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SMARTer Mouse TCR a/b Profiling Kit

Here we will discuss how to process TCR cDNA libraries obtained with SMARTer Mouse TCR a/b Profiling Kit.

Data libraries

This tutorial uses the data from the following publication: A T cell repertoire timestamp is at the core of responsiveness to CTLA-4 blockade Hagit Philip et al., , iScience, 2021 Feb; doi: 10.1016/j.isci.2021.102100

Samples were collected from 20 mice implanted with MC38 adenocarcinoma cells. The experiment contained 2 groups, of which the first group (5 animals with ids A - E) was dosed with 10 ml/kg/day of vehicle on day 1, 3 and 6 of the experiment. The second group (15 animals with ids F - T) was dosed with the investigational antibody anti-mCTLA4 (5/2.5 mg/kg/day at 1/3, 6). After treatment with the immune checkpoint inhibitor antibody anti-mCTLA4 blood samples for the preparation of PBMCs were taken at days 0,7,14 and 21. Tumors (where still visible) and spleens were collected immediately after euthanasia, and directly transferred to liquid nitrogen (snap-frozen samples). Total RNA was isolated from every sample and cDNA libraries were prepared using SMARTer Mouse TCR a/b Profiling Kit. TCR sequencing was performed on an Illumina Miseq sequencer using the 600-cycle Miseq reagent kit v3(Illumina) with pair-end, 2x300 base pair reads.

All data is available from SRA (PRJNA658348) 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/SRR124/021/SRR12487521/SRR12487521_1.fastq.gz
  out=raw/S_s_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/022/SRR12487522/SRR12487522_1.fastq.gz
  out=raw/S_7_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/023/SRR12487523/SRR12487523_1.fastq.gz
  out=raw/S_21_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/024/SRR12487524/SRR12487524_1.fastq.gz
  out=raw/S_14_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/025/SRR12487525/SRR12487525_1.fastq.gz
  out=raw/S_0_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/026/SRR12487526/SRR12487526_1.fastq.gz
  out=raw/I_s_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/027/SRR12487527/SRR12487527_1.fastq.gz
  out=raw/I_7_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/028/SRR12487528/SRR12487528_1.fastq.gz
  out=raw/I_21_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/029/SRR12487529/SRR12487529_1.fastq.gz
  out=raw/I_14_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/030/SRR12487530/SRR12487530_1.fastq.gz
  out=raw/I_0_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/031/SRR12487531/SRR12487531_1.fastq.gz
  out=raw/T_s_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/032/SRR12487532/SRR12487532_1.fastq.gz
  out=raw/J_s_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/033/SRR12487533/SRR12487533_1.fastq.gz
  out=raw/J_7_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/034/SRR12487534/SRR12487534_1.fastq.gz
  out=raw/J_21_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/035/SRR12487535/SRR12487535_1.fastq.gz
  out=raw/J_14_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/036/SRR12487536/SRR12487536_1.fastq.gz
  out=raw/J_0_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/037/SRR12487537/SRR12487537_1.fastq.gz
  out=raw/A_t_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/038/SRR12487538/SRR12487538_1.fastq.gz
  out=raw/A_7_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/039/SRR12487539/SRR12487539_1.fastq.gz
  out=raw/A_14_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/040/SRR12487540/SRR12487540_1.fastq.gz
  out=raw/A_0_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/041/SRR12487541/SRR12487541_1.fastq.gz
  out=raw/M_s_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/042/SRR12487542/SRR12487542_1.fastq.gz
  out=raw/T_7_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/043/SRR12487543/SRR12487543_1.fastq.gz
  out=raw/M_7_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/044/SRR12487544/SRR12487544_1.fastq.gz
  out=raw/M_21_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/045/SRR12487545/SRR12487545_1.fastq.gz
  out=raw/M_14_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/046/SRR12487546/SRR12487546_1.fastq.gz
  out=raw/M_0_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/047/SRR12487547/SRR12487547_1.fastq.gz
  out=raw/G_s_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/048/SRR12487548/SRR12487548_1.fastq.gz
  out=raw/G_t_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/049/SRR12487549/SRR12487549_1.fastq.gz
  out=raw/G_7_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/050/SRR12487550/SRR12487550_1.fastq.gz
  out=raw/G_21_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/051/SRR12487551/SRR12487551_1.fastq.gz
  out=raw/G_14_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/052/SRR12487552/SRR12487552_1.fastq.gz
  out=raw/G_0_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/053/SRR12487553/SRR12487553_1.fastq.gz
  out=raw/T_21_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/054/SRR12487554/SRR12487554_1.fastq.gz
  out=raw/O_s_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/055/SRR12487555/SRR12487555_1.fastq.gz
  out=raw/O_7_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/056/SRR12487556/SRR12487556_1.fastq.gz
  out=raw/O_21_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/057/SRR12487557/SRR12487557_1.fastq.gz
  out=raw/O_14_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/058/SRR12487558/SRR12487558_1.fastq.gz
  out=raw/O_0_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/059/SRR12487559/SRR12487559_1.fastq.gz
  out=raw/R_s_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/060/SRR12487560/SRR12487560_1.fastq.gz
  out=raw/R_7_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/061/SRR12487561/SRR12487561_1.fastq.gz
  out=raw/R_21_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/062/SRR12487562/SRR12487562_1.fastq.gz
  out=raw/R_14_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/063/SRR12487563/SRR12487563_1.fastq.gz
  out=raw/R_0_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/064/SRR12487564/SRR12487564_1.fastq.gz
  out=raw/T_14_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/065/SRR12487565/SRR12487565_1.fastq.gz
  out=raw/H_s_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/066/SRR12487566/SRR12487566_1.fastq.gz
  out=raw/H_7_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/067/SRR12487567/SRR12487567_1.fastq.gz
  out=raw/H_14_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/068/SRR12487568/SRR12487568_1.fastq.gz
  out=raw/H_0_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/069/SRR12487569/SRR12487569_1.fastq.gz
  out=raw/H_21_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/070/SRR12487570/SRR12487570_1.fastq.gz
  out=raw/P_s_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/071/SRR12487571/SRR12487571_1.fastq.gz
  out=raw/P_7_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/072/SRR12487572/SRR12487572_1.fastq.gz
  out=raw/P_21_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/073/SRR12487573/SRR12487573_1.fastq.gz
  out=raw/P_14_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/074/SRR12487574/SRR12487574_1.fastq.gz
  out=raw/P_0_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/075/SRR12487575/SRR12487575_1.fastq.gz
  out=raw/T_0_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/076/SRR12487576/SRR12487576_1.fastq.gz
  out=raw/E_t_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/077/SRR12487577/SRR12487577_1.fastq.gz
  out=raw/E_s_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/078/SRR12487578/SRR12487578_1.fastq.gz
  out=raw/E_7_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/079/SRR12487579/SRR12487579_1.fastq.gz
  out=raw/E_21_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/080/SRR12487580/SRR12487580_1.fastq.gz
  out=raw/E_14_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/081/SRR12487581/SRR12487581_1.fastq.gz
  out=raw/E_0_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/082/SRR12487582/SRR12487582_1.fastq.gz
  out=raw/D_s_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/083/SRR12487583/SRR12487583_1.fastq.gz
  out=raw/D_t_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/084/SRR12487584/SRR12487584_1.fastq.gz
  out=raw/D_7_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/085/SRR12487585/SRR12487585_1.fastq.gz
  out=raw/D_21_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/086/SRR12487586/SRR12487586_1.fastq.gz
  out=raw/Q_s_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/087/SRR12487587/SRR12487587_1.fastq.gz
  out=raw/D_14_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/088/SRR12487588/SRR12487588_1.fastq.gz
  out=raw/D_0_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/089/SRR12487589/SRR12487589_1.fastq.gz
  out=raw/K_s_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/090/SRR12487590/SRR12487590_1.fastq.gz
  out=raw/K_21_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/091/SRR12487591/SRR12487591_1.fastq.gz
  out=raw/K_14_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/092/SRR12487592/SRR12487592_1.fastq.gz
  out=raw/K_7_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/093/SRR12487593/SRR12487593_1.fastq.gz
  out=raw/L_s_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/094/SRR12487594/SRR12487594_1.fastq.gz
  out=raw/L_7_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/095/SRR12487595/SRR12487595_1.fastq.gz
  out=raw/L_21_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/096/SRR12487596/SRR12487596_1.fastq.gz
  out=raw/L_14_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/097/SRR12487597/SRR12487597_1.fastq.gz
  out=raw/Q_7_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/098/SRR12487598/SRR12487598_1.fastq.gz
  out=raw/L_0_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/099/SRR12487599/SRR12487599_1.fastq.gz
  out=raw/F_t_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/000/SRR12487600/SRR12487600_1.fastq.gz
  out=raw/F_s_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/001/SRR12487601/SRR12487601_1.fastq.gz
  out=raw/F_7_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/002/SRR12487602/SRR12487602_1.fastq.gz
  out=raw/F_21_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/003/SRR12487603/SRR12487603_1.fastq.gz
  out=raw/F_14_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/004/SRR12487604/SRR12487604_1.fastq.gz
  out=raw/F_0_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/005/SRR12487605/SRR12487605_1.fastq.gz
  out=raw/N_s_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/006/SRR12487606/SRR12487606_1.fastq.gz
  out=raw/N_7_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/007/SRR12487607/SRR12487607_1.fastq.gz
  out=raw/N_21_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/008/SRR12487608/SRR12487608_1.fastq.gz
  out=raw/Q_21_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/009/SRR12487609/SRR12487609_1.fastq.gz
  out=raw/N_14_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/010/SRR12487610/SRR12487610_1.fastq.gz
  out=raw/N_0_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/011/SRR12487611/SRR12487611_1.fastq.gz
  out=raw/C_s_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/012/SRR12487612/SRR12487612_1.fastq.gz
  out=raw/C_7_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/013/SRR12487613/SRR12487613_1.fastq.gz
  out=raw/C_21_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/014/SRR12487614/SRR12487614_1.fastq.gz
  out=raw/C_14_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/015/SRR12487615/SRR12487615_1.fastq.gz
  out=raw/C_0_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/016/SRR12487616/SRR12487616_1.fastq.gz
  out=raw/B_7_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/017/SRR12487617/SRR12487617_1.fastq.gz
  out=raw/B_14_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/018/SRR12487618/SRR12487618_1.fastq.gz
  out=raw/B_0_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/019/SRR12487619/SRR12487619_1.fastq.gz
  out=raw/Q_14_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/020/SRR12487620/SRR12487620_1.fastq.gz
  out=raw/Q_0_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/021/SRR12487521/SRR12487521_2.fastq.gz
  out=raw/S_s_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/022/SRR12487522/SRR12487522_2.fastq.gz
  out=raw/S_7_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/023/SRR12487523/SRR12487523_2.fastq.gz
  out=raw/S_21_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/024/SRR12487524/SRR12487524_2.fastq.gz
  out=raw/S_14_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/025/SRR12487525/SRR12487525_2.fastq.gz
  out=raw/S_0_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/026/SRR12487526/SRR12487526_2.fastq.gz
  out=raw/I_s_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/027/SRR12487527/SRR12487527_2.fastq.gz
  out=raw/I_7_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/028/SRR12487528/SRR12487528_2.fastq.gz
  out=raw/I_21_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/029/SRR12487529/SRR12487529_2.fastq.gz
  out=raw/I_14_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/030/SRR12487530/SRR12487530_2.fastq.gz
  out=raw/I_0_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/031/SRR12487531/SRR12487531_2.fastq.gz
  out=raw/T_s_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/032/SRR12487532/SRR12487532_2.fastq.gz
  out=raw/J_s_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/033/SRR12487533/SRR12487533_2.fastq.gz
  out=raw/J_7_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/034/SRR12487534/SRR12487534_2.fastq.gz
  out=raw/J_21_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/035/SRR12487535/SRR12487535_2.fastq.gz
  out=raw/J_14_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/036/SRR12487536/SRR12487536_2.fastq.gz
  out=raw/J_0_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/037/SRR12487537/SRR12487537_2.fastq.gz
  out=raw/A_t_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/038/SRR12487538/SRR12487538_2.fastq.gz
  out=raw/A_7_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/039/SRR12487539/SRR12487539_2.fastq.gz
  out=raw/A_14_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/040/SRR12487540/SRR12487540_2.fastq.gz
  out=raw/A_0_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/041/SRR12487541/SRR12487541_2.fastq.gz
  out=raw/M_s_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/042/SRR12487542/SRR12487542_2.fastq.gz
  out=raw/T_7_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/043/SRR12487543/SRR12487543_2.fastq.gz
  out=raw/M_7_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/044/SRR12487544/SRR12487544_2.fastq.gz
  out=raw/M_21_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/045/SRR12487545/SRR12487545_2.fastq.gz
  out=raw/M_14_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/046/SRR12487546/SRR12487546_2.fastq.gz
  out=raw/M_0_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/047/SRR12487547/SRR12487547_2.fastq.gz
  out=raw/G_s_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/048/SRR12487548/SRR12487548_2.fastq.gz
  out=raw/G_t_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/049/SRR12487549/SRR12487549_2.fastq.gz
  out=raw/G_7_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/050/SRR12487550/SRR12487550_2.fastq.gz
  out=raw/G_21_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/051/SRR12487551/SRR12487551_2.fastq.gz
  out=raw/G_14_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/052/SRR12487552/SRR12487552_2.fastq.gz
  out=raw/G_0_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/053/SRR12487553/SRR12487553_2.fastq.gz
  out=raw/T_21_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/054/SRR12487554/SRR12487554_2.fastq.gz
  out=raw/O_s_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/055/SRR12487555/SRR12487555_2.fastq.gz
  out=raw/O_7_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/056/SRR12487556/SRR12487556_2.fastq.gz
  out=raw/O_21_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/057/SRR12487557/SRR12487557_2.fastq.gz
  out=raw/O_14_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/058/SRR12487558/SRR12487558_2.fastq.gz
  out=raw/O_0_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/059/SRR12487559/SRR12487559_2.fastq.gz
  out=raw/R_s_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/060/SRR12487560/SRR12487560_2.fastq.gz
  out=raw/R_7_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/061/SRR12487561/SRR12487561_2.fastq.gz
  out=raw/R_21_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/062/SRR12487562/SRR12487562_2.fastq.gz
  out=raw/R_14_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/063/SRR12487563/SRR12487563_2.fastq.gz
  out=raw/R_0_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/064/SRR12487564/SRR12487564_2.fastq.gz
  out=raw/T_14_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/065/SRR12487565/SRR12487565_2.fastq.gz
  out=raw/H_s_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/066/SRR12487566/SRR12487566_2.fastq.gz
  out=raw/H_7_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/067/SRR12487567/SRR12487567_2.fastq.gz
  out=raw/H_14_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/068/SRR12487568/SRR12487568_2.fastq.gz
  out=raw/H_0_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/069/SRR12487569/SRR12487569_2.fastq.gz
  out=raw/H_21_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/070/SRR12487570/SRR12487570_2.fastq.gz
  out=raw/P_s_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/071/SRR12487571/SRR12487571_2.fastq.gz
  out=raw/P_7_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/072/SRR12487572/SRR12487572_2.fastq.gz
  out=raw/P_21_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/073/SRR12487573/SRR12487573_2.fastq.gz
  out=raw/P_14_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/074/SRR12487574/SRR12487574_2.fastq.gz
  out=raw/P_0_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/075/SRR12487575/SRR12487575_2.fastq.gz
  out=raw/T_0_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/076/SRR12487576/SRR12487576_2.fastq.gz
  out=raw/E_t_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/077/SRR12487577/SRR12487577_2.fastq.gz
  out=raw/E_s_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/078/SRR12487578/SRR12487578_2.fastq.gz
  out=raw/E_7_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/079/SRR12487579/SRR12487579_2.fastq.gz
  out=raw/E_21_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/080/SRR12487580/SRR12487580_2.fastq.gz
  out=raw/E_14_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/081/SRR12487581/SRR12487581_2.fastq.gz
  out=raw/E_0_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/082/SRR12487582/SRR12487582_2.fastq.gz
  out=raw/D_s_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/083/SRR12487583/SRR12487583_2.fastq.gz
  out=raw/D_t_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/084/SRR12487584/SRR12487584_2.fastq.gz
  out=raw/D_7_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/085/SRR12487585/SRR12487585_2.fastq.gz
  out=raw/D_21_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/086/SRR12487586/SRR12487586_2.fastq.gz
  out=raw/Q_s_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/087/SRR12487587/SRR12487587_2.fastq.gz
  out=raw/D_14_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/088/SRR12487588/SRR12487588_2.fastq.gz
  out=raw/D_0_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/089/SRR12487589/SRR12487589_2.fastq.gz
  out=raw/K_s_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/090/SRR12487590/SRR12487590_2.fastq.gz
  out=raw/K_21_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/091/SRR12487591/SRR12487591_2.fastq.gz
  out=raw/K_14_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/092/SRR12487592/SRR12487592_2.fastq.gz
  out=raw/K_7_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/093/SRR12487593/SRR12487593_2.fastq.gz
  out=raw/L_s_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/094/SRR12487594/SRR12487594_2.fastq.gz
  out=raw/L_7_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/095/SRR12487595/SRR12487595_2.fastq.gz
  out=raw/L_21_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/096/SRR12487596/SRR12487596_2.fastq.gz
  out=raw/L_14_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/097/SRR12487597/SRR12487597_2.fastq.gz
  out=raw/Q_7_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/098/SRR12487598/SRR12487598_2.fastq.gz
  out=raw/L_0_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/099/SRR12487599/SRR12487599_2.fastq.gz
  out=raw/F_t_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/000/SRR12487600/SRR12487600_2.fastq.gz
  out=raw/F_s_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/001/SRR12487601/SRR12487601_2.fastq.gz
  out=raw/F_7_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/002/SRR12487602/SRR12487602_2.fastq.gz
  out=raw/F_21_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/003/SRR12487603/SRR12487603_2.fastq.gz
  out=raw/F_14_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/004/SRR12487604/SRR12487604_2.fastq.gz
  out=raw/F_0_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/005/SRR12487605/SRR12487605_2.fastq.gz
  out=raw/N_s_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/006/SRR12487606/SRR12487606_2.fastq.gz
  out=raw/N_7_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/007/SRR12487607/SRR12487607_2.fastq.gz
  out=raw/N_21_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/008/SRR12487608/SRR12487608_2.fastq.gz
  out=raw/Q_21_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/009/SRR12487609/SRR12487609_2.fastq.gz
  out=raw/N_14_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/010/SRR12487610/SRR12487610_2.fastq.gz
  out=raw/N_0_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/011/SRR12487611/SRR12487611_2.fastq.gz
  out=raw/C_s_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/012/SRR12487612/SRR12487612_2.fastq.gz
  out=raw/C_7_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/013/SRR12487613/SRR12487613_2.fastq.gz
  out=raw/C_21_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/014/SRR12487614/SRR12487614_2.fastq.gz
  out=raw/C_14_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/015/SRR12487615/SRR12487615_2.fastq.gz
  out=raw/C_0_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/016/SRR12487616/SRR12487616_2.fastq.gz
  out=raw/B_7_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/017/SRR12487617/SRR12487617_2.fastq.gz
  out=raw/B_14_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/018/SRR12487618/SRR12487618_2.fastq.gz
  out=raw/B_0_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/019/SRR12487619/SRR12487619_2.fastq.gz
  out=raw/Q_14_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR124/020/SRR12487620/SRR12487620_2.fastq.gz
  out=raw/Q_0_R2.fastq.gz

