iRepertoire

Here we will discuss how to process the data obtained using iRepertoire TCR LR kit. This is a multiplex protocol designed in such a way that forward primers are located in FR1 region of V gene and reverse primers are complimentary to constant region.

Data libraries

This tutorial uses the data from the following publication: Longitudinal High-Throughput Sequencing of the T-Cell Receptor Repertoire Reveals Dynamic Change and Prognostic Significance of Peripheral Blood TCR Diversity in Metastatic Colorectal Cancer During Chemotherapy Yi-Tung Chen et al., Front. Immunol., 2022 Jan;12:743448 doi: 10.3389/fimmu.2021.743448

A total of 36 subjects, including 20 healthy controls and 16 metastatic CRC patients, were enrolled in this study.Peripheral blood samples were obtained from 20 age-matched healthy controls (62.6 ± 10.48 years old) and 16 CRC patients (62.38 ± 12.62 years old) before therapy. Among the 16 CRC patients, 67 peripheral blood samples were collected from 13 patients with follow-up every two months for approximately 98 to 452 days. 103 samples in total. Peripheral blood mononuclear cells (PBMCs) were isolated following the standard procedure, and total RNA from PBMCs was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol. A multiplex PCR amplification reaction was used to amplify the TCR immune repertoire. Human TCRα and TCRβ libraries were prepared using the HTAI-M and HTBI-M Kits (iRepertoire, Inc.) according to the manufacturer’s instructions and 2 × 250 bp paired-end sequenced was performed on the Illumina MiSeq platform.

All data is available from SRA (PRJNA754274) 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/SRR836/008/SRR8365468/SRR8365468_1.fastq.gz
  out=raw/SRR8365468_HIP2_male_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR836/008/SRR8365468/SRR8365468_2.fastq.gz
  out=raw/SRR8365468_HIP2_male_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR836/007/SRR8365457/SRR8365457_1.fastq.gz
  out=raw/SRR8365457_HIP1_female_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR836/007/SRR8365457/SRR8365457_2.fastq.gz
  out=raw/SRR8365457_HIP1_female_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR836/008/SRR8365458/SRR8365458_1.fastq.gz
  out=raw/SRR8365458_HIP1_female_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR836/008/SRR8365458/SRR8365458_2.fastq.gz
  out=raw/SRR8365458_HIP1_female_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR836/009/SRR8365459/SRR8365459_1.fastq.gz
  out=raw/SRR8365459_HIP1_female_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR836/009/SRR8365459/SRR8365459_2.fastq.gz
  out=raw/SRR8365459_HIP1_female_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR836/003/SRR8365463/SRR8365463_1.fastq.gz
  out=raw/SRR8365463_HIP1_female_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR836/003/SRR8365463/SRR8365463_2.fastq.gz
  out=raw/SRR8365463_HIP1_female_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR836/009/SRR8365469/SRR8365469_1.fastq.gz
  out=raw/SRR8365469_HIP2_male_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR836/009/SRR8365469/SRR8365469_2.fastq.gz
  out=raw/SRR8365469_HIP2_male_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR836/005/SRR8365465/SRR8365465_1.fastq.gz
  out=raw/SRR8365465_HIP1_female_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR836/005/SRR8365465/SRR8365465_2.fastq.gz
  out=raw/SRR8365465_HIP1_female_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR836/007/SRR8365467/SRR8365467_1.fastq.gz
  out=raw/SRR8365467_HIP2_male_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR836/007/SRR8365467/SRR8365467_2.fastq.gz
  out=raw/SRR8365467_HIP2_male_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR836/004/SRR8365464/SRR8365464_1.fastq.gz
  out=raw/SRR8365464_HIP1_female_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR836/004/SRR8365464/SRR8365464_2.fastq.gz
  out=raw/SRR8365464_HIP1_female_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR836/000/SRR8365450/SRR8365450_1.fastq.gz
  out=raw/SRR8365450_HIP3_male_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR836/000/SRR8365450/SRR8365450_2.fastq.gz
  out=raw/SRR8365450_HIP3_male_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR836/001/SRR8365461/SRR8365461_1.fastq.gz
  out=raw/SRR8365461_HIP1_female_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR836/001/SRR8365461/SRR8365461_2.fastq.gz
  out=raw/SRR8365461_HIP1_female_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR836/002/SRR8365462/SRR8365462_1.fastq.gz
  out=raw/SRR8365462_HIP1_female_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR836/002/SRR8365462/SRR8365462_2.fastq.gz
  out=raw/SRR8365462_HIP1_female_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR836/006/SRR8365456/SRR8365456_1.fastq.gz
  out=raw/SRR8365456_HIP1_female_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR836/006/SRR8365456/SRR8365456_2.fastq.gz
  out=raw/SRR8365456_HIP1_female_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR836/005/SRR8365475/SRR8365475_1.fastq.gz
  out=raw/SRR8365475_HIP2_male_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR836/005/SRR8365475/SRR8365475_2.fastq.gz
  out=raw/SRR8365475_HIP2_male_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR836/000/SRR8365460/SRR8365460_1.fastq.gz
  out=raw/SRR8365460_HIP1_female_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR836/000/SRR8365460/SRR8365460_2.fastq.gz
  out=raw/SRR8365460_HIP1_female_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR836/000/SRR8365470/SRR8365470_1.fastq.gz
  out=raw/SRR8365470_HIP2_male_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR836/000/SRR8365470/SRR8365470_2.fastq.gz
  out=raw/SRR8365470_HIP2_male_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR836/001/SRR8365471/SRR8365471_1.fastq.gz
  out=raw/SRR8365471_HIP2_male_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR836/001/SRR8365471/SRR8365471_2.fastq.gz
  out=raw/SRR8365471_HIP2_male_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR836/003/SRR8365473/SRR8365473_1.fastq.gz
  out=raw/SRR8365473_HIP2_male_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR836/003/SRR8365473/SRR8365473_2.fastq.gz
  out=raw/SRR8365473_HIP2_male_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR836/009/SRR8365449/SRR8365449_1.fastq.gz
  out=raw/SRR8365449_HIP3_male_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR836/009/SRR8365449/SRR8365449_2.fastq.gz
  out=raw/SRR8365449_HIP3_male_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR836/002/SRR8365482/SRR8365482_1.fastq.gz
  out=raw/SRR8365482_HIP1_female_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR836/002/SRR8365482/SRR8365482_2.fastq.gz
  out=raw/SRR8365482_HIP1_female_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR836/006/SRR8365446/SRR8365446_1.fastq.gz
  out=raw/SRR8365446_HIP3_male_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR836/006/SRR8365446/SRR8365446_2.fastq.gz
  out=raw/SRR8365446_HIP3_male_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR836/003/SRR8365483/SRR8365483_1.fastq.gz
  out=raw/SRR8365483_HIP1_female_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR836/003/SRR8365483/SRR8365483_2.fastq.gz
  out=raw/SRR8365483_HIP1_female_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR836/007/SRR8365447/SRR8365447_1.fastq.gz
  out=raw/SRR8365447_HIP3_male_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR836/007/SRR8365447/SRR8365447_2.fastq.gz
  out=raw/SRR8365447_HIP3_male_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR836/004/SRR8365484/SRR8365484_1.fastq.gz
  out=raw/SRR8365484_HIP1_female_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR836/004/SRR8365484/SRR8365484_2.fastq.gz
  out=raw/SRR8365484_HIP1_female_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR836/008/SRR8365448/SRR8365448_1.fastq.gz
  out=raw/SRR8365448_HIP3_male_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR836/008/SRR8365448/SRR8365448_2.fastq.gz
  out=raw/SRR8365448_HIP3_male_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR836/004/SRR8365424/SRR8365424_1.fastq.gz
  out=raw/SRR8365424_HIP3_male_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR836/004/SRR8365424/SRR8365424_2.fastq.gz
  out=raw/SRR8365424_HIP3_male_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR836/005/SRR8365485/SRR8365485_1.fastq.gz
  out=raw/SRR8365485_HIP1_female_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR836/005/SRR8365485/SRR8365485_2.fastq.gz
  out=raw/SRR8365485_HIP1_female_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR836/008/SRR8365488/SRR8365488_1.fastq.gz
  out=raw/SRR8365488_HIP1_female_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR836/008/SRR8365488/SRR8365488_2.fastq.gz
  out=raw/SRR8365488_HIP1_female_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR836/001/SRR8365421/SRR8365421_1.fastq.gz
  out=raw/SRR8365421_HIP3_male_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR836/001/SRR8365421/SRR8365421_2.fastq.gz
  out=raw/SRR8365421_HIP3_male_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR836/009/SRR8365489/SRR8365489_1.fastq.gz
  out=raw/SRR8365489_HIP1_female_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR836/009/SRR8365489/SRR8365489_2.fastq.gz
  out=raw/SRR8365489_HIP1_female_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR836/000/SRR8365490/SRR8365490_1.fastq.gz
  out=raw/SRR8365490_HIP1_female_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR836/000/SRR8365490/SRR8365490_2.fastq.gz
  out=raw/SRR8365490_HIP1_female_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR836/006/SRR8365246/SRR8365246_1.fastq.gz
  out=raw/SRR8365246_HIP1_female_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR836/006/SRR8365246/SRR8365246_2.fastq.gz
  out=raw/SRR8365246_HIP1_female_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR836/004/SRR8365474/SRR8365474_1.fastq.gz
  out=raw/SRR8365474_HIP1_female_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR836/004/SRR8365474/SRR8365474_2.fastq.gz
  out=raw/SRR8365474_HIP1_female_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR836/002/SRR8365422/SRR8365422_1.fastq.gz
  out=raw/SRR8365422_HIP3_male_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR836/002/SRR8365422/SRR8365422_2.fastq.gz
  out=raw/SRR8365422_HIP3_male_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR836/003/SRR8365423/SRR8365423_1.fastq.gz
  out=raw/SRR8365423_HIP3_male_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR836/003/SRR8365423/SRR8365423_2.fastq.gz
  out=raw/SRR8365423_HIP3_male_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR836/000/SRR8365420/SRR8365420_1.fastq.gz
  out=raw/SRR8365420_HIP3_male_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR836/000/SRR8365420/SRR8365420_2.fastq.gz
  out=raw/SRR8365420_HIP3_male_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR836/009/SRR8365419/SRR8365419_1.fastq.gz
  out=raw/SRR8365419_HIP3_male_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR836/009/SRR8365419/SRR8365419_2.fastq.gz
  out=raw/SRR8365419_HIP3_male_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR836/008/SRR8365248/SRR8365248_1.fastq.gz
  out=raw/SRR8365248_HIP1_female_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR836/008/SRR8365248/SRR8365248_2.fastq.gz
  out=raw/SRR8365248_HIP1_female_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR836/009/SRR8365249/SRR8365249_1.fastq.gz
  out=raw/SRR8365249_HIP1_female_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR836/009/SRR8365249/SRR8365249_2.fastq.gz
  out=raw/SRR8365249_HIP1_female_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR836/007/SRR8365247/SRR8365247_1.fastq.gz
  out=raw/SRR8365247_HIP1_female_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR836/007/SRR8365247/SRR8365247_2.fastq.gz
  out=raw/SRR8365247_HIP1_female_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR836/000/SRR8365250/SRR8365250_1.fastq.gz
  out=raw/SRR8365250_HIP1_female_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR836/000/SRR8365250/SRR8365250_2.fastq.gz
  out=raw/SRR8365250_HIP1_female_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR836/008/SRR8365348/SRR8365348_1.fastq.gz
  out=raw/SRR8365348_HIP1_female_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR836/008/SRR8365348/SRR8365348_2.fastq.gz
  out=raw/SRR8365348_HIP1_female_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR836/001/SRR8365251/SRR8365251_1.fastq.gz
  out=raw/SRR8365251_HIP1_female_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR836/001/SRR8365251/SRR8365251_2.fastq.gz
  out=raw/SRR8365251_HIP1_female_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR836/008/SRR8365418/SRR8365418_1.fastq.gz
  out=raw/SRR8365418_HIP3_male_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR836/008/SRR8365418/SRR8365418_2.fastq.gz
  out=raw/SRR8365418_HIP3_male_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR836/000/SRR8365310/SRR8365310_1.fastq.gz
  out=raw/SRR8365310_HIP1_female_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR836/000/SRR8365310/SRR8365310_2.fastq.gz
  out=raw/SRR8365310_HIP1_female_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR836/002/SRR8365252/SRR8365252_1.fastq.gz
  out=raw/SRR8365252_HIP1_female_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR836/002/SRR8365252/SRR8365252_2.fastq.gz
  out=raw/SRR8365252_HIP1_female_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR836/008/SRR8365308/SRR8365308_1.fastq.gz
  out=raw/SRR8365308_HIP1_female_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR836/008/SRR8365308/SRR8365308_2.fastq.gz
  out=raw/SRR8365308_HIP1_female_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR836/009/SRR8365309/SRR8365309_1.fastq.gz
  out=raw/SRR8365309_HIP1_female_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR836/009/SRR8365309/SRR8365309_2.fastq.gz
  out=raw/SRR8365309_HIP1_female_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR836/003/SRR8365253/SRR8365253_1.fastq.gz
  out=raw/SRR8365253_HIP1_female_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR836/003/SRR8365253/SRR8365253_2.fastq.gz
  out=raw/SRR8365253_HIP1_female_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR836/007/SRR8365307/SRR8365307_1.fastq.gz
  out=raw/SRR8365307_HIP1_female_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR836/007/SRR8365307/SRR8365307_2.fastq.gz
  out=raw/SRR8365307_HIP1_female_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR836/009/SRR8365259/SRR8365259_1.fastq.gz
  out=raw/SRR8365259_HIP2_male_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR836/009/SRR8365259/SRR8365259_2.fastq.gz
  out=raw/SRR8365259_HIP2_male_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR836/008/SRR8365258/SRR8365258_1.fastq.gz
  out=raw/SRR8365258_HIP2_male_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR836/008/SRR8365258/SRR8365258_2.fastq.gz
  out=raw/SRR8365258_HIP2_male_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR836/006/SRR8365306/SRR8365306_1.fastq.gz
  out=raw/SRR8365306_HIP1_female_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR836/006/SRR8365306/SRR8365306_2.fastq.gz
  out=raw/SRR8365306_HIP1_female_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR836/005/SRR8365305/SRR8365305_1.fastq.gz
  out=raw/SRR8365305_HIP1_female_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR836/005/SRR8365305/SRR8365305_2.fastq.gz
  out=raw/SRR8365305_HIP1_female_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR836/004/SRR8365304/SRR8365304_1.fastq.gz
  out=raw/SRR8365304_HIP1_female_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR836/004/SRR8365304/SRR8365304_2.fastq.gz
  out=raw/SRR8365304_HIP1_female_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR836/000/SRR8365260/SRR8365260_1.fastq.gz
  out=raw/SRR8365260_HIP2_male_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR836/000/SRR8365260/SRR8365260_2.fastq.gz
  out=raw/SRR8365260_HIP2_male_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR836/003/SRR8365303/SRR8365303_1.fastq.gz
  out=raw/SRR8365303_HIP1_female_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR836/003/SRR8365303/SRR8365303_2.fastq.gz
  out=raw/SRR8365303_HIP1_female_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR836/001/SRR8365261/SRR8365261_1.fastq.gz
  out=raw/SRR8365261_HIP2_male_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR836/001/SRR8365261/SRR8365261_2.fastq.gz
  out=raw/SRR8365261_HIP2_male_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR836/002/SRR8365262/SRR8365262_1.fastq.gz
  out=raw/SRR8365262_HIP2_male_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR836/002/SRR8365262/SRR8365262_2.fastq.gz
  out=raw/SRR8365262_HIP2_male_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR836/002/SRR8365302/SRR8365302_1.fastq.gz
  out=raw/SRR8365302_HIP1_female_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR836/002/SRR8365302/SRR8365302_2.fastq.gz
  out=raw/SRR8365302_HIP1_female_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR836/001/SRR8365301/SRR8365301_1.fastq.gz
  out=raw/SRR8365301_HIP1_female_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR836/001/SRR8365301/SRR8365301_2.fastq.gz
  out=raw/SRR8365301_HIP1_female_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR836/003/SRR8365263/SRR8365263_1.fastq.gz
  out=raw/SRR8365263_HIP2_male_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR836/003/SRR8365263/SRR8365263_2.fastq.gz
  out=raw/SRR8365263_HIP2_male_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR836/004/SRR8365264/SRR8365264_1.fastq.gz
  out=raw/SRR8365264_HIP1_female_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR836/004/SRR8365264/SRR8365264_2.fastq.gz
  out=raw/SRR8365264_HIP1_female_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR836/007/SRR8365267/SRR8365267_1.fastq.gz
  out=raw/SRR8365267_HIP1_female_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR836/007/SRR8365267/SRR8365267_2.fastq.gz
  out=raw/SRR8365267_HIP1_female_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR836/004/SRR8365274/SRR8365274_1.fastq.gz
  out=raw/SRR8365274_HIP1_female_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR836/004/SRR8365274/SRR8365274_2.fastq.gz
  out=raw/SRR8365274_HIP1_female_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR836/009/SRR8365269/SRR8365269_1.fastq.gz
  out=raw/SRR8365269_HIP1_female_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR836/009/SRR8365269/SRR8365269_2.fastq.gz
  out=raw/SRR8365269_HIP1_female_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR836/001/SRR8365271/SRR8365271_1.fastq.gz
  out=raw/SRR8365271_HIP1_female_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR836/001/SRR8365271/SRR8365271_2.fastq.gz
  out=raw/SRR8365271_HIP1_female_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR836/003/SRR8365273/SRR8365273_1.fastq.gz
  out=raw/SRR8365273_HIP1_female_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR836/003/SRR8365273/SRR8365273_2.fastq.gz
  out=raw/SRR8365273_HIP1_female_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR836/005/SRR8365265/SRR8365265_1.fastq.gz
  out=raw/SRR8365265_HIP1_female_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR836/005/SRR8365265/SRR8365265_2.fastq.gz
  out=raw/SRR8365265_HIP1_female_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR836/000/SRR8365270/SRR8365270_1.fastq.gz
  out=raw/SRR8365270_HIP1_female_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR836/000/SRR8365270/SRR8365270_2.fastq.gz
  out=raw/SRR8365270_HIP1_female_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR836/002/SRR8365272/SRR8365272_1.fastq.gz
  out=raw/SRR8365272_HIP1_female_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR836/002/SRR8365272/SRR8365272_2.fastq.gz
  out=raw/SRR8365272_HIP1_female_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR836/006/SRR8365266/SRR8365266_1.fastq.gz
  out=raw/SRR8365266_HIP1_female_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR836/006/SRR8365266/SRR8365266_2.fastq.gz
  out=raw/SRR8365266_HIP1_female_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR836/008/SRR8365268/SRR8365268_1.fastq.gz
  out=raw/SRR8365268_HIP1_female_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR836/008/SRR8365268/SRR8365268_2.fastq.gz
  out=raw/SRR8365268_HIP1_female_R2.fastq.gz

