RNA-Seq
Here we will discuss how to extract TCR repertoire from RNA-Seq data. In this particular case we extract only TCR data because only T-cells were used in cDNA library preparation, but this protocol with slight adjustments, that will be mentioned further, is applicable to BCR data also.
Data libraries
This tutorial uses the data from the following publication: Calibration of cell-intrinsic interleukin-2 response thresholds guides design of a regulatory T cell biased agonist. Glassman CR et al., Elife 2021 May 18;10. doi: 10.7554/eLife.65777
Tregs were isolated from B6-Foxp3EGFP mice using CD4+ T cell isolation kit followed by fluorescence-activated cell sorting (FACS) for GFP expression to isolate Tregs. CD8+ T cells were isolated from C57BL/6J mice using CD8+ T cell isolation kit. Isolated cells were stimulated with 200 nM IL-2 or IL-2-REH and total RNA was extracted. 500 ng of RNA was used for RNA-Seq library preparation with the Kapa mRNA HyperPrep Kit (KK8580, Kapa Biosystems). DNA fragments between 250 bp and 400 bp were recovered, purified, and sequenced on an Illumina HiSeq3000 system (50 bp, single end).
In this publication authors did not study immune repertoires. This is also an example of how MiXCR can retrieve data from experiments that were nor originally designed for obtaining TCR or BCR repertoires.
On the scheme bellow you can see structure of cDNA library. UMI is located in the first 12 bp of R2.
All data may be downloaded directly from SRA (PRJEB44566) using e.g. SRA Explorer.
Use aria2c for efficient download of the full dataset with the proper filenames:
download.sh
mkdir -p raw
aria2c -c -s 16 -x 16 -k 1M -j 8 -i download-list.txt
download-list.txt
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR132/028/SRR13228528/SRR13228528.fastq.gz
out=raw/CD8T_REH_4h_rep1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR132/010/SRR13228510/SRR13228510.fastq.gz
out=raw/Treg_IL2_1.5h_rep1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR132/027/SRR13228527/SRR13228527.fastq.gz
out=raw/CD8T_IL2_4h_rep1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR132/016/SRR13228516/SRR13228516.fastq.gz
out=raw/Treg_REH_1.5h_rep2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR132/009/SRR13228509/SRR13228509.fastq.gz
out=raw/Treg_0h_rep1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR132/015/SRR13228515/SRR13228515.fastq.gz
out=raw/Treg_IL2_1.5h_rep2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR132/017/SRR13228517/SRR13228517.fastq.gz
out=raw/Treg_IL2_4h_rep2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR132/011/SRR13228511/SRR13228511.fastq.gz
out=raw/Treg_REH_1.5h_rep1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR132/026/SRR13228526/SRR13228526.fastq.gz
out=raw/CD8T_REH_1.5h_rep1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR132/012/SRR13228512/SRR13228512.fastq.gz
out=raw/Treg_IL2_4h_rep1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR132/025/SRR13228525/SRR13228525.fastq.gz
out=raw/CD8T_IL2_1.5h_rep1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR132/013/SRR13228513/SRR13228513.fastq.gz
out=raw/Treg_REH_4h_rep1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR132/018/SRR13228518/SRR13228518.fastq.gz
out=raw/Treg_REH_4h_rep2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR132/024/SRR13228524/SRR13228524.fastq.gz
out=raw/CD8T_0h_rep1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR132/023/SRR13228523/SRR13228523.fastq.gz
out=raw/Treg_REH_4h_rep3.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR132/014/SRR13228514/SRR13228514.fastq.gz
out=raw/Treg_0h_rep2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR132/022/SRR13228522/SRR13228522.fastq.gz
out=raw/Treg_IL2_4h_rep3.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR132/019/SRR13228519/SRR13228519.fastq.gz
out=raw/Treg_0h_rep3.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR132/021/SRR13228521/SRR13228521.fastq.gz
out=raw/Treg_REH_1.5h_rep3.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR132/020/SRR13228520/SRR13228520.fastq.gz
out=raw/Treg_IL2_1.5h_rep3.fastq.gz
Upstream analysis
The easiest way to obtain results from this type of data is to use mixcr analyze rna-seq command:
MiXCR has a dedicated preset for RNA-seq data, thus running the pipeline is as easy as:
mixcr analyze rna-seq \
--species hsa \
raw/CD8T_REH_4h_rep1.fastq.gz \
results/CD8T_REH_4h_rep1
--species mmu- is a mix-in that specifies the name of the species. mmu for Mus Musculus in this case.
Running the command above will generate the following files:
> ls results/
# human-readable reports
CD8T_REH_4h_rep1.report
# raw alignments (highly compressed binary file)
CD8T_REH_4h_rep1.vdjca
# files for each mixcr assemblePartial
CD8T_REH_4h_rep1.passembled.0.vdjca
CD8T_REH_4h_rep1.passembled.1.vdjca
# `mixcr extend` outputfile with CDR3 region extended alignments
CD8T_REH_4h_rep1.extended.vdjca
# A binary file wich has all the information on the clonotypes
CD8T_REH_4h_rep1.clns
# TRA, TRB, TRG and TRD CDR3 clonotypes exported in tab-delimited txt
CD8T_REH_4h_rep1.clonotypes.TRA.tsv
CD8T_REH_4h_rep1.clonotypes.TRB.tsv
CD8T_REH_4h_rep1.clonotypes.TRG.tsv
CD8T_REH_4h_rep1.clonotypes.TRD.tsv
CD8T_REH_4h_rep1.clonotypes.TRAD.tsv
While .clns file holds all data and is used for downstream analysis using mixcr postanalisis, the output .txt clonotype table will contain exhaustive information about each clonotype as well:
See first 100 records from FebControl1.clones.IGH.tsv clonotype table
| cloneId | cloneCount | cloneFraction | targetSequences | targetQualities | allVHitsWithScore | allDHitsWithScore | allJHitsWithScore | allCHitsWithScore | allVAlignments | allDAlignments | allJAlignments | allCAlignments | nSeqFR1 | minQualFR1 | nSeqCDR1 | minQualCDR1 | nSeqFR2 | minQualFR2 | nSeqCDR2 | minQualCDR2 | nSeqFR3 | minQualFR3 | nSeqCDR3 | minQualCDR3 | nSeqFR4 | minQualFR4 | aaSeqFR1 | aaSeqCDR1 | aaSeqFR2 | aaSeqCDR2 | aaSeqFR3 | aaSeqCDR3 | aaSeqFR4 | refPoints |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 0 | 7 | 0.00798176 | TGTTTGCAAAGTGATAACATGCCATTCACGTTC | JJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJ | IGKV17-127*00(159.3) | nan | IGKJ4*00(90.7) | nan | 418|441|464|0|23||115.0 | nan | 20|30|58|23|33||50.0 | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | TGTTTGCAAAGTGATAACATGCCATTCACGTTC | 41 | nan | nan | nan | nan | nan | nan | nan | CLQSDNMPFTF | nan | :::::::::0:-3:23:::::23:0:33::: |
| 1 | 7 | 0.00798176 | TGTCAGCAAAGTAATGAGGATCCGTACACGTTC | JJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJ | IGKV3-400(160.7),IGKV3-500(160.7) | nan | IGKJ2*00(84) | nan | 433|456|479|0|23||115.0;433|456|479|0|23||115.0 | nan | 21|31|59|23|33||50.0 | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | TGTCAGCAAAGTAATGAGGATCCGTACACGTTC | 41 | nan | nan | nan | nan | nan | nan | nan | CQQSNEDPYTF | nan | :::::::::0:-3:23:::::23:-1:33::: |
| 2 | 6 | 0.00684151 | TGCCAGCAGTGGAGTAGTTACCCACGGACGTTC | JJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJ | IGKV4-55*00(160) | nan | IGKJ1*00(87.3) | nan | 424|449|472|0|25||125.0 | nan | 22|30|58|25|33||40.0 | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | TGCCAGCAGTGGAGTAGTTACCCACGGACGTTC | 41 | nan | nan | nan | nan | nan | nan | nan | CQQWSSYPRTF | nan | :::::::::0:-3:25:::::25:-2:33::: |
| 3 | 5 | 0.00570125 | TGTGCCAGCAGCTTAGGGGACTCCGACTACACCTTC | JJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJ | TRBV3*00(110) | nan | TRBJ1-2*00(125) | nan | 430|446|467|0|16||80.0 | nan | 23|40|68|19|36||85.0 | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | TGTGCCAGCAGCTTAGGGGACTCCGACTACACCTTC | 41 | nan | nan | nan | nan | nan | nan | nan | CASSLGDSDYTF | nan | :::::::::0:-1:16:::::19:-3:36::: |
| 4 | 5 | 0.00570125 | TGCTCTCAAAGTACACATGTTCCGTGGACGTTC | JJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJ | IGKV1-110*00(176.8) | nan | IGKJ1*00(75) | nan | 433|456|477|0|23||115.0 | nan | 19|30|58|22|33||55.0 | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | TGCTCTCAAAGTACACATGTTCCGTGGACGTTC | 41 | nan | nan | nan | nan | nan | nan | nan | CSQSTHVPWTF | nan | :::::::::0:-1:23:::::22:1:33::: |
| 5 | 5 | 0.00570125 | TGCCAGCAGTGGAGTAGTTACCCTTTCACGTTC | JJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJ | IGKV4-55*00(166) | nan | IGKJ4*00(79) | nan | 424|447|472|0|23||115.0 | nan | 21|30|58|24|33||45.0 | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | TGCCAGCAGTGGAGTAGTTACCCTTTCACGTTC | 41 | nan | nan | nan | nan | nan | nan | nan | CQQWSSYPFTF | nan | :::::::::0:-5:23:::::24:-1:33::: |