The project contains 100 FASTQ file pairs. For the purpose of this tutorial we assume that all fastq files are stored in fastq/ folder. Each file name encodes the information about mouse id and PMBC sample time-point (0,7,14,21 days) or tissue (s - spleen, t -tumor) - S - mouce id, experiment group - s - spleen tissue

The structure of the library is shown on the picture bellow.

Upstream analysis

MiXCR has a dedicated preset for this protocol, thus analysing the data is as easy as:

mixcr analyze takara-mouse-rna-tcr-smarter \
    raw/S_s_R1.fastq.gz \
    raw/S_s_R2.fastq.gz \
    results/S_s

One might also use GNU Parallel to process all samples at once:

#!/usr/bin/env bash

mkdir -p results

ls /raw/*R1* |
    parallel -j 2 --line-buffer \
    "mixcr analyze takara-mouse-rna-tcr-smarter \
    {} \
    {=s:R1:R2:=} \
    {=s:.*/:results/:;s:_R.*::=}"

Under the hood the command above actually executes the following pipeline:

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 generic-amplicon \
    -OvParameters.geneFeatureToAlign="VTranscriptWithout5UTRWithP" \
    -OvParameters.parameters.floatingLeftBound=false \
    -OjParameters.parameters.floatingRightBound=false \
    --report results/S_s.report \
    --json-report results/S_s.json \
     raw/S_s_R1.fastq.gz \
     raw/S_s_R2.fastq.gz \
     results/S_s.vdjca

Option --report is specified here explicitly.

--species mmu
determines the organism species (mmu for Mus Musculus).
-p generic-amplicon
a default preset of MiXCR parameters for amplicon data.
-OvParameters.geneFeatureToAlign="VTranscriptWithout5UTRWithP"
Sets a V gene feature to align. Check gene features for more info.
-OvParameters.parameters.floatingLeftBound=false
Results in a global alignment algorithm for V gene left bound. We use it because we don't have any primers covering V gene coding sequence.
-OjParameters.parameters.floatingRightBound=false
Results in a global alignment algorithm for J gene right bound, because reverse primers are located in C-gene region.

assemble

Assembles alignments into clonotypes and applies several layers of errors correction(ex. quality-dependent correction for sequencing errors, PCR-error correction by clustering). Check mixcr assemble for more information.

mixcr assemble \
    -OassemblingFeatures="CDR3"
    -OseparateByJ=true \
    -OseparateByV=true \
    --report results/S_s.report \
    --json-report results/S_s.json \
    results/S_s.vdjca \
    results/S_s.clns

Options --report and --json-report are specified here explicitly so that the report files will be appended with assembly report.

-OassemblingFeatures=CDR3
By default takara-mmu-tcr-cdr3 preset assembles clones by CDR3 sequence.

export

Exports clonotypes from .clns file into human-readable tables.

mixcr exportClones \
    -c TRA \
    results/S_s.clns \
    results/S_s.clonotypes.TRA.txt
-с <chain>
defines a specific chain to be exported.