The project contains 103 FASTQ file pairs. For the purpose of this tutorial we assume that all fastq files are stored in fastq/ folder. The structure of sequences is shown on the picture bellow. The data was obtained using multiplex primers for V and J genes. Below you can see the structure of cDNA library.

Upstream analysis

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

mixcr analyze irepertoire-human-rna-xcr-repseq-sr \
    raw/CRC016_preTherapy_R1.fastq.gz \
    raw/CRC016_preTherapy_R2.fastq.gz \
    results/CRC016_preTherapy

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 irepertoire-human-rna-xcr-repseq-sr \
    {} \
    {=s:R1:R2:=} \
    {=s:.*/:results/:;s:_R.*::=}"

Under the hood pipeline:

Under the hood irepertoire-human-rna-xcr-repseq-sr executes the following pipeline:

`align`

Alignment of raw sequencing reads against reference database of V-, D-, J- and C- gene segments.

mixcr align \
    --species hsa \
    -p default_4.0 \
    -OvParameters.geneFeatureToAlign="VTranscriptWithout5UTRWithP" \
    -OvParameters.parameters.floatingLeftBound=true \
    -OjParameters.parameters.floatingRightBound=false \
    -OcParameters.parameters.floatingRightBound=true \
    --report results/CRC016_preTherapy.report.txt \
    --json-report results/CRC016_preTherapy.report.json \
     raw/CRC016_preTherapy_R1.fastq.gz \
     raw/CRC016_preTherapy_R2.fastq.gz \
     results/CRC016_preTherapy.vdjca

Option --report is specified here explicitly.

--species hsa: determines the organism species.
-p: generic-amplicon a 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=true: Results in a local alignment algorithm for V gene left bound due to the presence of primer sequences in V-gene region.
-OjParameters.parameters.floatingRightBound=false -OcParameters.parameters.floatingRightBound=true: Results in a global alignment algorithm for J gene right bound and a local alignment algorithm for C-gene right bound due to the presence of primer sequences.

`assemble`

Assembles alignments into clonotypes and applies several layers of errors correction(ex. quality-awared correction for sequencing errors, clustering to correct for PCR errors). Check mixcr assemble for more information. By default, clones will be assembled by CDR3 gene feature.

-OseparateByJ=true: Split clones with the same CDR3 sequence and different J-genes

mixcr assemble \
    -OassemblingFeatures="CDR3" \
    -OseparateByJ=true \
    --report results/CRC016_preTherapy.report.txt \
    --json-report results/CRC016_preTherapy.report.json \
    results/CRC016_preTherapy.vdjca \
    results/CRC016_preTherapy.clns

`export`

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

mixcr exportClones \
    -c IGH \
    results/CRC016_preTherapy.clns \
    results/CRC016_preTherapy.clonotypes.TRA.tsv

mixcr exportClones \
    -c IGL \
    results/CRC016_preTherapy.clns \
    results/CRC016_preTherapy.clonotypes.TRB.tsv

-с <chain>: defines a specific chain to be exported.

After execution is complete the following list of files is generated for every sample:

# human-readable reports 
CRC016_preTherapy.report

# raw alignments (highly compressed binary file)
CRC016_preTherapy.vdjca

# TRA, TRB CDR3 clonotypes (highly compressed binary file)
CRC016_preTherapy.clns

# TRA, TRB CDR3 clonotypes exported in tab-delimited txt
CRC016_preTherapy.TRA.tsv
CRC016_preTherapy.TRB.tsv

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

See first 100 records from clonotype table CRC016_preTherapy:

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	32290	0.0900717	TGTGCCAGCAGCACCTGGACAGGGAGTGGGGATGAGCAGTTCTTC	HHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH	TRBV7-9*00(1141.7)	TRBD1*00(41)	TRBJ2-1*00(209.2)	TRBC2*00(258.2)	273\|285\|310\|0\|12\|\|60.0	10\|21\|36\|13\|24\|SG12T\|41.0	28\|42\|70\|31\|45\|\|70.0	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	TGTGCCAGCAGCACCTGGACAGGGAGTGGGGATGAGCAGTTCTTC	39	nan	nan	nan	nan	nan	nan	nan	CASSTWTGSGDEQFF	nan	:::::::::0:-5:12:13:2:-3:24:31:-8:45:::
1	25443	0.0709723	TGTGCCAGCAGCCCCTTTGAGGGACAGGGGCGCTTCGAGCAGTACTTC	HHHHHHHHHHHHHHHHHHHHHHHHHHHGGGGGGHGGHHHHHHHHHHHG	TRBV3-1*00(1156.4)	TRBD1*00(50)	TRBJ2-7*00(205.2)	TRBC2*00(258.1)	270\|283\|307\|0\|13\|\|65.0	12\|22\|36\|20\|30\|\|50.0	23\|39\|67\|32\|48\|SA25T\|66.0	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	TGTGCCAGCAGCCCCTTTGAGGGACAGGGGCGCTTCGAGCAGTACTTC	38	nan	nan	nan	nan	nan	nan	nan	CASSPFEGQGRFEQYF	nan	:::::::::0:-4:13:20:0:-2:30:32:-3:48:::
2	9071	0.0253032	TGTGCCAGCAGTTACTCGAACCCGGACAGTGTTGGGCCCTACGAGCAGTACTTC	HHHHHHHHHHHHHHHHHHGGGGGHHHHHGGGHGGGHGGGGGGHHHHHHHHHHHG	TRBV6-5*00(1207.6)	TRBD1*00(33)	TRBJ2-7*00(224.2)	TRBC2*00(258.1)	270\|289\|307\|0\|19\|\|95.0	9\|19\|36\|20\|29\|DG12\|33.0	22\|39\|67\|37\|54\|\|85.0	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	TGTGCCAGCAGTTACTCGAACCCGGACAGTGTTGGGCCCTACGAGCAGTACTTC	38	nan	nan	nan	nan	nan	nan	nan	CASSYSNPDSVGPYEQYF	nan	:::::::::0:2:19:20:3:-5:29:37:-2:54:::
3	7841	0.0218722	TGTGCCTGGACAAAACCGGGCCAGGGTATCGCTGAAGCTTTCTTT	HHHHHHHHHHHGGGGGGHHHHHHHHGGHGGHHHHHHHHHHHHHHH	TRBV30*00(1146.3)	TRBD1*00(41)	TRBJ1-1*00(209.1)	TRBC1*00(257.8)	270\|280\|304\|0\|10\|\|50.0	10\|21\|36\|15\|26\|SA15C\|41.0	26\|40\|68\|31\|45\|\|70.0	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	TGTGCCTGGACAAAACCGGGCCAGGGTATCGCTGAAGCTTTCTTT	38	nan	nan	nan	nan	nan	nan	nan	CAWTKPGQGIAEAFF	nan	:::::::::0:-4:10:15:2:-3:26:31:-6:45:::
4	6356	0.0177298	TGCAGTGCCCGGGGGGGCCTCCATAGCAATCAGCCCCAGCATTTT	HHHHGGGGGGGGGGGGGGHHHHHHHHHHHHGGGGGHHHHHHHHHH	TRBV20-1*00(1182.6)	TRBD2*00(39)	TRBJ1-5*00(254.2)	TRBC1*00(258)	279\|287\|313\|0\|8\|\|40.0	24\|38\|48\|9\|22\|DA28SC32G\|39.0	19\|42\|70\|22\|45\|\|115.0	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	TGCAGTGCCCGGGGGGGCCTCCATAGCAATCAGCCCCAGCATTTT	38	nan	nan	nan	nan	nan	nan	nan	CSARGGLHSNQPQHF	nan	:::::::::0:-6:8:9:-8:6:22:22:1:45:::
5	4823	0.0134536	TGTGCCTGGAGACGGAGCACAGATACGCAGTATTTT	HHHHHHHHHHHHGHHHHHHHHHGGGGHHHHHHHHHH	TRBV30*00(1160.5)	nan	TRBJ2-3*00(244.2)	TRBC2*00(257.8)	270\|281\|304\|0\|11\|\|55.0	nan	20\|41\|69\|15\|36\|\|105.0	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	TGTGCCTGGAGACGGAGCACAGATACGCAGTATTTT	38	nan	nan	nan	nan	nan	nan	nan	CAWRRSTDTQYF	nan	:::::::::0:-3:11:::::15:0:36:::
6	4392	0.0122513	TGTGCCTCGGGGACCTACGAGCAGTACTTC	HHGGHHHGHHHHHGGGGGHHHHHHHHHHHG	TRBV7-6*00(1117.5)	nan	TRBJ2-7*00(224.1)	TRBC2*00(258)	273\|279\|310\|0\|6\|\|30.0	nan	22\|39\|67\|13\|30\|\|85.0	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	TGTGCCTCGGGGACCTACGAGCAGTACTTC	38	nan	nan	nan	nan	nan	nan	nan	CASGTYEQYF	nan	:::::::::0:-11:6:::::13:-2:30:::
7	3785	0.0105581	TGTGCCAGCAGCTTCTCGGATAGCAGAGAGACCCAGTACTTC	HHHHHHHHHHHHGGGGGHHHHHHHHHHHHHHHHHHHHHHHHH	TRBV5-4*00(1159.7)	nan	TRBJ2-5*00(220.1)	TRBC2*00(257.9)	270\|284\|306\|0\|14\|\|70.0	nan	13\|40\|68\|13\|42\|ST16CI19ASC21GI24G\|83.0	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	TGTGCCAGCAGCTTCTCGGATAGCAGAGAGACCCAGTACTTC	38	nan	nan	nan	nan	nan	nan	nan	CASSFSDSRETQYF	nan	:::::::::0:-2:14:::::13:7:42:::
8	3443	0.00960412	TGTGCCAGCAGCGTAACCGGGACTTACCCCCCAGATACGCAGTATTTT	HHHHHHHGGGGGGGGGGGGHHHHHGHGGGHHHHHGGGGHHHHHHHHHH	TRBV9*00(1160.3)	TRBD200(40),TRBD100(35)	TRBJ2-3*00(224.1)	TRBC2*00(258.1)	270\|285\|306\|0\|15\|\|75.0	14\|22\|48\|16\|24\|\|40.0;10\|17\|36\|16\|23\|\|35.0	24\|41\|69\|31\|48\|\|85.0	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	TGTGCCAGCAGCGTAACCGGGACTTACCCCCCAGATACGCAGTATTTT	38	nan	nan	nan	nan	nan	nan	nan	CASSVTGTYPPDTQYF	nan	:::::::::0:-1:15:16:2:-10:24:31:-4:48:::
9	2785	0.00776865	TGCGCCAGCAGCTTGGAACAGGGGGCGCGGACTGAAAAACTGTTTTTT	HHHGHHHHHHGHHHHHHHGGGGGGGGGGHHHHHHGHHHHHHGGHHHHH	TRBV5-1*00(1209.1)	TRBD1*00(55)	TRBJ1-4*00(219.1)	TRBC1*00(258.2)	270\|286\|306\|0\|16\|\|80.0	15\|26\|36\|17\|28\|\|55.0	27\|43\|71\|32\|48\|\|80.0	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	TGCGCCAGCAGCTTGGAACAGGGGGCGCGGACTGAAAAACTGTTTTTT	38	nan	nan	nan	nan	nan	nan	nan	CASSLEQGARTEKLFF	nan	:::::::::0:0:16:17:-3:2:28:32:-7:48:::
10	2619	0.0073056	TGCGCCAGCAATGAGTGGGGGGTCGGCACTGAAGCTTTCTTT	HHHGHHHHHHHHHHHHHGHGGGGGHHHHHHHHHHHHHHHHHH	TRBV10-2*00(1183.2)	TRBD1*00(25)	TRBJ1-1*00(219.1)	TRBC1*00(257.8)	270\|286\|307\|0\|16\|SG280A\|66.0	18\|23\|36\|16\|21\|\|25.0	24\|40\|68\|26\|42\|\|80.0	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	TGCGCCAGCAATGAGTGGGGGGTCGGCACTGAAGCTTTCTTT	38	nan	nan	nan	nan	nan	nan	nan	CASNEWGVGTEAFF	nan	:::::::::0:-1:16:16:-6:-1:21:26:-4:42:::
11	2355	0.00656918	TGTGCCAGCTCACCGGGTCGTGGAACTGAAGCTTTCTTT	HHHHHHHHHGGGGGGGGGGHHHHHHHHHHHHHHHHHHHH	TRBV18*00(1172.8)	nan	TRBJ1-1*00(214.2)	TRBC1*00(258)	273\|287\|310\|0\|14\|\|70.0	nan	25\|40\|68\|24\|39\|\|75.0	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	TGTGCCAGCTCACCGGGTCGTGGAACTGAAGCTTTCTTT	38	nan	nan	nan	nan	nan	nan	nan	CASSPGRGTEAFF	nan	:::::::::0:-3:14:::::24:-5:39:::
12	2312	0.00644924	TGTGCCAGCTCTCAGCAAGCCCAGACCGGGGAGCTGTTTTTT	HHHHHHHHHHHHHHHGHHHHHHGGGGGGGHHHHHHGGHHHHH	TRBV28*00(1129.2)	nan	TRBJ2-2*00(229.1)	TRBC2*00(258.2)	270\|279\|307\|0\|9\|\|45.0	nan	25\|43\|71\|24\|42\|\|90.0	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	TGTGCCAGCTCTCAGCAAGCCCAGACCGGGGAGCTGTTTTTT	38	nan	nan	nan	nan	nan	nan	nan	CASSQQAQTGELFF	nan	:::::::::0:-8:9:::::24:-5:42:::
13	1965	0.00548129	TGTGCCAGCAGCGGACAGAGAACTATGAACACTGAAGCTTTCTTT	HHHHHHHGGGGGHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH	TRBV9*00(1151.4)	TRBD1*00(25)	TRBJ1-1*00(244.2)	TRBC1*00(257.4)	270\|283\|306\|0\|13\|\|65.0	14\|19\|36\|13\|18\|\|25.0	19\|40\|68\|24\|45\|\|105.0	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	TGTGCCAGCAGCGGACAGAGAACTATGAACACTGAAGCTTTCTTT	38	nan	nan	nan	nan	nan	nan	nan	CASSGQRTMNTEAFF	nan	:::::::::0:-3:13:13:-2:-5:18:24:1:45:::
14	1902	0.00530556	TGTGCCAGTTGGGGAGGCGAGCAGTACTTC	HHHHHHHGGHHGHGGGGGHHHHHHHGHHHG	TRBV6-5*00(1159.5)	TRBD2*00(30)	TRBJ2-7*00(204.1)	TRBC2*00(258.2)	270\|278\|307\|0\|8\|\|40.0	25\|31\|48\|11\|17\|\|30.0	26\|39\|67\|17\|30\|\|65.0	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	TGTGCCAGTTGGGGAGGCGAGCAGTACTTC	38	nan	nan	nan	nan	nan	nan	nan	CASWGGEQYF	nan	:::::::::0:-9:8:11:-9:-1:17:17:-6:30:::
15	1578	0.00440177	TGTGCCAGCAGCCATCGGGACAGAAACTACGAGCAGTACTTC	HHHHHHHGGHHGGGGGHHHHHHHHHGGGGGHHHHHHHHHHHG	TRBV7-9*00(1137.3)	TRBD1*00(40)	TRBJ2-7*00(219.3)	TRBC2*00(258)	273\|285\|310\|0\|12\|\|60.0	11\|19\|36\|15\|23\|\|40.0	23\|39\|67\|26\|42\|\|80.0	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	TGTGCCAGCAGCCATCGGGACAGAAACTACGAGCAGTACTTC	38	nan	nan	nan	nan	nan	nan	nan	CASSHRDRNYEQYF	nan	:::::::::0:-5:12:15:1:-5:23:26:-3:42:::
16	1003	0.00279783	TGTGCCAGCAGCTTAGGGACAGATACGCAGTATTTT	HHHHHHHHHHHHHHHHHHHHHHGGGGHHHHHHHHHH	TRBV13*00(1188)	nan	TRBJ2-3*00(229.2)	TRBC2*00(258.2)	270\|287\|307\|0\|17\|\|85.0	nan	23\|41\|69\|18\|36\|\|90.0	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	TGTGCCAGCAGCTTAGGGACAGATACGCAGTATTTT	38	nan	nan	nan	nan	nan	nan	nan	CASSLGTDTQYF	nan	:::::::::0:0:17:::::18:-3:36:::
17	992	0.00276715	TGTGCCTGGAGTGACAGGGTAGAGACCCAGTACTTC	HHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHG	TRBV30*00(1168.5)	nan	TRBJ2-5*00(219.3)	TRBC2*00(258.1)	270\|287\|304\|0\|16\|DT283\|68.0	nan	24\|40\|68\|20\|36\|\|80.0	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	TGTGCCTGGAGTGACAGGGTAGAGACCCAGTACTTC	38	nan	nan	nan	nan	nan	nan	nan	CAWSDRVETQYF	nan	:::::::::0:3:16:::::20:-4:36:::
18	962	0.00268346	TGTGCCTGGATGGAGTCTGAAGCTTTCTTT	HHHHHHHHHHHHHHHHHHHHHHHHHHHHHH	TRBV30*00(1154.9)	nan	TRBJ1-1*00(208.8)	TRBC1*00(257.7)	270\|280\|304\|0\|10\|\|50.0	nan	26\|40\|68\|16\|30\|\|70.0	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	TGTGCCTGGATGGAGTCTGAAGCTTTCTTT	39	nan	nan	nan	nan	nan	nan	nan	CAWMESEAFF	nan	:::::::::0:-4:10:::::16:-6:30:::
19	897	0.00250215	TGTGCCAGCTCACCACAGGGGGGGACTGAAGCTTTCTTT	HHHGHHHHHHGHHHHGGGGGHHHHHHHHHHHHHHHHHHH	TRBV18*00(1179.8)	nan	TRBJ1-1*00(208.1)	TRBC1*00(258.1)	273\|302\|310\|0\|28\|SC289AST292GST295GDG298\|86.0	nan	25\|40\|68\|24\|39\|\|75.0	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	TGTGCCAGCTCACCACAGGGGGGGACTGAAGCTTTCTTT	38	nan	nan	nan	nan	nan	nan	nan	CASSPQGGTEAFF	nan	:::::::::0:12:28:::::24:-5:39:::
