@ -733,10 +736,14 @@ elif [[ "$technology" == "quartz-seq2-1536" ]]; then
		barcodelength=15
		umilength=8
		minlength=15
		elif [[ "$technology" == "sciseq" ]]; then
		elif [[ "$technology" == "sciseq2" ]]; then
		barcodelength=30
		umilength=8
		minlength=30
		elif [[ "$technology" == "sciseq3" ]]; then
		barcodelength=40
		umilength=8
		minlength=40
		elif [[ "$technology" == "scrbseq" ]]; then
		barcodelength=6
		umilength=10
		@@ -945,7 +952,7 @@ fi
		#index 2
		if [[ $setup == "false" ]]; then
		#only check I2 for dual-indexed techniques
		if [[ "$technology" == "indrop-v3" ]] \|\| [[ "$technology" == "sci-seq" ]] \|\| [[ "$technology" == "smartseq"* ]]; then
		if [[ "$technology" == "indrop-v3" ]] \|\| [[ "$technology" == "sciseq2" ]] \|\| [[ "$technology" == "sciseq3" ]] \|\| [[ "$technology" == "smartseq"* ]]; then
		if [[ ${#index2[@]} -ne ${#read1[@]} ]]; then
		if [[ ${#index2[@]} -gt 0 ]]; then
		echo " Error: number of index1 files is not matching the number of index2 files"
		@@ -1472,7 +1479,7 @@ if [[ -n "$barcodefile" ]]; then
		#getting absolute path
		barcodefile=$(readlink -f $barcodefile)
		#allowing WellList from ICELL8 and other well-based techniques
		if [[ "$technology" == "icell8" ]] \|\| [[ "$technology" == "quartz-seq2" ]] \|\| [[ "$technology" == "splitseq" ]] \|\| [[ "$technology" == "smartseq" ]] \|\| [[ "$technology" == "seqwell" ]] \|\| [[ "$technology" == "sciseq" ]] \|\| [[ "$technology" == "custom" ]]; then
		if [[ "$technology" == "icell8" ]] \|\| [[ "$technology" == "quartz-seq2" ]] \|\| [[ "$technology" == "splitseq" ]] \|\| [[ "$technology" == "smartseq" ]] \|\| [[ "$technology" == "seqwell" ]] \|\| [[ "$technology" == "sciseq2" ]] \|\| [[ "$technology" == "sciseq3" ]] \|\| [[ "$technology" == "custom" ]]; then
		seg=$'\t'
		n_col=$(awk -F'\t' '{print NF}' $barcodefile \| sort -nu \| tail -n 1)
		if [[ $n_col -eq 1 ]]; then
		@@ -1527,7 +1534,12 @@ else
		barcodefile=${whitelistdir}/inDrop-v3_barcodes.txt
		echo "*WARNING: combination of list1 and list2 from indrop-v2 (https://github.com/indrops/indrops/issues/32)**"
		fi
		elif [[ "$technology" == "sciseq" ]]; then
		elif [[ "$technology" == "sciseq2" ]]; then
		barcodefile=${whitelistdir}/sciseq2_barcode.txt
		if [[ ! -f ${whitelistdir}/sciseq2_barcode.txt ]]; then
		echo " ...generating combination of I1, I2, and RT barcodes..."
		fi
		elif [[ "$technology" == "sciseq3" ]]; then
		barcodefile=${whitelistdir}/sciseq3_barcode.txt
		if [[ ! -f ${whitelistdir}/sciseq3_barcode.txt ]]; then
		echo " ...generating combination of I1, I2, and RT barcodes..."
		@@ -1581,9 +1593,19 @@ else
		#allow for barcodes in index (I1) and R1
		perl ${MAKEINDROPBARCODES} ${whitelistdir}/inDrop_gel_barcode1_list.txt ${whitelistdir}/inDrop_gel_barcode2_list.txt v3 ${whitelistdir}
		fi
		elif [[ "$technology" == "sciseq" ]]; then
		elif [[ "$technology" == "sciseq2" ]]; then
		#generates all combinations of I1-I2-R1 barcodes
		join -j 9999 ${whitelistdir}/sci-seq3_i5_barcodes.txt ${whitelistdir}/sci-seq3_i7_barcodes.txt \| sed "s/ //g" \| join -j 9999 - ${whitelistdir}/sci-seq3_rt_barcodes.txt \| sed "s/ //g" \| awk '!a[$0]++' > ${whitelistdir}/sciseq3_barcode.txt
		if [[ ! -f ${whitelistdir}/sciseq2_barcode.txt ]]; then
		join -j 9999 ${whitelistdir}/sci-seq3_i5_barcodes.txt ${whitelistdir}/sci-seq3_i7_barcodes.txt \| sed "s/ //g" \
		\| join -j 9999 - ${whitelistdir}/sci-seq3_rt_barcodes.txt \| sed "s/ //g" \| awk '!a[$0]++' > ${whitelistdir}/sciseq2_barcode.txt
		fi
		elif [[ "$technology" == "sciseq3" ]]; then
		if [[ ! -f ${whitelistdir}/sciseq3_barcode.txt ]]; then
		#generates all combinations of I1-I2-R1 barcodes
		join -j 9999 ${whitelistdir}/sci-seq3_i5_barcodes.txt ${whitelistdir}/sci-seq3_i7_barcodes.txt \| sed "s/ //g" \
		\| join -j 9999 - ${whitelistdir}/sci-seq3_hp_barcodes.txt \| sed "s/ //g" \| join -j 9999 - ${whitelistdir}/sci-seq3_rt_barcodes.txt \| sed "s/ //g" \
		\| awk '!a[$0]++' > ${whitelistdir}/sciseq3_barcode.txt
		fi
		else
		#generating permutations of ATCG of barcode length (non-standard evaluation required to run in script)
		echo $(eval echo $(for ii in $(eval echo {1..${barcodelength}}); do echo "{A,T,C,G}"; done \| tr "\n" " " \| sed "s/ //g" \| xargs -I {} echo {})) \| sed 's/ /\n/g' \| sort \| uniq > ${barcodefile}
		@@ -2372,16 +2394,16 @@ else
		fi