After executing the command above for every sample we will have a following set of output files: 

# human-readable reports 
S_s.report
# raw alignments (highly compressed binary file)
S_s.vdjca
# TCRα & TCRβ CDR3 clonotypes (highly compressed binary file)
S_s.clns
# TCRα & TCRβ CDR3 clonotypes exported in tab-delimited txt
S_s.TRB.txt
S_s.TRB.txt

While .clns file holds all data and is used for downstream analysis, the output .txt clonotype table will contain exhaustive information about each clonotype as well:

See first 100 records from S_s 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
0 194 0.0056568 TGTGTGGTGGGCGATAGAGGTTCAGCCTTAGGGAGGCTGCATTTT GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG TRAV1100(1349),TRAV11D00(1349) nan TRAJ18*00(314.5) nan 267|280|300|0|13||65.0;267|280|300|0|13||65.0 nan 22|55|86|12|45||165.0 nan nan nan nan nan nan nan nan nan nan nan TGTGTGGTGGGCGATAGAGGTTCAGCCTTAGGGAGGCTGCATTTT 38 nan nan nan nan nan nan nan CVVGDRGSALGRLHF nan :::::::::0:0:13:::::12:-2:45:::
1 135 0.00393643 TGTGTGGTGGGGGATAGAGGTTCAGCCTTAGGGAGGCTGCATTTT GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG TRAV1100(1363),TRAV11D00(1363) nan TRAJ18*00(316.6) nan 267|278|300|0|11||55.0;267|278|300|0|11||55.0 nan 22|55|86|12|45||165.0 nan nan nan nan nan nan nan nan nan nan nan TGTGTGGTGGGGGATAGAGGTTCAGCCTTAGGGAGGCTGCATTTT 38 nan nan nan nan nan nan nan CVVGDRGSALGRLHF nan :::::::::0:-2:11:::::12:-2:45:::
2 104 0.00303251 TGTGTGGTGGGAGATAGAGGTTCAGCCTTAGGGAGGCTGCATTTT GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG TRAV1100(1380.9),TRAV11D00(1380.9) nan TRAJ18*00(320.8) nan 267|278|300|0|11||55.0;267|278|300|0|11||55.0 nan 21|55|86|11|45||170.0 nan nan nan nan nan nan nan nan nan nan nan TGTGTGGTGGGAGATAGAGGTTCAGCCTTAGGGAGGCTGCATTTT 38 nan nan nan nan nan nan nan CVVGDRGSALGRLHF nan :::::::::0:-2:11:::::11:-1:45:::
3 66 0.00192448 TGTGCAGTGAATAGGGGTAGCAATAACAGAATCTTCTTT GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG TRAV7N-6*00(1062.4) nan TRAJ31*00(270.8) nan 264|274|297|0|10||50.0 nan 24|46|77|17|39||110.0 nan nan nan nan nan nan nan nan nan nan nan TGTGCAGTGAATAGGGGTAGCAATAACAGAATCTTCTTT 38 nan nan nan nan nan nan nan CAVNRGSNNRIFF nan :::::::::0:-3:10:::::17:-4:39:::
4 53 0.00154541 TGTGTGGTGGGTGATAGAGGTTCAGCCTTAGGGAGGCTGCATTTT GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG TRAV1100(1382.6),TRAV11D00(1382.6) nan TRAJ18*00(316.2) nan 267|278|300|0|11||55.0;267|278|300|0|11||55.0 nan 22|55|86|12|45||165.0 nan nan nan nan nan nan nan nan nan nan nan TGTGTGGTGGGTGATAGAGGTTCAGCCTTAGGGAGGCTGCATTTT 38 nan nan nan nan nan nan nan CVVGDRGSALGRLHF nan :::::::::0:-2:11:::::12:-2:45:::
5 34 0.000991398 TGTGCAGCAAGTAGGGATTATAACCAGGGGAAGCTTATCTTT GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG TRAV14N-3*00(1280.9) nan TRAJ23*00(280.2) nan 267|279|300|0|12||60.0 nan 23|49|80|16|42||130.0 nan nan nan nan nan nan nan nan nan nan nan TGTGCAGCAAGTAGGGATTATAACCAGGGGAAGCTTATCTTT 38 nan nan nan nan nan nan nan CAASRDYNQGKLIF nan :::::::::0:-1:12:::::16:-3:42:::
6 33 0.000962239 TGTGCTATGAGAGAGGGCAGCAACAACAGACTTACTTTG GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG TRAV16D-DV11*00(1370.6) nan TRAJ7*00(260.7) nan 273|291|310|0|18||90.0 nan 26|48|79|17|39||110.0 nan nan nan nan nan nan nan nan nan nan nan TGTGCTATGAGAGAGGGCAGCAACAACAGACTTACTTTG 38 nan nan nan nan nan nan nan CAMREGSNNRLTL nan :::::::::0:1:18:::::17:-6:39:::
7 32 0.000933081 TGTGCTTTGGAACATAATGCAGGTGCCAAGCTCACATTC GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG TRAV13-1*00(1047.1) nan TRAJ39*00(283.3) nan 258|271|291|0|13||65.0 nan 26|52|83|13|39||130.0 nan nan nan nan nan nan nan nan nan nan nan TGTGCTTTGGAACATAATGCAGGTGCCAAGCTCACATTC 38 nan nan nan nan nan nan nan CALEHNAGAKLTF nan :::::::::0:0:13:::::13:-6:39:::
8 29 0.000845604 TGTGCCAGCAGCCCCGGGACAGGGGTTGGAAATACGCTCTATTTT GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG TRBV5*00(1343.2) TRBD1*00(60) TRBJ1-3*00(228) nan 267|280|304|0|13||65.0 10|22|36|13|25||60.0 23|42|70|26|45||95.0 nan nan nan nan nan nan nan nan nan nan nan TGTGCCAGCAGCCCCGGGACAGGGGTTGGAAATACGCTCTATTTT 38 nan nan nan nan nan nan nan CASSPGTGVGNTLYF nan :::::::::0:-4:13:13:2:-2:25:26:-3:45:::
9 27 0.000787287 TGTGCAGCAAGTGAGGGAGGCAGAGCTCTGATATTT GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG TRAV14N-3*00(1179.1) nan TRAJ15*00(274) nan 267|280|300|0|13||65.0 nan 26|49|80|13|36||115.0 nan nan nan nan nan nan nan nan nan nan nan TGTGCAGCAAGTGAGGGAGGCAGAGCTCTGATATTT 38 nan nan nan nan nan nan nan CAASEGGRALIF nan :::::::::0:0:13:::::13:-6:36:::
10 25 0.000728969 TGTGCTTCCTTGAATAGCAATAACAGAATCTTCTTT GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG TRAV13-1*00(1018.5) nan TRAJ31*00(276.7) nan 258|265|291|0|7||35.0 nan 21|46|77|11|36||125.0 nan nan nan nan nan nan nan nan nan nan nan TGTGCTTCCTTGAATAGCAATAACAGAATCTTCTTT 38 nan nan nan nan nan nan nan CASLNSNNRIFF nan :::::::::0:-6:7:::::11:-1:36:::
11 25 0.000728969 TGTGCAGTGAGGTATAATACAGGAAACTACAAATACGTCTTT GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG TRAV7-5*00(1327) nan TRAJ40*00(304.1) nan 264|275|297|0|11||55.0 nan 22|50|81|14|42||140.0 nan nan nan nan nan nan nan nan nan nan nan TGTGCAGTGAGGTATAATACAGGAAACTACAAATACGTCTTT 38 nan nan nan nan nan nan nan CAVRYNTGNYKYVF nan :::::::::0:-2:11:::::14:-2:42:::
12 25 0.000728969 TGTGCAGCAAGCATATGGGATTATAACCAGGGGAAGCTTATCTTT GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG TRAV1000(1410.5),TRAV10D00(1380.8) nan TRAJ23*00(283.3) nan 267|281|300|0|14||70.0;267|281|300|0|14||70.0 nan 23|49|80|19|45||130.0 nan nan nan nan nan nan nan nan nan nan nan TGTGCAGCAAGCATATGGGATTATAACCAGGGGAAGCTTATCTTT 38 nan nan nan nan nan nan nan CAASIWDYNQGKLIF nan :::::::::0:1:14:::::19:-3:45:::
13 23 0.000670652 TGTGCCAGCTCTCTCGTGGGGGCAAACTCCGACTACACCTTC GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG TRBV12-1*00(1217.9) TRBD2*00(25) TRBJ1-2*00(234.5) nan 270|286|305|0|16||80.0 19|24|42|16|21||25.0 19|40|68|21|42||105.0 nan nan nan nan nan nan nan nan nan nan nan TGTGCCAGCTCTCTCGTGGGGGCAAACTCCGACTACACCTTC 38 nan nan nan nan nan nan nan CASSLVGANSDYTF nan :::::::::0:1:16:16:-5:-4:21:21:1:42:::
14 21 0.000612334 TGTGCTATGAGAGAGGGCTACAGCAACAACAGACTTACTTTG GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG TRAV16D-DV1100(1376.7),TRAV16N00(1362.7) nan TRAJ7*00(272.7) nan 273|291|310|0|18||90.0;273|291|310|0|18||90.0 nan 20|48|79|14|42|SA22G|126.0 nan nan nan nan nan nan nan nan nan nan nan TGTGCTATGAGAGAGGGCTACAGCAACAACAGACTTACTTTG 38 nan nan nan nan nan nan nan CAMREGYSNNRLTL nan :::::::::0:1:18:::::14:0:42:::
15 20 0.000583175 TGTGTGGTGGGCGATAGAGGTTCAGCCTTAGGGAGGCTGCATTTT GGGGGGGGGGGGGGGGGGGGGGGGGFGGGGGGGGGGGGGGGGGGG TRAV11D*00(1532.3) nan TRAJ18*00(307.2) nan 267|280|300|0|13||65.0 nan 22|55|86|12|45||165.0 nan nan nan nan nan nan nan nan nan nan nan TGTGTGGTGGGCGATAGAGGTTCAGCCTTAGGGAGGCTGCATTTT 37 nan nan nan nan nan nan nan CVVGDRGSALGRLHF nan :::::::::0:0:13:::::12:-2:45:::
16 20 0.000583175 TGTGCTATGGAAAGTGATGGAAATGAGAAAATAACTTTT GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG TRAV13-4-DV7*00(772.4) nan TRAJ48*00(270) nan 258|273|291|0|15|SC270A|61.0 nan 27|50|81|16|39||115.0 nan nan nan nan nan nan nan nan nan nan nan TGTGCTATGGAAAGTGATGGAAATGAGAAAATAACTTTT 38 nan nan nan nan nan nan nan CAMESDGNEKITF nan :::::::::0:2:15:::::16:-7:39:::
17 20 0.000583175 TGTGCAGCAAGTTCTAACAGTGCAGGGAACAAGCTAACTTTT GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG TRAV14N-3*00(1298.5) nan TRAJ17*00(299.5) nan 267|279|300|0|12||60.0 nan 23|52|83|13|42||145.0 nan nan nan nan nan nan nan nan nan nan nan TGTGCAGCAAGTTCTAACAGTGCAGGGAACAAGCTAACTTTT 38 nan nan nan nan nan nan nan CAASSNSAGNKLTF nan :::::::::0:-1:12:::::13:-3:42:::
18 20 0.000583175 TGTGGTGCTAGACTGGGGGGGCGCGGGTATGCTGAGCAGTTCTTC GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG TRBV20*00(1367.4) TRBD2*00(65) TRBJ2-1*00(225.8) nan 279|290|313|0|11||55.0 17|30|42|11|24||65.0 24|42|70|27|45||90.0 nan nan nan nan nan nan nan nan nan nan nan TGTGGTGCTAGACTGGGGGGGCGCGGGTATGCTGAGCAGTTCTTC 38 nan nan nan nan nan nan nan CGARLGGRGYAEQFF nan :::::::::0:-3:11:11:-3:2:24:27:-4:45:::
