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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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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

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 assemblePartial on 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 CDR3 sequence.
-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

chainUsage.svg

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:

  1. --write-alignments option is added to mixcr assemble command to preserve the alignments that were not used in CDR3 clone 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
  1. An additional assembleContigs step will be added after mixcr 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%)

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  "noCDR3PartsAlignments": 881,
  "partialAlignments": 6877,
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{
  "type": "extenderReport",
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{
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  "type": "partialAlignmentsAssemblerReport",
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