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Annotated sample pRESTO code.txt
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Annotated sample pRESTO code.txt
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#
#Create and enter working directory
#
mkdir DATE_Seq
#
cd DATE_Seq
#
#Create directories for samples
#
mkdir WT_1_5N_LN
#
mkdir RAG_1_5N_LN
#
mkdir RAG_1_5P_LN
#
mkdir RAG_1_5P_LP
#
mkdir VH12_1_5N_LN
#
mkdir VH12_1_5P_LP
#
mkdir VH12RAG_1_5N_LN
#
mkdir VH12RAG_1_5P_LP
#
#Enter directory (folder) containing sequencing files from MiSeq (uploaded via FileZilla)
#
#demultiplex data into mouse numbers
#
#Make sure to rename output directory!!!
#
cd DATE_SW_Seq
#
bcl2fastq -o ~/DATE_Seq --use-bases-mask Y*,I6N2,N2I6,Y* --barcode-mismatch 0
#
#Return to previous directory
#
cd ..
#
#Move files into corresponding directories
#
mv WT_1_5N_LN_*.fastq.gz WT_1_5N_LN
#
mv RAG_1_5N_LN_*.fastq.gz RAG_1_5N_LN
#
mv RAG_1_5P_LN_*.fastq.gz RAG_1_5P_LN
#
mv RAG_1_5P_LP_*.fastq.gz RAG_1_5P_LP
#
mv VH12_1_5N_LN_*.fastq.gz VH12_1_5N_LN
#
mv VH12_1_5P_LP_*.fastq.gz VH12_1_5P_LP
#
mv VH12RAG_1_5N_LN_*.fastq.gz VH12RAG_1_5N_LN
#
mv VH12RAG_1_5P_LP_*.fastq.gz VH12RAG_1_5P_LP
#
#Run pipeline for each sample within respective directory
#
#Sample WT_1_5N_LN
#
#Enter directory containing files for Sample WT_1_5N_LN
#
cd WT_1_5N_LN
#
#unzip files
#
gunzip *.gz
#
#Filter sequences by quality
#
FilterSeq.py quality -s WT_1_5N_LN_S1_L001_R1_001.fastq -q 20 --outname WT_1_5N_LN_R1 --log FS1.log
FilterSeq.py quality -s WT_1_5N_LN_S1_L001_R2_001.fastq -q 20 --outname WT_1_5N_LN_R2 --log FS2.log
#
#Mask primer squences
#
MaskPrimers.py score -s WT_1_5N_LN_R1_quality-pass.fastq -p ../V_primers.fasta --start 0 --mode mask --outname WT_1_5N_LN_R1 --log MP1.log
MaskPrimers.py score -s WT_1_5N_LN_R2_quality-pass.fastq -p ../C_primers.fasta --start 15 --mode cut --barcode --outname WT_1_5N_LN_R2 --log MP2.log
#
#Sorts and matches sequence records with matching coordinates across files
#
PairSeq.py -1 WT_1_5N_LN_R1_primers-pass.fastq -2 WT_1_5N_LN_R2_primers-pass.fastq --2f BARCODE --coord illumina
#
#Builds a consensus sequence for each set of input sequences
#
BuildConsensus.py -s WT_1_5N_LN_R1_primers-pass_pair-pass.fastq --bf BARCODE --pf PRIMER \
--prcons 0.6 --maxerror 0.1 --maxgap 0.5 --outname WT_1_5N_LN_R1 --log BC1.log
BuildConsensus.py -s WT_1_5N_LN_R2_primers-pass_pair-pass.fastq --bf BARCODE --pf PRIMER \
--maxerror 0.1 --maxgap 0.5 --outname WT_1_5N_LN_R2 --log BC2.log
#
#Sorts and matches sequence records with matching coordinates across files again
#
PairSeq.py -1 WT_1_5N_LN_R1_consensus-pass.fastq -2 WT_1_5N_LN_R2_consensus-pass.fastq --coord presto --1f CONSCOUNT --2f CONSCOUNT PRCONS
#
#Assembles paired-end reads into a single sequence
#
AssemblePairs.py align -1 WT_1_5N_LN_R1_consensus-pass_pair-pass.fastq -2 WT_1_5N_LN_R2_consensus-pass_pair-pass.fastq --coord presto --rc tail --1f CONSCOUNT --2f CONSCOUNT PRCONS --outname WT_1_5N_LN --log AP.log
#
#Parses pRESTO annotations in FASTA/FASTQ sequence headers
#
ParseHeaders.py collapse -s WT_1_5N_LN_assemble-pass.fastq -f CONSCOUNT --act min
#
#Removes duplicate sequences from FASTA/FASTQ file
#
CollapseSeq.py -s WT_1_5N_LN*reheader.fastq -n 20 --inner --uf PRCONS --cf CONSCOUNT --act sum --outname WT_1_5N_LN --failed
#
#duplicate and rename file for conversion to fasta
#
cp WT_1_5N_LN_collapse-unique.fastq WT_1_5N_LN_collapse-unique_all.fastq
#
#convert fastq to fasta to upload to IMGT
#
paste - - - - < WT_1_5N_LN_collapse-unique_all.fastq | cut -f 1,2 | sed 's/^@/>/' | tr "\t" "\n" > WT_1_5N_LN_collapse-unique_all.fasta
#
#Parses pRESTO annotations in FASTA/FASTQ sequence headers and parses records in the console log of pRESTO modules
#
ParseHeaders.py table -s WT_1_5N_LN_collapse-unique.fastq -f ID PRCONS CONSCOUNT DUPCOUNT
ParseLog.py -l FS1.log FS2.log -f ID QUALITY
ParseLog.py -l MP1.log MP2.log -f ID PRIMER BARCODE ERROR
ParseLog.py -l BC1.log BC2.log -f BARCODE SEQCOUNT CONSCOUNT PRIMER PRCONS PRCOUNT PRFREQ ERROR
ParseLog.py -l AP.log -f ID LENGTH OVERLAP ERROR PVALUE FIELDS1 FIELDS2
#
#Repeat for division based on UMI
#
#Sorts, samples, and splits FASTQ sequence files into groups with 2+ or one UMI
#
SplitSeq.py group -s WT_1_5N_LN_collapse-unique.fastq -f CONSCOUNT --num 2 --outname WT_1_5N_LN --fasta
#
cd ..