19 19 0.000554017 TGTGCAGTGAGCGACAATACAGAAGGTGCAGATAGACTCACCTTT GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG TRAV7-6*00(895.1) nan TRAJ45*00(326.1) nan 264|276|297|0|12||60.0 nan 20|49|80|16|45||145.0 nan nan nan nan nan nan nan nan nan nan nan TGTGCAGTGAGCGACAATACAGAAGGTGCAGATAGACTCACCTTT 38 nan nan nan nan nan nan nan CAVSDNTEGADRLTF nan :::::::::0:-1:12:::::16:0:45:::
20 18 0.000524858 TGTGCTTCCATGTCTAATTACAACGTGCTTTACTTC GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG TRAV13N-4*00(734.3) nan TRAJ21*00(295) nan 258|264|291|0|6||30.0 nan 18|46|77|8|36||140.0 nan nan nan nan nan nan nan nan nan nan nan TGTGCTTCCATGTCTAATTACAACGTGCTTTACTTC 38 nan nan nan nan nan nan nan CASMSNYNVLYF nan :::::::::0:-7:6:::::8:2:36:::
21 18 0.000524858 TGTGCTGCCCAAGGAGGGTCTGCGAAGCTCATCTTT GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG TRAV4-4-DV10*00(1181.1) nan TRAJ57*00(282.3) nan 258|266|291|0|8||40.0 nan 25|52|83|9|36||135.0 nan nan nan nan nan nan nan nan nan nan nan TGTGCTGCCCAAGGAGGGTCTGCGAAGCTCATCTTT 38 nan nan nan nan nan nan nan CAAQGGSAKLIF nan :::::::::0:-5:8:::::9:-5:36:::
22 17 0.000495699 TGTGCTTTGGAGCGTGGAGGCAGCAATTACAAACTGACATTT GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG TRAV13-1*00(851.6) nan TRAJ53*00(303.5) nan 258|273|291|0|15|SA269G|61.0 nan 26|55|86|13|42||145.0 nan nan nan nan nan nan nan nan nan nan nan TGTGCTTTGGAGCGTGGAGGCAGCAATTACAAACTGACATTT 38 nan nan nan nan nan nan nan CALERGGSNYKLTF nan :::::::::0:2:15:::::13:-6:42:::
23 17 0.000495699 TGTGCTACTCCTATAACAGGCAATACCGGAAAACTCATCTTT GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG TRAV8-1*00(1333.3) nan TRAJ37*00(303.5) nan 264|273|297|0|9||45.0 nan 18|49|80|11|42||155.0 nan nan nan nan nan nan nan nan nan nan nan TGTGCTACTCCTATAACAGGCAATACCGGAAAACTCATCTTT 38 nan nan nan nan nan nan nan CATPITGNTGKLIF nan :::::::::0:-4:9:::::11:2:42:::
24 17 0.000495699 TGTGCAGTGAGCATGAGGGTTATGGGAGCAGTGGCAACAAGCTCATCTTT GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG TRAV7-5*00(1195.4) nan TRAJ32*00(309.1) nan 264|279|297|0|15||75.0 nan 24|55|86|19|50||155.0 nan nan nan nan nan nan nan nan nan nan nan TGTGCAGTGAGCATGAGGGTTATGGGAGCAGTGGCAACAAGCTCATCTTT 38 nan nan nan nan nan nan nan CAVSMRVM_SSGNKLIF nan :::::::::0:2:15:::::19:-4:50:::
25 17 0.000495699 TGCTCCAGCAGCCCCGGGAGGGACACAGAAGTCTTCTTT GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG TRBV23*00(628.2) TRBD100(35),TRBD200(35) TRBJ1-1*00(218.3) nan 273|284|310|0|11||55.0 9|16|36|12|19||35.0;11|18|42|12|19||35.0 23|40|68|22|39||85.0 nan nan nan nan nan nan nan nan nan nan nan TGCTCCAGCAGCCCCGGGAGGGACACAGAAGTCTTCTTT 38 nan nan nan nan nan nan nan CSSSPGRDTEVFF nan :::::::::0:-6:11:12:3:-8:19:22:-3:39:::
26 16 0.00046654 TGTGCTTTTGGAGGCAGCAATTACAAACTGACATTT GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG TRAV13-1*00(318.3) nan TRAJ53*00(289.1) nan 258|269|291|0|12|I263T|43.0 nan 27|55|86|8|36||140.0 nan nan nan nan nan nan nan nan nan nan nan TGTGCTTTTGGAGGCAGCAATTACAAACTGACATTT 38 nan nan nan nan nan nan nan CAFGGSNYKLTF nan :::::::::0:-2:12:::::8:-7:36:::
27 16 0.00046654 TGTGCTTTGGGTTATAACACCAATACAGGCAAATTAACCTTT GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG TRAV13-1*00(305.8) nan TRAJ27*00(329.7) nan 258|268|291|0|10||50.0 nan 11|48|79|6|42|DT15|168.0 nan nan nan nan nan nan nan nan nan nan nan TGTGCTTTGGGTTATAACACCAATACAGGCAAATTAACCTTT 38 nan nan nan nan nan nan nan CALGYNTNTGKLTF nan :::::::::0:-3:10:::::6:9:42:::
28 16 0.00046654 TGTGCTATGGAACAAGAAAGAACACCAATACAGGCAAATTAACCTTT GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG TRAV13N-4*00(391) nan TRAJ27*00(307.8) nan 258|271|291|0|13||65.0 nan 21|48|79|20|47||135.0 nan nan nan nan nan nan nan nan nan nan nan TGTGCTATGGAACAAGAAAGAACACCAATACAGGCAAATTAACCTTT 38 nan nan nan nan nan nan nan CAMEQERT_NTGKLTF nan :::::::::0:0:13:::::20:-1:47:::
29 16 0.00046654 TGTGCTACTGATGGAGGGACTGGAGGCTATAAAGTGGTCTTT GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG TRAV8-1*00(1394.9) nan TRAJ12*00(287.4) nan 264|277|297|0|13||65.0 nan 21|48|79|15|42||135.0 nan nan nan nan nan nan nan nan nan nan nan TGTGCTACTGATGGAGGGACTGGAGGCTATAAAGTGGTCTTT 38 nan nan nan nan nan nan nan CATDGGTGGYKVVF nan :::::::::0:0:13:::::15:-1:42:::
30 16 0.00046654 TGTGCAGCAAGGACTAATTACAACGTGCTTTACTTC GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG TRAV1000(1384.8),TRAV10D00(1356.8) nan TRAJ21*00(264.8) nan 267|278|300|0|11||55.0;267|278|300|0|11||55.0 nan 23|46|77|13|36||115.0 nan nan nan nan nan nan nan nan nan nan nan TGTGCAGCAAGGACTAATTACAACGTGCTTTACTTC 38 nan nan nan nan nan nan nan CAARTNYNVLYF nan :::::::::0:-2:11:::::13:-3:36:::
31 16 0.00046654 TGCACCTGCAGTGCAGAGGGACAGGGGTGGGATGCAGAAACGCTGTATTTT GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG TRBV1*00(1107.6) TRBD1*00(50) TRBJ2-3*00(222.5) nan 270|287|307|0|17||85.0 12|22|36|17|27||50.0 22|41|69|32|51||95.0 nan nan nan nan nan nan nan nan nan nan nan TGCACCTGCAGTGCAGAGGGACAGGGGTGGGATGCAGAAACGCTGTATTTT 38 nan nan nan nan nan nan nan CTCSAEGQGWDAETLYF nan :::::::::0:0:17:17:0:-2:27:32:-2:51:::
32 15 0.000437382 TGTGCTATGGATTCCGGGACTGGAGGCTATAAAGTGGTCTTT GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG TRAV13D-100(330.2),TRAV13N-100(330.2) nan TRAJ12*00(287.5) nan 258|269|291|0|11||55.0;258|269|291|0|11||55.0 nan 21|48|79|15|42||135.0 nan nan nan nan nan nan nan nan nan nan nan TGTGCTATGGATTCCGGGACTGGAGGCTATAAAGTGGTCTTT 38 nan nan nan nan nan nan nan CAMDSGTGGYKVVF nan :::::::::0:-2:11:::::15:-1:42:::
33 15 0.000437382 TGTGCTATGGATGACACCAATACAGGCAAATTAACCTTT GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG TRAV13N-4*00(389.7) nan TRAJ27*00(285.1) nan 258|269|291|0|11||55.0 nan 19|48|79|10|39|SA21G|131.0 nan nan nan nan nan nan nan nan nan nan nan TGTGCTATGGATGACACCAATACAGGCAAATTAACCTTT 38 nan nan nan nan nan nan nan CAMDDTNTGKLTF nan :::::::::0:-2:11:::::10:1:39:::
34 15 0.000437382 TGTGCTATGGAACCTAATTACAACGTGCTTTACTTC GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG TRAV13D-100(291.9),TRAV13N-100(291.9) nan TRAJ21*00(275.9) nan 258|271|291|0|13||65.0;258|271|291|0|13||65.0 nan 23|46|77|13|36||115.0 nan nan nan nan nan nan nan nan nan nan nan TGTGCTATGGAACCTAATTACAACGTGCTTTACTTC 38 nan nan nan nan nan nan nan CAMEPNYNVLYF nan :::::::::0:0:13:::::13:-3:36:::
35 15 0.000437382 TGTGCAGCAAGCTTTAGCAATAACAGAATCTTCTTT GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG TRAV14-1*00(1353.3) nan TRAJ31*00(265) nan 267|278|300|0|11||55.0 nan 24|46|77|14|36||110.0 nan nan nan nan nan nan nan nan nan nan nan TGTGCAGCAAGCTTTAGCAATAACAGAATCTTCTTT 38 nan nan nan nan nan nan nan CAASFSNNRIFF nan :::::::::0:-2:11:::::14:-4:36:::
36 15 0.000437382 TGTGCAGCAAGCATGAACAGTGCAGGGAACAAGCTAACTTTT GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG TRAV7-200(980.4),TRAV7D-200(980.4) nan TRAJ17*00(289) nan 264|279|297|0|15||75.0;264|279|297|0|15||75.0 nan 25|52|83|15|42||135.0 nan nan nan nan nan nan nan nan nan nan nan TGTGCAGCAAGCATGAACAGTGCAGGGAACAAGCTAACTTTT 38 nan nan nan nan nan nan nan CAASMNSAGNKLTF nan :::::::::0:2:15:::::15:-5:42:::
37 14 0.000408223 TGTGCTGCTGAGGCCAACTATGGAAATGAGAAAATAACTTTT GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG TRAV4-4-DV10*00(1078.3) nan TRAJ48*00(292.2) nan 258|273|291|0|15||75.0 nan 20|50|81|12|42||150.0 nan nan nan nan nan nan nan nan nan nan nan TGTGCTGCTGAGGCCAACTATGGAAATGAGAAAATAACTTTT 38 nan nan nan nan nan nan nan CAAEANYGNEKITF nan :::::::::0:2:15:::::12:0:42:::
38 14 0.000408223 TGTGCTATGGCGGGCACAAATGCTTACAAAGTCATCTTT GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG TRAV13N-4*00(1087.2) nan TRAJ30*00(289.8) nan 258|268|291|0|10||50.0 nan 23|48|79|14|39||125.0 nan nan nan nan nan nan nan nan nan nan nan TGTGCTATGGCGGGCACAAATGCTTACAAAGTCATCTTT 38 nan nan nan nan nan nan nan CAMAGTNAYKVIF nan :::::::::0:-3:10:::::14:-3:39:::
