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UR:/lustre/scratch117/core/sciops_repository/references/Homo_sapiens/GRCh37_53/all/fasta/Homo_sapiens.GRCh37.dna.all.fa AS:GRCh37 53 M5:5d9ec007868d517e73543b005ba48535 SP:Homo sapiens @SQ SN:HSCHRUN_RANDOM_CTG2 LN:186858 UR:/lustre/scratch117/core/sciops_repository/references/Homo_sapiens/GRCh37_53/all/fasta/Homo_sapiens.GRCh37.dna.all.fa AS:GRCh37 53 M5:563531689f3dbd691331fd6c5730a88b SP:Homo sapiens @SQ SN:HSCHRUN_RANDOM_CTG14 LN:186861 UR:/lustre/scratch117/core/sciops_repository/references/Homo_sapiens/GRCh37_53/all/fasta/Homo_sapiens.GRCh37.dna.all.fa AS:GRCh37 53 M5:6fe9abac455169f50470f5a6b01d0f59 SP:Homo sapiens @SQ SN:HSCHR9_RANDOM_CTG4 LN:187035 UR:/lustre/scratch117/core/sciops_repository/references/Homo_sapiens/GRCh37_53/all/fasta/Homo_sapiens.GRCh37.dna.all.fa AS:GRCh37 53 M5:75e4c8d17cd4addf3917d1703cacaf25 SP:Homo sapiens @SQ SN:HSCHR4_RANDOM_CTG2 LN:189789 UR:/lustre/scratch117/core/sciops_repository/references/Homo_sapiens/GRCh37_53/all/fasta/Homo_sapiens.GRCh37.dna.all.fa AS:GRCh37 53 M5:dbb6e8ece0b5de29da56601613007c2a SP:Homo sapiens @SQ SN:HSCHR4_RANDOM_CTG3 LN:191469 UR:/lustre/scratch117/core/sciops_repository/references/Homo_sapiens/GRCh37_53/all/fasta/Homo_sapiens.GRCh37.dna.all.fa AS:GRCh37 53 M5:6ac8f815bf8e845bb3031b73f812c012 SP:Homo sapiens @SQ SN:HSCHRUN_RANDOM_CTG17 LN:211173 UR:/lustre/scratch117/core/sciops_repository/references/Homo_sapiens/GRCh37_53/all/fasta/Homo_sapiens.GRCh37.dna.all.fa AS:GRCh37 53 M5:63945c3e6962f28ffd469719a747e73c SP:Homo sapiens @SQ SN:HSCHR1_RANDOM_CTG12 LN:547496 UR:/lustre/scratch117/core/sciops_repository/references/Homo_sapiens/GRCh37_53/all/fasta/Homo_sapiens.GRCh37.dna.all.fa AS:GRCh37 53 M5:325ba9e808f669dfeee210fdd7b470ac SP:Homo sapiens @RG ID:24022_8#45 PL:ILLUMINA PU:171010_HS37_24022_B_CBFF0ANXX_8#45 LB:20059730 DS:EGAS00001002611: Regulatory T-cells (Treg) are a major obstacle of efficient cancer immune-surveillance, as they prevent tumor eradication by tumor-antigen (TA)-specific conventional T cells (Tconv), supporting cancer development. Indeed, Treg numbers are increased in tumors and peripheral blood of cancer patients correlating with poor prognosis. Thus, understanding the mechanisms of Treg repertoire formation in cancer patients is of major importance for the design of efficient cancer immunotherapies. In tumor-bearing mice, Treg accumulation results predominantly from the proliferation of pre-existing natural Treg (nTreg) but also from the conversion of tumor-killing Tconv into tumor-promoting induced Treg (iTreg) under suppressive conditions within the tumor-microenvironment. Nevertheless, Treg origin in tumor patients remains largely unknown. This study investigates the existence of TA-specific iTreg in tumor patients and their relevant contribution to the TCR repertoire of the Treg pool in the periphery. A few highly-expanded clonotypes were detected among both Treg and Tconv from cancer patients in contrast to the highly polyclonal populations in healthy individuals. However, no major overlap was observed between Treg and Tconv in the blood of tumor patients and healthy individuals, arguing against the existence of tumor-specific iTreg in the