20	888	0.00247704	TGCGCCAGCAAGACACAGGGCTCGAAGGCTGAAGCTTTCTTT	HHHGHHHHHHHHHHHHHGGGHHHHHHHHHHHHHHHHHHHHHH	TRBV5-1*00(1181.4)	TRBD1*00(30)	TRBJ1-1*00(208.5)	TRBC1*00(257.6)	270\|280\|306\|0\|10\|\|50.0	15\|21\|36\|14\|20\|\|30.0	26\|40\|68\|28\|42\|\|70.0	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	TGCGCCAGCAAGACACAGGGCTCGAAGGCTGAAGCTTTCTTT	38	nan	nan	nan	nan	nan	nan	nan	CASKTQGSKAEAFF	nan	:::::::::0:-6:10:14:-3:-3:20:28:-6:42:::
21	877	0.00244636	TGTGCCTGGAGTGATCGGGTGGAGACCCAGTACTTC	HHHHHHHHHHHGGGGGGHHHHHHHHHHHHHHHHHHG	TRBV30*00(1169.1)	nan	TRBJ2-5*00(213.9)	TRBC2*00(258.1)	270\|283\|304\|0\|13\|\|65.0	nan	25\|40\|68\|21\|36\|\|75.0	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	TGTGCCTGGAGTGATCGGGTGGAGACCCAGTACTTC	38	nan	nan	nan	nan	nan	nan	nan	CAWSDRVETQYF	nan	:::::::::0:-1:13:::::21:-5:36:::
22	734	0.00204747	TGCAGCGTTGTGGACAGGGGAGAGACCCAGTACTTC	HHHHHHGHHHHHHHHHGHHHHHHHHHHHHHHHHHHH	TRBV29-1*00(1210)	TRBD1*00(45)	TRBJ2-5*00(219.2)	TRBC2*00(257.8)	276\|286\|310\|0\|10\|\|50.0	13\|22\|36\|11\|20\|\|45.0	24\|40\|68\|20\|36\|\|80.0	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	TGCAGCGTTGTGGACAGGGGAGAGACCCAGTACTTC	38	nan	nan	nan	nan	nan	nan	nan	CSVVDRGETQYF	nan	:::::::::0:-4:10:11-2:20:20:-4:36:::
23	693	0.0019331	TGTGCCAGTAGTATAAGCTCGAACGGGAACACTGAAGCTTTCTTT	HHHHHHHHHHHHHHHHGHHGHHHGHHHHHHHHHHHHHHHHHHHHH	TRBV19*00(1159.9)	nan	TRBJ1-1*00(233.8)	TRBC1*00(258)	270\|285\|307\|0\|15\|\|75.0	nan	21\|40\|68\|26\|45\|\|95.0	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	TGTGCCAGTAGTATAAGCTCGAACGGGAACACTGAAGCTTTCTTT	38	nan	nan	nan	nan	nan	nan	nan	CASSISSNGNTEAFF	nan	:::::::::0:-2:15:::::26:-1:45:::
24	692	0.00193031	TGTGCCTGGAGTGTACTAGGGGGTAGTCAGCCCCAGCATTTT	HHHHHHHHHHHHHHHHGGGHHHHHHHHGGGGGHHHHHHHHHH	TRBV30*00(1187.8)	TRBD1*00(30)	TRBJ1-5*00(218.5)	TRBC1*00(258)	270\|286\|304\|0\|16\|\|80.0	17\|23\|36\|17\|23\|\|30.0	26\|42\|70\|26\|42\|\|80.0	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	TGTGCCTGGAGTGTACTAGGGGGTAGTCAGCCCCAGCATTTT	38	nan	nan	nan	nan	nan	nan	nan	CAWSVLGGSQPQHF	nan	:::::::::0:2:16:17:-5:-1:23:26:-6:42:::
25	678	0.00189126	TGTGCCTGGACCAAGGGACTAGCGGGGGTCAATGAGCAGTTCTTC	HHHHHHHHHHHHHHHHHHGGGGGGGHHHHHHHHHHHHHHHGGGGG	TRBV30*00(1154)	TRBD2*00(60)	TRBJ2-1*00(219.1)	TRBC2*00(256.8)	270\|280\|304\|0\|10\|\|50.0	16\|28\|48\|14\|26\|\|60.0	26\|42\|70\|29\|45\|\|80.0	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	TGTGCCTGGACCAAGGGACTAGCGGGGGTCAATGAGCAGTTCTTC	38	nan	nan	nan	nan	nan	nan	nan	CAWTKGLAGVNEQFF	nan	:::::::::0:-4:10:14:0:-4:26:29:-6:45:::
26	667	0.00186057	TGTGCCTGGAGTGTGGGGGCCAGGCCATATAGCAATCAGCCCCAGCATTTT	HHHHHHHHHHGGGGGGHHHHGHHHHHHHHHHHHHHHGGGGGHHHHHHHHHH	TRBV30*00(1167.5)	TRBD1*00(30)	TRBJ1-5*00(259.1)	TRBC1*00(257.3)	270\|284\|304\|0\|14\|\|70.0	18\|24\|36\|14\|20\|\|30.0	18\|42\|70\|27\|51\|\|120.0	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	TGTGCCTGGAGTGTGGGGGCCAGGCCATATAGCAATCAGCCCCAGCATTTT	38	nan	nan	nan	nan	nan	nan	nan	CAWSVGARPYSNQPQHF	nan	:::::::::0:0:14:14:-6:0:20:27:2:51:::
27	649	0.00181036	TGTGCCAGCAGCCAAGACGGGGGATCCAGCTCCTACGAGCAGTACTTC	HHHHHHHHGHHHHHHHHHGGHHHHHHHHHHHGGGGGHHHHHHHGGHGG	TRBV3-1*00(1172.3)	TRBD1*00(25)	TRBJ2-7*00(244.1)	TRBC2*00(257.8)	270\|287\|307\|0\|17\|\|85.0	18\|23\|36\|18\|23\|\|25.0	18\|39\|67\|27\|48\|\|105.0	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	TGTGCCAGCAGCCAAGACGGGGGATCCAGCTCCTACGAGCAGTACTTC	38	nan	nan	nan	nan	nan	nan	nan	CASSQDGGSSSYEQYF	nan	:::::::::0:0:17:18:-6:-1:23:27:2:48:::
28	638	0.00177968	TGCAGCGTCTGGGACAGGGAGGCCTACACCTTC	HHHHGGGHHHHHHHHHGHHHHHHHHHHHHHHHH	TRBV29-1*00(1214.7)	TRBD1*00(48)	TRBJ1-2*00(189)	TRBC1*00(258.1)	276\|284\|310\|0\|8\|\|40.0	12\|24\|36\|10\|23\|I21A\|48.0	30\|40\|68\|23\|33\|\|50.0	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	TGCAGCGTCTGGGACAGGGAGGCCTACACCTTC	38	nan	nan	nan	nan	nan	nan	nan	CSVWDREAYTF	nan	:::::::::0:-6:8:10:0:0:23:23:-10:33:::
29	626	0.0017462	TGTGCCTGGAGTGGGGGGGGCGCGGCGCGGGGCACTGAAGCTTTCTTT	HHHHHHHHHHGGGGGGGGGGGGGGGGGGGHHHHHHHHHHHHHHHHHHH	TRBV30*00(1170.2)	TRBD1*00(40)	TRBJ1-1*00(219)	TRBC1*00(257.7)	270\|283\|304\|0\|13\|\|65.0	18\|26\|36\|15\|23\|\|40.0	24\|40\|68\|32\|48\|\|80.0	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	TGTGCCTGGAGTGGGGGGGGCGCGGCGCGGGGCACTGAAGCTTTCTTT	38	nan	nan	nan	nan	nan	nan	nan	CAWSGGGAARGTEAFF	nan	:::::::::0:-1:13:15:-6:2:23:32:-4:48:::
30	622	0.00173505	TGCGCCAGCAGTATAGAAGACGCCCGTAATGAAAAACTGTTTTTT	HGGGHHHHHHHHHHHHGGGGGGGGGHHHHHHGHHHHHHGGHHHHH	TRBV5-1*00(1189.2)	TRBD1*00(25)	TRBJ1-4*00(234)	TRBC1*00(257.8)	270\|281\|306\|0\|11\|\|55.0	23\|28\|36\|20\|25\|\|25.0	24\|43\|71\|26\|45\|\|95.0	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	TGCGCCAGCAGTATAGAAGACGCCCGTAATGAAAAACTGTTTTTT	38	nan	nan	nan	nan	nan	nan	nan	CASSIEDARNEKLFF	nan	:::::::::0:-5:11:20:-11:4:25:26:-4:45:::
31	599	0.00167089	TGTGCCAGCAGCGAGATCGGGGGTCCGAGCTCCTACGAGCAGTACTTC	HHHHHHHGGGGHHHGGGGGGGGGGGGHHHHHGGGGGHHHHHHHGHHHG	TRBV5-4*00(1144.3)	TRBD1*00(25)	TRBJ2-7*00(249.4)	TRBC2*00(258.1)	270\|282\|306\|0\|12\|\|60.0	18\|23\|36\|18\|23\|\|25.0	17\|39\|67\|26\|48\|\|110.0	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	TGTGCCAGCAGCGAGATCGGGGGTCCGAGCTCCTACGAGCAGTACTTC	38	nan	nan	nan	nan	nan	nan	nan	CASSEIGGPSSYEQYF	nan	:::::::::0:-4:12:18:-6:-1:23:26:3:48:::
32	594	0.00165694	TGTGCCTGGACTGGTGGGGCTAGCACAGATACGCAGTATTTT	HHHHHHHHHHHHGGGGHHHHHHHHHHHHGGGGHHHHHHHHHH	TRBV30*00(1157)	nan	TRBJ2-3*00(259.3)	TRBC2*00(258.2)	270\|283\|304\|0\|13\|SG280C\|51.0	nan	17\|41\|69\|18\|42\|\|120.0	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	TGTGCCTGGACTGGTGGGGCTAGCACAGATACGCAGTATTTT	38	nan	nan	nan	nan	nan	nan	nan	CAWTGGASTDTQYF	nan	:::::::::0:-1:13:::::18:3:42:::
33	552	0.00153978	TGTGCCTGGAGTGTCCAGGGCGTCTACGAGCAGTACTTC	HHHHHHHHHHHHHHHHGGGGGHGGGGGHHHHHHHGHHHH	TRBV30*00(1162.2)	TRBD1*00(25)	TRBJ2-7*00(219.2)	TRBC2*00(258.3)	270\|287\|304\|0\|17\|SA284C\|71.0	20\|25\|36\|17\|22\|\|25.0	23\|39\|67\|23\|39\|\|80.0	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	TGTGCCTGGAGTGTCCAGGGCGTCTACGAGCAGTACTTC	38	nan	nan	nan	nan	nan	nan	nan	CAWSVQGVYEQYF	nan	:::::::::0:3:17:17:-8:1:22:23:-3:39:::
34	520	0.00145052	TGTGCCAGCAGCCACCTTGAAACAACCCAAGAGACCCAGTACTTC	HHHHHHHGGHHGHHHHHHHHHGGGGHHHHHHHHHHHHHHHGHHHG	TRBV7-8*00(1138.4)	nan	TRBJ2-5*00(234.1)	TRBC2*00(257.6)	273\|285\|310\|0\|12\|\|60.0	nan	21\|40\|68\|26\|45\|\|95.0	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	TGTGCCAGCAGCCACCTTGAAACAACCCAAGAGACCCAGTACTTC	38	nan	nan	nan	nan	nan	nan	nan	CASSHLETTQETQYF	nan	:::::::::0:-5:12:::::26:-1:45:::
35	519	0.00144773	TGTGCCTGGAGTGTGACAGTGATGAATCAGCCCCAGCATTTT	HHHHHHHHHHHHHHHHHHHHHHHHHHHGGGGGHHHHHHHHHH	TRBV30*00(1168.3)	nan	TRBJ1-5*00(229)	TRBC1*00(256)	270\|287\|304\|0\|18\|I284G\|73.0	nan	24\|42\|70\|24\|42\|\|90.0	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	TGTGCCTGGAGTGTGACAGTGATGAATCAGCCCCAGCATTTT	38	nan	nan	nan	nan	nan	nan	nan	CAWSVTVMNQPQHF	nan	:::::::::0:3:18:::::24:-4:42:::
36	517	0.00144215	TGTGCCTGGAGTACGGGACCGGCGAACACTGAAGCTTTCTTT	HHHHHHHHHHGGHGHGGGGGGGGHHHHHHHHHHHHHHHHHHH	TRBV30*00(1163.4)	TRBD100(31),TRBD200(30)	TRBJ1-1*00(234.2)	TRBC1*00(258.6)	270\|282\|304\|0\|12\|\|60.0	11\|20\|36\|13\|22\|SA17C\|31.0;15\|21\|48\|13\|19\|\|30.0	21\|40\|68\|23\|42\|\|95.0	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	TGTGCCTGGAGTACGGGACCGGCGAACACTGAAGCTTTCTTT	38	nan	nan	nan	nan	nan	nan	nan	CAWSTGPANTEAFF	nan	:::::::::0:-2:12:13:1:-4:22:2342:::
37	487	0.00135847	TGTGCCTGGACGGTCCGACAGGCCCAGTACTTC	HHHHHHGGGGGGGGGGHHGGHHHHHHHHHHHHG	TRBV30*00(1150.9)	nan	TRBJ2-5*00(195.4)	TRBC2*00(258)	270\|280\|304\|0\|10\|\|50.0	nan	26\|40\|68\|19\|33\|SA28G\|56.0	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	TGTGCCTGGACGGTCCGACAGGCCCAGTACTTC	38	nan	nan	nan	nan	nan	nan	nan	CAWTVRQAQYF	nan	:::::::::0:-4:10:::::19:-6:33:::
38	474	0.00132221	TGTGCCTGGAGTCCGGACGGGACGCAAGAGACCCAGTACTTC	HHHHHHHHHGGGGGGGHGGGGGGHHHHHHHHHHHHHHHHHHG	TRBV30*00(1160.9)	TRBD100(30),TRBD200(30)	TRBJ2-5*00(228.9)	TRBC2*00(258.1)	270\|282\|304\|0\|12\|\|60.0	11\|17\|36\|17\|23\|\|30.0;15\|21\|48\|17\|23\|\|30.0	22\|40\|68\|24\|42\|\|90.0	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	TGTGCCTGGAGTCCGGACGGGACGCAAGAGACCCAGTACTTC	38	nan	nan	nan	nan	nan	nan	nan	CAWSPDGTQETQYF	nan	:::::::::0:-2:12:17:1:-7:23:24:-2:42:::
39	458	0.00127757	TGTGCCTGGAAGGCGGATACGCAGTATTTT	HHHHHHHHHGGGGGHHGGGGHHHHHHHHHH	TRBV30*00(1155.4)	nan	TRBJ2-3*00(214.1)	TRBC2*00(257.3)	270\|280\|304\|0\|10\|\|50.0	nan	26\|41\|69\|15\|30\|\|75.0	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	TGTGCCTGGAAGGCGGATACGCAGTATTTT	38	nan	nan	nan	nan	nan	nan	nan	CAWKADTQYF	nan	:::::::::0:-4:10:::::15:-6:30:::