39 14 0.000408223 TGTGCTATAGACTACAGAAGGTGCAGATAGACTCACCTTT GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG TRAV13-2*00(1339.2) nan TRAJ45*00(284.2) nan 258|269|291|0|11||55.0 nan 21|49|80|12|40||140.0 nan nan nan nan nan nan nan nan nan nan nan TGTGCTATAGACTACAGAAGGTGCAGATAGACTCACCTTT 38 nan nan nan nan nan nan nan CAIDYRR_ADRLTF nan :::::::::0:-2:11:::::12:-1:40:::
40 14 0.000408223 TGTGCAGTGTACACAAATGCTTACAAAGTCATCTTT GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG TRAV7-6*00(772.7) nan TRAJ30*00(314.8) nan 264|273|297|0|9||45.0 nan 13|48|79|0|36|DT17I20GI22T|134.0 nan nan nan nan nan nan nan nan nan nan nan TGTGCAGTGTACACAAATGCTTACAAAGTCATCTTT 38 nan nan nan nan nan nan nan CAVYTNAYKVIF nan :::::::::0:-4:9:::::0:7:36:::
41 14 0.000408223 TGTGCAGTGAGATCTGGCAGCTGGCAACTCATCTTT GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG TRAV7-5*00(1385.9) nan TRAJ22*00(269.8) nan 264|275|297|0|11||55.0 nan 25|49|80|12|36||120.0 nan nan nan nan nan nan nan nan nan nan nan TGTGCAGTGAGATCTGGCAGCTGGCAACTCATCTTT 38 nan nan nan nan nan nan nan CAVRSGSWQLIF nan :::::::::0:-2:11:::::12:-5:36:::
42 14 0.000408223 TGCACCTGCAGTGCAGCCGGGACTTTTAACCAAGACACCCAGTACTTT GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG TRBV1*00(1088.8) TRBD200(40),TRBD100(35) TRBJ2-5*00(245.4) nan 270|286|307|0|16||80.0 12|20|42|16|24||40.0;10|17|36|16|23||35.0 18|41|69|25|48||115.0 nan nan nan nan nan nan nan nan nan nan nan TGCACCTGCAGTGCAGCCGGGACTTTTAACCAAGACACCCAGTACTTT 38 nan nan nan nan nan nan nan CTCSAAGTFNQDTQYF nan :::::::::0:-1:16:16:2:-8:24:25:2:48:::
43 13 0.000379064 TGTGTGGTTAGGAATACCAACAAAGTCGTCTTT GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG TRAV1100(1236),TRAV11D00(1236) nan TRAJ34*00(260) nan 267|275|300|0|8||40.0;267|275|300|0|8||40.0 nan 26|47|78|12|33||105.0 nan nan nan nan nan nan nan nan nan nan nan TGTGTGGTTAGGAATACCAACAAAGTCGTCTTT 38 nan nan nan nan nan nan nan CVVRNTNKVVF nan :::::::::0:-5:8:::::12:-6:33:::
44 13 0.000379064 TGTGCTATGGGGGCTATAACAGGCAATACCGGAAAACTCATCTTT GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG TRAV13D-1*00(419.1) nan TRAJ37*00(310) nan 258|268|291|0|10||50.0 nan 18|49|80|14|45||155.0 nan nan nan nan nan nan nan nan nan nan nan TGTGCTATGGGGGCTATAACAGGCAATACCGGAAAACTCATCTTT 38 nan nan nan nan nan nan nan CAMGAITGNTGKLIF nan :::::::::0:-3:10:::::14:2:45:::
45 13 0.000379064 TGTGCTATGGGGATGAATTATAACCAGGGGAAGCTTATCTTT GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG TRAV13D-100(503.5),TRAV13N-100(502.3) nan TRAJ23*00(328.2) nan 258|268|291|0|10||50.0;258|268|291|0|10||50.0 nan 19|49|80|12|42||150.0 nan nan nan nan nan nan nan nan nan nan nan TGTGCTATGGGGATGAATTATAACCAGGGGAAGCTTATCTTT 38 nan nan nan nan nan nan nan CAMGMNYNQGKLIF nan :::::::::0:-3:10:::::12:1:42:::
46 13 0.000379064 TGTGCTATAGACCTGGACACAAATGCTTACAAAGTCATCTTT GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG TRAV13D-200(1029.8),TRAV13N-200(1029.8) nan TRAJ30*00(285.7) nan 258|269|291|0|11||55.0;258|269|291|0|11||55.0 nan 21|48|79|15|42||135.0 nan nan nan nan nan nan nan nan nan nan nan TGTGCTATAGACCTGGACACAAATGCTTACAAAGTCATCTTT 38 nan nan nan nan nan nan nan CAIDLDTNAYKVIF nan :::::::::0:-2:11:::::15:-1:42:::
47 13 0.000379064 TGTGCCAGCTCTCTCGACTGGGGGGACACCGGGCAGCTCTACTTT GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG TRBV12-2*00(1099.8) TRBD2*00(40) TRBJ2-2*00(247.5) nan 270|287|305|0|17||85.0 18|26|42|17|25||40.0 23|43|71|25|45||100.0 nan nan nan nan nan nan nan nan nan nan nan TGTGCCAGCTCTCTCGACTGGGGGGACACCGGGCAGCTCTACTTT 38 nan nan nan nan nan nan nan CASSLDWGDTGQLYF nan :::::::::0:2:17:17:-4:-2:25:25:-3:45:::
48 13 0.000379064 TGTGCCAGCGGTGAATACTGGGGGACTAGTCAAAACACCTTGTACTTT GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG TRBV13-2*00(1240.5) TRBD2*00(35) TRBJ2-4*00(251.6) nan 270|284|306|0|14||70.0 17|24|42|16|23||35.0 16|41|69|23|48||125.0 nan nan nan nan nan nan nan nan nan nan nan TGTGCCAGCGGTGAATACTGGGGGACTAGTCAAAACACCTTGTACTTT 38 nan nan nan nan nan nan nan CASGEYWGTSQNTLYF nan :::::::::0:-2:14:16:-3:-4:23:23:4:48:::
49 13 0.000379064 TGTGCAGCAAAGAGTGGCAGTGGTGGAAAACTCACTTTG GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG TRAV1000(1270.1),TRAV10D00(1246.8) nan TRAJ44*00(276.3) nan 267|277|300|0|10||50.0;267|277|300|0|10||50.0 nan 25|50|81|14|39||125.0 nan nan nan nan nan nan nan nan nan nan nan TGTGCAGCAAAGAGTGGCAGTGGTGGAAAACTCACTTTG 38 nan nan nan nan nan nan nan CAAKSGSGGKLTL nan :::::::::0:-3:10:::::14:-5:39:::
50 12 0.000349905 TGTGTGGTGGGAGATAGAGGTTCAGCCTTAGGGAGGCTGCATTTT GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG TRAV11D*00(1547.7) nan TRAJ18*00(317.7) nan 267|278|300|0|11||55.0 nan 21|55|86|11|45||170.0 nan nan nan nan nan nan nan nan nan nan nan TGTGTGGTGGGAGATAGAGGTTCAGCCTTAGGGAGGCTGCATTTT 38 nan nan nan nan nan nan nan CVVGDRGSALGRLHF nan :::::::::0:-2:11:::::11:-1:45:::
51 12 0.000349905 TGTGCTTTGGGCCGTTATGGGAGCAGTGGCAACAAGCTCATCTTT GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG TRAV13-1*00(1041) nan TRAJ32*00(305.5) nan 258|274|291|0|16|SA268GSA269C|52.0 nan 24|55|86|14|45||155.0 nan nan nan nan nan nan nan nan nan nan nan TGTGCTTTGGGCCGTTATGGGAGCAGTGGCAACAAGCTCATCTTT 38 nan nan nan nan nan nan nan CALGRYGSSGNKLIF nan :::::::::0:3:16:::::14:-4:45:::
52 12 0.000349905 TGTGCTATCCTTGACACAAATGCTTACAAAGTCATCTTT GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG TRAV13N-4*00(391.4) nan TRAJ30*00(290.3) nan 258|266|291|0|8||40.0 nan 20|48|79|11|39||140.0 nan nan nan nan nan nan nan nan nan nan nan TGTGCTATCCTTGACACAAATGCTTACAAAGTCATCTTT 38 nan nan nan nan nan nan nan CAILDTNAYKVIF nan :::::::::0:-5:8:::::11:0:39:::
53 12 0.000349905 TGTGCTATGGTCCTAAATGCTTACAAAGTCATCTTT GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG TRAV13N-4*00(318.9) nan TRAJ30*00(262.2) nan 258|268|291|0|10||50.0 nan 26|48|79|14|36||110.0 nan nan nan nan nan nan nan nan nan nan nan TGTGCTATGGTCCTAAATGCTTACAAAGTCATCTTT 38 nan nan nan nan nan nan nan CAMVLNAYKVIF nan :::::::::0:-3:10:::::14:-6:36:::
54 12 0.000349905 TGTGCTATGGAACGGGGAGGGTCTGCGAAGCTCATCTTT GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG TRAV13D-100(368.5),TRAV13N-100(368.5) nan TRAJ57*00(272.5) nan 258|272|291|0|14||70.0;258|272|291|0|14||70.0 nan 28|52|83|15|39||120.0 nan nan nan nan nan nan nan nan nan nan nan TGTGCTATGGAACGGGGAGGGTCTGCGAAGCTCATCTTT 38 nan nan nan nan nan nan nan CAMERGGSAKLIF nan :::::::::0:1:14:::::15:-8:39:::
55 12 0.000349905 TGTGCAGTGTCCACTACTGGAGGCAATAATAAGCTGACTTTT GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG TRAV7-5*00(1348.5) nan TRAJ56*00(298.7) nan 264|273|297|0|9||45.0 nan 23|52|83|13|42||145.0 nan nan nan nan nan nan nan nan nan nan nan TGTGCAGTGTCCACTACTGGAGGCAATAATAAGCTGACTTTT 38 nan nan nan nan nan nan nan CAVSTTGGNNKLTF nan :::::::::0:-4:9:::::13:-3:42:::
56 12 0.000349905 TGTGCAGCCCAGACAGGCTTTGCAAGTGCGCTGACATTT GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG TRAV7D-400(1165.7),TRAV7-400(1151.7) nan TRAJ35*00(317.9) nan 264|272|301|0|8||40.0;264|272|301|0|8||40.0 nan 18|51|85|6|39|SG20C|151.0 nan nan nan nan nan nan nan nan nan nan nan TGTGCAGCCCAGACAGGCTTTGCAAGTGCGCTGACATTT 38 nan nan nan nan nan nan nan CAAQTGFASALTF nan :::::::::0:-9:8:::::6:2:39:::
57 12 0.000349905 TGTGCAGCAAGTGACTACAGCAACAACAGACTTACTTTG GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG TRAV14N-3*00(1182.3) nan TRAJ7*00(288.4) nan 267|284|300|0|16|DC280|68.0 nan 21|48|79|12|39||135.0 nan nan nan nan nan nan nan nan nan nan nan TGTGCAGCAAGTGACTACAGCAACAACAGACTTACTTTG 38 nan nan nan nan nan nan nan CAASDYSNNRLTL nan :::::::::0:4:16:::::12:-1:39:::
58 11 0.000320746 TGTGCTTTGAGTGACCGCCAGGGAGGCAGAGCTCTGATATTT GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG TRAV12-2*00(1046.2) nan TRAJ15*00(275.3) nan 267|281|301|0|14||70.0 nan 24|49|80|17|42||125.0 nan nan nan nan nan nan nan nan nan nan nan TGTGCTTTGAGTGACCGCCAGGGAGGCAGAGCTCTGATATTT 38 nan nan nan nan nan nan nan CALSDRQGGRALIF nan :::::::::0:0:14:::::17:-4:42:::
59 11 0.000320746 TGTGCTTTAACAGGCAATACCGGAAAACTCATCTTT GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG TRAV13-1*00(1035.5) nan TRAJ37*00(296.2) nan 258|266|291|0|8||40.0 nan 20|49|80|7|36||145.0 nan nan nan nan nan nan nan nan nan nan nan TGTGCTTTAACAGGCAATACCGGAAAACTCATCTTT 38 nan nan nan nan nan nan nan CALTGNTGKLIF nan :::::::::0:-5:8:::::7:0:36:::