#
#Run pipeline for each sample within respective directory
#
#Sample RAG_1_5N_LN
#
#Enter directory containing files for Sample RAG_1_5N_LN
#
cd RAG_1_5N_LN
#
#unzip files
#
gunzip *.gz
#
#Filter sequences by quality
#
FilterSeq.py quality -s RAG_1_5N_LN_S2_L001_R1_001.fastq -q 20 --outname RAG_1_5N_LN_R1 --log FS1.log
FilterSeq.py quality -s RAG_1_5N_LN_S2_L001_R2_001.fastq -q 20 --outname RAG_1_5N_LN_R2 --log FS2.log
#
#Mask primer squences
#
MaskPrimers.py score -s RAG_1_5N_LN_R1_quality-pass.fastq -p ../V_primers.fasta --start 0 --mode mask --outname RAG_1_5N_LN_R1 --log MP1.log
MaskPrimers.py score -s RAG_1_5N_LN_R2_quality-pass.fastq -p ../C_primers.fasta --start 15 --mode cut --barcode --outname RAG_1_5N_LN_R2 --log MP2.log
#
#Sorts and matches sequence records with matching coordinates across files
#
PairSeq.py -1 RAG_1_5N_LN_R1_primers-pass.fastq -2 RAG_1_5N_LN_R2_primers-pass.fastq --2f BARCODE --coord illumina
#
#Builds a consensus sequence for each set of input sequences
#
BuildConsensus.py -s RAG_1_5N_LN_R1_primers-pass_pair-pass.fastq --bf BARCODE --pf PRIMER \
--prcons 0.6 --maxerror 0.1 --maxgap 0.5 --outname RAG_1_5N_LN_R1 --log BC1.log
BuildConsensus.py -s RAG_1_5N_LN_R2_primers-pass_pair-pass.fastq --bf BARCODE --pf PRIMER \
--maxerror 0.1 --maxgap 0.5 --outname RAG_1_5N_LN_R2 --log BC2.log
#
#Sorts and matches sequence records with matching coordinates across files again
#
PairSeq.py -1 RAG_1_5N_LN_R1_consensus-pass.fastq -2 RAG_1_5N_LN_R2_consensus-pass.fastq --coord presto --1f CONSCOUNT --2f CONSCOUNT PRCONS
#
#Assembles paired-end reads into a single sequence
#
AssemblePairs.py align -1 RAG_1_5N_LN_R1_consensus-pass_pair-pass.fastq -2 RAG_1_5N_LN_R2_consensus-pass_pair-pass.fastq --coord presto --rc tail --1f CONSCOUNT --2f CONSCOUNT PRCONS --outname RAG_1_5N_LN --log AP.log
#
#Parses pRESTO annotations in FASTA/FASTQ sequence headers
#
ParseHeaders.py collapse -s RAG_1_5N_LN_assemble-pass.fastq -f CONSCOUNT --act min
#
#Removes duplicate sequences from FASTA/FASTQ file
#
CollapseSeq.py -s RAG_1_5N_LN*reheader.fastq -n 20 --inner --uf PRCONS --cf CONSCOUNT --act sum --outname RAG_1_5N_LN --failed
#
#duplicate and rename file for conversion to fasta
#
cp RAG_1_5N_LN_collapse-unique.fastq RAG_1_5N_LN_collapse-unique_all.fastq
#
#convert fastq to fasta to upload to IMGT
#
paste - - - - < RAG_1_5N_LN_collapse-unique_all.fastq | cut -f 1,2 | sed 's/^@/>/' | tr "\t" "\n" > RAG_1_5N_LN_collapse-unique_all.fasta
#
#Parses pRESTO annotations in FASTA/FASTQ sequence headers and parses records in the console log of pRESTO modules
#
ParseHeaders.py table -s RAG_1_5N_LN_collapse-unique.fastq -f ID PRCONS CONSCOUNT DUPCOUNT
ParseLog.py -l FS1.log FS2.log -f ID QUALITY
ParseLog.py -l MP1.log MP2.log -f ID PRIMER BARCODE ERROR
ParseLog.py -l BC1.log BC2.log -f BARCODE SEQCOUNT CONSCOUNT PRIMER PRCONS PRCOUNT PRFREQ ERROR
ParseLog.py -l AP.log -f ID LENGTH OVERLAP ERROR PVALUE FIELDS1 FIELDS2
#
#Repeat for division based on UMI
#
#Sorts, samples, and splits FASTQ sequence files into groups with 2+ or one UMI
#
SplitSeq.py group -s RAG_1_5N_LN_collapse-unique.fastq -f CONSCOUNT --num 2 --outname RAG_1_5N_LN --fasta
#
cd ..