		#Sci-Seq: remove adapter and swap barcode and UMI
		if [[ "$technology" == "sciseq" ]]; then
		if [[ "$technology" == "sciseq2" ]]; then
		echo " ...remove adapter for ${technology}"
		for convFile in "${convFiles[@]}"; do
		#remove adapter if detected (and hairpin/tn5 barcode)
		#remove adapter if detected (two-level indexing)
		sed -E '
		/^ACGACGCTCTTCCGATCT(.{10})CAGAGC/ {
		s/^ACGACGCTCTTCCGATCT(.{10})CAGAGC(.{18})/\2/g
		/^ACGACGCTCTTCCGATCT/ {
		s/^ACGACGCTCTTCCGATCT(.{18})/\1/g
		n
		n
		s/^(.{26})(.{18})/\2/g
		s/^(.{18})(.{18})/\2/g
		}' $convFile > ${crIN}/.temp
		mv ${crIN}/.temp $convFile
		#swap barcode and UMI
		@@ -2402,7 +2424,40 @@ else

		#returns a combined R1 file with I1-I2-R1 concatenated (I1 and I2 are R1 barcode)
		mv $crIN/Concatenated_File.fastq ${convR1}
		done
		fi


		if [[ "$technology" == "sciseq3" ]]; then
		echo " ...remove adapter for ${technology}"
		for convFile in "${convFiles[@]}"; do
		#remove adapter if detected (and keep hairpin/tn5 barcode)
		sed -E '
		/^ACGACGCTCTTCCGATCT(.{10})CAGAGC/ {
		s/^ACGACGCTCTTCCGATCT(.{10})CAGAGC(.{18})/\1\2/g
		n
		n
		s/^(.{26})(.{10})(.{6})(.{18})/\2\4/g
		}' $convFile > ${crIN}/.temp
		mv ${crIN}/.temp $convFile
		#swap barcode and UMI
		echo " ...barcode and UMI swapped for ${technology}"
		sed -E '2~2s/(.{10})(.{8})(.{10})/\3\1\2/' $convFile > ${crIN}/.temp
		mv ${crIN}/.temp $convFile

		read=$convFile
		convR1=$read
		convR2=$(echo $read \| perl -pne 's/(.*)_R1/$1_R2/' )
		convI1=$(echo $read \| perl -pne 's/(.*)_R1/$1_I1/' )
		convI2=$(echo $read \| perl -pne 's/(.*)_R1/$1_I2/' )

		echo " ...concatencate barcodes to R1 from I1 and I2 index files"
		# concatenate barcocdes from dual indexes to R1 as (bases 1-20 of the) barcode, moving RT barcode (21-30) UMI to (31-38)
		# filter UMI reads by matching tag sequence ATTGCGCAATG (bases 1-11 of R1) and remove as an adapters
		perl sub/ConcatenateDualIndexBarcodes.pl --additive=${convI1} --additive=${convI2} --ref_fastq=${convR1} --out_dir $crIN

		#returns a combined R1 file with I1-I2-R1 concatenated (I1 and I2 are R1 barcode)
		mv $crIN/Concatenated_File.fastq ${convR1}
		done
		fi

Admin message