60 11 0.000320746 TGTGCTGCCTACCAGGGAGGCAGAGCTCTGATATTT GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG TRAV13D-100(266.8),TRAV13N-100(266.8) nan TRAJ15*00(303.4) nan 258|264|291|0|6||30.0;258|264|291|0|6||30.0 nan 19|49|80|6|36||150.0 nan nan nan nan nan nan nan nan nan nan nan TGTGCTGCCTACCAGGGAGGCAGAGCTCTGATATTT 38 nan nan nan nan nan nan nan CAAYQGGRALIF nan :::::::::0:-7:6:::::6:1:36:::
61 11 0.000320746 TGTGCTATGGTAGCAAATTCTGGGACTTACCAGAGGTTT GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG TRAV13D-100(395.1),TRAV13N-100(395.1) nan TRAJ13*00(287.2) nan 258|268|291|0|10||50.0;258|268|291|0|10||50.0 nan 19|46|77|12|39||135.0 nan nan nan nan nan nan nan nan nan nan nan TGTGCTATGGTAGCAAATTCTGGGACTTACCAGAGGTTT 38 nan nan nan nan nan nan nan CAMVANSGTYQRF nan :::::::::0:-3:10:::::12:1:39:::
62 11 0.000320746 TGTGCTATGGGGCCCTCTTCTGGCAGCTGGCAACTCATCTTT GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG TRAV13D-100(375.8),TRAV13N-100(375.8) nan TRAJ22*00(282.4) nan 258|268|291|0|10||50.0;258|268|291|0|10||50.0 nan 22|49|80|15|42||135.0 nan nan nan nan nan nan nan nan nan nan nan TGTGCTATGGGGCCCTCTTCTGGCAGCTGGCAACTCATCTTT 38 nan nan nan nan nan nan nan CAMGPSSGSWQLIF nan :::::::::0:-3:10:::::15:-2:42:::
63 11 0.000320746 TGTGCTATGGAACACGGCAGGGAGGCAGAGCTCTGATATTT GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG TRAV13D-100(336.8),TRAV13N-100(336.8) nan TRAJ15*00(294.1) nan 258|271|291|0|13||65.0;258|271|291|0|13||65.0 nan 25|49|80|17|41||120.0 nan nan nan nan nan nan nan nan nan nan nan TGTGCTATGGAACACGGCAGGGAGGCAGAGCTCTGATATTT 38 nan nan nan nan nan nan nan CAMEHGR_GRALIF nan :::::::::0:0:13:::::17:-5:41:::
64 11 0.000320746 TGTGCCAGCAGCCAAGACGGGGGGGCCCACTCCTATGAACAGTACTTC GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG TRBV5*00(1313.2) TRBD200(40),TRBD100(36) TRBJ2-7*00(235) nan 267|284|304|0|17||85.0 20|28|42|18|26||40.0;18|28|36|18|28|SC23G|36.0 20|39|67|29|48||95.0 nan nan nan nan nan nan nan nan nan nan nan TGTGCCAGCAGCCAAGACGGGGGGGCCCACTCCTATGAACAGTACTTC 38 nan nan nan nan nan nan nan CASSQDGGAHSYEQYF nan :::::::::0:0:17:18:-6:0:26:29:0:48:::
65 11 0.000320746 TGTGCAGTGAGCATTCGTGGAGCTAACACTGGAAAGCTCACGTTT GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG TRAV7N-6*00(788.5) nan TRAJ52*00(289.1) nan 264|278|297|0|14||70.0 nan 27|55|86|17|45||140.0 nan nan nan nan nan nan nan nan nan nan nan TGTGCAGTGAGCATTCGTGGAGCTAACACTGGAAAGCTCACGTTT 38 nan nan nan nan nan nan nan CAVSIRGANTGKLTF nan :::::::::0:1:14:::::17:-7:45:::
66 11 0.000320746 TGTGCAGCAAGTGCAGAGACTGGAGGCTATAAAGTGGTCTTT GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG TRAV14D-200(1282.1),TRAV14N-200(1282.1) nan TRAJ12*00(280) nan 267|282|300|0|15||75.0;267|282|300|0|15||75.0 nan 23|48|79|17|42||125.0 nan nan nan nan nan nan nan nan nan nan nan TGTGCAGCAAGTGCAGAGACTGGAGGCTATAAAGTGGTCTTT 38 nan nan nan nan nan nan nan CAASAETGGYKVVF nan :::::::::0:2:15:::::17:-3:42:::
67 11 0.000320746 TGTGCAGATAGCAACTATCAGTTGATCTGG GGGGGGGGGGGGGGGGGGGGGGGGGGGGGG TRAV7-1*00(1269.2) nan TRAJ33*00(245.7) nan 264|277|298|0|13|ST271ASG272T|37.0 nan 22|46|77|6|30||120.0 nan nan nan nan nan nan nan nan nan nan nan TGTGCAGATAGCAACTATCAGTTGATCTGG 38 nan nan nan nan nan nan nan CADSNYQLIW nan :::::::::0:-1:13:::::6:-2:30:::
68 11 0.000320746 CCCCACATAATGCAGGTGCCAAGCTCACATTC GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG TRAV7D-500(1196.3),TRAV7N-500(1196.3) nan TRAJ39*00(282.5) nan 264|264|297|0|0||0.0;264|264|297|0|0||0.0 nan 26|52|83|6|32||130.0 nan nan nan nan nan nan nan nan nan nan nan CCCCACATAATGCAGGTGCCAAGCTCACATTC 38 nan nan nan nan nan nan nan PHIMQ_AKLTF nan :::::::::0:-13:0:::::6:-6:32:::
69 10 0.000291588 TGTCCGTCTAATTACAACGTGCTTTACTTC GGGGGGGGGGGGGGGGGGGGGGGGGGGGGG TRAV13-1*00(1006.3) nan TRAJ21*00(280) nan 258|261|291|0|3||15.0 nan 21|46|77|5|30||125.0 nan nan nan nan nan nan nan nan nan nan nan TGTCCGTCTAATTACAACGTGCTTTACTTC 38 nan nan nan nan nan nan nan CPSNYNVLYF nan :::::::::0:-10:3:::::5:-1:30:::
70 10 0.000291588 TGTGCTTTGGAACGGGGCAATAACAGAATCTTCTTT GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG TRAV13-1*00(371) nan TRAJ31*00(255) nan 258|272|291|0|14||70.0 nan 26|46|77|16|36||100.0 nan nan nan nan nan nan nan nan nan nan nan TGTGCTTTGGAACGGGGCAATAACAGAATCTTCTTT 38 nan nan nan nan nan nan nan CALERGNNRIFF nan :::::::::0:1:14:::::16:-6:36:::
71 10 0.000291588 TGTGCTATGGTCATGAATTATAACCAGGGGAAGCTTATCTTT GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG TRAV13D-100(1060.6),TRAV13N-100(1050.1) nan TRAJ23*00(311.5) nan 258|268|291|0|10||50.0;258|268|291|0|10||50.0 nan 17|49|80|10|42||160.0 nan nan nan nan nan nan nan nan nan nan nan TGTGCTATGGTCATGAATTATAACCAGGGGAAGCTTATCTTT 38 nan nan nan nan nan nan nan CAMVMNYNQGKLIF nan :::::::::0:-3:10:::::10:3:42:::
72 10 0.000291588 TGTGCTATGGAATACCTACCAGGCACTGGGAGTAACAGGCTCACTTTT GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG TRAV13D-100(521.3),TRAV13N-100(521.3) nan TRAJ28*00(316.9) nan 258|270|291|0|12||60.0;258|270|291|0|12||60.0 nan 21|54|85|15|48||165.0 nan nan nan nan nan nan nan nan nan nan nan TGTGCTATGGAATACCTACCAGGCACTGGGAGTAACAGGCTCACTTTT 38 nan nan nan nan nan nan nan CAMEYLPGTGSNRLTF nan :::::::::0:-1:12:::::15:-1:48:::
73 10 0.000291588 TGTGCTATGGAACCTGGTCTTCTGGCAGCTGGCAACTCATCTTT GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG TRAV13N-4*00(309.6) nan TRAJ22*00(288.6) nan 258|271|291|0|13||65.0 nan 22|49|80|17|44||135.0 nan nan nan nan nan nan nan nan nan nan nan TGTGCTATGGAACCTGGTCTTCTGGCAGCTGGCAACTCATCTTT 38 nan nan nan nan nan nan nan CAMEPGL_GSWQLIF nan :::::::::0:0:13:::::17:-2:44:::
74 10 0.000291588 TGTGCCAGCAGTTTGACGGCCAACACCGGGCAGCTCTACTTT GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG TRBV14*00(1433.4) nan TRBJ2-2*00(234.8) nan 273|287|309|0|14||70.0 nan 19|43|71|18|42|SA21C|106.0 nan nan nan nan nan nan nan nan nan nan nan TGTGCCAGCAGTTTGACGGCCAACACCGGGCAGCTCTACTTT 38 nan nan nan nan nan nan nan CASSLTANTGQLYF nan :::::::::0:-2:14:::::18:1:42:::
75 10 0.000291588 TGTGCCAGCAGTGATAAGGGGAACTATGCTGAGCAGTTCTTC GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG TRBV13-1*00(1341.9) TRBD1*00(25) TRBJ2-1*00(239.4) nan 270|285|306|0|15||75.0 17|22|36|16|21||25.0 21|42|70|21|42||105.0 nan nan nan nan nan nan nan nan nan nan nan TGTGCCAGCAGTGATAAGGGGAACTATGCTGAGCAGTTCTTC 38 nan nan nan nan nan nan nan CASSDKGNYAEQFF nan :::::::::0:-1:15:16:-5:-2:21:21👎42:::
76 10 0.000291588 TGTGCGGCCAATACAGGCAAATTAACCTTT GGGGGGGGGGGGGGGGGGGGGGGGGGGGGG TRAV13N-4*00(280.1) nan TRAJ27*00(270) nan 258|263|291|0|5||25.0 nan 25|48|79|7|30||115.0 nan nan nan nan nan nan nan nan nan nan nan TGTGCGGCCAATACAGGCAAATTAACCTTT 38 nan nan nan nan nan nan nan CAANTGKLTF nan :::::::::0:-8:5:::::7:-5:30:::
77 10 0.000291588 TGTGCAGTGAGCCTTGTTAATACAGGAAACTACAAATACGTCTTT GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG TRAV7-6*00(723.2) nan TRAJ40*00(322.4) nan 264|276|297|0|12||60.0 nan 20|50|81|15|45||150.0 nan nan nan nan nan nan nan nan nan nan nan TGTGCAGTGAGCCTTGTTAATACAGGAAACTACAAATACGTCTTT 38 nan nan nan nan nan nan nan CAVSLVNTGNYKYVF nan :::::::::0:-1:12:::::15:0:45:::
78 10 0.000291588 TGTGCAGTGAGCATGGACTCGGGATACAACAAACTCACTTTT GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG TRAV7D-500(1367.6),TRAV7N-500(1367.6) nan TRAJ11*00(290) nan 264|279|297|0|15||75.0;264|279|297|0|15||75.0 nan 14|48|79|8|42|DT17I22G|141.0 nan nan nan nan nan nan nan nan nan nan nan TGTGCAGTGAGCATGGACTCGGGATACAACAAACTCACTTTT 38 nan nan nan nan nan nan nan CAVSMDSGYNKLTF nan :::::::::0:2:15:::::8:6:42:::
79 10 0.000291588 TGTGCAGCCGCAGGCTTTGCAAGTGCGCTGACATTT GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG TRAV7-200(1335.4),TRAV7D-200(1335.4) nan TRAJ35*00(276.6) nan 264|282|297|0|17|DA272SA273CST277G|45.0;264|282|297|0|17|DA272SA273CST277G|45.0 nan 25|51|85|10|36||130.0 nan nan nan nan nan nan nan nan nan nan nan TGTGCAGCCGCAGGCTTTGCAAGTGCGCTGACATTT 38 nan nan nan nan nan nan nan CAAAGFASALTF nan :::::::::0:5:17:::::10:-5:36:::