#
#Run pipeline for each sample within respective directory
#
#Sample RAG_1_5P_LN
#
#Enter directory containing files for Sample RAG_1_5P_LN
#
cd RAG_1_5P_LN
#
#unzip files
#
gunzip *.gz
#
#Filter sequences by quality
#
FilterSeq.py quality -s RAG_1_5P_LN_S3_L001_R1_001.fastq -q 20 --outname RAG_1_5P_LN_R1 --log FS1.log
FilterSeq.py quality -s RAG_1_5P_LN_S3_L001_R2_001.fastq -q 20 --outname RAG_1_5P_LN_R2 --log FS2.log
#
#Mask primer squences
#
MaskPrimers.py score -s RAG_1_5P_LN_R1_quality-pass.fastq -p ../V_primers.fasta --start 0 --mode mask --outname RAG_1_5P_LN_R1 --log MP1.log
MaskPrimers.py score -s RAG_1_5P_LN_R2_quality-pass.fastq -p ../C_primers.fasta --start 15 --mode cut --barcode --outname RAG_1_5P_LN_R2 --log MP2.log
#
#Sorts and matches sequence records with matching coordinates across files
#
PairSeq.py -1 RAG_1_5P_LN_R1_primers-pass.fastq -2 RAG_1_5P_LN_R2_primers-pass.fastq --2f BARCODE --coord illumina
#
#Builds a consensus sequence for each set of input sequences
#
BuildConsensus.py -s RAG_1_5P_LN_R1_primers-pass_pair-pass.fastq --bf BARCODE --pf PRIMER \
--prcons 0.6 --maxerror 0.1 --maxgap 0.5 --outname RAG_1_5P_LN_R1 --log BC1.log
BuildConsensus.py -s RAG_1_5P_LN_R2_primers-pass_pair-pass.fastq --bf BARCODE --pf PRIMER \
--maxerror 0.1 --maxgap 0.5 --outname RAG_1_5P_LN_R2 --log BC2.log
#
#Sorts and matches sequence records with matching coordinates across files again
#
PairSeq.py -1 RAG_1_5P_LN_R1_consensus-pass.fastq -2 RAG_1_5P_LN_R2_consensus-pass.fastq --coord presto --1f CONSCOUNT --2f CONSCOUNT PRCONS
#
#Assembles paired-end reads into a single sequence
#
AssemblePairs.py align -1 RAG_1_5P_LN_R1_consensus-pass_pair-pass.fastq -2 RAG_1_5P_LN_R2_consensus-pass_pair-pass.fastq --coord presto --rc tail --1f CONSCOUNT --2f CONSCOUNT PRCONS --outname RAG_1_5P_LN --log AP.log
#
#Parses pRESTO annotations in FASTA/FASTQ sequence headers
#
ParseHeaders.py collapse -s RAG_1_5P_LN_assemble-pass.fastq -f CONSCOUNT --act min
#
#Removes duplicate sequences from FASTA/FASTQ file
#
CollapseSeq.py -s RAG_1_5P_LN*reheader.fastq -n 20 --inner --uf PRCONS --cf CONSCOUNT --act sum --outname RAG_1_5P_LN --failed
#
#duplicate and rename file for conversion to fasta
#
cp RAG_1_5P_LN_collapse-unique.fastq RAG_1_5P_LN_collapse-unique_all.fastq
#
#convert fastq to fasta to upload to IMGT
#
paste - - - - < RAG_1_5P_LN_collapse-unique_all.fastq | cut -f 1,2 | sed 's/^@/>/' | tr "\t" "\n" > RAG_1_5P_LN_collapse-unique_all.fasta
#
#Parses pRESTO annotations in FASTA/FASTQ sequence headers and parses records in the console log of pRESTO modules
#
ParseHeaders.py table -s RAG_1_5P_LN_collapse-unique.fastq -f ID PRCONS CONSCOUNT DUPCOUNT
ParseLog.py -l FS1.log FS2.log -f ID QUALITY
ParseLog.py -l MP1.log MP2.log -f ID PRIMER BARCODE ERROR
ParseLog.py -l BC1.log BC2.log -f BARCODE SEQCOUNT CONSCOUNT PRIMER PRCONS PRCOUNT PRFREQ ERROR
ParseLog.py -l AP.log -f ID LENGTH OVERLAP ERROR PVALUE FIELDS1 FIELDS2
#
#Repeat for division based on UMI
#
#Sorts, samples, and splits FASTQ sequence files into groups with 2+ or one UMI
#
SplitSeq.py group -s RAG_1_5P_LN_collapse-unique.fastq -f CONSCOUNT --num 2 --outname RAG_1_5P_LN --fasta
#
cd ..