| 6 | 4 | 0.004561 | TGTCAACAGTGGAGTAGTTACCCACTCACGTTC | JJJJJJJNNNNNNNNNNNNNNNNNNNNNNNNNN | IGKV4-53*00(215) | nan | IGKJ2*00(137) | nan | 430|458|478|0|28||140.0 | nan | 24|31|59|26|33||35.0 | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | TGTCAACAGTGGAGTAGTTACCCACTCACGTTC | 41 | nan | nan | nan | nan | nan | nan | nan | CQQWSSYPLTF | nan | :::::::::0:0:28:::::26:-4:33::: |
| 7 | 4 | 0.004561 | TGTGCTCTGAGTGACTAGCAACATGGGCTACAAACTTACCTTC | JJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJFFF | TRAV12D-300(95),TRAV12N-100(95),TRAV12N-300(95),TRAV6-500(95),TRAV6-7-DV9*00(95) | nan | TRAJ9*00(155) | nan | 433|447|467|0|14||70.0;427|441|461|0|14||70.0;433|447|467|0|14||70.0;424|438|458|0|14||70.0;424|438|458|0|14||70.0 | nan | 18|47|78|14|43||145.0 | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | TGTGCTCTGAGTGACTAGCAACATGGGCTACAAACTTACCTTC | 37 | nan | nan | nan | nan | nan | nan | nan | CALSD*Q_MGYKLTF | nan | :::::::::0:0:14:::::14:2:43::: |
| 8 | 4 | 0.004561 | TGTGCCAGCAGTTTAGACTTAAACACCTTGTACTTT | JJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJ | TRBV15*00(115) | nan | TRBJ2-4*00(99) | nan | 430|446|467|0|16||80.0 | nan | 25|41|69|20|36||80.0 | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | TGTGCCAGCAGTTTAGACTTAAACACCTTGTACTTT | 41 | nan | nan | nan | nan | nan | nan | nan | CASSLDLNTLYF | nan | :::::::::0:-1:16:::::20:-5:36::: |
| 9 | 4 | 0.004561 | TGTGCCAGCAGCCTCTATGAACAGTACTTC | JJJJJJJJJJJJJJJJJJJJJJJJJJJJJJ | TRBV14*00(115) | nan | TRBJ2-7*00(120) | nan | 430|441|466|0|11||55.0 | nan | 23|39|67|14|30||80.0 | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | TGTGCCAGCAGCCTCTATGAACAGTACTTC | 41 | nan | nan | nan | nan | nan | nan | nan | CASSLYEQYF | nan | :::::::::0:-5:11:::::14:-3:30::: |
| 10 | 4 | 0.004561 | TGTGCAGTGGCAAATTCTGGGACTTACCAGAGGTTT | JJJJJJJJJJJJJJJJJJJJJJJJJFFFAA?????? | TRAV7-300(141),TRAV7D-300(141),TRAV7D-500(141),TRAV7N-500(141) | nan | TRAJ13*00(105) | nan | 424|433|457|0|9||45.0;424|433|455|0|9||45.0;424|433|457|0|9||45.0;424|433|457|0|9||45.0 | nan | 19|46|77|9|36||135.0 | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | TGTGCAGTGGCAAATTCTGGGACTTACCAGAGGTTT | 30 | nan | nan | nan | nan | nan | nan | nan | CAVANSGTYQRF | nan | :::::::::0:-4:9:::::9:1:36::: |
| 11 | 4 | 0.004561 | TGTGCAGGACCCTACCAGGGAGGCAGAGCTCTGATATTT | JJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJFFFA | TRAV7-300(85),TRAV7D-200(85),TRAV7D-300(85),TRAV7D-500(85),TRAV7N-5*00(85) | nan | TRAJ15*00(150) | nan | 424|431|457|0|7||35.0;424|431|457|0|7||35.0;424|431|455|0|7||35.0;424|431|457|0|7||35.0;424|431|457|0|7||35.0 | nan | 20|49|80|10|39||145.0 | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | TGTGCAGGACCCTACCAGGGAGGCAGAGCTCTGATATTT | 32 | nan | nan | nan | nan | nan | nan | nan | CAGPYQGGRALIF | nan | :::::::::0:-6:7:::::10:0:39::: |
| 12 | 4 | 0.004561 | TGCTGGCAAGGTACACATTTTCCGTGGACGTTC | JJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJ | IGKV1-135*00(157.5) | nan | IGKJ1*00(93.5) | nan | 436|459|482|0|23||115.0 | nan | 19|30|58|22|33||55.0 | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | TGCTGGCAAGGTACACATTTTCCGTGGACGTTC | 41 | nan | nan | nan | nan | nan | nan | nan | CWQGTHFPWTF | nan | :::::::::0:-3:23:::::22:1:33::: |
| 13 | 3 | 0.00342075 | TGTCAGCAAAGTAATGAGGATCCTCGGACGTTC | JJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJ | IGKV3-400(134),IGKV3-500(134) | nan | IGKJ1*00(100) | nan | 433|458|479|0|25||125.0;433|458|479|0|25||125.0 | nan | 22|30|58|25|33||40.0 | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | TGTCAGCAAAGTAATGAGGATCCTCGGACGTTC | 41 | nan | nan | nan | nan | nan | nan | nan | CQQSNEDPRTF | nan | :::::::::0:-1:25:::::25:-2:33::: |
| 14 | 3 | 0.00342075 | TGTCAACATCATTATGGTACTCCGTACACGTTC | JJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJ | IGKV12-44*00(161.7) | nan | IGKJ2*00(83) | nan | 421|444|467|0|23||115.0 | nan | 21|31|59|23|33||50.0 | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | TGTCAACATCATTATGGTACTCCGTACACGTTC | 41 | nan | nan | nan | nan | nan | nan | nan | CQHHYGTPYTF | nan | :::::::::0:-3:23:::::23:-1:33::: |
| 15 | 3 | 0.00342075 | TGTGCTCCTCCAAGAATTATGGGAGCAGTGGCAACAAGCTCATCTTT | JJJJJJJJJJJJJJJJJJJJJJJJJJJFFFAA??????????????? | TRAV13D-300(120),TRAV13N-100(120),TRAV13N-200(120),TRAV13N-300(120),TRAV13N-4*00(120) | nan | TRAJ32*00(95) | nan | 424|430|457|0|6||30.0;418|424|451|0|6||30.0;418|424|451|0|6||30.0;424|430|457|0|6||30.0;418|424|451|0|6||30.0 | nan | 21|55|86|13|47||170.0 | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | TGTGCTCCTCCAAGAATTATGGGAGCAGTGGCAACAAGCTCATCTTT | 30 | nan | nan | nan | nan | nan | nan | nan | CAPPRIMG_SGNKLIF | nan | :::::::::0:-7:6:::::13:-1:47::: |
| 16 | 3 | 0.00342075 | TGTGCTGCAACCCGGAATGCAGGTGCCAAGCTCACATTC | JJJJJJJJJJJJJJJJJJJJJJJJJJJJJJFFFAA???? | TRAV5-400(125),TRAV5D-400(125),TRAV5N-4*00(125) | nan | TRAJ39*00(100) | nan | 430|440|463|0|10||50.0;430|440|463|0|10||50.0;430|440|463|0|10||50.0 | nan | 28|52|83|15|39||120.0 | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | TGTGCTGCAACCCGGAATGCAGGTGCCAAGCTCACATTC | 30 | nan | nan | nan | nan | nan | nan | nan | CAATRNAGAKLTF | nan | :::::::::0:-3:10:::::15:-8:39::: |
| 17 | 3 | 0.00342075 | TGTGCTGCAAGTATCACGGGTTACCAGAACTTCTATTTT | JJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJ | TRAV5-400(80),TRAV5D-400(80),TRAV5N-4*00(80) | nan | TRAJ49*00(160) | nan | 430|442|463|0|12||60.0;430|442|463|0|12||60.0;430|442|463|0|12||60.0 | nan | 23|48|79|14|39||125.0 | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | TGTGCTGCAAGTATCACGGGTTACCAGAACTTCTATTTT | 41 | nan | nan | nan | nan | nan | nan | nan | CAASITGYQNFYF | nan | :::::::::0:-1:12:::::14:-3:39::: |
| 18 | 3 | 0.00342075 | TGTGCTACTGATGCGACTGGAGGCTATAAAGTGGTCTTT | JJJJJJJJJJJJJJJJJJJJJJJJJFFFAA????????? | TRAV8-100(170),TRAV8D-100(170) | nan | TRAJ12*00(80) | nan | 415|429|448|0|14||70.0;424|438|457|0|14||70.0 | nan | 23|48|79|14|39||125.0 | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | TGTGCTACTGATGCGACTGGAGGCTATAAAGTGGTCTTT | 30 | nan | nan | nan | nan | nan | nan | nan | CATDATGGYKVVF | nan | :::::::::0:1:14:::::14:-3:39::: |
| 19 | 3 | 0.00342075 | TGTGCTACTGATACTAATTACAACGTGCTTTACTTC | JJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJFFF | TRAV8-100(120),TRAV8-200(120),TRAV8D-100(120),TRAV8N-200(120) | nan | TRAJ21*00(125) | nan | 415|427|448|0|12||60.0;424|436|457|0|12||60.0;424|436|457|0|12||60.0;424|436|457|0|12||60.0 | nan | 23|46|77|13|36||115.0 | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | TGTGCTACTGATACTAATTACAACGTGCTTTACTTC | 37 | nan | nan | nan | nan | nan | nan | nan | CATDTNYNVLYF | nan | :::::::::0:-1:12:::::13:-3:36::: |
| 20 | 3 | 0.00342075 | TGTGCTAGCAGTAGCTCACAACCAAGACACCCAGTACTTT | JJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJF | TRBV17*00(100) | nan | TRBJ2-5*00(125) | nan | 427|441|464|0|14||70.0 | nan | 20|41|69|19|40||105.0 | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | TGTGCTAGCAGTAGCTCACAACCAAGACACCCAGTACTTT | 37 | nan | nan | nan | nan | nan | nan | nan | CASSSSQ_QDTQYF | nan | :::::::::0:-3:14:::::19:0:40::: |
| 21 | 3 | 0.00342075 | TGTGCCTGGAGTCCACAGGGGAACCAAGACACCCAGTACTTT | JJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJFFF | TRBV31*00(95) | TRBD1*00(35) | TRBJ2-5*00(115) | nan | 421|434|454|0|13||65.0 | 15|22|36|14|21||35.0 | 20|41|69|21|42||105.0 | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | TGTGCCTGGAGTCCACAGGGGAACCAAGACACCCAGTACTTT | 37 | nan | nan | nan | nan | nan | nan | nan | CAWSPQGNQDTQYF | nan | :::::::::0:0:13:14:-3:-2:21:21:0:42::: |
| 22 | 3 | 0.00342075 | TGTGCCAGCTCTCTCGGGACGAACTATGCTGAGCAGTTCTTC | JJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJFFF | TRBV12-100(110),TRBV12-200(110) | nan | TRBJ2-1*00(115) | nan | 457|473|492|0|16||80.0;457|473|492|0|16||80.0 | nan | 21|42|70|21|42||105.0 | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | TGTGCCAGCTCTCTCGGGACGAACTATGCTGAGCAGTTCTTC | 37 | nan | nan | nan | nan | nan | nan | nan | CASSLGTNYAEQFF | nan | :::::::::0:1:16:::::21:-1:42::: |
| 23 | 3 | 0.00342075 | TGTGCCAGCTCTCTCGACCACTCCTATGAACAGTACTTC | JJJJJJJJJJJJJJJJJJJFJJJJJJJJJJJJJJJJJJJ | TRBV12-100(100),TRBV12-200(100) | nan | TRBJ2-7*00(135) | nan | 457|474|492|0|17||85.0;457|474|492|0|17||85.0 | nan | 20|39|67|20|39||95.0 | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | TGTGCCAGCTCTCTCGACCACTCCTATGAACAGTACTTC | 37 | nan | nan | nan | nan | nan | nan | nan | CASSLDHSYEQYF | nan | :::::::::0:2:17:::::20:0:39::: |