80 10 0.000291588 TGTGCAGCAAGTGTGTCTAATTACAACGTGCTTTACTTC GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG TRAV14D-100(1387.6),TRAV14N-100(1387.6) nan TRAJ21*00(281.9) nan 267|280|300|0|13||65.0;267|280|300|0|13||65.0 nan 20|46|77|13|39||130.0 nan nan nan nan nan nan nan nan nan nan nan TGTGCAGCAAGTGTGTCTAATTACAACGTGCTTTACTTC 38 nan nan nan nan nan nan nan CAASVSNYNVLYF nan :::::::::0:0:13:::::13:0:39:::
81 10 0.000291588 TGTGCAGCAAGTGACCCTTCTGGCAGCTGGCAACTCATCTTT GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG TRAV14N-3*00(330.2) nan TRAJ22*00(263.8) nan 267|289|300|0|22|I280ASA281CDG285|67.0 nan 23|49|80|16|42||130.0 nan nan nan nan nan nan nan nan nan nan nan TGTGCAGCAAGTGACCCTTCTGGCAGCTGGCAACTCATCTTT 38 nan nan nan nan nan nan nan CAASDPSGSWQLIF nan :::::::::0:9:22:::::16:-3:42:::
82 10 0.000291588 TGTGCAGCAAGTAGGGGGGGCAGTGCAGGGAACAAGCTAACTTTT GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG TRAV14D-100(1369.9),TRAV14N-100(1369.9) nan TRAJ17*00(278.6) nan 267|279|300|0|12||60.0;267|279|300|0|12||60.0 nan 27|52|83|20|45||125.0 nan nan nan nan nan nan nan nan nan nan nan TGTGCAGCAAGTAGGGGGGGCAGTGCAGGGAACAAGCTAACTTTT 38 nan nan nan nan nan nan nan CAASRGGSAGNKLTF nan :::::::::0:-1:12:::::20:-7:45:::
83 10 0.000291588 TGCGCAGTCACCCTAACTATGCCCAGGGATTAACCTTC GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG TRAV3D-300(661.9),TRAV3N-300(661.9) nan TRAJ26*00(277.6) nan 267|277|300|0|10||50.0;267|277|300|0|10||50.0 nan 24|49|80|13|38||125.0 nan nan nan nan nan nan nan nan nan nan nan TGCGCAGTCACCCTAACTATGCCCAGGGATTAACCTTC 38 nan nan nan nan nan nan nan CAVTLT_AQGLTF nan :::::::::0:-3:10:::::13:-4:38:::
84 9 0.000262429 TGTGTGGTGGCCGATAGAGGTTCAGCCTTAGGGAGGCTGCATTTT GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG TRAV1100(1301.4),TRAV11D00(1301.4) nan TRAJ18*00(316.9) nan 267|280|300|0|13|SG277C|51.0;267|280|300|0|13|SG277C|51.0 nan 22|55|86|12|45||165.0 nan nan nan nan nan nan nan nan nan nan nan TGTGTGGTGGCCGATAGAGGTTCAGCCTTAGGGAGGCTGCATTTT 38 nan nan nan nan nan nan nan CVVADRGSALGRLHF nan :::::::::0:0:13:::::12:-2:45:::
85 9 0.000262429 TGTGTGGTGGGCGACAGAGGTTCAGCCTTAGGGAGGCTGCATTTT GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG TRAV1100(1410),TRAV11D00(1410) nan TRAJ18*00(306) nan 267|280|300|0|13||65.0;267|280|300|0|13||65.0 nan 22|55|86|12|45|ST24C|151.0 nan nan nan nan nan nan nan nan nan nan nan TGTGTGGTGGGCGACAGAGGTTCAGCCTTAGGGAGGCTGCATTTT 38 nan nan nan nan nan nan nan CVVGDRGSALGRLHF nan :::::::::0:0:13:::::12:-2:45:::
86 9 0.000262429 TGTGCTTTGGGGGGCAATAACAGAATCTTCTTT GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG TRAV13-1*00(384.7) nan TRAJ31*00(247.2) nan 258|268|291|0|10||50.0 nan 26|46|77|13|33||100.0 nan nan nan nan nan nan nan nan nan nan nan TGTGCTTTGGGGGGCAATAACAGAATCTTCTTT 38 nan nan nan nan nan nan nan CALGGNNRIFF nan :::::::::0:-3:10:::::13:-6:33:::
87 9 0.000262429 TGTGCTTTGGAACGTGACTCGGGATACAACAAACTCACTTTT GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG TRAV13-1*00(760.4) nan TRAJ11*00(317.3) nan 258|273|291|0|15||75.0 nan 20|48|79|14|42||140.0 nan nan nan nan nan nan nan nan nan nan nan TGTGCTTTGGAACGTGACTCGGGATACAACAAACTCACTTTT 38 nan nan nan nan nan nan nan CALERDSGYNKLTF nan :::::::::0:2:15:::::14:0:42:::
88 9 0.000262429 TGTGCTCTCTGGGAGCTGGATAGCAATAACAGAATCTTCTTT GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG TRAV15-1-DV6-1*00(1175.3) nan TRAJ31*00(266.9) nan 273|292|312|0|19||95.0 nan 23|46|77|19|42||115.0 nan nan nan nan nan nan nan nan nan nan nan TGTGCTCTCTGGGAGCTGGATAGCAATAACAGAATCTTCTTT 38 nan nan nan nan nan nan nan CALWELDSNNRIFF nan :::::::::0:0:19:::::19:-3:42:::
89 9 0.000262429 TGTGCTCTGGGTGATGATAGCAATAACAGAATCTTCTTT GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG TRAV6-600(1355.6),TRAV6D-600(1355.6) nan TRAJ31*00(259) nan 273|288|307|0|15||75.0;273|288|307|0|15||75.0 nan 23|46|77|16|39||115.0 nan nan nan nan nan nan nan nan nan nan nan TGTGCTCTGGGTGATGATAGCAATAACAGAATCTTCTTT 38 nan nan nan nan nan nan nan CALGDDSNNRIFF nan :::::::::0:1:15:::::16:-3:39:::
90 9 0.000262429 TGTGCTCTGAGCTCCGTTAATACAGGAAACTACAAATACGTCTTT GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG TRAV6-7-DV9*00(1252.2) nan TRAJ40*00(341.8) nan 267|278|301|0|11||55.0 nan 19|50|81|14|45||155.0 nan nan nan nan nan nan nan nan nan nan nan TGTGCTCTGAGCTCCGTTAATACAGGAAACTACAAATACGTCTTT 38 nan nan nan nan nan nan nan CALSSVNTGNYKYVF nan :::::::::0:-3:11:::::14:1:45:::
91 9 0.000262429 TGTGCTCCCGACACAAATGCTTACAAAGTCATCTTT GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG TRAV13-1*00(1211.1) nan TRAJ30*00(288) nan 258|264|291|0|6||30.0 nan 21|48|79|9|36||135.0 nan nan nan nan nan nan nan nan nan nan nan TGTGCTCCCGACACAAATGCTTACAAAGTCATCTTT 38 nan nan nan nan nan nan nan CAPDTNAYKVIF nan :::::::::0:-7:6:::::9:-1:36:::
92 9 0.000262429 TGTGCTGCTTACAACGTGCTTTACTTC GGGGGGGGGGGGGGGGGGGGGGGGGGG TRAV13N-4*00(237) nan TRAJ21*00(244.8) nan 258|264|291|0|6||30.0 nan 27|46|77|8|27||95.0 nan nan nan nan nan nan nan nan nan nan nan TGTGCTGCTTACAACGTGCTTTACTTC 38 nan nan nan nan nan nan nan CAAYNVLYF nan :::::::::0:-7:6:::::8:-7:27:::
93 9 0.000262429 TGTGCTGCTGAGGCGGGAGGCTATAAAGTGGTCTTT GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG TRAV4D-300(1355.3),TRAV4N-300(1355.3) nan TRAJ12*00(258.3) nan 258|272|291|0|14||70.0;258|272|291|0|14||70.0 nan 27|48|79|15|36||105.0 nan nan nan nan nan nan nan nan nan nan nan TGTGCTGCTGAGGCGGGAGGCTATAAAGTGGTCTTT 38 nan nan nan nan nan nan nan CAAEAGGYKVVF nan :::::::::0:1:14:::::15:-7:36:::
94 9 0.000262429 TGTGCTATGGCGGCTTCAAGTGGCCAGAAGCTGGTTTTT GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG TRAV13D-100(411.9),TRAV13N-100(411.9) nan TRAJ16*00(268.5) nan 258|268|291|0|10||50.0;258|268|291|0|10||50.0 nan 24|50|81|13|39||130.0 nan nan nan nan nan nan nan nan nan nan nan TGTGCTATGGCGGCTTCAAGTGGCCAGAAGCTGGTTTTT 38 nan nan nan nan nan nan nan CAMAASSGQKLVF nan :::::::::0:-3:10:::::13:-4:39:::
95 9 0.000262429 TGTGCTATGGACAAAAATACTGGAGGACTAAGTGGTAAATTAACATTC GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG TRAV13N-300(1073.5),TRAV13D-300(1057.8) nan TRAJ2*00(307.4) nan 264|275|297|0|11||55.0;264|275|297|0|11||55.0 nan 22|55|86|15|48||165.0 nan nan nan nan nan nan nan nan nan nan nan TGTGCTATGGACAAAAATACTGGAGGACTAAGTGGTAAATTAACATTC 38 nan nan nan nan nan nan nan CAMDKNTGGLSGKLTF nan :::::::::0:-2:11:::::15:-2:48:::
96 9 0.000262429 TGTGCTATGGAACGTAATACAGGAAACTACAAATACGTCTTT GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG TRAV13D-100(299.7),TRAV13N-100(299.7) nan TRAJ40*00(295) nan 258|273|291|0|15||75.0;258|273|291|0|15||75.0 nan 17|50|81|10|42|DT22|148.0 nan nan nan nan nan nan nan nan nan nan nan TGTGCTATGGAACGTAATACAGGAAACTACAAATACGTCTTT 38 nan nan nan nan nan nan nan CAMERNTGNYKYVF nan :::::::::0:2:15:::::10:3:42:::
97 9 0.000262429 TGTGCTATGGAACGAATAATGCAGGTGCCAAGCTCACATTC GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG TRAV13N-4*00(1043.7) nan TRAJ39*00(288.4) nan 258|272|291|0|14||70.0 nan 25|52|83|14|41||135.0 nan nan nan nan nan nan nan nan nan nan nan TGTGCTATGGAACGAATAATGCAGGTGCCAAGCTCACATTC 38 nan nan nan nan nan nan nan CAMERIM_GAKLTF nan :::::::::0:1:14:::::14:-5:41:::
98 9 0.000262429 TGTGCTATAGGGTAGCAATAACAGAATCTTCTTT GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG TRAV13N-4*00(264.4) nan TRAJ31*00(265) nan 258|266|291|0|8||40.0 nan 24|46|77|12|34||110.0 nan nan nan nan nan nan nan nan nan nan nan TGTGCTATAGGGTAGCAATAACAGAATCTTCTTT 38 nan nan nan nan nan nan nan CAIG*Q_NRIFF nan :::::::::0:-5:8:::::12:-4:34:::
99 9 0.000262429 TGTGCCTGGAGTACTACAGGGGGAATCTATGCTGAGCAGTTCTTC GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG TRBV31*00(1236.1) nan TRBJ2-1*00(211.7) nan 270|292|303|0|20|DC282DG283SC287A|62.0 nan 23|42|70|26|45||95.0 nan nan nan nan nan nan nan nan nan nan nan TGTGCCTGGAGTACTACAGGGGGAATCTATGCTGAGCAGTTCTTC 38 nan nan nan nan nan nan nan CAWSTTGGIYAEQFF nan :::::::::0:9:20:::::26:-3:45:::