#
#Run pipeline for each sample within respective directory
#
#Sample RAG_1_5P_LP
#
#Enter directory containing files for Sample RAG_1_5P_LP
#
cd RAG_1_5P_LP
#
#unzip files
#
gunzip *.gz
#
#Filter sequences by quality
#
FilterSeq.py quality -s RAG_1_5P_LP_S4_L001_R1_001.fastq -q 20 --outname RAG_1_5P_LP_R1 --log FS1.log
FilterSeq.py quality -s RAG_1_5P_LP_S4_L001_R2_001.fastq -q 20 --outname RAG_1_5P_LP_R2 --log FS2.log
#
#Mask primer squences
#
MaskPrimers.py score -s RAG_1_5P_LP_R1_quality-pass.fastq -p ../V_primers.fasta --start 0 --mode mask --outname RAG_1_5P_LP_R1 --log MP1.log
MaskPrimers.py score -s RAG_1_5P_LP_R2_quality-pass.fastq -p ../C_primers.fasta --start 15 --mode cut --barcode --outname RAG_1_5P_LP_R2 --log MP2.log
#
#Sorts and matches sequence records with matching coordinates across files
#
PairSeq.py -1 RAG_1_5P_LP_R1_primers-pass.fastq -2 RAG_1_5P_LP_R2_primers-pass.fastq --2f BARCODE --coord illumina
#
#Builds a consensus sequence for each set of input sequences
#
BuildConsensus.py -s RAG_1_5P_LP_R1_primers-pass_pair-pass.fastq --bf BARCODE --pf PRIMER \
--prcons 0.6 --maxerror 0.1 --maxgap 0.5 --outname RAG_1_5P_LP_R1 --log BC1.log
BuildConsensus.py -s RAG_1_5P_LP_R2_primers-pass_pair-pass.fastq --bf BARCODE --pf PRIMER \
--maxerror 0.1 --maxgap 0.5 --outname RAG_1_5P_LP_R2 --log BC2.log
#
#Sorts and matches sequence records with matching coordinates across files again
#
PairSeq.py -1 RAG_1_5P_LP_R1_consensus-pass.fastq -2 RAG_1_5P_LP_R2_consensus-pass.fastq --coord presto --1f CONSCOUNT --2f CONSCOUNT PRCONS
#
#Assembles paired-end reads into a single sequence
#
AssemblePairs.py align -1 RAG_1_5P_LP_R1_consensus-pass_pair-pass.fastq -2 RAG_1_5P_LP_R2_consensus-pass_pair-pass.fastq --coord presto --rc tail --1f CONSCOUNT --2f CONSCOUNT PRCONS --outname RAG_1_5P_LP --log AP.log
#
#Parses pRESTO annotations in FASTA/FASTQ sequence headers
#
ParseHeaders.py collapse -s RAG_1_5P_LP_assemble-pass.fastq -f CONSCOUNT --act min
#
#Removes duplicate sequences from FASTA/FASTQ file
#
CollapseSeq.py -s RAG_1_5P_LP*reheader.fastq -n 20 --inner --uf PRCONS --cf CONSCOUNT --act sum --outname RAG_1_5P_LP --failed
#
#duplicate and rename file for conversion to fasta
#
cp RAG_1_5P_LP_collapse-unique.fastq RAG_1_5P_LP_collapse-unique_all.fastq
#
#convert fastq to fasta to upload to IMGT
#
paste - - - - < RAG_1_5P_LP_collapse-unique_all.fastq | cut -f 1,2 | sed 's/^@/>/' | tr "\t" "\n" > RAG_1_5P_LP_collapse-unique_all.fasta
#
#Parses pRESTO annotations in FASTA/FASTQ sequence headers and parses records in the console log of pRESTO modules
#
ParseHeaders.py table -s RAG_1_5P_LP_collapse-unique.fastq -f ID PRCONS CONSCOUNT DUPCOUNT
ParseLog.py -l FS1.log FS2.log -f ID QUALITY
ParseLog.py -l MP1.log MP2.log -f ID PRIMER BARCODE ERROR
ParseLog.py -l BC1.log BC2.log -f BARCODE SEQCOUNT CONSCOUNT PRIMER PRCONS PRCOUNT PRFREQ ERROR
ParseLog.py -l AP.log -f ID LENGTH OVERLAP ERROR PVALUE FIELDS1 FIELDS2
#
#Repeat for division based on UMI
#
#Sorts, samples, and splits FASTQ sequence files into groups with 2+ or one UMI
#
SplitSeq.py group -s RAG_1_5P_LP_collapse-unique.fastq -f CONSCOUNT --num 2 --outname RAG_1_5P_LP --fasta
#
cd ..