| 24 | 3 | 0.00342075 | TGTGCCAGCTCTCTCGACACTAACTATGCTGAGCAGTTCTTC | JJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJFFFA | TRBV12-100(120),TRBV12-200(120) | nan | TRBJ2-1*00(115) | nan | 457|474|492|0|17||85.0;457|474|492|0|17||85.0 | nan | 20|42|70|20|42||110.0 | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | TGTGCCAGCTCTCTCGACACTAACTATGCTGAGCAGTTCTTC | 32 | nan | nan | nan | nan | nan | nan | nan | CASSLDTNYAEQFF | nan | :::::::::0:2:17:::::20:0:42::: |
| 25 | 3 | 0.00342075 | TGTGCCAGCTCTCCTCGACAACAAGACACCCAGTACTTT | JJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJ | TRBV12-1*00(80) | nan | TRBJ2-5*00(130) | nan | 457|470|492|0|13||65.0 | nan | 23|41|69|21|39||90.0 | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | TGTGCCAGCTCTCCTCGACAACAAGACACCCAGTACTTT | 41 | nan | nan | nan | nan | nan | nan | nan | CASSPRQQDTQYF | nan | :::::::::0:-2:13:::::21:-3:39::: |
| 26 | 3 | 0.00342075 | TGTGCCAGCTCTCATAACAACCAGGCTCCGCTTTTT | JJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJ | TRBV12-100(90),TRBV12-200(90) | nan | TRBJ1-5*00(160) | nan | 457|470|492|0|13||65.0;457|470|492|0|13||65.0 | nan | 19|42|70|13|36||115.0 | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | TGTGCCAGCTCTCATAACAACCAGGCTCCGCTTTTT | 41 | nan | nan | nan | nan | nan | nan | nan | CASSHNNQAPLF | nan | :::::::::0:-2:13:::::13:1:36::: |
| 27 | 3 | 0.00342075 | TGTGCCAGCTCCACCGGGCCCGACTCCGACTACACCTTC | JJJJJJFJJJJJFJJJJJJJJJJJJJJJJJJJJJJJFJJ | TRBV18*00(80) | TRBD100(25),TRBD200(25) | TRBJ1-2*00(120) | nan | 429|441|466|0|12||60.0 | 10|15|36|13|18||25.0;12|17|42|13|18||25.0 | 23|40|68|22|39||85.0 | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | TGTGCCAGCTCCACCGGGCCCGACTCCGACTACACCTTC | 37 | nan | nan | nan | nan | nan | nan | nan | CASSTGPDSDYTF | nan | :::::::::0:-5:12:13:2:-9:18:22:-3:39::: |
| 28 | 3 | 0.00342075 | TGTGCCAGCCCAGTTTCTGGAAATACGCTCTATTTT | JJJJJJJJJJJJJJJJJJJJJJJFFFAA???????? | TRBV13-3*00(155) | TRBD2*00(25) | TRBJ1-3*00(70) | nan | 424|433|460|0|9||45.0 | 6|11|42|9|14||25.0 | 20|42|70|14|36||110.0 | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | TGTGCCAGCCCAGTTTCTGGAAATACGCTCTATTTT | 30 | nan | nan | nan | nan | nan | nan | nan | CASPVSGNTLYF | nan | :::::::::0:-7:9:9:8:-17:14:14:0:36::: |
| 29 | 3 | 0.00342075 | TGTGCCAGCGGTGATCGGGACAGGGAGGAAAGATTATTTTTC | JJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJFFFAA? | TRBV13-2*00(120) | TRBD1*00(50) | TRBJ1-4*00(70) | nan | 424|439|460|0|15||75.0 | 11|21|36|15|25||50.0 | 28|43|71|27|42||75.0 | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | TGTGCCAGCGGTGATCGGGACAGGGAGGAAAGATTATTTTTC | 30 | nan | nan | nan | nan | nan | nan | nan | CASGDRDREERLFF | nan | :::::::::0:-1:15:15:1:-3:25:27:-8:42::: |
| 30 | 3 | 0.00342075 | TGTGCCAGCGGTGATGTGGGGGCTTCCTATGAACAGTACTTC | JJFJJJFJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJ | TRBV13-2*00(80) | TRBD100(30),TRBD200(30) | TRBJ2-7*00(130) | nan | 424|440|460|0|16||80.0 | 18|24|36|17|23||30.0;19|25|42|16|22||30.0 | 21|39|67|24|42||90.0 | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | TGTGCCAGCGGTGATGTGGGGGCTTCCTATGAACAGTACTTC | 37 | nan | nan | nan | nan | nan | nan | nan | CASGDVGASYEQYF | nan | :::::::::0:0:16:17:-6:0:23:24 42::: |
| 31 | 3 | 0.00342075 | TGTGCCAGCAGTCCTAACAAGGCTGAGCAGTTCTTC | JJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJFFFA | TRBV13-1*00(125) | nan | TRBJ2-1*00(83) | nan | 421|433|457|0|12||60.0 | nan | 20|42|70|14|36|ST24AST26G|78.0 | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | TGTGCCAGCAGTCCTAACAAGGCTGAGCAGTTCTTC | 32 | nan | nan | nan | nan | nan | nan | nan | CASSPNKAEQFF | nan | :::::::::0:-4:12:::::14:0:36::: |
| 32 | 3 | 0.00342075 | TGTGCCAGCAGTCCAGGGGGAGGCGAAAGATTATTTTTC | JJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJFFFA | TRBV19*00(110) | TRBD1*00(35) | TRBJ1-4*00(85) | nan | 436|448|472|0|12||60.0 | 16|23|36|13|20||35.0 | 27|43|71|23|39||80.0 | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | TGTGCCAGCAGTCCAGGGGGAGGCGAAAGATTATTTTTC | 32 | nan | nan | nan | nan | nan | nan | nan | CASSPGGGERLFF | nan | :::::::::0:-4:12:13:-4:-1:20:23:-7:39::: |
| 33 | 3 | 0.00342075 | TGTGCCAGCAGTCCAAGACAGGGAGGGGAGCTCCTATGAACAGTACTTC | JJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJFFFAA???????? | TRBV13-3*00(105) | TRBD1*00(35) | TRBJ2-7*00(75) | nan | 424|436|460|0|12||60.0 | 14|21|36|16|23||35.0 | 16|39|67|26|49||115.0 | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | TGTGCCAGCAGTCCAAGACAGGGAGGGGAGCTCCTATGAACAGTACTTC | 30 | nan | nan | nan | nan | nan | nan | nan | CASSPRQG_GSSYEQYF | nan | :::::::::0:-4:12:16:-2:-3:23:26:4:49::: |
| 34 | 3 | 0.00342075 | TGTGCCAGCAGTGACAGGGTGGACTCCGACTACACCTTC | JJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJFFF | TRBV13-3*00(115) | TRBD1*00(25) | TRBJ1-2*00(95) | nan | 424|438|460|0|14||70.0 | 16|21|36|14|19||25.0 | 23|40|68|22|39||85.0 | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | TGTGCCAGCAGTGACAGGGTGGACTCCGACTACACCTTC | 37 | nan | nan | nan | nan | nan | nan | nan | CASSDRVDSDYTF | nan | :::::::::0:-2:14:14:-4:-3:19:22:-3:39::: |
| 35 | 3 | 0.00342075 | TGTGCCAGCAGTATAGCTGGGGGGAGTGCAGAAACGCTGTATTTT | JJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJFFFAA?????? | TRBV19*00(145) | TRBD2*00(30) | TRBJ2-3*00(75) | nan | 436|454|472|0|18||90.0 | 20|26|42|18|24||30.0 | 20|41|69|24|45||105.0 | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | TGTGCCAGCAGTATAGCTGGGGGGAGTGCAGAAACGCTGTATTTT | 30 | nan | nan | nan | nan | nan | nan | nan | CASSIAGGSAETLYF | nan | :::::::::0:2:18:18:-6:-2:24:24:0:45::: |
| 36 | 3 | 0.00342075 | TGTGCCAGCAGTATAGACTGGGTTAACCAAGACACCCAGTACTTT | FJFJJJJJJJJJJFJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJF | TRBV19*00(85) | nan | TRBJ2-5*00(145) | nan | 436|452|472|0|16||80.0 | nan | 16|41|69|20|45||125.0 | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | TGTGCCAGCAGTATAGACTGGGTTAACCAAGACACCCAGTACTTT | 37 | nan | nan | nan | nan | nan | nan | nan | CASSIDWVNQDTQYF | nan | :::::::::0:0:16:::::20:4:45::: |
| 37 | 3 | 0.00342075 | TGTGCCAGCAGCTCCGACAGGGTAAACTCCGACTACACCTTC | JJJJJJJJJJJJJJJJJJJJJJJJJFJJJJJJJJJJJJJJJJ | TRBV25*00(80) | TRBD1*00(35) | TRBJ1-2*00(109) | nan | 406|420|441|0|14||70.0 | 14|21|36|15|22||35.0 | 21|40|68|23|42||95.0 | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | TGTGCCAGCAGCTCCGACAGGGTAAACTCCGACTACACCTTC | 37 | nan | nan | nan | nan | nan | nan | nan | CASSSDRVNSDYTF | nan | :::::::::0:-1:14:15:-2:-3:22:2342::: |
| 38 | 3 | 0.00342075 | TGTGCCAGCAGGAGAGGGGGGGACGCAGAAACGCTGTATTTT | JJJJJJJJJJJJFJJJJJJJJJJJJJJJJJJJJJJJJJJFFF | TRBV13-100(85),TRBV200(85),TRBV3*00(85) | TRBD200(35),TRBD100(30) | TRBJ2-3*00(100) | nan | 421|432|457|0|11||55.0;427|438|464|0|11||55.0;430|441|467|0|11||55.0 | 20|27|42|15|22||35.0;17|23|36|14|20||30.0 | 23|41|69|24|42||90.0 | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | TGTGCCAGCAGGAGAGGGGGGGACGCAGAAACGCTGTATTTT | 37 | nan | nan | nan | nan | nan | nan | nan | CASRRGGDAETLYF | nan | :::::::::0:-5:11:15:-6:-1:22:24:-3:42::: |
| 39 | 3 | 0.00342075 | TGTGCAAGCAGCTTAGAAGGGGTGGCCAACGAAAGATTATTTTTC | JJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJFFFA | TRBV16*00(105) | TRBD1*00(25) | TRBJ1-4*00(105) | nan | 430|447|467|0|17||85.0 | 17|22|36|17|22||25.0 | 23|43|71|25|45||100.0 | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | TGTGCAAGCAGCTTAGAAGGGGTGGCCAACGAAAGATTATTTTTC | 32 | nan | nan | nan | nan | nan | nan | nan | CASSLEGVANERLFF | nan | :::::::::0:0:17:17:-5:-2:22:25:-3:45::: |
| 40 | 3 | 0.00342075 | TGCCATCAGTGGAGTAGTCACCCATTCACGTTC | JJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJ | IGKV4-79*00(154) | nan | IGKJ4*00(85) | nan | 430|454|478|0|24|ST448C|104.0 | nan | 20|30|58|23|33||50.0 | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | TGCCATCAGTGGAGTAGTCACCCATTCACGTTC | 41 | nan | nan | nan | nan | nan | nan | nan | CHQWSSHPFTF | nan | :::::::::0:-4:24:::::23:0:33::: |
| 41 | 3 | 0.00342075 | TGCACCTGCAGTGCAGACAGGGGGGGTGCTGAGCAGTTCTTC | JJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJFFFA | TRBV1*00(118.3) | TRBD100(35),TRBD200(35) | TRBJ2-1*00(85) | nan | 433|450|470|0|17||85.0 | 16|23|36|17|24||35.0;20|27|42|19|26||35.0 | 26|42|70|26|42||80.0 | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | TGCACCTGCAGTGCAGACAGGGGGGGTGCTGAGCAGTTCTTC | 32 | nan | nan | nan | nan | nan | nan | nan | CTCSADRGGAEQFF | nan | :::::::::0:0:17:17:-4:-1:24:26:-6:42::: |