40	456	0.00127199	TGTGCCAGCGTGAACAGATACCTCAACTCTGGGGCCAACGTCCTGACTTTC	HHHGGHHHHHHHHHHHHHHHHHHHHHHHGGGHHHHHGGGHHHHHHHHHHHG	TRBV6-5*00(1148)	nan	TRBJ2-6*00(264.1)	TRBC2*00(258)	270\|279\|307\|0\|9\|\|45.0	nan	20\|45\|73\|26\|51\|\|125.0	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	TGTGCCAGCGTGAACAGATACCTCAACTCTGGGGCCAACGTCCTGACTTTC	38	nan	nan	nan	nan	nan	nan	nan	CASVNRYLNSGANVLTF	nan	:::::::::0:-8:9:::::26:0:51:::
41	450	0.00125526	TGTGCCTGGGTCAAGATCCAGGGGGGTACTGAAGCTTTCTTT	HHHHGHHHHHHHHHHHHHGGGGHHHHHHHHHHHHHHHHHHHH	TRBV30*00(1149.6)	TRBD1*00(35)	TRBJ1-1*00(214)	TRBC1*00(258.6)	270\|279\|304\|0\|9\|\|45.0	16\|23\|36\|18\|25\|\|35.0	25\|40\|68\|27\|42\|\|75.0	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	TGTGCCTGGGTCAAGATCCAGGGGGGTACTGAAGCTTTCTTT	38	nan	nan	nan	nan	nan	nan	nan	CAWVKIQGGTEAFF	nan	:::::::::0:-5:9:18:-4:-1:25:27:-5:42:::
42	411	0.00114647	TGTGCCAGCAGCCAAGAGGTAAGGGGTCGGGGCCACAATCAGCCCCAGCATTTT	HHHHHHHHGHHHHHHHHHHHHGGGGGGGGHHHGHHHHHHGGGGGHHHHHHHHHH	TRBV4-2*00(1136.5)	TRBD100(26),TRBD200(25)	TRBJ1-5*00(234.5)	TRBC1*00(257)	270\|287\|307\|0\|17\|\|85.0	17\|25\|36\|21\|29\|SG22T\|26.0;29\|34\|48\|29\|34\|\|25.0	23\|42\|70\|35\|54\|\|95.0	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	TGTGCCAGCAGCCAAGAGGTAAGGGGTCGGGGCCACAATCAGCCCCAGCATTTT	38	nan	nan	nan	nan	nan	nan	nan	CASSQEVRGRGHNQPQHF	nan	:::::::::0:0:17:21:-5:1:29:35:-3:54:::
43	407	0.00113531	TGTGCCAGCAGTTACGCAGAATCCTACGAGCAGTACTTC	HHHHHHHHHHGGGGHHHHHHHHGGGGGHHHHHHHGHHHG	TRBV6-5*00(1190.3)	nan	TRBJ2-7*00(226.3)	TRBC2*00(257.7)	270\|285\|307\|0\|15\|\|75.0	nan	21\|39\|67\|21\|39\|\|90.0	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	TGTGCCAGCAGTTACGCAGAATCCTACGAGCAGTACTTC	38	nan	nan	nan	nan	nan	nan	nan	CASSYAESYEQYF	nan	:::::::::0:-2:15:::::21:-1:39:::
44	404	0.00112694	TGCAGTGCTAGGAACCGGGACAGGAACACTGAAGCTTTCTTT	HHHHHHHHHHHGGGGGHHHHHHHHHHHHHHHHHHHHHHHHHH	TRBV20-1*00(1199.4)	TRBD100(30),TRBD200(30)	TRBJ1-1*00(234.6)	TRBC1*00(256.9)	279\|290\|313\|0\|11\|\|55.0	10\|16\|36\|14\|20\|\|30.0;14\|20\|48\|14\|20\|\|30.0	18\|40\|68\|20\|42\|ST20G\|96.0	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	TGCAGTGCTAGGAACCGGGACAGGAACACTGAAGCTTTCTTT	38	nan	nan	nan	nan	nan	nan	nan	CSARNRDRNTEAFF	nan	:::::::::0:-3:11:14:2:-8:20:20:2:42:::
45	402	0.00112136	TGTGCCTGGAGCCGCGGGACAGAAACAGATACGCAGTATTTT	HHHHHHHGGGGGGGGHHHHHHHHHHHHHGGGGHHHHHHHHHH	TRBV30*00(1156.6)	TRBD1*00(40)	TRBJ2-3*00(228.7)	TRBC2*00(258.1)	270\|281\|304\|0\|11\|\|55.0	11\|19\|36\|14\|22\|\|40.0	23\|41\|69\|24\|42\|\|90.0	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	TGTGCCTGGAGCCGCGGGACAGAAACAGATACGCAGTATTTT	38	nan	nan	nan	nan	nan	nan	nan	CAWSRGTETDTQYF	nan	:::::::::0:-3:11:14:1:-5:22:24:-3:42:::
46	401	0.00111857	TGTGCCAGCAGCTCAGGAGAGACCCAGTACTTC	HHHGHHHHHHHHHHHHHHHHHHHHHHHHHHHHG	TRBV7-6*00(1148.8)	nan	TRBJ2-5*00(219.4)	TRBC2*00(258.7)	273\|289\|310\|0\|16\|ST286C\|66.0	nan	24\|40\|68\|17\|33\|\|80.0	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	TGTGCCAGCAGCTCAGGAGAGACCCAGTACTTC	38	nan	nan	nan	nan	nan	nan	nan	CASSSGETQYF	nan	:::::::::0:-1:16:::::17:-4:33:::
47	386	0.00107673	TGTGCCAGCAGCTTGGGAGGGAACACTGAAGCTTTCTTT	HHHHHHHHHHGHHHGGHHHHHHHHHHHHHHHHHHHHHHH	TRBV5-8*00(1160.7)	nan	TRBJ1-1*00(233.6)	TRBC1*00(257.2)	270\|286\|306\|0\|16\|\|80.0	nan	21\|40\|68\|20\|39\|\|95.0	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	TGTGCCAGCAGCTTGGGAGGGAACACTGAAGCTTTCTTT	38	nan	nan	nan	nan	nan	nan	nan	CASSLGGNTEAFF	nan	:::::::::0:0:16:::::20:-1:39:::
48	382	0.00106557	TGTGCCAGCAGCTCCGGGGGGGCTAAGGAGCAGTTCTTC	HHHHHHHHHHGGGGGGGGGHHHHHHHHHHHHHHHHHHHG	TRBV9*00(1143.1)	TRBD200(31),TRBD100(30)	TRBJ2-1*00(199.7)	TRBC2*00(256.5)	270\|282\|306\|0\|12\|\|60.0	24\|33\|48\|14\|23\|SA28G\|31.0;18\|24\|36\|17\|23\|\|30.0	27\|42\|70\|24\|39\|ST29G\|61.0	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	TGTGCCAGCAGCTCCGGGGGGGCTAAGGAGCAGTTCTTC	38	nan	nan	nan	nan	nan	nan	nan	CASSSGGAKEQFF	nan	:::::::::0:-4:12:14:-8:1:23:24:-7:39:::
49	376	0.00104884	TGTGCCTGGGTGCAAAAAGACGCTAGCACAGATACGCAGTATTTT	HHHHGHGGHHHHGHHHGGGGGHHHHHHHHHHGGGGHHHHHHHHHG	TRBV30*00(1154)	nan	TRBJ2-3*00(259.2)	TRBC2*00(257.5)	270\|283\|304\|0\|12\|DA279\|48.0	nan	17\|41\|69\|21\|45\|\|120.0	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	TGTGCCTGGGTGCAAAAAGACGCTAGCACAGATACGCAGTATTTT	38	nan	nan	nan	nan	nan	nan	nan	CAWVQKDASTDTQYF	nan	:::::::::0:-1:12:::::21:3:45:::
50	374	0.00104326	TGTGCCAGCAGCTCGAAGCGTGGGACAAACTACGAGCAGTACTTC	HHHHHHHHHGGHHGGGGGGHHHHHHHHHGGGGGHHHHHHHGGGGG	TRBV11-2*00(1147.1)	TRBD1*00(30)	TRBJ2-7*00(219.3)	TRBC2*00(257.3)	273\|286\|310\|0\|13\|\|65.0	12\|18\|36\|21\|27\|\|30.0	23\|39\|67\|29\|45\|\|80.0	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	TGTGCCAGCAGCTCGAAGCGTGGGACAAACTACGAGCAGTACTTC	38	nan	nan	nan	nan	nan	nan	nan	CASSSKRGTNYEQYF	nan	:::::::::0:-4:13:21:0:-6:27:29:-3:45:::
51	368	0.00102652	TGTGGCGCGGGCTCCTACAATGAGCAGTTCTTC	HGGGGGGGGGHHHHHHHHHHHHHHHHHHGGGGG	TRBV30*00(1121.8)	nan	TRBJ2-1*00(254.1)	TRBC2*00(258.3)	270\|274\|304\|0\|4\|\|20.0	nan	19\|42\|70\|10\|33\|\|115.0	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	TGTGGCGCGGGCTCCTACAATGAGCAGTTCTTC	38	nan	nan	nan	nan	nan	nan	nan	CGAGSYNEQFF	nan	:::::::::0:-10:4:::::10:1:33:::
52	364	0.00101536	TGTGCCAGCAGCCCTCGCCGGTCCATGAACACTGAAGCTTTCTTT	HHHHHHHGGHGGGGGGGGGHHHHHHHHHHHHHHHHHHHHHHHHHH	TRBV5-4*00(1149.6)	nan	TRBJ1-1*00(249.7)	TRBC1*00(255.8)	270\|282\|306\|0\|12\|\|60.0	nan	15\|40\|68\|20\|45\|ST17C\|111.0	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	TGTGCCAGCAGCCCTCGCCGGTCCATGAACACTGAAGCTTTCTTT	38	nan	nan	nan	nan	nan	nan	nan	CASSPRRSMNTEAFF	nan	:::::::::0:-4:12:::::20:5:45:::
53	352	0.000981891	TGCAGCGTTAAAACAGCCGCGAAAAGGTACGAGCAGTACTTC	HGHHHHHHHHHHHHHGHHGGHHHHHGHGGHHHHHHHHGGGGG	TRBV29-1*00(1212.6)	nan	TRBJ2-7*00(214.2)	TRBC2*00(258.1)	276\|288\|310\|0\|12\|SG285A\|46.0	nan	24\|39\|67\|27\|42\|\|75.0	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	TGCAGCGTTAAAACAGCCGCGAAAAGGTACGAGCAGTACTTC	38	nan	nan	nan	nan	nan	nan	nan	CSVKTAAKRYEQYF	nan	:::::::::0:-2:12:::::27:-4:42:::
54	347	0.000967943	TGTGCCAGCAGCTTAGTGATAGGGAACACCGGGGAGCTGTTTTTT	HHHGHHHHHHHHHHHHHHHHHHHHGGGGGGGGHHHHHHGGHHHHH	TRBV7-7*00(1144.4)	nan	TRBJ2-2*00(249.4)	TRBC2*00(257.7)	273\|289\|310\|0\|16\|\|80.0	nan	21\|43\|71\|23\|45\|\|110.0	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	TGTGCCAGCAGCTTAGTGATAGGGAACACCGGGGAGCTGTTTTTT	38	nan	nan	nan	nan	nan	nan	nan	CASSLVIGNTGELFF	nan	:::::::::0:-1:16:::::23:-1:45:::
55	345	0.000962365	TGTGCCTGGAGAAATGGAGGGGCTCCCGGTTTTGCCTACGAGCAGTACTTC	HHHHHHHHHHHHHHHGGHGGGGGGGGGGHHHHHHGGGGGHHHHHHHGGHGG	TRBV30*00(1161.3)	TRBD2*00(39)	TRBJ2-7*00(224.5)	TRBC2*00(257.7)	270\|281\|304\|0\|11\|\|55.0	26\|40\|48\|15\|28\|DC32SC33G\|39.0	22\|39\|67\|34\|51\|\|85.0	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	TGTGCCTGGAGAAATGGAGGGGCTCCCGGTTTTGCCTACGAGCAGTACTTC	38	nan	nan	nan	nan	nan	nan	nan	CAWRNGGAPGFAYEQYF	nan	:::::::::0:-3:11:15:-10:8:28:34:-2:51:::
56	335	0.00093447	TGTGCCAGCAGCGTAGATTGGGCCGTCGGCAATGAGCAGTTCTTC	HHHHHHHGGGGGHHHHHHGGGGGGGGGHHHHHHHHHHHHHGGHGG	TRBV9*00(1168)	TRBD2*00(26)	TRBJ2-1*00(219.4)	TRBC2*00(256.2)	270\|286\|306\|0\|16\|\|80.0	29\|37\|48\|19\|27\|SC34G\|26.0	26\|42\|70\|29\|45\|\|80.0	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	TGTGCCAGCAGCGTAGATTGGGCCGTCGGCAATGAGCAGTTCTTC	38	nan	nan	nan	nan	nan	nan	nan	CASSVDWAVGNEQFF	nan	:::::::::0:0:16:19:-13:5:27:29:-6:45:::
57	334	0.00093168	TGTGCCAGCAGCGTAGGCTACAGCCAAGAGACCCAGTACTTC	HHHHHHHGGGGGHHHHHHHHGHHHHHHHHHHHHHHHHHHHHH	TRBV9*00(1178.4)	nan	TRBJ2-5*00(234.3)	TRBC2*00(258.4)	270\|290\|306\|0\|21\|I285G\|88.0	nan	21\|40\|68\|23\|42\|\|95.0	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	TGTGCCAGCAGCGTAGGCTACAGCCAAGAGACCCAGTACTTC	38	nan	nan	nan	nan	nan	nan	nan	CASSVGYSQETQYF	nan	:::::::::0:4:21:::::23:-1:42:::
58	332	0.000926102	TGCAGCGTTGACGAGGGGGCAGGACAGCCCCAGCATTTT	HHGHGGHGHGHGGGGGHHHHHHHHGGGGGHHHHHHHHHH	TRBV29-1*00(1233.3)	TRBD1*00(30)	TRBJ1-5*00(214.2)	TRBC1*00(257.8)	276\|290\|310\|0\|14\|SA287C\|56.0	18\|24\|36\|14\|20\|\|30.0	27\|42\|70\|24\|39\|\|75.0	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	TGCAGCGTTGACGAGGGGGCAGGACAGCCCCAGCATTTT	38	nan	nan	nan	nan	nan	nan	nan	CSVDEGAGQPQHF	nan	:::::::::0:0:14:14:-6:0:20:24:-7:39:::
59	329	0.000917733	TGTGCCAGCAGCTTAGAGAGGGGGACAGCTAACTATGGCTACACCTTC	HHHGHHHHHHHHHHHHHGGGHHHHHHHHHHHHHHHHHHHHHHHGGGGG	TRBV7-6*00(1157.9)	TRBD1*00(30)	TRBJ1-2*00(249.2)	TRBC1*00(257.4)	273\|289\|310\|0\|16\|\|80.0	17\|23\|36\|18\|24\|\|30.0	18\|40\|68\|26\|48\|\|110.0	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	TGTGCCAGCAGCTTAGAGAGGGGGACAGCTAACTATGGCTACACCTTC	38	nan	nan	nan	nan	nan	nan	nan	CASSLERGTANYGYTF	nan	:::::::::0:-1:16:18:-5:-1:24:26:2:48:::