#
#Run pipeline for each sample within respective directory
#
#Sample VH12_1_5N_LN
#
#Enter directory containing files for Sample VH12_1_5N_LN
#
cd VH12_1_5N_LN
#
#unzip files
#
gunzip *.gz
#
#Filter sequences by quality
#
FilterSeq.py quality -s VH12_1_5N_LN_S5_L001_R1_001.fastq -q 20 --outname VH12_1_5N_LN_R1 --log FS1.log
FilterSeq.py quality -s VH12_1_5N_LN_S5_L001_R2_001.fastq -q 20 --outname VH12_1_5N_LN_R2 --log FS2.log
#
#Mask primer squences
#
MaskPrimers.py score -s VH12_1_5N_LN_R1_quality-pass.fastq -p ../V_primers.fasta --start 0 --mode mask --outname VH12_1_5N_LN_R1 --log MP1.log
MaskPrimers.py score -s VH12_1_5N_LN_R2_quality-pass.fastq -p ../C_primers.fasta --start 15 --mode cut --barcode --outname VH12_1_5N_LN_R2 --log MP2.log
#
#Sorts and matches sequence records with matching coordinates across files
#
PairSeq.py -1 VH12_1_5N_LN_R1_primers-pass.fastq -2 VH12_1_5N_LN_R2_primers-pass.fastq --2f BARCODE --coord illumina
#
#Builds a consensus sequence for each set of input sequences
#
BuildConsensus.py -s VH12_1_5N_LN_R1_primers-pass_pair-pass.fastq --bf BARCODE --pf PRIMER \
--prcons 0.6 --maxerror 0.1 --maxgap 0.5 --outname VH12_1_5N_LN_R1 --log BC1.log
BuildConsensus.py -s VH12_1_5N_LN_R2_primers-pass_pair-pass.fastq --bf BARCODE --pf PRIMER \
--maxerror 0.1 --maxgap 0.5 --outname VH12_1_5N_LN_R2 --log BC2.log
#
#Sorts and matches sequence records with matching coordinates across files again
#
PairSeq.py -1 VH12_1_5N_LN_R1_consensus-pass.fastq -2 VH12_1_5N_LN_R2_consensus-pass.fastq --coord presto --1f CONSCOUNT --2f CONSCOUNT PRCONS
#
#Assembles paired-end reads into a single sequence
#
AssemblePairs.py align -1 VH12_1_5N_LN_R1_consensus-pass_pair-pass.fastq -2 VH12_1_5N_LN_R2_consensus-pass_pair-pass.fastq --coord presto --rc tail --1f CONSCOUNT --2f CONSCOUNT PRCONS --outname VH12_1_5N_LN --log AP.log
#
#Parses pRESTO annotations in FASTA/FASTQ sequence headers
#
ParseHeaders.py collapse -s VH12_1_5N_LN_assemble-pass.fastq -f CONSCOUNT --act min
#
#Removes duplicate sequences from FASTA/FASTQ file
#
CollapseSeq.py -s VH12_1_5N_LN*reheader.fastq -n 20 --inner --uf PRCONS --cf CONSCOUNT --act sum --outname VH12_1_5N_LN --failed
#
#duplicate and rename file for conversion to fasta
#
cp VH12_1_5N_LN_collapse-unique.fastq VH12_1_5N_LN_collapse-unique_all.fastq
#
#convert fastq to fasta to upload to IMGT
#
paste - - - - < VH12_1_5N_LN_collapse-unique_all.fastq | cut -f 1,2 | sed 's/^@/>/' | tr "\t" "\n" > VH12_1_5N_LN_collapse-unique_all.fasta
#
#Parses pRESTO annotations in FASTA/FASTQ sequence headers and parses records in the console log of pRESTO modules
#
ParseHeaders.py table -s VH12_1_5N_LN_collapse-unique.fastq -f ID PRCONS CONSCOUNT DUPCOUNT
ParseLog.py -l FS1.log FS2.log -f ID QUALITY
ParseLog.py -l MP1.log MP2.log -f ID PRIMER BARCODE ERROR
ParseLog.py -l BC1.log BC2.log -f BARCODE SEQCOUNT CONSCOUNT PRIMER PRCONS PRCOUNT PRFREQ ERROR
ParseLog.py -l AP.log -f ID LENGTH OVERLAP ERROR PVALUE FIELDS1 FIELDS2
#
#Repeat for division based on UMI
#
#Sorts, samples, and splits FASTQ sequence files into groups with 2+ or one UMI
#
SplitSeq.py group -s VH12_1_5N_LN_collapse-unique.fastq -f CONSCOUNT --num 2 --outname VH12_1_5N_LN --fasta
#
cd ..