circulation. Interestingly, within tumors of the same patients we detected highly expanded clones not only among Treg but also within an intermediate Activated (Act Tconv) population. Both Tconv and Act Tconv subsets shared dominant highly expanded clonotypes with Treg, shaping of the tumor-infiltrating Treg repertoire but with no effect in Treg from peripheral blood. Taken together, our data support the hypothesis that within the tumor dominant tumor-killing TA-specific Tconv may convert to tumor-promoting iTreg with a major impact on the tumor-infiltrating Treg population. To prove this hypothesis, we need to identify whether two different cells, one of a Treg suppressive profile and one of a non-suppressive tumor-killing Tconv phenotype, share the same TCR and, thus, have a common clonal origin due to peripheral iTreg generation. To this direction, we have already isolated tumor-infiltrating Treg and Tconv from freshly resected tumors and blood of breast cancer patients using single-cell FACS sorting and plan to apply the G&T method that combines total mRNA sequencing and methylation analysis on the same single-cell, as already established by the group of Prof. Thierry Voet (Macaulay et al 2015). 1) This data is part of a pre-publication release. For information on the proper use of pre-publication data shared by the Wellcome Trust Sanger Institute please see http://www.sanger.ac.uk/datasharing/ DT:2017-10-10T01:00:00+0100 PG:SCS SM:4909STDY7100308 CN:SC @PG ID:SCS PN:RTA VN:1.18.66.3 DS:Controlling software on instrument @PG ID:basecalling PN:RTA PP:SCS VN:1.18.66.3 DS:Basecalling Package @PG ID:bambi PN:bambi PP:basecalling VN:0.9.11 DS:Convert Illumina BCL to BAM or SAM file CL:/software/solexa/pkg/bambi/0.9.11/bin/bambi i2b --intensity-dir=/nfs/sf30/ILorHSany_sf30/analysis/171010_HS37_24022_B_CBFF0ANXX/Data/Intensities --basecalls-dir=/nfs/sf30/ILorHSany_sf30/analysis/171010_HS37_24022_B_CBFF0ANXX/Data/Intensities/BaseCalls --lane=8 --platform-unit=171010_HS37_24022_B_CBFF0ANXX_8 --read-group-id=24022_8 --study-name="EGAS00001002611: Regulatory T-cells (Treg) are a major obstacle of efficient cancer immune-surveillance, as they prevent tumor eradication by tumor-antigen (TA)-specific conventional T cells (Tconv), supporting cancer development. Indeed, Treg numbers are increased in tumors and peripheral blood of cancer patients correlating with poor prognosis. Thus, understanding the mechanisms of Treg repertoire formation in cancer patients is of major importance for the design of efficient cancer immunotherapies. In tumor-bearing mice, Treg accumulation results predominantly from the proliferation of pre-existing natural Treg (nTreg) but also from the conversion of tumor-killing Tconv into tumor-promoting induced Treg (iTreg) under suppressive conditions within the tumor-microenvironment. Nevertheless, Treg origin in tumor patients remains largely unknown. This study investigates the existence of TA-specific iTreg in tumor patients and their relevant contribution to the TCR repertoire of the Treg pool in the periphery. A few highly-expanded clonotypes were detected among both Treg and Tconv from cancer patients in contrast to the highly polyclonal populations in healthy individuals. However, no