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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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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

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
      }
    }
  }
}