#
#Run pipeline for each sample within respective directory
#
#Sample VH12_1_5P_LP
#
#Enter directory containing files for Sample VH12_1_5P_LP
#
cd VH12_1_5P_LP
#
#unzip files
#
gunzip *.gz
#
#Filter sequences by quality
#
FilterSeq.py quality -s VH12_1_5P_LP_S6_L001_R1_001.fastq -q 20 --outname VH12_1_5P_LP_R1 --log FS1.log
FilterSeq.py quality -s VH12_1_5P_LP_S6_L001_R2_001.fastq -q 20 --outname VH12_1_5P_LP_R2 --log FS2.log
#
#Mask primer squences
#
MaskPrimers.py score -s VH12_1_5P_LP_R1_quality-pass.fastq -p ../V_primers.fasta --start 0 --mode mask --outname VH12_1_5P_LP_R1 --log MP1.log
MaskPrimers.py score -s VH12_1_5P_LP_R2_quality-pass.fastq -p ../C_primers.fasta --start 15 --mode cut --barcode --outname VH12_1_5P_LP_R2 --log MP2.log
#
#Sorts and matches sequence records with matching coordinates across files
#
PairSeq.py -1 VH12_1_5P_LP_R1_primers-pass.fastq -2 VH12_1_5P_LP_R2_primers-pass.fastq --2f BARCODE --coord illumina
#
#Builds a consensus sequence for each set of input sequences
#
BuildConsensus.py -s VH12_1_5P_LP_R1_primers-pass_pair-pass.fastq --bf BARCODE --pf PRIMER \
--prcons 0.6 --maxerror 0.1 --maxgap 0.5 --outname VH12_1_5P_LP_R1 --log BC1.log
BuildConsensus.py -s VH12_1_5P_LP_R2_primers-pass_pair-pass.fastq --bf BARCODE --pf PRIMER \
--maxerror 0.1 --maxgap 0.5 --outname VH12_1_5P_LP_R2 --log BC2.log
#
#Sorts and matches sequence records with matching coordinates across files again
#
PairSeq.py -1 VH12_1_5P_LP_R1_consensus-pass.fastq -2 VH12_1_5P_LP_R2_consensus-pass.fastq --coord presto --1f CONSCOUNT --2f CONSCOUNT PRCONS
#
#Assembles paired-end reads into a single sequence
#
AssemblePairs.py align -1 VH12_1_5P_LP_R1_consensus-pass_pair-pass.fastq -2 VH12_1_5P_LP_R2_consensus-pass_pair-pass.fastq --coord presto --rc tail --1f CONSCOUNT --2f CONSCOUNT PRCONS --outname VH12_1_5P_LP --log AP.log
#
#Parses pRESTO annotations in FASTA/FASTQ sequence headers
#
ParseHeaders.py collapse -s VH12_1_5P_LP_assemble-pass.fastq -f CONSCOUNT --act min
#
#Removes duplicate sequences from FASTA/FASTQ file
#
CollapseSeq.py -s VH12_1_5P_LP*reheader.fastq -n 20 --inner --uf PRCONS --cf CONSCOUNT --act sum --outname VH12_1_5P_LP --failed
#
#duplicate and rename file for conversion to fasta
#
cp VH12_1_5P_LP_collapse-unique.fastq VH12_1_5P_LP_collapse-unique_all.fastq
#
#convert fastq to fasta to upload to IMGT
#
paste - - - - < VH12_1_5P_LP_collapse-unique_all.fastq | cut -f 1,2 | sed 's/^@/>/' | tr "\t" "\n" > VH12_1_5P_LP_collapse-unique_all.fasta
#
#Parses pRESTO annotations in FASTA/FASTQ sequence headers and parses records in the console log of pRESTO modules
#
ParseHeaders.py table -s VH12_1_5P_LP_collapse-unique.fastq -f ID PRCONS CONSCOUNT DUPCOUNT
ParseLog.py -l FS1.log FS2.log -f ID QUALITY
ParseLog.py -l MP1.log MP2.log -f ID PRIMER BARCODE ERROR
ParseLog.py -l BC1.log BC2.log -f BARCODE SEQCOUNT CONSCOUNT PRIMER PRCONS PRCOUNT PRFREQ ERROR
ParseLog.py -l AP.log -f ID LENGTH OVERLAP ERROR PVALUE FIELDS1 FIELDS2
#
#Repeat for division based on UMI
#
#Sorts, samples, and splits FASTQ sequence files into groups with 2+ or one UMI
#
SplitSeq.py group -s VH12_1_5P_LP_collapse-unique.fastq -f CONSCOUNT --num 2 --outname VH12_1_5P_LP --fasta
#
cd ..