60	327	0.000912154	TGTGCCAGCAGCGGAAACAGGGGCCGCACAGATACGCAGTATTTT	HHHHHHHGGGGGHHHHHGHGGGGGGHHHHHHGGGGHHHHHHHHHH	TRBV5-4*00(1157.4)	TRBD1*00(35)	TRBJ2-3*00(239.2)	TRBC2*00(258.1)	270\|282\|306\|0\|12\|\|60.0	15\|22\|36\|16\|23\|\|35.0	21\|41\|69\|25\|45\|\|100.0	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	TGTGCCAGCAGCGGAAACAGGGGCCGCACAGATACGCAGTATTTT	38	nan	nan	nan	nan	nan	nan	nan	CASSGNRGRTDTQYF	nan	:::::::::0:-4:12:16:-3:-2:23:2545:::
61	320	0.000892628	TGTGCCTGGAGCAAACAGGGCACTGAAGCTTTCTTT	HHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH	TRBV30*00(1146.4)	TRBD1*00(30)	TRBJ1-1*00(219.3)	TRBC1*00(258.5)	270\|281\|304\|0\|11\|\|55.0	15\|21\|36\|14\|20\|\|30.0	24\|40\|68\|20\|36\|\|80.0	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	TGTGCCTGGAGCAAACAGGGCACTGAAGCTTTCTTT	39	nan	nan	nan	nan	nan	nan	nan	CAWSKQGTEAFF	nan	:::::::::0:-3:11:14:-3:-3:20:20:-4:36:::
62	319	0.000889839	TGTGCCTGGAGTATGGCAGGGGCAGGAAACACCATATATTTT	HHHHHHHHHHHHHHHHGHGHHHHHHHHGHHHHHHHHHHHHHH	TRBV30*00(1165.9)	TRBD1*00(30)	TRBJ1-3*00(228.7)	TRBC1*00(256.9)	270\|282\|304\|0\|12\|\|60.0	16\|22\|36\|16\|22\|\|30.0	24\|42\|70\|24\|42\|\|90.0	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	TGTGCCTGGAGTATGGCAGGGGCAGGAAACACCATATATTTT	38	nan	nan	nan	nan	nan	nan	nan	CAWSMAGAGNTIYF	nan	:::::::::0:-2:12:16:-4:-2:22:24:-4:42:::
63	315	0.000878681	TGTGCCTGGAGTGTACAGGGTTTCACCCTCCACTTT	HHHHHHHHHHHHHHHHHGHHHHHGGGGHHHHHHHHH	TRBV30*00(1183.1)	nan	TRBJ1-6*00(188)	TRBC1*00(255.8)	270\|287\|304\|0\|17\|\|85.0	nan	29\|45\|73\|21\|36\|DC36\|63.0	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	TGTGCCTGGAGTGTACAGGGTTTCACCCTCCACTTT	38	nan	nan	nan	nan	nan	nan	nan	CAWSVQGFTLHF	nan	:::::::::0:3:17:::::21:-9:36:::
64	315	0.000878681	TGTGCCTGGAGTAGGGAACAGAACACCGGGGAGCTGTTTTTT	HHHHHHHHHHHHHHHHHHHHHGGGGGGGGHHHHHHGGHHHHH	TRBV30*00(1166.4)	nan	TRBJ2-2*00(248.7)	TRBC2*00(257.8)	270\|282\|304\|0\|12\|\|60.0	nan	21\|43\|71\|20\|42\|\|110.0	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	TGTGCCTGGAGTAGGGAACAGAACACCGGGGAGCTGTTTTTT	38	nan	nan	nan	nan	nan	nan	nan	CAWSREQNTGELFF	nan	:::::::::0:-2:12:::::20:-1:42:::
65	313	0.000873102	TGTGCCTGGAAGTATGCCCCATTGACCCGGGCGAGGAACACTGAAGCTTTCTTT	HHHHHHHHHHHHHHGGGHHHHHHGGGGGGGGGHHHHHHHHHHHHHHHHHHHHHH	TRBV30*00(1155.7)	TRBD100(35),TRBD200(30)	TRBJ1-1*00(234.1)	TRBC1*00(258.3)	270\|280\|304\|0\|10\|\|50.0	0\|15\|36\|15\|31\|I5ASC5TST8A\|35.0;13\|19\|48\|25\|31\|\|30.0	21\|40\|68\|35\|54\|\|95.0	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	TGTGCCTGGAAGTATGCCCCATTGACCCGGGCGAGGAACACTGAAGCTTTCTTT	38	nan	nan	nan	nan	nan	nan	nan	CAWKYAPLTRARNTEAFF	nan	:::::::::0:-4:10:15:12:-9:31:3554:::
66	302	0.000842418	TGTGCCTGGAGTGACCGGACAGGGAAGGACACTGAAGCTTTCTTT	HHHHHHHHHHHGGGGGHHHHHHHHHHHHHHHHHHHHHHHHHHHHH	TRBV30*00(1161.5)	TRBD1*00(40)	TRBJ1-1*00(224.2)	TRBC1*00(256.4)	270\|283\|304\|0\|13\|\|65.0	13\|21\|36\|16\|24\|\|40.0	23\|40\|68\|28\|45\|\|85.0	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	TGTGCCTGGAGTGACCGGACAGGGAAGGACACTGAAGCTTTCTTT	38	nan	nan	nan	nan	nan	nan	nan	CAWSDRTGKDTEAFF	nan	:::::::::0:-1:13:16-3:24:28:-3:45:::
67	296	0.000825681	TGTGCCTTACAGAGGGCTAGGTACGAGCAGTACTTC	HHHHHHHHHHHHHHHHHHHGHGGGHHHHHHHGGGGG	TRBV30*00(1138.4)	TRBD100(31),TRBD200(30)	TRBJ2-7*00(214.3)	TRBC2*00(257.3)	270\|277\|304\|0\|7\|\|35.0	15\|24\|36\|8\|17\|SG19A\|31.0;27\|33\|48\|11\|17\|\|30.0	24\|39\|67\|21\|36\|\|75.0	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	TGTGCCTTACAGAGGGCTAGGTACGAGCAGTACTTC	38	nan	nan	nan	nan	nan	nan	nan	CALQRARYEQYF	nan	:::::::::0:-7:7:8:-3:0:17:21:-4:36:::
68	294	0.000820102	TGTGCCTGGAGTGCTGGAAACTATGGCTACACCTTC	HHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHGHHH	TRBV30*00(1173.3)	nan	TRBJ1-2*00(215.1)	TRBC1*00(257.7)	270\|283\|304\|0\|13\|\|65.0	nan	22\|40\|68\|18\|36\|\|90.0	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	TGTGCCTGGAGTGCTGGAAACTATGGCTACACCTTC	38	nan	nan	nan	nan	nan	nan	nan	CAWSAGNYGYTF	nan	:::::::::0:-1:13:::::18:-2:36:::
69	293	0.000817313	TGTGCCTGGACTGGCGGGAGCAGCACAGATACGCAGTATTTT	HHHHHHHHHHGGGGGGHHHHHHHHHHHHGGGGHHHHHHHHHH	TRBV30*00(1288.1)	TRBD2*00(35)	TRBJ2-3*00(243.7)	TRBC2*00(257.8)	270\|283\|304\|0\|13\|SG280C\|51.0	23\|30\|48\|13\|20\|\|35.0	20\|41\|69\|21\|42\|\|105.0	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	TGTGCCTGGACTGGCGGGAGCAGCACAGATACGCAGTATTTT	38	nan	nan	nan	nan	nan	nan	nan	CAWTGGSSTDTQYF	nan	:::::::::0:-1:13:13:-7:-2:20:21:0:42:::
70	290	0.000808944	TGCAGCGGGGCCGGGAGCAGGGATTACCAAGAGACCCAGTACTTC	HGGGGGGGGGGGGHHHHHHHHHHHHHHHHHHHHHHHHHHHGGGGG	TRBV29-1*00(1209.4)	TRBD1*00(43)	TRBJ2-5*00(244.1)	TRBC2*00(258.4)	276\|283\|310\|0\|7\|\|35.0	10\|21\|36\|10\|22\|I16G\|43.0	19\|40\|68\|24\|45\|\|105.0	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	TGCAGCGGGGCCGGGAGCAGGGATTACCAAGAGACCCAGTACTTC	38	nan	nan	nan	nan	nan	nan	nan	CSGAGSRDYQETQYF	nan	:::::::::0:-7:7:10:2:-3:22:24:1:45:::
71	282	0.000786628	TGTGCCAGCAGCGTAGAGGCCTCCACAGATACGCAGTATTTT	HHHHHHHGGGGGHHHHHGGGHHHHHHHHGGGGHHHHHHHHHH	TRBV9*00(1167.2)	nan	TRBJ2-3*00(233.8)	TRBC2*00(258)	270\|286\|306\|0\|16\|\|80.0	nan	22\|41\|69\|23\|42\|\|95.0	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	TGTGCCAGCAGCGTAGAGGCCTCCACAGATACGCAGTATTTT	38	nan	nan	nan	nan	nan	nan	nan	CASSVEASTDTQYF	nan	:::::::::0:0:16:::::23:-2:42:::
72	280	0.00078105	TGCAGCGTTGAATCGCGATCTCCGGGACTTTTT	HHGHGGHHHHHGGGGHHHGGGGGHHHHGHHHHH	TRBV29-1*00(1231)	TRBD200(40),TRBD100(35)	TRBJ1-1*00(153.9)	TRBC1*00(257.3)	276\|288\|310\|0\|12\|\|60.0	14\|22\|48\|21\|29\|\|40.0;10\|17\|36\|21\|28\|\|35.0	37\|40\|68\|30\|33\|\|15.0	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	TGCAGCGTTGAATCGCGATCTCCGGGACTTTTT	38	nan	nan	nan	nan	nan	nan	nan	CSVESRSPGLF	nan	:::::::::0:-2:12:21:2:-10:29:30:-17:33:::
73	272	0.000758734	TGTGCCAGCCAGGGGAGCCATGAGCAGTTCTTC	HHHHGGHHHGHGGGHHHHHHHHHHHHHHGGGGG	TRBV19*00(1125.3)	TRBD1*00(30)	TRBJ2-1*00(209.2)	TRBC2*00(256.8)	270\|278\|307\|0\|8\|\|40.0	16\|22\|36\|9\|15\|\|30.0	28\|42\|70\|19\|33\|\|70.0	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	TGTGCCAGCCAGGGGAGCCATGAGCAGTTCTTC	38	nan	nan	nan	nan	nan	nan	nan	CASQGSHEQFF	nan	:::::::::0:-9:8:9:-4:-2:15:19:-8:33:::
74	271	0.000755944	TGCAGCGTTGGCCCCGACTTCAATCAGCCCCAGCATTTT	HGGGGGGHGGGGGGGHHHHHHHHHGGGGGHHHHHHHHHH	TRBV29-1*00(1219.3)	TRBD1*00(25)	TRBJ1-5*00(233.8)	TRBC1*00(257.2)	276\|286\|310\|0\|10\|\|50.0	0\|5\|36\|10\|15\|\|25.0	23\|42\|70\|20\|39\|\|95.0	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	TGCAGCGTTGGCCCCGACTTCAATCAGCCCCAGCATTTT	38	nan	nan	nan	nan	nan	nan	nan	CSVGPDFNQPQHF	nan	:::::::::0:-4:10:10:12:-19:15:20:-3:39:::
75	269	0.000750365	TGTGCCTGGAGTGTGGACTCCCCGGGCAATCAGCCCCAGCATTTT	HHHHHHHHHHGHHHHHGGGGGGGHHHHHHHGGGGGHHHHHHHHHH	TRBV30*00(1182.6)	nan	TRBJ1-5*00(239.1)	TRBC1*00(256.5)	270\|291\|304\|0\|21\|SA284GSC285G\|77.0	nan	22\|42\|70\|25\|45\|\|100.0	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	TGTGCCTGGAGTGTGGACTCCCCGGGCAATCAGCCCCAGCATTTT	38	nan	nan	nan	nan	nan	nan	nan	CAWSVDSPGNQPQHF	nan	:::::::::0:7:21:::::25:-2:45:::
76	269	0.000750365	TGTGCCAGCAGCCGCGGGACTCTCCTGTTCTTC	HHHGHHHGGGGGGGGHHHHGHGHHHHHHHGGGG	TRBV7-6*00(1139)	TRBD200(35),TRBD100(30)	TRBJ2-1*00(174.4)	TRBC2*00(258.8)	273\|285\|310\|0\|12\|\|60.0	15\|22\|48\|14\|21\|\|35.0;11\|17\|36\|14\|20\|\|30.0	35\|42\|70\|26\|33\|\|35.0	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	TGTGCCAGCAGCCGCGGGACTCTCCTGTTCTTC	38	nan	nan	nan	nan	nan	nan	nan	CASSRGTLLFF	nan	:::::::::0:-5:12:14:1:-10:21:26:-15:33:::
77	267	0.000744786	TGTGCCAGCAGTTACGGCGGGGGGCCCTCCTACGAGCAGTACTTC	HHHGHHHHHHGHGGHGGGGGGGGGGGHHGGGGGHHHHHHHGGGGG	TRBV6-5*00(1178.4)	TRBD2*00(38)	TRBJ2-7*00(234)	TRBC2*00(252.6)	270\|285\|307\|0\|15\|\|75.0	23\|34\|48\|16\|26\|DA28\|38.0	20\|39\|67\|26\|45\|\|95.0	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	TGTGCCAGCAGTTACGGCGGGGGGCCCTCCTACGAGCAGTACTTC	38	nan	nan	nan	nan	nan	nan	nan	CASSYGGGPSYEQYF	nan	:::::::::0:-2:15:16:-7:2:26:26:0:45:::
78	266	0.000741997	TGTGCCTGGGCCACGGTACGGACGGGAGACACTGAAGCTTTCTTT	HHHHGHGHHGGHGGGGHHGGGHGHHHHHHHHHHHHHHHHHHHHHH	TRBV30*00(1154.1)	TRBD2*00(30)	TRBJ1-1*00(224)	TRBC1*00(256.9)	270\|279\|304\|0\|9\|\|45.0	24\|30\|48\|22\|28\|\|30.0	23\|40\|68\|28\|45\|\|85.0	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	TGTGCCTGGGCCACGGTACGGACGGGAGACACTGAAGCTTTCTTT	38	nan	nan	nan	nan	nan	nan	nan	CAWATVRTGDTEAFF	nan	:::::::::0:-5:9:22:-8:-2:28:28:-3:45:::
79	263	0.000733629	TGTGCCGTGAAGGGTAGCGGGAGATACGAGCAGTACTTC	HGGGGHHHHHHHHGGGGGHHHHHHGGGHHHHHHHGGHGG	TRBV30*00(1150.1)	TRBD2*00(35)	TRBJ2-7*00(213.9)	TRBC2*00(258.1)	270\|285\|304\|0\|16\|I276GSG278AST281G\|35.0	23\|30\|48\|16\|23\|\|35.0	24\|39\|67\|24\|39\|\|75.0	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	TGTGCCGTGAAGGGTAGCGGGAGATACGAGCAGTACTTC	38	nan	nan	nan	nan	nan	nan	nan	CAVKGSGRYEQYF	nan	:::::::::0:1:16:16:-7:-2:23:24:-4:39:::