| 42 | 3 | 0.00342075 | TGCACAGCTAACTTTGACTACTGG | JJJJJJJJJJJJJJJJJJJJJFFF | IGHV6-3*00(161.7) | nan | IGHJ2*00(73.3) | nan | 448|455|476|0|7||35.0 | nan | 23|37|68|10|24||70.0 | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | TGCACAGCTAACTTTGACTACTGG | 37 | nan | nan | nan | nan | nan | nan | nan | CTANFDYW | nan | :::::::::0:-1:7:::::10:-3:24::: |
| 43 | 2 | 0.0022805 | TGTCTAACTATGGAAATGAGAAAATAACTTTT | JJJJJJJJJJJJJJJJJJJJJJJJJJJJJJFF | TRAV4-4-DV1000(90),TRAV4D-300(90),TRAV4D-400(90),TRAV4N-300(90),TRAV4N-4*00(90) | nan | TRAJ48*00(150) | nan | 418|421|451|0|3||15.0;415|418|448|0|3||15.0;418|421|451|0|3||15.0;418|421|451|0|3||15.0;418|421|451|0|3||15.0 | nan | 23|50|81|5|32||135.0 | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | TGTCTAACTATGGAAATGAGAAAATAACTTTT | 37 | nan | nan | nan | nan | nan | nan | nan | CLTME_EKITF | nan | :::::::::0:-10:3:::::5:-3:32::: |
| 44 | 2 | 0.0022805 | TGTCATCAATACCTCTCCTCGTGGACGTTC | JJJJJJJJFJAJJJJJJJJJJJJJJJJJJJ | IGKV8-27*00(170) | nan | IGKJ1*00(90) | nan | 439|460|480|0|21||105.0 | nan | 15|30|58|15|30|SA18T|59.0 | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | TGTCATCAATACCTCTCCTCGTGGACGTTC | 32 | nan | nan | nan | nan | nan | nan | nan | CHQYLSSWTF | nan | :::::::::0:0:21:::::15:5:30::: |
| 45 | 2 | 0.0022805 | TGTCAGCAGTATTATAAGCTTCCGTGGACGTTC | JJJJJJJJJJJJJJJJFJJJJJJJJJJJJJJJJ | IGKV10-9400(118),IGKV10-9500(118) | nan | IGKJ1*00(105) | nan | 421|444|467|0|23|SA433TSG434A|83.0;421|444|467|0|23|SA433TSG434A|83.0 | nan | 19|30|58|22|33||55.0 | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | TGTCAGCAGTATTATAAGCTTCCGTGGACGTTC | 37 | nan | nan | nan | nan | nan | nan | nan | CQQYYKLPWTF | nan | :::::::::0:-3:23:::::22:1:33::: |
| 46 | 2 | 0.0022805 | TGTGCTTTGGATAGCAACTATCAGTTGATCTGG | JJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJ | TRAV13-1*00(80) | nan | TRAJ33*00(185) | nan | 418|429|451|0|11||55.0 | nan | 20|46|77|7|33||130.0 | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | TGTGCTTTGGATAGCAACTATCAGTTGATCTGG | 41 | nan | nan | nan | nan | nan | nan | nan | CALDSNYQLIW | nan | :::::::::0:-2:11:::::7:0:33::: |
| 47 | 2 | 0.0022805 | TGTGCTTTGGAACACAACACGGGTTACCAGAACTTCTATTTT | JJAJJJJJJJJJJJJJJJFJJJJJJJJJJJJJJJJJJJJFFF | TRAV13-1*00(95) | nan | TRAJ49*00(145) | nan | 418|431|451|0|13||65.0 | nan | 21|48|79|15|42||135.0 | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | TGTGCTTTGGAACACAACACGGGTTACCAGAACTTCTATTTT | 32 | nan | nan | nan | nan | nan | nan | nan | CALEHNTGYQNFYF | nan | :::::::::0:0:13:::::15:-1:42::: |
| 48 | 2 | 0.0022805 | TGTGCTTTGAGTGAATCTTCCAATACCAACAAAGTCGTCTTT | JJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJ | TRAV12-2*00(85) | nan | TRAJ34*00(165) | nan | 430|444|464|0|14||70.0 | nan | 19|47|78|14|42||140.0 | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | TGTGCTTTGAGTGAATCTTCCAATACCAACAAAGTCGTCTTT | 41 | nan | nan | nan | nan | nan | nan | nan | CALSESSNTNKVVF | nan | :::::::::0:0:14:::::14:1:42::: |
| 49 | 2 | 0.0022805 | TGTGCTTTGAGGCCTTATACAGAAGGTGCAGATAGACTCACCTTT | JJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJFFFAA?????? | TRAV12-2*00(110) | nan | TRAJ45*00(120) | nan | 430|441|464|0|11||55.0 | nan | 19|49|80|15|45||150.0 | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | TGTGCTTTGAGGCCTTATACAGAAGGTGCAGATAGACTCACCTTT | 30 | nan | nan | nan | nan | nan | nan | nan | CALRPYTEGADRLTF | nan | :::::::::0:-3:11:::::15:1:45::: |
| 50 | 2 | 0.0022805 | TGTGCTCTCTTATCTGGTAGCTTCAATAAGTTGACCTTT | JJJJJJJJJJJJJJJJJJJJJJJJJJJFFFAA??????? | TRAV13D-300(120),TRAV13N-100(120),TRAV13N-200(120),TRAV13N-300(120),TRAV13N-4*00(120) | nan | TRAJ4*00(115) | nan | 424|430|457|0|6||30.0;418|424|451|0|6||30.0;418|424|451|0|6||30.0;424|430|457|0|6||30.0;418|424|451|0|6||30.0 | nan | 22|52|83|9|39||150.0 | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | TGTGCTCTCTTATCTGGTAGCTTCAATAAGTTGACCTTT | 30 | nan | nan | nan | nan | nan | nan | nan | CALLSGSFNKLTF | nan | :::::::::0:-7:6:::::9:-2:39::: |
| 51 | 2 | 0.0022805 | TGTGCTCTGGGTGATACAGGAAACTACAAATACGTCTTT | JJJJJJJJJJJJJJJJJJJJJJJJJJJJJFFAAA????? | TRAV6-600(155),TRAV6D-600(155),TRAV6D-700(155),TRAV6N-500(155),TRAV6N-7*00(155) | nan | TRAJ40*00(105) | nan | 430|445|464|0|15||75.0;430|445|464|0|15||75.0;424|439|458|0|15||75.0;427|442|461|0|15||75.0;454|469|488|0|15||75.0 | nan | 24|50|81|13|39||130.0 | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | TGTGCTCTGGGTGATACAGGAAACTACAAATACGTCTTT | 30 | nan | nan | nan | nan | nan | nan | nan | CALGDTGNYKYVF | nan | :::::::::0:1:15:::::13:-4:39::: |
| 52 | 2 | 0.0022805 | TGTGCTCTGGGTGAGGCACAGGTTGTGGGGCAGCTCACTTTC | JJJJJJAJJJJJJFJJJJJJJJJJJJJJJJJJJJJJJJJJJJ | TRAV6-600(80),TRAV6D-600(80),TRAV6N-5*00(80) | nan | TRAJ5*00(160) | nan | 430|444|464|0|14||70.0;430|444|464|0|14||70.0;427|441|461|0|14||70.0 | nan | 25|51|82|16|42||130.0 | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | TGTGCTCTGGGTGAGGCACAGGTTGTGGGGCAGCTCACTTTC | 32 | nan | nan | nan | nan | nan | nan | nan | CALGEAQVVGQLTF | nan | :::::::::0:0:14:::::16:-5:42::: |
| 53 | 2 | 0.0022805 | TGTGCTCTGAGTGATCGTAGAAATAGCAATAACAGAATCTTCTTT | JJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJFFFAA????????? | TRAV6-500(150),TRAV6-7-DV900(150) | nan | TRAJ31*00(75) | nan | 424|440|458|0|16||80.0;424|440|458|0|16||80.0 | nan | 22|46|77|21|45||120.0 | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | TGTGCTCTGAGTGATCGTAGAAATAGCAATAACAGAATCTTCTTT | 30 | nan | nan | nan | nan | nan | nan | nan | CALSDRRNSNNRIFF | nan | :::::::::0:2:16:::::21:-2:45::: |
| 54 | 2 | 0.0022805 | TGTGCTCTGAGTGATCAGGACTATGCAAACAAGATGATCTTT | JJJJJJJJJJJJJFJJJJJJJJJJJJJJJJJJJJJJJJJJJJ | TRAV12D-300(90),TRAV12N-100(90),TRAV12N-300(90),TRAV6-500(90),TRAV6-7-DV9*00(90) | nan | TRAJ47*00(155) | nan | 433|450|467|0|17||85.0;427|444|461|0|17||85.0;433|450|467|0|17||85.0;424|441|458|0|17||85.0;424|441|458|0|17||85.0 | nan | 20|44|75|18|42||120.0 | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | TGTGCTCTGAGTGATCAGGACTATGCAAACAAGATGATCTTT | 37 | nan | nan | nan | nan | nan | nan | nan | CALSDQDYANKMIF | nan | :::::::::0:3:17:::::18:0:42::: |
| 55 | 2 | 0.0022805 | TGTGCTCTGAGTGACCTGGGCTACAAACTTACCTTC | JJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJAF | TRAV12-300(125),TRAV12D-100(125),TRAV12D-300(125),TRAV12N-100(125),TRAV12N-3*00(125) | nan | TRAJ9*00(99) | nan | 433|447|467|0|14||70.0;427|441|461|0|14||70.0;433|447|467|0|14||70.0;427|441|461|0|14||70.0;433|447|467|0|14||70.0 | nan | 27|47|78|16|36||100.0 | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | TGTGCTCTGAGTGACCTGGGCTACAAACTTACCTTC | 32 | nan | nan | nan | nan | nan | nan | nan | CALSDLGYKLTF | nan | :::::::::0:0:14:::::16:-7:36::: |
| 56 | 2 | 0.0022805 | TGTGCTCTGAACAACTATCAGTTGATCTGG | JJJJJJJJJJJJJJJJJJJJJJJJJJJJJJ | TRAV12-300(105),TRAV12D-100(105),TRAV12D-300(105),TRAV12N-100(105),TRAV12N-3*00(105) | nan | TRAJ33*00(140) | nan | 433|443|467|0|10||50.0;427|437|461|0|10||50.0;433|443|467|0|10||50.0;427|437|461|0|10||50.0;433|443|467|0|10||50.0 | nan | 27|46|77|11|30||95.0 | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | TGTGCTCTGAACAACTATCAGTTGATCTGG | 41 | nan | nan | nan | nan | nan | nan | nan | CALNNYQLIW | nan | :::::::::0:-4:10:::::11:-7:30::: |
| 57 | 2 | 0.0022805 | TGTGCTGTGAGTTCCAATACCAACAAAGTCGTCTTT | JJJJJJJJJFJJJJJJJJJJJJJFFFAA???????? | TRAV9-100(165),TRAV9-400(165),TRAV9D-1*00(165) | nan | TRAJ34*00(85) | nan | 424|435|457|0|11||55.0;427|438|458|0|11||55.0;424|435|456|0|11||55.0 | nan | 22|47|78|11|36||125.0 | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | TGTGCTGTGAGTTCCAATACCAACAAAGTCGTCTTT | 30 | nan | nan | nan | nan | nan | nan | nan | CAVSSNTNKVVF | nan | :::::::::0:-2:11:::::11:-2:36::: |
| 58 | 2 | 0.0022805 | TGTGCTGCAAGTGCTGTGAGAGGCAGCAATTACAAACTGACATTT | JJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJFFFAA?????? | TRAV5-400(125),TRAV5D-400(125),TRAV5N-4*00(125) | nan | TRAJ53*00(100) | nan | 430|444|463|0|14||70.0;430|444|463|0|14||70.0;430|444|463|0|14||70.0 | nan | 29|55|86|19|45||130.0 | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | TGTGCTGCAAGTGCTGTGAGAGGCAGCAATTACAAACTGACATTT | 30 | nan | nan | nan | nan | nan | nan | nan | CAASAVRGSNYKLTF | nan | :::::::::0:1:14:::::19:-9:45::: |