#
#Run pipeline for each sample within respective directory
#
#Sample VH12_1_RAG_1_5N_LN
#
#Enter directory containing files for Sample VH12_1_RAG_1_5N_LN
#
cd VH12_1_RAG_1_5N_LN
#
#unzip files
#
gunzip *.gz
#
#Filter sequences by quality
#
FilterSeq.py quality -s VH12_1_RAG_1_5N_LN_S7_L001_R1_001.fastq -q 20 --outname VH12_1_RAG_1_5N_LN_R1 --log FS1.log
FilterSeq.py quality -s VH12_1_RAG_1_5N_LN_S7_L001_R2_001.fastq -q 20 --outname VH12_1_RAG_1_5N_LN_R2 --log FS2.log
#
#Mask primer squences
#
MaskPrimers.py score -s VH12_1_RAG_1_5N_LN_R1_quality-pass.fastq -p ../V_primers.fasta --start 0 --mode mask --outname VH12_1_RAG_1_5N_LN_R1 --log MP1.log
MaskPrimers.py score -s VH12_1_RAG_1_5N_LN_R2_quality-pass.fastq -p ../C_primers.fasta --start 15 --mode cut --barcode --outname VH12_1_RAG_1_5N_LN_R2 --log MP2.log
#
#Sorts and matches sequence records with matching coordinates across files
#
PairSeq.py -1 VH12_1_RAG_1_5N_LN_R1_primers-pass.fastq -2 VH12_1_RAG_1_5N_LN_R2_primers-pass.fastq --2f BARCODE --coord illumina
#
#Builds a consensus sequence for each set of input sequences
#
BuildConsensus.py -s VH12_1_RAG_1_5N_LN_R1_primers-pass_pair-pass.fastq --bf BARCODE --pf PRIMER \
--prcons 0.6 --maxerror 0.1 --maxgap 0.5 --outname VH12_1_RAG_1_5N_LN_R1 --log BC1.log
BuildConsensus.py -s VH12_1_RAG_1_5N_LN_R2_primers-pass_pair-pass.fastq --bf BARCODE --pf PRIMER \
--maxerror 0.1 --maxgap 0.5 --outname VH12_1_RAG_1_5N_LN_R2 --log BC2.log
#
#Sorts and matches sequence records with matching coordinates across files again
#
PairSeq.py -1 VH12_1_RAG_1_5N_LN_R1_consensus-pass.fastq -2 VH12_1_RAG_1_5N_LN_R2_consensus-pass.fastq --coord presto --1f CONSCOUNT --2f CONSCOUNT PRCONS
#
#Assembles paired-end reads into a single sequence
#
AssemblePairs.py align -1 VH12_1_RAG_1_5N_LN_R1_consensus-pass_pair-pass.fastq -2 VH12_1_RAG_1_5N_LN_R2_consensus-pass_pair-pass.fastq --coord presto --rc tail --1f CONSCOUNT --2f CONSCOUNT PRCONS --outname VH12_1_RAG_1_5N_LN --log AP.log
#
#Parses pRESTO annotations in FASTA/FASTQ sequence headers
#
ParseHeaders.py collapse -s VH12_1_RAG_1_5N_LN_assemble-pass.fastq -f CONSCOUNT --act min
#
#Removes duplicate sequences from FASTA/FASTQ file
#
CollapseSeq.py -s VH12_1_RAG_1_5N_LN*reheader.fastq -n 20 --inner --uf PRCONS --cf CONSCOUNT --act sum --outname VH12_1_RAG_1_5N_LN --failed
#
#duplicate and rename file for conversion to fasta
#
cp VH12_1_RAG_1_5N_LN_collapse-unique.fastq VH12_1_RAG_1_5N_LN_collapse-unique_all.fastq
#
#convert fastq to fasta to upload to IMGT
#
paste - - - - < VH12_1_RAG_1_5N_LN_collapse-unique_all.fastq | cut -f 1,2 | sed 's/^@/>/' | tr "\t" "\n" > VH12_1_RAG_1_5N_LN_collapse-unique_all.fasta
#
#Parses pRESTO annotations in FASTA/FASTQ sequence headers and parses records in the console log of pRESTO modules
#
ParseHeaders.py table -s VH12_1_RAG_1_5N_LN_collapse-unique.fastq -f ID PRCONS CONSCOUNT DUPCOUNT
ParseLog.py -l FS1.log FS2.log -f ID QUALITY
ParseLog.py -l MP1.log MP2.log -f ID PRIMER BARCODE ERROR
ParseLog.py -l BC1.log BC2.log -f BARCODE SEQCOUNT CONSCOUNT PRIMER PRCONS PRCOUNT PRFREQ ERROR
ParseLog.py -l AP.log -f ID LENGTH OVERLAP ERROR PVALUE FIELDS1 FIELDS2
#
#Repeat for division based on UMI
#
#Sorts, samples, and splits FASTQ sequence files into groups with 2+ or one UMI
#
SplitSeq.py group -s VH12_1_RAG_1_5N_LN_collapse-unique.fastq -f CONSCOUNT --num 2 --outname VH12_1_RAG_1_5N_LN --fasta
#
cd ..
