add subroutine for non-UMI Smart-Seq2 technology (13a27721) · Commits · github_fork / Universc

launch_universc.sh

+94 −11

Original line number	Diff line number	Diff line
		@@ -1501,7 +1501,7 @@ 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" == "smartseq3" ]]; then
		elif [[ "$technology" == "smartseq2" ]] \|\| [[ "$technology" == "smartseq3" ]]; then
		barcodefile=${whitelistdir}/SmartSeq3_barcode.txt
		else
		echo "*WARNING: whitelist for ${technology} will be all possible combinations of ${minlength}bp. valid barcode will be 100% as a result*"
		@@ -2362,8 +2362,94 @@ else
		done
		fi

		#Smart-Seq2 (or Smart-Seq)
		if [[ "$technology" == "smartseq2" ]];then
		echo " ...processsing for ${technology}"
		if [[ $verbose ]]; then
		echo "Note: SmartSeq-2 does not contain UMIs"
		fi
		for convFile in "${convFiles[@]}"; do
		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/' )

		#remove R1 adapter if detected
		sed -E '
		/^AGATGTGTATAAGAGACAG/ {
		s/^(.{19})//g
		n
		n
		s/^(.{19})//g
		}' $convR1 > ${crIN}/.temp
		mv ${crIN}/.temp $convR1
		#remove R2 adapter if detected
		sed -E '
		/CTGTCTCTTATACACATCT$/ {
		s/(.{19})$//g
		n
		n
		s/(.{19})$//g
		}' $convR2 > ${crIN}/.temp
		mv ${crIN}/.temp $convR2

		echo" ...remove internal reads for ${technology} by matching TSO sequence for UMI reads"
		# filter UMI reads by matching tag sequence ATTGCGCAATG (bases 1-11 of R1) and remove as an adapters

		perl sub/FilterSmartSeqReadUMI.pl --r1=${convR1} --r2=${convR2} --i1=${convI1} --i2=${convI2} --tag="AAGCAGTGGTATCAACGCAGAGTAC" --out_dir $crIN
		echo " ...trim tag sequence from R1"

		# returns R1 with tag sequence removed (left trim) starting with 8pbp UMI and corresponding reads for I1, I2, and R2
		mv $crIN/parsed_R1.fastq ${convR1}
		mv $crIN/parsed_R2.fastq ${convR2}
		mv $crIN/parsed_I1.fastq ${convI1}
		mv $crIN/parsed_I2.fastq ${convI2}

		echo " ...concatencate barcodes to R1 from I1 and I2 index files"
		# concatenate barcocdes from dual indexes to R1 as barcode (bases 1-16)
		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)
		## 16 bp barcode, GGG for TSO, no UMI
		mv $crIN/Concatenated_File.fastq ${convR1}

		# add mock UMI (count reads instead of UMI) barcodelength=16, umi_default=10
		perl sub/AddMockUMI.pl --fastq=${convR1} --out_dir $crIN --head_length=$barcodelength --umi_length=$umi_default
		umilength=$umi_default
		umiadjust=0
		chemistry="SC3Pv2"

		#returns a combined R1 file with barcode and mock UMI
		## 16 bp barcode, 10 bp UMI, GGG for TSO
		mv $crIN/mock_UMI.fastq ${convR1}

		#convert TSO to expected length for 10x 5' (TSS in R1 from base 39)
		echo " handling $convFile ..."
		tsoS="TTTCTTATATGGG"
		tsoQ="IIIIIIIIIIIII"
		#Add 10x TSO characters to the end of the sequence
		cmd=$(echo 'sed -E "2~4s/(.{'$barcodelength'})(.{'${umilength}'})(.{3})/\1\2'$tsoS'/" '$convFile' > '${crIN}'/.temp')
		if [[ $verbose ]]; then
		echo technology $technology
		echo barcode: $barcodelength
		echo umi: $umilength
		echo $cmd
		fi
		# run command with barcode and umi length, e.g.,: sed -E "2~4s/(.{16})(.{8})(.{3})(.*)/\1\2$tsoS\4/" $convFile > ${crIN}/.temp
		eval $cmd
		mv ${crIN}/.temp $convFile
		#Add n characters to the end of the quality
		cmd=$(echo 'sed -E "4~4s/(.{'$barcodelength'})(.{'${umilength}'})(.{3})/\1\2'$tsoQ'/" '$convFile' > '${crIN}'/.temp')
		# run command with barcode and umi length, e.g.,: sed -E "4~4s/(.{16})(.{8})(.{3})(.*)/\1\2$tsoQ\4/" $convFile > ${crIN}/.temp
		eval $cmd
		mv ${crIN}/.temp $convFile
		echo " ${convFile} adjusted"
		done
		fi

		#Smart-Seq3
		if [[ "$technology" == "smartseq"* ]];then
		if [[ "$technology" == "smartseq3" ]];then
		echo " ...processsing for ${technology}"
		if [[ $verbose ]]; then
		echo "Note: SmartSeq-3 requires additional filtering for UMIs"
		@@ -2377,18 +2463,18 @@ else

		echo " ...remove internal for ${technology} by matching tag sequence for UMI reads"
		# filter UMI reads by matching tag sequence ATTGCGCAATG (bases 1-11 of R1) and remove as an adapters
		perl sub/FilterSmartSeqReadUMI.pl --r1=${convR1} --r2=${convR2} --i1=${convI1} --i2=${convI2} --out_dir $crIN
		perl sub/FilterSmartSeqReadUMI.pl --r1=${convR1} --r2=${convR2} --i1=${convI1} --i2=${convI2} --out_dir=$crIN --tag=
		echo " ...trim tag sequence from R1"

		# returns R1 with tag sequence removed (left trim) starting with 8pbp UMI and corresponding reads for I1, I2, and R2
		mv $crIN/SmartSeq3_parsed_R1.fastq ${convR1}
		mv $crIN/SmartSeq3_parsed_R2.fastq ${convR2}
		mv $crIN/SmartSeq3_parsed_I1.fastq ${convI1}
		mv $crIN/SmartSeq3_parsed_I2.fastq ${convI2}
		mv $crIN/parsed_R1.fastq ${convR1}
		mv $crIN/parsed_R2.fastq ${convR2}
		mv $crIN/parsed_I1.fastq ${convI1}
		mv $crIN/parsed_I2.fastq ${convI2}

		echo " ...concatencate barcodes to R1 from I1 and I2 index files"
		# concatenate barcocdes from dual indexes to R1 as barcode (bases 1-16)
		perl sub/ConcatenateDualIndexBarcodes.pl --additive=${convI1} --additive=${convI2} --ref_fastq=${convR1} --out_dir $crIN
		perl sub/ConcatenateDualIndexBarcodes.pl --additive=${convI1} --additive=${convI2} --ref_fastq=${convR1} --tag="ATTGCGCAATG" --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}
		@@ -2416,9 +2502,6 @@ else
		echo " ${convFile} adjusted"
		done
		fi
		#Smart-Seq2
		#echo 'TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG'
		#echo 'CTGTCTCTTATACACATCTCCGAGCCCACGAGAC'

		#converting barcodes
		echo " adjusting barcodes of R1 files"

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