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| Status |
Public on Nov 21, 2018 |
| Title |
P07_TPRstim_CD8_beta |
| Sample type |
SRA |
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| Source name |
peripheral blood mononuclear cells
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| Organism |
Homo sapiens |
| Characteristics |
cmv status: positive
cmv peptide: TPRVTGGGAM
cell subpopulation: CD8-enriched
culture time (days): 10
productive reads: 5359364
clonotype count: 119764
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| Treatment protocol |
Libraries were prepared from ex vivo peripheral blood mononuclear cells (labelled "unstim") or after 10-day stimulation with one out of four immunogenic, HLA-matched CMV peptides
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| Growth protocol |
Four different CMV-derived peptides, namely CRV (HLA-C*07:02), FRC (HLA-C*07:02), RPH (HLA-B*07:02), and TPR (HLA-B*07:02), were separately used to stimulate and selectively expand virus- specific T cells from CMV-positive donors P01-P08 in the presence of IL-2.
At 6±1 days, the cells of each well were resuspended, distributed to two wells, and 1 ml of fresh culture medium supplemented with IL-2 was added to each well. Cells were harvested at day 10 of culture.
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| Extracted molecule |
total RNA |
| Extraction protocol |
RNA extraction was performed on bulk cells (either unsorted or CD8-enriched using MACS) with the Qiagen RNeasy kit.
1 μg RNA per sample was reversely transcribed to cDNA with the QuantiTect Reverse Transcription Kit (Qiagen) using a primer designed to target both the Cβ1 and Cβ2 regions of the TCR RNA. cDNA was amplified in two subsequent PCRs using Pfu DNA polymerase (Thermo Scientific).
The first PCR was a multiplex PCR comprising 42 distinct forward primers that bind to the Vβ region and cover all possible human TCR Vβ segments, and a reverse primer that anneals to the Cβ1 and Cβ2 regions. Forward and reverse primers carried sequences complementary to Illumina Read 2 and Read 1 primer sequences.
In the second PCR, index primers (NEBNext Multiplex Oligos for Illumina; New England BioLabs) were used to attach barcodes and the i5 and i7 adapters for binding to the Illumina flow cell (Supplementary Fig. 2).
After each PCR step, the PCR product was purified using Agencourt AMPure XP magnetic beads (Beckman Coulter). The correct length and amount of the PCR products for sequencing was confirmed using the Agilent DNA 1000 Kit and the Bioanalyzer 2100 (Agilent).
To enhance the nucleotide diversity of TCR reads facilitating cluster recognition and avoiding artifacts in q-score calibration and base calling, three different forms of the reverse primer, with 0, 1 or 2 arbitrary nucleotides between the Cβ-binding sequence and the Illumina Read 1 sequence were used.
All primers were used in equimolar amounts (a total of 10μM; for primer sequences see Supplementary Table 1). The first PCR consisted of only 10 amplification cycles to minimize PCR amplification bias.
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| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 1500 |
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| Data processing |
Demultiplex with Je-Demultiplex-Illu (Galaxy Version 1.2.1.), with 0 or 1 barcode tolerance for single and dual indexed samples, respectively.
Quality filter with custom fastqFilter software (Built under Galaxy Version 1.0 by LAFUGA), minimum read length after trimming 100 nt, quality threshold 20
TCR clonotype alignment and assembly with MiXCR (Version 1.8.1.) run on Java (Version 1.8.0_60). Command lines: mixcr align --loci TRB --species hsa --report ALIGN_REPORT_samplename.txt -OminSumScore=258 -OvParameters.geneFeatureToAlign={FR3End\(-26\):FR3End\(+3\)} -OcParameters.geneFeatureToAlign={CBegin:CBegin\(+20\)} -OjParameters.geneFeatureToAlign={FR4Begin\(-3\):FR4End} -OjParameters.parameters.floatingRightBound=false -OvParameters.parameters.minAlignmentLength=25 -OjParameters.parameters.minAlignmentLength=25 -OvParameters.parameters.absoluteMinScore=115 -OjParameters.parameters.absoluteMinScore=125 -OvParameters.parameters.relativeMinScore=0.8 -OjParameters.parameters.relativeMinScore=0.8 -OcParameters.parameters.relativeMinScore=0.8 -OvParameters.parameters.maxHits=5 -OjParameters.parameters.maxHits=5 -OvParameters.parameters.scoring.gapPenalty=-28 -OjParameters.parameters.scoring.gapPenalty=-28 File_with_Galaxy_forward_reads.fastqsanger File_with_Galaxy_reverse_reads.fastqsanger align_samplename.vdjca mixcr exportAlignmentsPretty --skip 10000 --limit 50 align_samplename.vdjca pretty_alignment_samplename.txt mixcr assemble --report ASSEBMBLE_REPORT_samplename.txt -OassemblingFeatures=[CDR3] -OminimalClonalSequenceLength=6 -OqualityAggregationType=Average -ObadQualityThreshold=20 -OmaxBadPointsPercent=0 -OaddReadsCountOnClustering=true -OcloneClusteringParameters.searchDepth=2 -OcloneClusteringParameters.searchParameters=oneMismatch -OcloneClusteringParameters.clusteringFilter.specificMutationProbability=1E-4 align_samplename.vdjca clones_samplename.clns mixcr exportClones --filter-out-of-frames --filter-stops -count -fraction -sequence -aaFeature CDR3 -vHit -vHitsWithScore -jHit -jHitsWithScore -dHit -cHit -lengthOf CDR3 -quality -minFeatureQuality CDR3 clones_samplename.clns clones_samplename.txt Replace "samplename" with the name of the sample and "input" with the forward and reverse read file names downloaded from Galaxy
Reformatting MiXCR output in R (Version 3.4.2.) with a custom Script written in R Studio (Version 1.1.383): Remove irrelevant columns, add sample metadata, replace V gene names by V gene group names in cases where distinction of closely related V genes is not possible
Supplementary_files_format_and_content: tab-delimited .txt file containing TCR clonotype data and sample metadata.
Supplementary_files_format_and_content: Columns: V.gene | CDR3.sequence (translated CDR3 amino acid sequence) | J.gene | Read.count (Reads per clonotype) | Clone.fraction (Proportion of reads on all sample reads) | CDR3.nt.seq | CDR3.nt.length | Donor | Treatment | Cell.type | Serostatus | HLA.class.I (Donor HLA type)
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| Submission date |
May 25, 2018 |
| Last update date |
Nov 21, 2018 |
| Contact name |
Andreas Moosmann |
| E-mail(s) |
andreas.moosmann@helmholtz-muenchen.de
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| Phone |
+49 89 3187 1202
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| Organization name |
Helmholtz Center Munich
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| Department |
AGV
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| Street address |
Marchioninistr. 25
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| City |
Munich |
| ZIP/Postal code |
81377 |
| Country |
Germany |
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| Platform ID |
GPL18460 |
| Series (1) |
| GSE114931 |
Antigen-specific T-cell receptor signatures of cytomegalovirus infection |
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| Relations |
| BioSample |
SAMN09261100 |
| SRA |
SRX4125958 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3155124_P07_TPRstim_CD8_beta.txt.gz |
1.8 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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