#
#Run pipeline for each sample within respective directory
#
#Sample "X"
#
#Enter directory containing files for Sample "X"
#
cd "X"
#
#unzip files
#
gunzip *.gz
#
#Filter sequences by quality
#
FilterSeq.py quality -s "X"_S8_L001_R1_001.fastq -q 20 --outname "X"_R1 --log FS1.log
FilterSeq.py quality -s "X"_S8_L001_R2_001.fastq -q 20 --outname "X"_R2 --log FS2.log
#
#Mask primer squences
#
MaskPrimers.py score -s "X"_R1_quality-pass.fastq -p ../V_primers.fasta --start 0 --mode mask --outname "X"_R1 --log MP1.log
MaskPrimers.py score -s "X"_R2_quality-pass.fastq -p ../C_primers.fasta --start 15 --mode cut --barcode --outname "X"_R2 --log MP2.log
#
#Sorts and matches sequence records with matching coordinates across files
#
PairSeq.py -1 "X"_R1_primers-pass.fastq -2 "X"_R2_primers-pass.fastq --2f BARCODE --coord illumina
#
#Builds a consensus sequence for each set of input sequences
#
BuildConsensus.py -s "X"_R1_primers-pass_pair-pass.fastq --bf BARCODE --pf PRIMER \
--prcons 0.6 --maxerror 0.1 --maxgap 0.5 --outname "X"_R1 --log BC1.log
BuildConsensus.py -s "X"_R2_primers-pass_pair-pass.fastq --bf BARCODE --pf PRIMER \
--maxerror 0.1 --maxgap 0.5 --outname "X"_R2 --log BC2.log
#
#Sorts and matches sequence records with matching coordinates across files again
#
PairSeq.py -1 "X"_R1_consensus-pass.fastq -2 "X"_R2_consensus-pass.fastq --coord presto --1f CONSCOUNT --2f CONSCOUNT PRCONS
#
#Assembles paired-end reads into a single sequence
#
AssemblePairs.py align -1 "X"_R1_consensus-pass_pair-pass.fastq -2 "X"_R2_consensus-pass_pair-pass.fastq --coord presto --rc tail --1f CONSCOUNT --2f CONSCOUNT PRCONS --outname "X" --log AP.log
#
#Parses pRESTO annotations in FASTA/FASTQ sequence headers
#
ParseHeaders.py collapse -s "X"_assemble-pass.fastq -f CONSCOUNT --act min
#
#Removes duplicate sequences from FASTA/FASTQ file
#
CollapseSeq.py -s "X"*reheader.fastq -n 20 --inner --uf PRCONS --cf CONSCOUNT --act sum --outname "X" --failed
#
#duplicate and rename file for conversion to fasta
#
cp "X"_collapse-unique.fastq "X"_collapse-unique_all.fastq
#
#convert fastq to fasta to upload to IMGT
#
paste - - - - < "X"_collapse-unique_all.fastq | cut -f 1,2 | sed 's/^@/>/' | tr "\t" "\n" > "X"_collapse-unique_all.fasta
#
#Parses pRESTO annotations in FASTA/FASTQ sequence headers and parses records in the console log of pRESTO modules
#
ParseHeaders.py table -s "X"_collapse-unique.fastq -f ID PRCONS CONSCOUNT DUPCOUNT
ParseLog.py -l FS1.log FS2.log -f ID QUALITY
ParseLog.py -l MP1.log MP2.log -f ID PRIMER BARCODE ERROR
ParseLog.py -l BC1.log BC2.log -f BARCODE SEQCOUNT CONSCOUNT PRIMER PRCONS PRCOUNT PRFREQ ERROR
ParseLog.py -l AP.log -f ID LENGTH OVERLAP ERROR PVALUE FIELDS1 FIELDS2
#
#Repeat for division based on UMI
#
#Sorts, samples, and splits FASTQ sequence files into groups with 2+ or one UMI
#
SplitSeq.py group -s "X"_collapse-unique.fastq -f CONSCOUNT --num 2 --outname "X" --fasta
#
cd ..
#
#Run pipeline for each sample within respective directory
#
#Using FileZilla, download the 3 FASTA files that were created in each directory (3/sample) and upload them to IMGT High-V Quest for annotation
#
#When uplaoding to IMGT, use the follwoing guide for sample entry to ensure compatibility with file names in subsequent processing steps
#
#Samples must be named as follows for IMGT runs:
#Sample_collapse-unique_all.fasta--> "Sample all",
#Sample_atleast-2.fasta --> "Sample 2",
#Sample_under-2.fasta --> "Sample 1"