major overlap was observed between Treg and Tconv in the blood of tumor patients and healthy individuals, arguing against the existence of tumor-specific iTreg in the circulation. Interestingly, within tumors of the same patients we detected highly expanded clones not only among Treg but also within an intermediate Activated (Act Tconv) population. Both Tconv and Act Tconv subsets shared dominant highly expanded clonotypes with Treg, shaping of the tumor-infiltrating Treg repertoire but with no effect in Treg from peripheral blood. Taken together, our data support the hypothesis that within the tumor dominant tumor-killing TA-specific Tconv may convert to tumor-promoting iTreg with a major impact on the tumor-infiltrating Treg population. To prove this hypothesis, we need to identify whether two different cells, one of a Treg suppressive profile and one of a non-suppressive tumor-killing Tconv phenotype, share the same TCR and, thus, have a common clonal origin due to peripheral iTreg generation. To this direction, we have already isolated tumor-infiltrating Treg and Tconv from freshly resected tumors and blood of breast cancer patients using single-cell FACS sorting and plan to apply the G&T method that combines total mRNA sequencing and methylation analysis on the same single-cell, as already established by the group of Prof. Thierry Voet (Macaulay et al 2015). 1) This data is part of a pre-publication release. For information on the proper use of pre-publication data shared by the Wellcome Trust Sanger Institute please see http://www.sanger.ac.uk/datasharing/" 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/nfs/sf30/ILorHSany_sf30/analysis/171010_HS37_24022_B_CBFF0ANXX/Data/Intensities/BAM_basecalls_20171015-153502/no_cal/archive/qc/tileviz/24022_8 --region_size 200 --region_mismatch_threshold 0.0160 --region_insertion_threshold 0.0160 --region_deletion_threshold 0.0160 /dev/stdin ; /software/solexa/pkg/pb_calibration/10.28/bin/spatial_filter -a -f -u -l /nfs/sf30/ILorHSany_sf30/analysis/171010_HS37_24022_B_CBFF0ANXX/Data/Intensities/BAM_basecalls_20171015-153502/no_cal/24022_8.bam.filter.stats -F /tmp/i7R0BGLqeV/tee_post_filter_creation:__APPLY_FILTER_OUT___out /dev/stdin @PG ID:samtools PN:samtools PP:spf VN:1.5 CL:/software/solexa/pkg/samtools/1.5/bin/samtools split -f /nfs/sf30/ILorHSany_sf30/analysis/171010_HS37_24022_B_CBFF0ANXX/Data/Intensities/BAM_basecalls_20171015-153502/no_cal/lane8/%!.bam - @PG ID:bamcollate2' PN:bamcollate2 PP:samtools VN:2.0.76 CL:/software/solexa/pkg/biobambam/2.0.76/bin/bamcollate2 collate=1 level=0 @PG ID:bamreset PN:bamreset PP:bamcollate2' VN:2.0.76 CL:/software/solexa/pkg/biobambam/2.0.76/bin/bamreset resetaux=0 level=0 verbose=0 @PG ID:bamadapterclip PN:bamadapterclip PP:bamreset VN:2.0.76 CL:/software/solexa/pkg/biobambam/2.0.76/bin/bamadapterclip verbose=0 level=0 @PG ID:bwa' PN:bwa PP:bamadapterclip VN:0.7.16a-r1181 CL:/software/solexa/pkg/bwa/0.7.16a/bwa mem -t 8 -p -Y -K 100000000 /lustre/scratch117/core/sciops_repository/references/Homo_sapiens/GRCh37_53/all/bwa0_6/Homo_sapiens.GRCh37.dna.all.fa /tmp/NqwCyUtgzn/alntgt_bamtofastq_out @PG ID:scramble' PN:scramble PP:bwa' VN:1.14.9 CL:/software/solexa/pkg/scramble/1.14.9/bin/scramble -0 -I sam -O