| 59 | 2 | 0.0022805 | TGTGCTATTTCCTCCTCCTTCAGCAAGCTGGTGTTT | JJJJJJFJJJJJJJJJJJFFFFAA???????????? | TRAV16N*00(170) | nan | TRAJ50*00(75) | nan | 433|441|470|0|8||40.0 | nan | 25|52|83|9|36||135.0 | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | TGTGCTATTTCCTCCTCCTTCAGCAAGCTGGTGTTT | 30 | nan | nan | nan | nan | nan | nan | nan | CAISSSFSKLVF | nan | :::::::::0:-9:8:::::9:-5:36::: |
| 60 | 2 | 0.0022805 | TGTGCTATGGTGGATAGCAACTATCAGTTGATCTGG | FJJJFJFJJJJJJJJJJJJFFFFJJFJFFJJJJJJJ | TRAV13D-100(80),TRAV13D-300(80),TRAV13N-100(80),TRAV13N-300(80),TRAV13N-4*00(80) | nan | TRAJ33*00(170) | nan | 418|428|451|0|10||50.0;424|434|457|0|10||50.0;418|428|451|0|10||50.0;424|434|457|0|10||50.0;418|428|451|0|10||50.0 | nan | 20|46|77|10|36||130.0 | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | TGTGCTATGGTGGATAGCAACTATCAGTTGATCTGG | 37 | nan | nan | nan | nan | nan | nan | nan | CAMVDSNYQLIW | nan | :::::::::0:-3:10:::::10:0:36::: |
| 61 | 2 | 0.0022805 | TGTGCTATGGGCCCCACCCGGGTTACCAGAACTTCTATTTT | JJJJJJJJJJJJJJJJJJJJJJJJJJJFFFAA????????? | TRAV13D-100(140),TRAV13D-300(140),TRAV13N-100(140),TRAV13N-300(140),TRAV13N-4*00(140) | nan | TRAJ49*00(70) | nan | 418|428|451|0|10||50.0;424|434|457|0|10||50.0;418|428|451|0|10||50.0;424|434|457|0|10||50.0;418|428|451|0|10||50.0 | nan | 25|48|79|18|41||115.0 | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | TGTGCTATGGGCCCCACCCGGGTTACCAGAACTTCTATTTT | 30 | nan | nan | nan | nan | nan | nan | nan | CAMGPTR_YQNFYF | nan | :::::::::0:-3:10:::::18:-5:41::: |
| 62 | 2 | 0.0022805 | TGTGCTATGGACTATGCAAACAAGATGATCTTT | JJJJJJJJJJJJJJJJJJJJJFFJJJJJJJJJJ | TRAV13D-100(85),TRAV13D-300(85),TRAV13N-100(85),TRAV13N-300(85),TRAV13N-4*00(85) | nan | TRAJ47*00(175) | nan | 418|429|451|0|11||55.0;424|435|457|0|11||55.0;418|429|451|0|11||55.0;424|435|457|0|11||55.0;418|429|451|0|11||55.0 | nan | 20|44|75|9|33||120.0 | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | TGTGCTATGGACTATGCAAACAAGATGATCTTT | 37 | nan | nan | nan | nan | nan | nan | nan | CAMDYANKMIF | nan | :::::::::0:-2:11:::::9:0:33::: |
| 63 | 2 | 0.0022805 | TGTGCTATGGACCGAAATAACTATGCCCAGGGATTAACCTTC | JJFJJJJJJJJJJJJJJJJJJJJFFFAA?????????????? | TRAV13D-100(165),TRAV13D-300(165),TRAV13N-100(165),TRAV13N-300(165),TRAV13N-4*00(165) | nan | TRAJ26*00(80) | nan | 418|429|451|0|11||55.0;424|435|457|0|11||55.0;418|429|451|0|11||55.0;424|435|457|0|11||55.0;418|429|451|0|11||55.0 | nan | 19|49|80|12|42||150.0 | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | TGTGCTATGGACCGAAATAACTATGCCCAGGGATTAACCTTC | 30 | nan | nan | nan | nan | nan | nan | nan | CAMDRNNYAQGLTF | nan | :::::::::0:-2:11:::::12:1:42::: |
| 64 | 2 | 0.0022805 | TGTGCTATGAGAGATCGGGCGTCTAATTACAACGTGCTTTACTTC | JJJJJJJFFFJJJAJJJJJJJJJJJJJJJJJJJJJJJJJFJJFFF | TRAV6-3*00(95) | nan | TRAJ21*00(135) | nan | 424|440|458|0|16||80.0 | nan | 21|46|77|20|45||125.0 | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | TGTGCTATGAGAGATCGGGCGTCTAATTACAACGTGCTTTACTTC | 32 | nan | nan | nan | nan | nan | nan | nan | CAMRDRASNYNVLYF | nan | :::::::::0:2:16:::::20:-1:45::: |
| 65 | 2 | 0.0022805 | TGTGCTATAGAAGCATCTTCTGGCAGCTGGCAACTCATCTTT | JJJJJJJJJJJJJJJFJJFJJJJJJFJJJFFFAA???????? | TRAV13-2*00(135) | nan | TRAJ22*00(114) | nan | 418|429|451|0|11||55.0 | nan | 15|49|80|8|42|ST18A|154.0 | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | TGTGCTATAGAAGCATCTTCTGGCAGCTGGCAACTCATCTTT | 30 | nan | nan | nan | nan | nan | nan | nan | CAIEASSGSWQLIF | nan | :::::::::0:-2:11:::::8:5:42::: |
| 66 | 2 | 0.0022805 | TGTGCTACTGATAATAGCAATAACAGAATCTTCTTT | JJJJJJFFFJFJFJJFJJJJJJFFFAA????????? | TRAV8N-2*00(175) | nan | TRAJ31*00(75) | nan | 424|436|457|0|12||60.0 | nan | 22|46|77|12|36||120.0 | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | TGTGCTACTGATAATAGCAATAACAGAATCTTCTTT | 30 | nan | nan | nan | nan | nan | nan | nan | CATDNSNNRIFF | nan | :::::::::0:-1:12:::::12:-2:36::: |
| 67 | 2 | 0.0022805 | TGTGCTAGCAGTTTCGACAGAAACACCTTGTACTTT | JJFJJJJJJJJJJJJJJJJJJJJJJJJJJJJJFFFA | TRBV29*00(135) | TRBD1*00(25) | TRBJ2-4*00(85) | nan | 427|441|464|0|14||70.0 | 14|19|36|15|20||25.0 | 25|41|69|20|36||80.0 | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | TGTGCTAGCAGTTTCGACAGAAACACCTTGTACTTT | 32 | nan | nan | nan | nan | nan | nan | nan | CASSFDRNTLYF | nan | :::::::::0:-3:14:15:-2:-5:20:20:-5:36::: |
| 68 | 2 | 0.0022805 | TGTGCTAGCAGTTTAGCAGGCGGAGACACCCAGTACTTT | JJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJ | TRBV29*00(85) | nan | TRBJ2-5*00(125) | nan | 427|442|464|0|15||75.0 | nan | 25|41|69|23|39||80.0 | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | TGTGCTAGCAGTTTAGCAGGCGGAGACACCCAGTACTTT | 41 | nan | nan | nan | nan | nan | nan | nan | CASSLAGGDTQYF | nan | :::::::::0:-2:15:::::23:-5:39::: |
| 69 | 2 | 0.0022805 | TGTGCTAGCAGTTTAACTCGCAACTCCGACTACACCTTC | JJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJ | TRBV29*00(85) | nan | TRBJ1-2*00(135) | nan | 427|442|464|0|15||75.0 | nan | 22|40|68|21|39||90.0 | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | TGTGCTAGCAGTTTAACTCGCAACTCCGACTACACCTTC | 41 | nan | nan | nan | nan | nan | nan | nan | CASSLTRNSDYTF | nan | :::::::::0:-2:15:::::21:-2:39::: |
| 70 | 2 | 0.0022805 | TGTGCTAGCAGTGTACTGGGGGGGATGTACTTC | JJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJ | TRBV17*00(90) | TRBD2*00(50) | TRBJ2-7*00(90) | nan | 427|439|464|0|12||60.0 | 17|27|42|14|24||50.0 | 32|39|67|26|33||35.0 | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | TGTGCTAGCAGTGTACTGGGGGGGATGTACTTC | 41 | nan | nan | nan | nan | nan | nan | nan | CASSVLGGMYF | nan | :::::::::0:-5:12:14:-3:-1:24:26:-12:33::: |
| 71 | 2 | 0.0022805 | TGTGCTAGCAGTACCAGTAACCAAGACACCCAGTACTTT | JJJJJJJJJJJJJJJJJJJJJJJJJFJJJJJJJJJJJJJ | TRBV17*00(80) | nan | TRBJ2-5*00(150) | nan | 427|440|464|0|13||65.0 | nan | 19|41|69|17|39||110.0 | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | TGTGCTAGCAGTACCAGTAACCAAGACACCCAGTACTTT | 37 | nan | nan | nan | nan | nan | nan | nan | CASSTSNQDTQYF | nan | :::::::::0:-4:13:::::17:1:39::: |
| 72 | 2 | 0.0022805 | TGTGCTAGCAGTAGTCAGGGGTTCTATGAACAGTACTTC | JJJJJJFJFJJJJJJJJJJJJJJJJJJJJFJJJJJJFFF | TRBV17*00(115) | TRBD1*00(30) | TRBJ2-7*00(90) | nan | 427|441|464|0|14||70.0 | 16|22|36|15|21||30.0 | 23|39|67|23|39||80.0 | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | TGTGCTAGCAGTAGTCAGGGGTTCTATGAACAGTACTTC | 37 | nan | nan | nan | nan | nan | nan | nan | CASSSQGFYEQYF | nan | :::::::::0:-3:14:15:-4:-2:21:23:-3:39::: |
| 73 | 2 | 0.0022805 | TGTGCTAGCAGTAGAGAGCACCAAGACACCCAGTACTTT | JJJJJJJJJJJJFJFJAJJJJJJJJJJJJJJFFJFJFJF | TRBV17*00(132.5) | nan | TRBJ2-5*00(107.5) | nan | 427|444|464|0|17||85.0 | nan | 21|41|69|19|39||100.0 | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | TGTGCTAGCAGTAGAGAGCACCAAGACACCCAGTACTTT | 32 | nan | nan | nan | nan | nan | nan | nan | CASSREHQDTQYF | nan | :::::::::0:0:17:::::19:-1:39::: |
| 74 | 2 | 0.0022805 | TGTGCCTGGAGTCTATTATATGAACAGTACTTC | JJJJFJJJJFJJJJFJJFJJJFFJJJJJJJJJJ | TRBV31*00(110) | nan | TRBJ2-7*00(115) | nan | 421|434|454|0|13||65.0 | nan | 24|39|67|18|33||75.0 | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | TGTGCCTGGAGTCTATTATATGAACAGTACTTC | 37 | nan | nan | nan | nan | nan | nan | nan | CAWSLLYEQYF | nan | :::::::::0:0:13:::::18:-4:33::: |
| 75 | 2 | 0.0022805 | TGTGCCCCTGCATCTTCTGGCAGCTGGCAACTCATCTTT | JJJJJJJJJJJJJJJJJJJJJJJFFFAA??????????? | TRAV12-300(135),TRAV12D-100(135),TRAV12D-300(135),TRAV12N-100(135),TRAV12N-3*00(135) | nan | TRAJ22*00(100) | nan | 433|438|467|0|5||25.0;427|432|461|0|5||25.0;433|438|467|0|5||25.0;427|432|461|0|5||25.0;433|438|467|0|5||25.0 | nan | 18|49|80|8|39||155.0 | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | TGTGCCCCTGCATCTTCTGGCAGCTGGCAACTCATCTTT | 30 | nan | nan | nan | nan | nan | nan | nan | CAPASSGSWQLIF | nan | :::::::::0:-9:5:::::8:2:39::: |
| 76 | 2 | 0.0022805 | TGTGCCAGCTCTCTCTGGGGGGATAACTATGCTGAGCAGTTCTTC | JJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJFFFA | TRBV12-100(95),TRBV12-200(95) | TRBD2*00(35) | TRBJ2-1*00(120) | nan | 457|472|492|0|15||75.0;457|472|492|0|15||75.0 | 19|26|42|15|22||35.0 | 19|42|70|22|45||115.0 | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | TGTGCCAGCTCTCTCTGGGGGGATAACTATGCTGAGCAGTTCTTC | 32 | nan | nan | nan | nan | nan | nan | nan | CASSLWGDNYAEQFF | nan | :::::::::0:0:15:15:-5:-2:22:22:1:45::: |