Quality control

Now when we have processed all samples, we can proceed to quality control. First thing that we want to look at is the alignment quality. This can be easily done with the following command:

mixcr exportQc align results/*.clns figs/alignQc.pdf

alignQC.svg

From the plot above we see, that only about 50% of reads from every sample have been successfully aligned. The major reason why the alignment failed is that a lot of reads lack TCR sequences. In that case, if we want to dig a bit deeper into the issue we can realign one of the samples and save not-aligned reads into separate files for manual inspection. Let's pick one of the samples, ex. S_7, and realign it.

mkdir -p debug
mixcr align -f \
    --species mmu \
    -OvParameters.geneFeatureToAlign="VTranscriptWithout5UTRWithP" \
    -OvParameters.parameters.floatingLeftBound=false \
    -OjParameters.parameters.floatingRightBound=false \
    --not-aligned-R1 debug/S_s_R1.fastq \
    --not-aligned-R2 debug/S_s_R2.fastq \
    --report debug/S_s.report \
    --json-report debug/S_s.json \
     raw/S_s_R1.fastq.gz \
     raw/S_s_R2.fastq.gz \
     debug/S_s.vdjca

Now one can use BLAST to determine the origin of the reads in S_7_notAligned_R1.fastq and S_7_notAligned_R2.fastq. In this particular case BLAST showed that major part of the non-aligned reads aligns with DNA sequence in between TCR gene segments which may be consequences of DNA contamination.

Finally, we can look at the chain distribution among all clones. 

mixcr exportQc chainUsage results/*.clns figs/chainUsage.pdf

On the plot bellow we see, despite some TRB dominance, both (TCR alpha and TCR beta) chains equally present in each sample.

chainUsage.svg

Full-length clonotype assembly

SMARTer Mouse TCR a/b Profiling Kit allows to recover a broader TCR receptor sequence then just CDR3 region. Since this protocol is based on 5'RACE method, the full V-gene sequence should present. Reverse sequence specific primers are located in C-gene region, thus the read fully covers J-gene.

The longest possible assembling feature for this protocol is "{FR1Begin:FR4End}".

MiXCR has a specific preset to obtain full-length TCR clones with SMARTer Mouse TCR a/b Profiling protocol:

mixcr analyze takara-mouse-rna-tcr-smarter \
    raw/S_s_R1.fastq.gz \
    raw/S_s_R2.fastq.gz \
    results/S_s.vdjca

The mixcr assemble step in this preset differs from the one above in the following manner:

mixcr assemble \
    -OassemblingFeatures="{FR1Begin:FR4End}" \
    --report results/S_s.report \
    --json-report results/S_s.json \
    results/S_s.vdjca \
    results/S_s.clns
-OassemblingFeatures="{FR1Begin:FR4End}"
sets the assembling feature to the region which starts from FR1Begin and ends at the end of FR4.

Notice that we do not use -OseparateByV=true and -OseparateByJ=true in this case because assembling feature already covers full V and J sequences, thus in case several clones have identical CDR3 they will still be separated.

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/S_s.clns \
    results/S_s.report.txt

mixcr exportReports \
    --json \
    results/S_s.clns \
    results/S_s.report.json
Show report file 
============== Align Report ==============
Input file(s): /raw/PRJNA658348_TakaraMouseTCR/S_s_R1.fastq.gz,/raw/PRJNA658348_TakaraMouseTCR/S_s_R2.fastq.gz
Output file(s): results/S_s.vdjca
Version: ; built=Mon Sep 26 10:55:18 CEST 2022; rev=8c998df1ab; lib=repseqio.v2.0
Command line arguments: --report results/S_s.align.report.txt --json-report results/S_s.align.report.json --preset local:takara-smarter-mouse-tcr-cdr3 +limitInput 100000 /raw/PRJNA658348_TakaraMouseTCR/S_s_R1.fastq.gz /raw/PRJNA658348_TakaraMouseTCR/S_s_R2.fastq.gz results/S_s.vdjca
Analysis time: 0ns
Total sequencing reads: 100000
Successfully aligned reads: 48077 (48.08%)
Paired-end alignment conflicts eliminated: 6461 (6.46%)
Alignment failed, no hits (not TCR/IG?): 35335 (35.34%)
Alignment failed because of absence of V hits: 10019 (10.02%)
Alignment failed because of absence of J hits: 6103 (6.1%)
No target with both V and J alignments: 400 (0.4%)
Alignment failed because of low total score: 66 (0.07%)
Overlapped: 72072 (72.07%)
Overlapped and aligned: 34890 (34.89%)
Alignment-aided overlaps: 1306 (3.74%)
Overlapped and not aligned: 37182 (37.18%)
No CDR3 parts alignments, percent of successfully aligned: 135 (0.28%)
Partial aligned reads, percent of successfully aligned: 1862 (3.87%)
V gene chimeras: 118 (0.12%)
J gene chimeras: 25 (0.03%)
TRA chains: 30220 (62.86%)
TRA non-functional: 4116 (13.62%)
TRB chains: 17849 (37.13%)
TRB non-functional: 1382 (7.74%)
TRD chains: 8 (0.02%)
TRD non-functional: 2 (25%)
Realigned with forced non-floating bound: 58468 (58.47%)
Realigned with forced non-floating right bound in left read: 7379 (7.38%)
Realigned with forced non-floating left bound in right read: 7379 (7.38%)
============== Assemble Report ==============
Input file(s): results/S_s.vdjca
Output file(s): results/S_s.clns
Version: ; built=Mon Sep 26 10:55:18 CEST 2022; rev=8c998df1ab; lib=repseqio.v2.0
Command line arguments: --report results/S_s.assemble.report.txt --json-report results/S_s.assemble.report.json results/S_s.vdjca results/S_s.clns
Analysis time: 0ns
Final clonotype count: 25533
Average number of reads per clonotype: 1.34
Reads used in clonotypes, percent of total: 34295 (34.3%)
Reads used in clonotypes before clustering, percent of total: 34336 (34.34%)
Number of reads used as a core, percent of used: 33568 (97.76%)
Mapped low quality reads, percent of used: 768 (2.24%)
Reads clustered in PCR error correction, percent of used: 41 (0.12%)
Reads pre-clustered due to the similar VJC-lists, percent of used: 85 (0.25%)
Reads dropped due to the lack of a clone sequence, percent of total: 2655 (2.65%)
Reads dropped due to a too short clonal sequence, percent of total: 0 (0%)
Reads dropped due to low quality, percent of total: 45 (0.04%)
Reads dropped due to failed mapping, percent of total: 10984 (10.98%)
Reads dropped with low quality clones, percent of total: 77 (0.08%)
Clonotypes eliminated by PCR error correction: 22
Clonotypes dropped as low quality: 45
Clonotypes pre-clustered due to the similar VJC-lists: 72
TRA chains: 15308 (59.95%)
TRA non-functional: 2060 (13.46%)
TRB chains: 10224 (40.04%)
TRB non-functional: 635 (6.21%)
TRD chains: 1 (0%)
TRD non-functional: 1 (100%)

{
  "type": "alignerReport",
  "commandLine": "--report results/S_s.align.report.txt --json-report results/S_s.align.report.json --preset local:takara-smarter-mouse-tcr-cdr3 +limitInput 100000 /raw/PRJNA658348_TakaraMouseTCR/S_s_R1.fastq.gz /raw/PRJNA658348_TakaraMouseTCR/S_s_R2.fastq.gz results/S_s.vdjca",
  "inputFiles": [
    "/raw/PRJNA658348_TakaraMouseTCR/S_s_R1.fastq.gz",
    "/raw/PRJNA658348_TakaraMouseTCR/S_s_R2.fastq.gz"
  ],
  "outputFiles": [
    "results/S_s.vdjca"
  ],
  "version": "; built=Mon Sep 26 10:55:18 CEST 2022; rev=8c998df1ab; lib=repseqio.v2.0",
  "trimmingReport": null,
  "totalReadsProcessed": 100000,
  "aligned": 48077,
  "notAligned": 51923,
  "notAlignedReasons": {
    "NoVHits": 10019,
    "LowTotalScore": 66,
    "NoCDR3Parts": 0,
    "VAndJOnDifferentTargets": 400,
    "NoBarcode": 0,
    "NoJHits": 6103,
    "NoHits": 35335
  },
  "chimeras": 0,
  "overlapped": 72072,
  "alignmentAidedOverlaps": 1306,
  "overlappedAligned": 34890,
  "overlappedNotAligned": 37182,
  "pairedEndAlignmentConflicts": 6461,
  "vChimeras": 118,
  "jChimeras": 25,
  "chainUsage": {
    "type": "chainUsage",
    "chimeras": 0,
    "total": 48077,
    "chains": {
      "TRA": {
        "total": 30220,
        "nonFunctional": 4116,
        "isOOF": 3698,
        "hasStops": 418
      },
      "TRB": {
        "total": 17849,
        "nonFunctional": 1382,
        "isOOF": 1035,
        "hasStops": 347
      },
      "TRD": {
        "total": 8,
        "nonFunctional": 2,
        "isOOF": 2,
        "hasStops": 0
      }
    }
  },
  "realignedWithForcedNonFloatingBound": 58468,
  "realignedWithForcedNonFloatingRightBoundInLeftRead": 7379,
  "realignedWithForcedNonFloatingLeftBoundInRightRead": 7379,
  "noCDR3PartsAlignments": 135,
  "partialAlignments": 1862,
  "tagReport": null
}
{
  "type": "assemblerReport",
  "commandLine": "--report results/S_s.assemble.report.txt --json-report results/S_s.assemble.report.json results/S_s.vdjca results/S_s.clns",
  "inputFiles": [
    "results/S_s.vdjca"
  ],
  "outputFiles": [
    "results/S_s.clns"
  ],
  "version": "; built=Mon Sep 26 10:55:18 CEST 2022; rev=8c998df1ab; lib=repseqio.v2.0",
  "preCloneAssemblerReport": null,
  "totalReadsProcessed": 100000,
  "initialClonesCreated": 25672,
  "readsDroppedNoTargetSequence": 2655,
  "readsDroppedTooShortClonalSequence": 0,
  "readsDroppedLowQuality": 25,
  "coreReads": 33568,
  "readsDroppedFailedMapping": 10984,
  "lowQualityRescued": 768,
  "clonesClustered": 22,
  "readsClustered": 41,
  "clones": 25533,
  "clonesDroppedAsLowQuality": 45,
  "clonesPreClustered": 72,
  "readsPreClustered": 85,
  "readsInClones": 34295,
  "readsInClonesBeforeClustering": 34336,
  "readsDroppedWithLowQualityClones": 77,
  "clonalChainUsage": {
    "type": "chainUsage",
    "chimeras": 0,
    "total": 25533,
    "chains": {
      "TRA": {
        "total": 15308,
        "nonFunctional": 2060,
        "isOOF": 2035,
        "hasStops": 25
      },
      "TRB": {
        "total": 10224,
        "nonFunctional": 635,
        "isOOF": 595,
        "hasStops": 40
      },
      "TRD": {
        "total": 1,
        "nonFunctional": 1,
        "isOOF": 1,
        "hasStops": 0
      }
    }
  }
}