80	261	0.00072805	TGTGCCTGGAGTCTGCTAGCGGGAGGGAACAATGAGCAGTTCTTC	HHHHHHHHHHHHHHHGGGGGGGHHHHHHHHHHHHHHHHHHGGGGG	TRBV30*00(1161.7)	TRBD2*00(60)	TRBJ2-1*00(223.4)	TRBC2*00(258.3)	270\|282\|304\|0\|12\|\|60.0	20\|32\|48\|15\|27\|\|60.0	25\|42\|70\|28\|45\|\|85.0	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	TGTGCCTGGAGTCTGCTAGCGGGAGGGAACAATGAGCAGTTCTTC	38	nan	nan	nan	nan	nan	nan	nan	CAWSLLAGGNNEQFF	nan	:::::::::0:-2:12:15:-4:0:27:28:-5:45:::
81	261	0.00072805	TGTGCCTGGAGTCCCAGGATGAACACTGAAGCTTTCTTT	HHHHHHHHHHHHGHHHHHHHHHHHHHHHHHHHHHHHHHH	TRBV30*00(1157.6)	nan	TRBJ1-1*00(244.3)	TRBC1*00(258.2)	270\|282\|304\|0\|12\|\|60.0	nan	19\|40\|68\|18\|39\|\|105.0	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	TGTGCCTGGAGTCCCAGGATGAACACTGAAGCTTTCTTT	38	nan	nan	nan	nan	nan	nan	nan	CAWSPRMNTEAFF	nan	:::::::::0:-2:12:::::18:1:39:::
82	259	0.000722471	TGTGCCTGGAGCGGGACAGGGATCAATGAGCAGTTCTTC	HHHHHHHGGGGGHHHHHHHHHHHHHHHHHHHHHHGGHGG	TRBV30*00(1154.6)	TRBD1*00(50)	TRBJ2-1*00(218.5)	TRBC2*00(258.4)	270\|281\|304\|0\|11\|\|55.0	11\|21\|36\|11\|21\|\|50.0	26\|42\|70\|23\|39\|\|80.0	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	TGTGCCTGGAGCGGGACAGGGATCAATGAGCAGTTCTTC	38	nan	nan	nan	nan	nan	nan	nan	CAWSGTGINEQFF	nan	:::::::::0:-3:11:11:1:-3:21:23:-6:39:::
83	256	0.000714102	TGTGCCTGGAGTGGCGGACTGAGTAGCAATCAGCCCCAGCATTTT	HHHHHHHHHHGGGGGHHHHHHHHHHHHHHHGGGGGHHHHHHHHHH	TRBV30*00(1257.6)	TRBD2*00(25)	TRBJ1-5*00(248.2)	TRBC1*00(257.9)	270\|283\|304\|0\|13\|\|65.0	17\|22\|48\|15\|20\|\|25.0	20\|42\|70\|23\|45\|\|110.0	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	TGTGCCTGGAGTGGCGGACTGAGTAGCAATCAGCCCCAGCATTTT	38	nan	nan	nan	nan	nan	nan	nan	CAWSGGLSSNQPQHF	nan	:::::::::0:-1:13:15-10:20:23:0:45:::
84	255	0.000711313	TGTGCCTGGAGTACCCTCGACAGGGCGAACTATGGCTACACCTTC	HHHHHHHHHHHHGGGGHGHHHGGGGGHHHHHHHHHHHHHHHHHHH	TRBV30*00(1165.5)	TRBD1*00(30)	TRBJ1-2*00(216.5)	TRBC1*00(258)	270\|282\|304\|0\|12\|\|60.0	14\|20\|36\|18\|24\|\|30.0	19\|40\|68\|24\|45\|ST21G\|91.0	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	TGTGCCTGGAGTACCCTCGACAGGGCGAACTATGGCTACACCTTC	38	nan	nan	nan	nan	nan	nan	nan	CAWSTLDRANYGYTF	nan	:::::::::0:-2:12:18:-2:-4:24:24:1:45:::
85	254	0.000708523	TGTGCCTGGAGGGACGCTTCTGGAAACACCATATATTTT	HHHHHHHHHHGGGGGHHHHHHHHHGHHHHHHHHHHHHHH	TRBV30*00(1158)	nan	TRBJ1-3*00(244.6)	TRBC1*00(258.5)	270\|281\|304\|0\|11\|\|55.0	nan	21\|42\|70\|18\|39\|\|105.0	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	TGTGCCTGGAGGGACGCTTCTGGAAACACCATATATTTT	38	nan	nan	nan	nan	nan	nan	nan	CAWRDASGNTIYF	nan	:::::::::0:-3:11:::::18:-1:39:::
86	254	0.000708523	TGTGCCTGGAGACGACAGGGGGCTAACTATGGCTACACCTTC	HHHHHHHHHHGGGHHGGGGHHHHHHHHHHHHHHHHHHGGHHH	TRBV30*00(1163.9)	TRBD1*00(40)	TRBJ1-2*00(243.9)	TRBC1*00(258.1)	270\|281\|304\|0\|11\|\|55.0	14\|22\|36\|13\|21\|\|40.0	19\|40\|68\|21\|42\|\|105.0	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	TGTGCCTGGAGACGACAGGGGGCTAACTATGGCTACACCTTC	38	nan	nan	nan	nan	nan	nan	nan	CAWRRQGANYGYTF	nan	:::::::::0:-3:11:13:-2:-2:21:21:1:42:::
87	251	0.000700155	TGTGCCAGCAGCTTGGGGGGGGGTACGGAAGAGCAGTTCTTC	HHHHHHHHHHGHGGGGGGGHHGGHHGHHHHHHHHHHHHHHHG	TRBV5-6*00(1166.5)	TRBD1*00(25)	TRBJ2-1*00(199.1)	TRBC2*00(257.6)	270\|286\|306\|0\|16\|\|80.0	18\|23\|36\|16\|21\|\|25.0	30\|42\|70\|30\|42\|\|60.0	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	TGTGCCAGCAGCTTGGGGGGGGGTACGGAAGAGCAGTTCTTC	38	nan	nan	nan	nan	nan	nan	nan	CASSLGGGTEEQFF	nan	:::::::::0:0:16:16:-6:-1:21:30:-10:42:::
88	250	0.000697366	TGTGCCAGCAGTTACTCGAAGGTTTCAGACCCCGGACCTGGAAACACCATATATTTT	HHHGHHHHHHHHHHHHHHHHHHHHHHHGGGGGGHHHHHHHHHGHHHHHHHHHHHHHH	TRBV6-5*00(1206.9)	TRBD200(27),TRBD100(26)	TRBJ1-3*00(239)	TRBC1*00(257.1)	270\|289\|307\|0\|19\|\|95.0	10\|21\|48\|26\|37\|ST12ASG16C\|27.0;9\|17\|36\|29\|37\|SG12C\|26.0	22\|42\|70\|37\|57\|\|100.0	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	TGTGCCAGCAGTTACTCGAAGGTTTCAGACCCCGGACCTGGAAACACCATATATTTT	38	nan	nan	nan	nan	nan	nan	nan	CASSYSKVSDPGPGNTIYF	nan	:::::::::0:2:19:26:6:-11:37:37:-2:57:::
89	249	0.000694576	TGTGCCTGCCCAAGAGACAGGGTCTATGGCTACACCTTC	HHHGGHHHHHHHHHHHHHHHHHHHHHHHHHHHHHGGGGG	TRBV30*00(1142.4)	TRBD1*00(35)	TRBJ1-2*00(219.1)	TRBC1*00(257.5)	270\|278\|304\|0\|8\|\|40.0	14\|21\|36\|15\|22\|\|35.0	24\|40\|68\|23\|39\|\|80.0	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	TGTGCCTGCCCAAGAGACAGGGTCTATGGCTACACCTTC	38	nan	nan	nan	nan	nan	nan	nan	CACPRDRVYGYTF	nan	:::::::::0:-6:8:15:-2:-3:22:23:-4:39:::
90	245	0.000683418	TGTGCCAGCAGCTTATATTACAGGGTTGGGGCTTTCTTT	HHHHHHHHHHHHHHHHHHHHHHGGGGGGHHHHHHHHHHH	TRBV5-8*00(1131.2)	TRBD1*00(30)	TRBJ1-1*00(184.3)	TRBC1*00(257.8)	270\|284\|306\|0\|14\|\|70.0	15\|21\|36\|19\|25\|\|30.0	31\|40\|68\|30\|39\|\|45.0	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	TGTGCCAGCAGCTTATATTACAGGGTTGGGGCTTTCTTT	38	nan	nan	nan	nan	nan	nan	nan	CASSLYYRVGAFF	nan	:::::::::0:-2:14:19:-3:-3:25:30:-11:39:::
91	244	0.000680629	TGTGCCTGGAGTGTATCGGCATCTGGAAACACCATATATTTT	HHHHHHHHHHHHGGGGGHHHHHHHHHHGHHHHHHHHHHHHHH	TRBV30*00(1173.4)	nan	TRBJ1-3*00(243.4)	TRBC1*00(257)	270\|285\|304\|0\|15\|\|75.0	nan	21\|42\|70\|21\|42\|\|105.0	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	TGTGCCTGGAGTGTATCGGCATCTGGAAACACCATATATTTT	38	nan	nan	nan	nan	nan	nan	nan	CAWSVSASGNTIYF	nan	:::::::::0:1:15:::::21:-1:42:::
92	241	0.00067226	TGTGCCAGTAGTCCGGGCATTTCCTACGAGCAGTACTTC	HHHHHHHHHGGGGGHHHHHHHHGGGGGHHHHHHHGGGGG	TRBV19*00(1143.2)	TRBD100(25),TRBD200(25)	TRBJ2-7*00(229.4)	TRBC2*00(257.9)	270\|282\|307\|0\|12\|\|60.0	10\|15\|36\|12\|17\|\|25.0;14\|19\|48\|12\|17\|\|25.0	21\|39\|67\|21\|39\|\|90.0	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	TGTGCCAGTAGTCCGGGCATTTCCTACGAGCAGTACTTC	38	nan	nan	nan	nan	nan	nan	nan	CASSPGISYEQYF	nan	:::::::::0:-5:12:12:2:-9:17:2139:::
93	241	0.00067226	TGCAGCGTGCTGGACAGGGGGTTGGACAATGAGCAGTTCTTC	HGGHGGHHHHHHHHGGGGGGHHHHHHHHHHHHHHHHHGGHGG	TRBV29-1*00(1214.6)	TRBD1*00(50)	TRBJ2-1*00(223.6)	TRBC2*00(258)	276\|284\|310\|0\|8\|\|40.0	13\|23\|36\|11\|21\|\|50.0	25\|42\|70\|25\|42\|\|85.0	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	TGCAGCGTGCTGGACAGGGGGTTGGACAATGAGCAGTTCTTC	38	nan	nan	nan	nan	nan	nan	nan	CSVLDRGLDNEQFF	nan	:::::::::0:-6:8:11-1:21:25:-5:42:::
94	235	0.000655524	TGCCTTCAGAGCCACATGAACACTGAAGCTTTCTTT	HHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH	TRBV20-1*00(1153.2)	nan	TRBJ1-1*00(249.5)	TRBC1*00(258.1)	279\|282\|313\|0\|3\|\|15.0	nan	18\|40\|68\|14\|36\|\|110.0	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	TGCCTTCAGAGCCACATGAACACTGAAGCTTTCTTT	39	nan	nan	nan	nan	nan	nan	nan	CLQSHMNTEAFF	nan	:::::::::0:-11:3:::::14:2:36:::
95	234	0.000652734	TGCAGCGTTGAAGCTTCGGCAGGGCAAAAAGCTTTCTTT	HHHHHHHHHHHHHHHHHHHHHHHHGHHHHHHHHHHHHHH	TRBV29-1*00(1223.9)	TRBD2*00(31)	TRBJ1-1*00(194.1)	TRBC1*00(257.7)	276\|289\|310\|0\|13\|\|65.0	24\|33\|48\|16\|25\|SG27C\|31.0	29\|40\|68\|28\|39\|\|55.0	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	TGCAGCGTTGAAGCTTCGGCAGGGCAAAAAGCTTTCTTT	38	nan	nan	nan	nan	nan	nan	nan	CSVEASAGQKAFF	nan	:::::::::0:-1:13:16:-8:1:25:28:-9:39:::
96	228	0.000635997	TGTGCCATCAGTGAGTTGGGGGGGCTCGTTTTAAAGGCTTCCGAAGCTTTCTTT	HHHHHHHHHHHHHHGGGGGGGGGGGGGHHHHHHHHHHGGGGGHHHHHHHHHHHH	TRBV10-3*00(1143.7)	TRBD100(30),TRBD200(26)	TRBJ1-1*00(198.7)	TRBC1*00(256.8)	270\|286\|307\|0\|16\|\|80.0	18\|24\|36\|19\|25\|\|30.0;25\|33\|48\|17\|25\|SA28G\|26.0	28\|40\|68\|42\|54\|\|60.0	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	TGTGCCATCAGTGAGTTGGGGGGGCTCGTTTTAAAGGCTTCCGAAGCTTTCTTT	38	nan	nan	nan	nan	nan	nan	nan	CAISELGGLVLKASEAFF	nan	:::::::::0:-1:16:19:-6:0:25:42:-8:54:::
97	227	0.000633208	TGCAGCGTTGAGGCGGGACGCCGTTACAATGAGCAGTTCTTC	HHHHHHHHHGHHHGGGGGGGGGHHHHHHHHHHHHHHHHHHHG	TRBV29-1*00(1219.9)	TRBD100(30),TRBD200(30)	TRBJ2-1*00(229.1)	TRBC2*00(258.2)	276\|287\|310\|0\|11\|\|55.0	11\|17\|36\|13\|19\|\|30.0;15\|21\|48\|13\|19\|\|30.0	24\|42\|70\|24\|42\|\|90.0	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	TGCAGCGTTGAGGCGGGACGCCGTTACAATGAGCAGTTCTTC	38	nan	nan	nan	nan	nan	nan	nan	CSVEAGRRYNEQFF	nan	:::::::::0:-3:11:13:1:-7:19:24:-4:42:::
98	226	0.000630419	TGTGCCAGCAGCGATGACAGCTCCTACGAGCAGTACTTC	HHHHHHHHGHGHHHHHHHHHHHGGGGGHHHHHHHGGGGG	TRBV14*00(1146.8)	nan	TRBJ2-7*00(244)	TRBC2*00(258.3)	273\|285\|310\|0\|12\|\|60.0	nan	18\|39\|67\|18\|39\|\|105.0	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	TGTGCCAGCAGCGATGACAGCTCCTACGAGCAGTACTTC	38	nan	nan	nan	nan	nan	nan	nan	CASSDDSSYEQYF	nan	:::::::::0:-5:12:::::18:2:39:::
99	225	0.000627629	TGTGCCTGGAGCGGAAGCACTGAAGCTTTCTTT	HHHHHHHGGGGGHHHHHHHHHHHHHHHHHHHHH	TRBV30*00(1162.7)	nan	TRBJ1-1*00(222.1)	TRBC1*00(254.9)	270\|281\|304\|0\|11\|\|55.0	nan	21\|40\|68\|13\|33\|I24G\|83.0	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	nan	TGTGCCTGGAGCGGAAGCACTGAAGCTTTCTTT	38	nan	nan	nan	nan	nan	nan	nan	CAWSGSTEAFF	nan	:::::::::0:-3:11:::::13:-1:33:::