| 77 | 2 | 0.0022805 | TGTGCCAGCTCTCTCGCTAGTGCAGAAACGCTGTATTTT | JJFJJJFJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJ | TRBV12-100(110),TRBV12-200(110) | nan | TRBJ2-3*00(140) | nan | 457|473|492|0|16||80.0;457|473|492|0|16||80.0 | nan | 18|41|69|16|39||115.0 | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | TGTGCCAGCTCTCTCGCTAGTGCAGAAACGCTGTATTTT | 37 | nan | nan | nan | nan | nan | nan | nan | CASSLASAETLYF | nan | :::::::::0:1:16:::::16:2:39::: |
| 78 | 2 | 0.0022805 | TGTGCCAGCTCTCTCGCGGGGGGGGAAGTCTTCTTT | JJ<JJJFJJJJJJFJJJJJJJJJJJJJJJJJJJJJJ | TRBV12-100(115),TRBV12-200(115) | TRBD2*00(35) | TRBJ1-1*00(95) | nan | 457|473|492|0|16||80.0;457|473|492|0|16||80.0 | 20|27|42|17|24||35.0 | 28|40|68|24|36||60.0 | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | TGTGCCAGCTCTCTCGCGGGGGGGGAAGTCTTCTTT | 27 | nan | nan | nan | nan | nan | nan | nan | CASSLAGGEVFF | nan | :::::::::0:1:16:17:-6:-1:24:24:-8:36::: |
| 79 | 2 | 0.0022805 | TGTGCCAGCTCTCTCGGGTATGCTGAGCAGTTCTTC | JJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJF | TRBV12-2*00(130) | nan | TRBJ2-1*00(110) | nan | 457|473|492|0|16||80.0 | nan | 24|42|70|18|36||90.0 | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | TGTGCCAGCTCTCTCGGGTATGCTGAGCAGTTCTTC | 37 | nan | nan | nan | nan | nan | nan | nan | CASSLGYAEQFF | nan | :::::::::0:1:16:::::18:-4:36::: |
| 80 | 2 | 0.0022805 | TGTGCCAGCTCTCTCGACCTTGCAAACACCGGGCAGCTCTACTTT | JJJJJJJJJJJJJJJJJJJJJJJJJJJJFFFAA???????????? | TRBV12-1*00(170) | nan | TRBJ2-2*00(70) | nan | 457|474|492|0|17||85.0 | nan | 17|43|71|19|45||130.0 | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | TGTGCCAGCTCTCTCGACCTTGCAAACACCGGGCAGCTCTACTTT | 30 | nan | nan | nan | nan | nan | nan | nan | CASSLDLANTGQLYF | nan | :::::::::0:2:17:::::19:3:45::: |
| 81 | 2 | 0.0022805 | TGTGCCAGCTCTCTCGACAGGGGGCAAAACACCTTGTACTTT | JJJJJJAJJJJJJJJJJJFJJJJJJJJJFJFJJJJJJJJFFA | TRBV12-100(115),TRBV12-200(115) | TRBD1*00(35) | TRBJ2-4*00(100) | nan | 457|474|492|0|17||85.0;457|474|492|0|17||85.0 | 16|23|36|17|24||35.0 | 23|41|69|24|42||90.0 | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | TGTGCCAGCTCTCTCGACAGGGGGCAAAACACCTTGTACTTT | 32 | nan | nan | nan | nan | nan | nan | nan | CASSLDRGQNTLYF | nan | :::::::::0:2:17:17:-4:-1:24:24:-3:42::: |
| 82 | 2 | 0.0022805 | TGTGCCAGCTCTCTCGAACGATTTAACCAAGACACCCAGTACTTT | JJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJFFFAA????????? | TRBV12-2*00(155) | nan | TRBJ2-5*00(70) | nan | 457|474|492|0|17||85.0 | nan | 18|41|69|22|45||115.0 | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | TGTGCCAGCTCTCTCGAACGATTTAACCAAGACACCCAGTACTTT | 30 | nan | nan | nan | nan | nan | nan | nan | CASSLERFNQDTQYF | nan | :::::::::0:2:17:::::22:2:45::: |
| 83 | 2 | 0.0022805 | TGTGCCAGCTCTCTGCCGGGACAACAAGACACCCAGTACTTT | JJJJJJFJJJJJJJJJJJJJFJJJJJJJJJJJJJJJJJJJFF | TRBV12-100(95),TRBV12-200(95) | TRBD100(40),TRBD200(35) | TRBJ2-5*00(105) | nan | 457|471|492|0|14||70.0;457|471|492|0|14||70.0 | 10|18|36|15|23||40.0;12|19|42|15|22||35.0 | 23|41|69|24|42||90.0 | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | TGTGCCAGCTCTCTGCCGGGACAACAAGACACCCAGTACTTT | 37 | nan | nan | nan | nan | nan | nan | nan | CASSLPGQQDTQYF | nan | :::::::::0:-1:14:15:2:-6:23:24:-3:42::: |
| 84 | 2 | 0.0022805 | TGTGCCAGCTCTCCGACAGGGGATAACTATGCTGAGCAGTTCTTC | JJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJFFFA | TRBV12-100(85),TRBV12-200(85) | TRBD1*00(40) | TRBJ2-1*00(120) | nan | 457|470|492|0|13||65.0;457|470|492|0|13||65.0 | 14|22|36|14|22||40.0 | 19|42|70|22|45||115.0 | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | TGTGCCAGCTCTCCGACAGGGGATAACTATGCTGAGCAGTTCTTC | 32 | nan | nan | nan | nan | nan | nan | nan | CASSPTGDNYAEQFF | nan | :::::::::0:-2:13:14:-2:-2:22:22:1:45::: |
| 85 | 2 | 0.0022805 | TGTGCCAGCTCCCGGGGACAGGACCTCCTATGAACAGTACTTC | JJJJJJFJJJJJJJJJJJJJJJJJJJJJJJJJJJJFFFAA??? | TRBV12-2*00(105) | TRBD100(25),TRBD200(25) | TRBJ2-7*00(89) | nan | 457|468|492|0|11||55.0 | 10|15|36|11|16||25.0;12|17|42|11|16||25.0 | 15|39|67|19|43|SG19C|104.0 | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | TGTGCCAGCTCCCGGGGACAGGACCTCCTATGAACAGTACTTC | 30 | nan | nan | nan | nan | nan | nan | nan | CASSRGQ_TSYEQYF | nan | :::::::::0:-4:11:11:2:-9:16:19:5:43::: |
| 86 | 2 | 0.0022805 | TGTGCCAGCTCACCGGGAGGAGGGCAAGACACCCAGTACTTT | JJFJJJJJJFJAJJJJJJJJAJJJJJJJJJJJJJJJJJJFFF | TRBV12-200(85),TRBV1800(85) | TRBD100(30),TRBD200(30) | TRBJ2-5*00(100) | nan | 457|468|492|0|11||55.0;429|440|466|0|11||55.0 | 10|16|36|12|18||30.0;12|18|42|12|18||30.0 | 23|41|69|24|42||90.0 | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | TGTGCCAGCTCACCGGGAGGAGGGCAAGACACCCAGTACTTT | 32 | nan | nan | nan | nan | nan | nan | nan | CASSPGGGQDTQYF | nan | :::::::::0:-4:11:12:2:-8:18:24:-3:42::: |
| 87 | 2 | 0.0022805 | TGTGCCAGCGGTGGGGGCCAAGACACCCAGTACTTT | JJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJFFF | TRBV13-2*00(125) | nan | TRBJ2-5*00(105) | nan | 424|437|460|0|13||65.0 | nan | 22|41|69|17|36||95.0 | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | TGTGCCAGCGGTGGGGGCCAAGACACCCAGTACTTT | 37 | nan | nan | nan | nan | nan | nan | nan | CASGGGQDTQYF | nan | :::::::::0:-3:13:::::17:-2:36::: |
| 88 | 2 | 0.0022805 | TGTGCCAGCGGTGGGGGACTGGGGGTTAACCAAGACACCCAGTACTTT | JF<JJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJFFFAA???????? | TRBV13-2*00(115) | TRBD2*00(45) | TRBJ2-5*00(85) | nan | 424|437|460|0|13||65.0 | 14|23|42|14|23||45.0 | 16|41|69|23|48||125.0 | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | TGTGCCAGCGGTGGGGGACTGGGGGTTAACCAAGACACCCAGTACTTT | 27 | nan | nan | nan | nan | nan | nan | nan | CASGGGLGVNQDTQYF | nan | :::::::::0:-3:13:14:0:-5:23:23:4:48::: |
| 89 | 2 | 0.0022805 | TGTGCCAGCGGTGATGTCTGGGGTGAAGACACCCAGTACTTT | JJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJF | TRBV13-2*00(50) | TRBD2*00(30) | TRBJ2-5*00(105) | nan | 424|440|460|0|16||80.0 | 18|24|42|17|23||30.0 | 24|41|69|25|42||85.0 | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | TGTGCCAGCGGTGATGTCTGGGGTGAAGACACCCAGTACTTT | 37 | nan | nan | nan | nan | nan | nan | nan | CASGDVWGEDTQYF | nan | :::::::::0:0:16:17:-4:-4:23:25:-4:42::: |
| 90 | 2 | 0.0022805 | TGTGCCAGCGGTGATGTGGAGGACCAAGACACCCAGTACTTT | FJJJJJFJJJJJFJJJJJJFJJJJJJJJJJJJJJJJJJJJJJ | TRBV13-2*00(80) | nan | TRBJ2-5*00(140) | nan | 424|440|460|0|16||80.0 | nan | 21|41|69|22|42||100.0 | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | TGTGCCAGCGGTGATGTGGAGGACCAAGACACCCAGTACTTT | 37 | nan | nan | nan | nan | nan | nan | nan | CASGDVEDQDTQYF | nan | :::::::::0:0:16:::::22:-1:42::: |
| 91 | 2 | 0.0022805 | TGTGCCAGCGGTGATGCTTACAAAAATAACTATGCTGAGCAGTTCTTC | JJJJJFFJJJJJJJJJJJJJJJJJJJJJJJJJJJJFFAAA???????? | TRBV13-2*00(135) | nan | TRBJ2-1*00(75) | nan | 424|441|460|0|17||85.0 | nan | 19|42|70|25|48||115.0 | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | TGTGCCAGCGGTGATGCTTACAAAAATAACTATGCTGAGCAGTTCTTC | 30 | nan | nan | nan | nan | nan | nan | nan | CASGDAYKNNYAEQFF | nan | :::::::::0:1:17:::::25:1:48::: |
| 92 | 2 | 0.0022805 | TGTGCCAGCGGTGATGCGACTGGGGGACAAGACACCCAGTACTTT | JJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJFF | TRBV13-2*00(95) | TRBD2*00(45) | TRBJ2-5*00(105) | nan | 424|441|460|0|17||85.0 | 16|25|42|17|26||45.0 | 23|41|69|27|45||90.0 | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | TGTGCCAGCGGTGATGCGACTGGGGGACAAGACACCCAGTACTTT | 37 | nan | nan | nan | nan | nan | nan | nan | CASGDATGGQDTQYF | nan | :::::::::0:1:17:17:-2:-3:26:27:-3:45::: |
| 93 | 2 | 0.0022805 | TGTGCCAGCGGTGATGCAGGACAGGGGAATTCTGGAAATACGCTCTATTTT | JJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJFFFAA?????????? | TRBV13-2*00(127) | TRBD1*00(40) | TRBJ1-3*00(75) | nan | 424|442|460|0|18||90.0 | 13|21|36|18|26||40.0 | 17|42|70|26|51||125.0 | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | TGTGCCAGCGGTGATGCAGGACAGGGGAATTCTGGAAATACGCTCTATTTT | 30 | nan | nan | nan | nan | nan | nan | nan | CASGDAGQGNSGNTLYF | nan | :::::::::0:2:18:18-3:26:26:3:51::: |
| 94 | 2 | 0.0022805 | TGTGCCAGCGGTGATAGGGCAGGGGGACCAGAAGTCTTCTTT | JJFJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJFFF | TRBV13-2*00(105) | TRBD1*00(35) | TRBJ1-1*00(80) | nan | 424|439|460|0|15||75.0 | 16|23|36|19|26||35.0 | 26|40|68|28|42||70.0 | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | TGTGCCAGCGGTGATAGGGCAGGGGGACCAGAAGTCTTCTTT | 37 | nan | nan | nan | nan | nan | nan | nan | CASGDRAGGPEVFF | nan | :::::::::0:-1:15:19:-4:-1:26:28:-6:42::: |