bam @PG ID:samtools' PN:samtools PP:scramble' VN:1.5 CL:/software/solexa/pkg/samtools/1.5/bin/samtools reheader /tmp/ZpNGJQXbLK/postalntgt_alterSQ_headerSQfix_out /tmp/Ai8rtncJWo/postalntgt_mbuffer_headerSQfix_out @PG ID:bam12split PN:bam12split PP:samtools' VN:2.0.76 CL:/software/solexa/pkg/biobambam/2.0.76/bin/bam12split verbose=0 level=0 @PG ID:bamsormadup PN:bamsormadup PP:bam12split VN:2.0.76 CL:/software/solexa/pkg/biobambam/2.0.76/bin/bamsormadup threads=8 SO=queryname level=0 @PG ID:bam12auxmerge' PN:bam12auxmerge PP:bamsormadup VN:2.0.76 CL:/software/solexa/pkg/biobambam/2.0.76/bin/bam12auxmerge level=0 rankstrip=1 ranksplit=0 zztoname=0 clipreinsert=1 /tmp/e0x76h4lBA/amp_bamadapterclip_pre_auxmerge_out @PG ID:AlignmentFilter PN:AlignmentFilter PP:bam12auxmerge' DS:Give a list of SAM/BAM files with the same set of records and in the same order but aligned with different references, split reads into different files according to alignments. You have option to put unaligned reads into one of output files or a separate file VN:V1.19 CL:uk.ac.sanger.npg.picard.AlignmentFilter INPUT_ALIGNMENT=[./initial_phix_aln_24022_8#45.bam, /tmp/wfmhWLaKFM/postalntgt_bam12auxmerge_out] OUTPUT_ALIGNMENT=[/tmp/KaKmuKhF7u/alignment_filter:__PHIX_BAM_OUT___out, /dev/stdout] METRICS_FILE=24022_8#45.bam_alignment_filter_metrics.json VERBOSITY=INFO QUIET=false VALIDATION_STRINGENCY=SILENT COMPRESSION_LEVEL=0 MAX_RECORDS_IN_RAM=500000 CREATE_INDEX=false CREATE_MD5_FILE=false @PG ID:bamsormadup' PN:bamsormadup CL:/software/solexa/pkg/biobambam/2.0.76/bin/bamsormadup threads=8 SO=coordinate level=0 verbose=0 fixmate=1 adddupmarksupport=1 tmpfile=/nfs/sf30/ILorHSany_sf30/analysis/171010_HS37_24022_B_CBFF0ANXX/Data/Intensities/BAM_basecalls_20171015-153502/no_cal/archive/lane8/bsfopt_24022_8#45.tmp PP:AlignmentFilter VN:2.0.76 @PG ID:bamstreamingmarkduplicates PN:bamstreamingmarkduplicates CL:/software/solexa/pkg/biobambam/2.0.76/bin/bamstreamingmarkduplicates level=0 verbose=0 tmpfile=/nfs/sf30/ILorHSany_sf30/analysis/171010_HS37_24022_B_CBFF0ANXX/Data/Intensities/BAM_basecalls_20171015-153502/no_cal/archive/lane8/bmdfopt_24022_8#45.tmp M=/nfs/sf30/ILorHSany_sf30/analysis/171010_HS37_24022_B_CBFF0ANXX/Data/Intensities/BAM_basecalls_20171015-153502/no_cal/archive/lane8/24022_8#45.markdups_metrics.txt PP:bamsormadup' VN:2.0.76 @PG ID:scramble.3 PN:scramble PP:bamstreamingmarkduplicates VN:1.14.9 CL:/software/solexa/pkg/scramble/1.14.9/bin/scramble -t 3 -7 -I bam -O cram -r /lustre/scratch117/core/sciops_repository/references/Homo_sapiens/GRCh37_53/all/fasta/Homo_sapiens.GRCh37.dna.all.fa @CO TY:checksum ST:all PA:all HA:crc32prod CO:1591906 BS:5421b181 NS:591d8437 SQ:324feb0d ST:BC,FI,QT,RT,TC:5c0c8c62 @CO TY:checksum ST:all PA:pass HA:crc32prod CO:1591906 BS:5421b181 NS:591d8437 SQ:324feb0d ST:BC,FI,QT,RT,TC:5c0c8c62 @CO TY:checksum ST: PA:all HA:crc32prod CO:0 BS:1 NS:1 SQ:1 ST:BC,FI,QT,RT,TC:1 @CO TY:checksum ST: PA:pass HA:crc32prod CO:0 BS:1 NS:1 SQ:1 ST:BC,FI,QT,RT,TC:1 @CO TY:checksum ST:24022_8#45 PA:all HA:crc32prod CO:1591906 BS:5421b181 NS:591d8437 SQ:324feb0d ST:BC,FI,QT,RT,TC:5c0c8c62 @CO TY:checksum ST:24022_8#45 PA:pass HA:crc32prod CO:1591906 BS:5421b181 NS:591d8437 SQ:324feb0d ST:BC,FI,QT,RT,TC:5c0c8c62