| 95 | 2 | 0.0022805 | TGTGCCAGCGGTGACTGGGGGCACGGCGACTACACCTTC | JJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJ | TRBV13-2*00(95) | TRBD200(35),TRBD100(30) | TRBJ1-2*00(95) | nan | 424|438|460|0|14||70.0 | 18|25|42|14|21||35.0;18|24|36|16|22||30.0 | 27|40|68|26|39||65.0 | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | TGTGCCAGCGGTGACTGGGGGCACGGCGACTACACCTTC | 41 | nan | nan | nan | nan | nan | nan | nan | CASGDWGHGDYTF | nan | :::::::::0:-2:14:14:-4:-3:21:26:-7:39::: |
| 96 | 2 | 0.0022805 | TGTGCCAGCGGTAGAAACACAGAAGTCTTCTTT | JJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJ | TRBV13-2*00(85) | nan | TRBJ1-1*00(155) | nan | 424|436|460|0|12||60.0 | nan | 21|40|68|14|33||95.0 | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | TGTGCCAGCGGTAGAAACACAGAAGTCTTCTTT | 41 | nan | nan | nan | nan | nan | nan | nan | CASGRNTEVFF | nan | :::::::::0:-4:12:::::14:-1:33::: |
| 97 | 2 | 0.0022805 | TGTGCCAGCAGTTTTGACAGAAAAGACACCCAGTACTTT | JJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJ | TRBV14*00(85) | TRBD1*00(25) | TRBJ2-5*00(125) | nan | 430|444|466|0|14||70.0 | 14|19|36|15|20||25.0 | 24|41|69|22|39||85.0 | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | TGTGCCAGCAGTTTTGACAGAAAAGACACCCAGTACTTT | 41 | nan | nan | nan | nan | nan | nan | nan | CASSFDRKDTQYF | nan | :::::::::0:-2:14:15:-2:-5:20:22:-4:39::: |
| 98 | 2 | 0.0022805 | TGTGCCAGCAGTTTCTGGGGTGCAAACACCGGGCAGCTCTACTTT | AJJJJJJJJJJJJJJJJJJJJJJJJAJJJJJJJJJJJJJJFJJJJ | TRBV14*00(80) | nan | TRBJ2-2*00(150) | nan | 430|446|466|0|16||80.0 | nan | 18|43|71|20|45||125.0 | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | TGTGCCAGCAGTTTCTGGGGTGCAAACACCGGGCAGCTCTACTTT | 32 | nan | nan | nan | nan | nan | nan | nan | CASSFWGANTGQLYF | nan | :::::::::0:0:16:::::20:2:45::: |
| 99 | 2 | 0.0022805 | TGTGCCAGCAGTTTAGACGTTGCAAACACAGAAGTCTTCTTT | JJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJF | TRBV15*00(100) | nan | TRBJ1-1*00(135) | nan | 430|446|467|0|16||80.0 | nan | 17|40|68|19|42||115.0 | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | nan | TGTGCCAGCAGTTTAGACGTTGCAAACACAGAAGTCTTCTTT | 37 | nan | nan | nan | nan | nan | nan | nan | CASSLDVANTEVFF | nan | :::::::::0:-1:16:::::19:3:42::: |
In order to run the analysis for all samples in the project on Linux we can use GNU Parallel in the following way:
#!/usr/bin/env bash
mkdir -p results
ls /raw/*.gz |
parallel -j 2 --line-buffer \
"mixcr analyze rna-seq \
--species hsa \
{} \
{=s:.*/:results/:;s:\.fastq\.gz::=}"
Under the hood pipeline:
Under the hood the command above actually executes the following pipeline:
align
Alignment of raw sequencing reads against reference database of V-, D-, J- and C- gene segments.
mixcr align \
--species mmu \
-p shotgun-base \
-OvParameters.geneFeatureToAlign="VTranscriptWithout5UTRWithP" \
-OvParameters.parameters.floatingLeftBound=false \
-OjParameters.parameters.floatingRightBound=false \
-OсParameters.parameters.floatingRightBound=false \
-OallowPartialAlignments=true \
--report results/CD8T_REH_4h_rep1.report.txt \
--json-report results/CD8T_REH_4h_rep1.report.json \
raw/CD8T_REH_4h_rep1.fastq.gz \
results/CD8T_REH_4h_rep1.vdjca
Option --report is specified here explicitly.
--species mmu- mmu for Mus Musculus
-p rna-seq- defines a set of aligner parameters specifically for RNASeq data.
-OvParameters.geneFeatureToAlign="VTranscriptWithout5UTRWithP"- defines V gene feature to align, which includes the full V-gene sequence excluding 5'UTR.
-OvParameters.parameters.floatingLeftBound=false -OjParameters.parameters.floatingRightBound=false -OсParameters.parameters.floatingRightBound=false- a global alignment will be used on every gene-segment bound due to the absence of primers
-OallowPartialAlignments=true- preserves partial alignments to be assembled with
assemblePartialon the next step.
assemblePartial
Assembles alignments that only partially cover CDR3 region. This is a mandatory step for RNA-Seq data, as reads randomly cover all TCR / BCR segments. This function works with pairs of alignments (assembles two alignments at a time), thus it is usually recommended to perform two rounds of assemblePartial for better yield. For more information check mixcr assemblePartial.
# assemble overlapping fragmented sequencing reads
# First round
mixcr assemblePartial \
--report results/CD8T_REH_4h_rep1.report.txt \
--json-report results/CD8T_REH_4h_rep1.report.json \
results/CD8T_REH_4h_rep1.vdjca \
results/CD8T_REH_4h_rep1.passembled.1.vdjca
#Second round
mixcr assemblePartial \
--report results/CD8T_REH_4h_rep1.report.txt \
--json-report results/CD8T_REH_4h_rep1.report.json \
results/CD8T_REH_4h_rep1.passembled.1.vdjca \
results/CD8T_REH_4h_rep1.passembled.2.vdjca
Note that we specify the same report file on every step, thus reports will be appended to the same file.
extend
If V- and/or J- segments are uniquely determined, but CDR3 edges lack nucleotides, mixcr extend will impute those from germline.
Note
This step is only applicable to T-cells due to the absence of hypermutations! In case of BCR data this step is omitted.
mixcr extend \
--report results/CD8T_REH_4h_rep1.report.txt \
--json-report results/CD8T_REH_4h_rep1.report.json \
results/CD8T_REH_4h_rep1.passembled.2.vdjca \
results/CD8T_REH_4h_rep1.extended.vdjca
assemble
Assembles alignments into clonotypes and applies several layers of errors correction(ex. quality-awared correction for sequencing errors, clustering to correct for PCR errors). Check mixcr assemble for more information.
mixcr assemble \
-OassemblingFeatures="CDR3" \
-OseparateByJ=true \
-OseparateByV=true \
--report results/CD8T_REH_4h_rep1.report.txt \
--json-report results/CD8T_REH_4h_rep1.report.json \
results/CD8T_REH_4h_rep1.extended.vdjca \
results/CD8T_REH_4h_rep1.clns
-OassemblingFeatures="CDR3"- clones will be assembled by
CDR3sequence. -OseparateByV=true- Separate clones by V-gene
-OseparateByJ=true- Separate clones by J-gene
export
Exports clonotypes from .clns file into human-readable tables.
mixcr exportClones \
results/CD8T_REH_4h_rep1.clns \
results/CD8T_REH_4h_rep1.clones.tsv
Check mixcr export for more additional fields.
Quality control
Now, when the analysis is complete, lets visualize quality report data. Looking at the alignment report in this case won't be of much help, because the cDNA library was not enriched with TCR sequences, thus we already know that only a small part of reads has been successfully aligned. What is going to be more descriptive is to look at the chain usage among samples.
mixcr exportQc chainUsage results/*.clns figs/chainUsage.pdf
This plot reveals a mild contamination by B-cells, since IGH and IGKL chains present in the samples despite Treg and CD8+ cells have been isolated for library preparation.
Full length receptor assembly
Because RNA-Seq reads randomly cover the whole receptor gene region it is possible to assemble longer clone sequences then just CDR3. To do that we can use another RNA-seq preset specifically tuned to assemble the longest clone sequence possible.
mixcr analyze rna-seq \
--species hsa \
raw/CD8T_REH_4h_rep1.fastq.gz \
results/CD8T_REH_4h_rep1
Under the hood this preset differs from the one used previously in the following manner:
--write-alignmentsoption is added tomixcr assemblecommand to preserve the alignments that were not used inCDR3clone assembly.
Note
With this option MiXCR will output .clna (instead of .clns) file that preserves original alignments.
mixcr assemble \
-OassemblingFeatures="CDR3" \
-OseparateByV=true \
-OseparateByJ=true \
--write-alignments \
--report results/FebControl1.report.txt \
--json-report results/FebControl1.report.json \
results/CD8T_REH_4h_rep1.extended.vdjca \
results/CD8T_REH_4h_rep1.clna
- An additional
assembleContigsstep will be added aftermixcr assemble. On this step MiXCR will use previously preserved alignments to build the longest possible clonal sequence.
mixcr assembleContigs \
--report results/CD8T_REH_4h_rep1.report.txt \
--json-report results/CD8T_REH_4h_rep1.report.json \
results/CD8T_REH_4h_rep1.clna \
results/CD8T_REH_4h_rep1.clns
Reports
Finally, MiXCR provides a very convenient way to look at the reports generated at ech step. Every .vdjca, .clns and .clna file holds all the reports for every MiXCR function that has been applied to this sample. E.g. in our case .clns file contains reports for mixcr align and mixcr assemble. To output this report use mixcr exportReports as shown bellow. Note --json parameter will output a JSON-formatted report.
mixcr exportReports \
results/CD8T_REH_4h_rep1.clns \
results/CD8T_REH_4h_rep1.report.txt
mixcr exportReports \
--json \
results/CD8T_REH_4h_rep1.clns \
results/CD8T_REH_4h_rep1.report.json
Show report file
============== Align Report ==============
Input file(s): /raw/PRJNA683867_Treg_CD8_RNAseq/CD8T_REH_4h_rep1.fastq.gz
Output file(s): results/CD8T_REH_4h_rep1.vdjca
Version: 4.0.0-331-protocols; built=Thu Oct 06 19:33:27 CEST 2022; rev=83ef6ba9c4; lib=repseqio.v2.0
Command line arguments: --report results/CD8T_REH_4h_rep1.align.report.txt --json-report results/CD8T_REH_4h_rep1.align.report.json --preset local:rnaseq-tcr-cdr3 +species mmu /raw/PRJNA683867_Treg_CD8_RNAseq/CD8T_REH_4h_rep1.fastq.gz results/CD8T_REH_4h_rep1.vdjca
Analysis time: 0ns
Total sequencing reads: 78755048
Successfully aligned reads: 8509 (0.01%)
Alignment failed, no hits (not TCR/IG?): 78421282 (99.58%)
Alignment failed because of absence of CDR3 parts: 32282 (0.04%)
Alignment failed because of low total score: 292975 (0.37%)
Overlapped: 0 (0%)
Overlapped and aligned: 0 (0%)
Alignment-aided overlaps: 0 (NaN%)
Overlapped and not aligned: 0 (0%)
No CDR3 parts alignments, percent of successfully aligned: 881 (10.35%)
Partial aligned reads, percent of successfully aligned: 6877 (80.82%)
TRA chains: 1659 (19.5%)
TRA non-functional: 22 (1.33%)
TRB chains: 6107 (71.77%)
TRB non-functional: 28 (0.46%)
TRD chains: 53 (0.62%)
TRD non-functional: 0 (0%)
TRG chains: 17 (0.2%)
TRG non-functional: 0 (0%)
TRA,TRD chains: 343 (4.03%)
TRA,TRD non-functional: 0 (0%)
IGH chains: 143 (1.68%)
IGH non-functional: 0 (0%)
IGK chains: 181 (2.13%)
IGK non-functional: 0 (0%)
IGL chains: 6 (0.07%)
IGL non-functional: 0 (0%)
Realigned with forced non-floating bound: 0 (0%)
Realigned with forced non-floating right bound in left read: 0 (0%)
Realigned with forced non-floating left bound in right read: 0 (0%)
============== Extend Report ==============
Input file(s): results/CD8T_REH_4h_rep1.vdjca
Output file(s): results/CD8T_REH_4h_rep1.extended.vdjca
Version: 4.0.0-331-protocols; built=Thu Oct 06 19:33:27 CEST 2022; rev=83ef6ba9c4; lib=repseqio.v2.0
Command line arguments: --report results/CD8T_REH_4h_rep1.extend.report.txt --json-report results/CD8T_REH_4h_rep1.extend.report.json results/CD8T_REH_4h_rep1.vdjca results/CD8T_REH_4h_rep1.extended.vdjca
Analysis time: 0ns
Extended alignments count: 265 (3.11%)
V extensions total: 0 (0%)
V extensions with merged targets: 0 (0%)
J extensions total: 265 (3.11%)
J extensions with merged targets: 0 (0%)
V+J extensions: 0 (0%)
Mean V extension length: NaN
Mean J extension length: 7.796226415094339
============== AssemblePartial Report ==============
Input file(s): results/CD8T_REH_4h_rep1.extended.vdjca
Output file(s): results/CD8T_REH_4h_rep1.passembled.0.vdjca
Version: 4.0.0-331-protocols; built=Thu Oct 06 19:33:27 CEST 2022; rev=83ef6ba9c4; lib=repseqio.v2.0
Command line arguments: --report results/CD8T_REH_4h_rep1.assemblePartial.report.txt --json-report results/CD8T_REH_4h_rep1.assemblePartial.report.json results/CD8T_REH_4h_rep1.extended.vdjca results/CD8T_REH_4h_rep1.passembled.0.vdjca
Analysis time: 0ns
Total alignments analysed: 8509
Number of output alignments: 8506 (99.96%)
Alignments already with CDR3 (no overlapping is performed): 1016 (11.94%)
Successfully overlapped alignments: 3 (0.04%)
Left parts with too small N-region (failed to extract k-mer): 1542 (18.12%)
Extracted k-mer diversity: 2889
Dropped due to wildcard in k-mer: 0 (0%)
Dropped due to too short NRegion parts in overlap: 89 (1.05%)
Dropped overlaps with empty N region due to no complete NDN coverage: 33 (0.39%)
Number of left-side alignments: 797 (9.37%)
Number of right-side alignments: 5154 (60.57%)
Complex overlaps: 0 (0%)
Over-overlaps: 0 (0%)
Partial alignments written to output: 7487 (87.99%)
============== AssemblePartial Report ==============
Input file(s): results/CD8T_REH_4h_rep1.passembled.0.vdjca
Output file(s): results/CD8T_REH_4h_rep1.passembled.1.vdjca
Version: 4.0.0-331-protocols; built=Thu Oct 06 19:33:27 CEST 2022; rev=83ef6ba9c4; lib=repseqio.v2.0
Command line arguments: --report results/CD8T_REH_4h_rep1.assemblePartial.report.txt --json-report results/CD8T_REH_4h_rep1.assemblePartial.report.json results/CD8T_REH_4h_rep1.passembled.0.vdjca results/CD8T_REH_4h_rep1.passembled.1.vdjca
Analysis time: 0ns
Total alignments analysed: 8506
Number of output alignments: 8506 (100%)
Alignments already with CDR3 (no overlapping is performed): 1019 (11.98%)
Successfully overlapped alignments: 0 (0%)
Left parts with too small N-region (failed to extract k-mer): 1542 (18.13%)
Extracted k-mer diversity: 2889
Dropped due to wildcard in k-mer: 0 (0%)
Dropped due to too short NRegion parts in overlap: 91 (1.07%)
Dropped overlaps with empty N region due to no complete NDN coverage: 30 (0.35%)
Number of left-side alignments: 794 (9.33%)
Number of right-side alignments: 5151 (60.56%)
Complex overlaps: 0 (0%)
Over-overlaps: 0 (0%)
Partial alignments written to output: 7487 (88.02%)
============== Assemble Report ==============
Input file(s): results/CD8T_REH_4h_rep1.passembled.1.vdjca
Output file(s): results/CD8T_REH_4h_rep1.clns
Version: 4.0.0-331-protocols; built=Thu Oct 06 19:33:27 CEST 2022; rev=83ef6ba9c4; lib=repseqio.v2.0
Command line arguments: --report results/CD8T_REH_4h_rep1.assemble.report.txt --json-report results/CD8T_REH_4h_rep1.assemble.report.json results/CD8T_REH_4h_rep1.passembled.1.vdjca results/CD8T_REH_4h_rep1.clns
Analysis time: 0ns
Final clonotype count: 632
Average number of reads per clonotype: 1.39
Reads used in clonotypes, percent of total: 877 (0%)
Reads used in clonotypes before clustering, percent of total: 877 (0%)
Number of reads used as a core, percent of used: 868 (98.97%)
Mapped low quality reads, percent of used: 9 (1.03%)
Reads clustered in PCR error correction, percent of used: 0 (0%)
Reads pre-clustered due to the similar VJC-lists, percent of used: 0 (0%)
Reads dropped due to the lack of a clone sequence, percent of total: 7487 (0.01%)
Reads dropped due to a too short clonal sequence, percent of total: 0 (0%)
Reads dropped due to low quality, percent of total: 0 (0%)
Reads dropped due to failed mapping, percent of total: 145 (0%)
Reads dropped with low quality clones, percent of total: 0 (0%)
Clonotypes eliminated by PCR error correction: 0
Clonotypes dropped as low quality: 0
Clonotypes pre-clustered due to the similar VJC-lists: 0
TRA chains: 196 (31.01%)
TRA non-functional: 29 (14.8%)
TRB chains: 398 (62.97%)
TRB non-functional: 18 (4.52%)
TRG chains: 1 (0.16%)
TRG non-functional: 0 (0%)
IGH chains: 8 (1.27%)
IGH non-functional: 0 (0%)
IGK chains: 28 (4.43%)
IGK non-functional: 0 (0%)
IGL chains: 1 (0.16%)
IGL non-functional: 0 (0%)
{
"type": "alignerReport",
"commandLine": "--report results/CD8T_REH_4h_rep1.align.report.txt --json-report results/CD8T_REH_4h_rep1.align.report.json --preset local:rnaseq-tcr-cdr3 +species mmu /raw/PRJNA683867_Treg_CD8_RNAseq/CD8T_REH_4h_rep1.fastq.gz results/CD8T_REH_4h_rep1.vdjca",
"inputFiles": [
"/raw/PRJNA683867_Treg_CD8_RNAseq/CD8T_REH_4h_rep1.fastq.gz"
],
"outputFiles": [
"results/CD8T_REH_4h_rep1.vdjca"
],
"version": "4.0.0-331-protocols; built=Thu Oct 06 19:33:27 CEST 2022; rev=83ef6ba9c4; lib=repseqio.v2.0",
"trimmingReport": null,
"totalReadsProcessed": 78755048,
"aligned": 8509,
"notAligned": 78746539,
"notAlignedReasons": {
"NoBarcode": 0,
"NoJHits": 0,
"NoVHits": 0,
"NoHits": 78421282,
"NoCDR3Parts": 32282,
"VAndJOnDifferentTargets": 0,
"LowTotalScore": 292975
},
"chimeras": 0,
"overlapped": 0,
"alignmentAidedOverlaps": 0,
"overlappedAligned": 0,
"overlappedNotAligned": 0,
"pairedEndAlignmentConflicts": 0,
"vChimeras": 0,
"jChimeras": 0,
"chainUsage": {
"type": "chainUsage",
"chimeras": 0,
"total": 8509,
"chains": {
"TRA": {
"total": 1659,
"nonFunctional": 22,
"isOOF": 22,
"hasStops": 0
},
"TRB": {
"total": 6107,
"nonFunctional": 28,
"isOOF": 28,
"hasStops": 0
},
"TRD": {
"total": 53,
"nonFunctional": 0,
"isOOF": 0,
"hasStops": 0
},
"TRG": {
"total": 17,
"nonFunctional": 0,
"isOOF": 0,
"hasStops": 0
},
"TRA,TRD": {
"total": 343,
"nonFunctional": 0,
"isOOF": 0,
"hasStops": 0
},
"IGH": {
"total": 143,
"nonFunctional": 0,
"isOOF": 0,
"hasStops": 0
},
"IGK": {
"total": 181,
"nonFunctional": 0,
"isOOF": 0,
"hasStops": 0
},
"IGL": {
"total": 6,
"nonFunctional": 0,
"isOOF": 0,
"hasStops": 0
}
}
},
"realignedWithForcedNonFloatingBound": 0,
"realignedWithForcedNonFloatingRightBoundInLeftRead": 0,
"realignedWithForcedNonFloatingLeftBoundInRightRead": 0,
"noCDR3PartsAlignments": 881,
"partialAlignments": 6877,
"tagReport": null
}
{
"type": "extenderReport",
"commandLine": "--report results/CD8T_REH_4h_rep1.extend.report.txt --json-report results/CD8T_REH_4h_rep1.extend.report.json results/CD8T_REH_4h_rep1.vdjca results/CD8T_REH_4h_rep1.extended.vdjca",
"inputFiles": [
"results/CD8T_REH_4h_rep1.vdjca"
],
"outputFiles": [
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],
"version": "4.0.0-331-protocols; built=Thu Oct 06 19:33:27 CEST 2022; rev=83ef6ba9c4; lib=repseqio.v2.0",
"totalProcessed": 8509,
"totalExtended": 265,
"vExtended": 0,
"vExtendedMerged": 0,
"jExtended": 265,
"jExtendedMerged": 0,
"vjExtended": 0,
"meanVExtensionLength": "NaN",
"meanJExtensionLength": 7.796226415094339
}
{
"type": "partialAlignmentsAssemblerReport",
"commandLine": "--report results/CD8T_REH_4h_rep1.assemblePartial.report.txt --json-report results/CD8T_REH_4h_rep1.assemblePartial.report.json results/CD8T_REH_4h_rep1.extended.vdjca results/CD8T_REH_4h_rep1.passembled.0.vdjca",
"inputFiles": [
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],
"outputFiles": [
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],
"version": "4.0.0-331-protocols; built=Thu Oct 06 19:33:27 CEST 2022; rev=83ef6ba9c4; lib=repseqio.v2.0",
"independentRuns": 1,
"totalProcessed": 8509,
"outputAlignments": 8506,
"withCDR3": 1016,
"overlapped": 3,
"leftTooShortNRegion": 1542,
"kMerDiversity": 2889,
"droppedWildcardsInKMer": 0,
"droppedSmallOverlapNRegion": 89,
"droppedNoNRegion": 33,
"leftParts": 797,
"rightParts": 5154,
"complexOverlaps": 0,
"overOverlaps": 0,
"partialAlignmentsAsIs": 7487
}
{
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