Quality control

Now when we have all files processed lets perform Quality Control. The first thing to check is the alignment rate. That can be easily done using mixcr exportQc align function.

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

From this plot we can clearly see some issues with the libraries. A lot of the samples have a relatively big fraction of not aligned reads primarily due to the absence of J hits.

MiXCR is a powerful tool that allows us to investigate further. Let's pick one of the samples where the issue is most obvious. (ex. CRC00308 ). To look at the reads' alignments for that sample we first will run mixcr align command for that sample once again, but this time we will specify additional options - -OallowPartialAlignments=true -OallowNoCDR3PartAlignments=true, that will preserve partially aligned reads (ex. reads that may lack J gene) and reads that lack CDR3 sequence.

mkdir -p debug
mixcr align \
    -s hsa \
    -p generic-amplicon \
    -OvParameters.geneFeatureToAlign="VTranscriptWithout5UTRWithP" \
    -OvParameters.parameters.floatingLeftBound=true \
    -OjParameters.parameters.floatingRightBound=false \
    -OcParameters.parameters.floatingRightBound=true \
    -OallowPartialAlignments=true \
    -OallowNoCDR3PartAlignments=true \
    raw/CRC003_therapy-08_R1.fastq.gz raw/CRC003_therapy-08_R2.fastq.gz \
    debug/CRC003_therapy-08_debug.vdjca

Now we can look at raw alignments itself using mixcr exportAlignmentsPretty.

The function bellow will generate a .txt human-readable file with alignments. We use parameter --skip 1000 to skip first 1000 reads, as first reads usually have bad quality, and --limit 100 will export only 100 alignments as we usually don't need to examine every alignment to see the issue.

mixcr exportAlignmentsPretty
    --skip 1000 \
    --limit 100 \
    debug/CRC003_therapy-08_debug.vdjca \
    debug/CRC003_therapy-08_debug.alignments.txt

Bellow you can see a few alignments from the generated file. The first one is an example of well aligned reads.

>>> Read ids: 1113


                                            FR1><CDR1     CDR1><FR2
              _ E  K  P  V  T  L  S  C  S  Q  T  L  N  H  N  V  M  Y  W  Y  Q  Q  K  S  S  Q
  Quality     66666767777777777777777777777777777777777777777777777777777777777777777777777777
  Target0   0 AGAAAAGCCAGTGACCCTGAGTTGTTCTCAGACTTTGAACCATAACGTCATGTACTGGTACCAGCAGAAGTCAAGTCAGG 79   Score
TRBV15*00 104    aaagccagtgaccctgagttgttctcagactttgaaccataacgtcatgtactggtaccagcagaagtcaagtcagg 180  1148

                              FR2><CDR2        CDR2><FR3
              A  P  K  L  L  F  H  Y  Y  D  K  D  F  N  N  E  A  D  T  P  D  N  F  Q  S  R  R
  Quality     77777777777777777777777777777777777777777777777777777777777777777777777777777777
  Target0  80 CCCCAAAGCTGCTGTTCCACTACTATGACAAAGATTTTAACAATGAAGCAGACACCCCTGATAACTTCCAATCCAGGAGG 159  Score
TRBV15*00 181 ccccaaagctgctgttccactactatgacaaagattttaacaatgaagcagacacccctgataacttccaatccaggagg 260  1148

                                                                            FR3><CDR3        V
               P  N  I  S  F  C  F  L  D  I  R  S  P  G  L  G  D  A  A  M  Y  L  C  A  T  S  G
  Quality     77777777777777777777777777777777777777777777777777777777777777777777777777777777
  Target0 160 CCGAACATTTCTTTCTGCTTTCTTGACATCCGCTCACCAGGCCTGGGGGACGCAGCCATGTACCTGTGTGCCACCAGCGG 239  Score
TRBV15*00 261 ccgaacaCttctttctgctttcttgacatccgctcaccaggcctgggggacAcagccatgtacctgtgtgccaccagcAg 340  1148

               >      <J        CDR3><FR4                    FR4><C
                 L  G  D  T  Q  Y  F  G  P  G  T  R  L  T  V  L  E  D  L  K  N  V  F  P  P  E
   Quality     77777777777777777777777777777777777777777777777777777777777777777777777777777777
   Target0 240 ACTAGGGGATACGCAGTATTTTGGCCCAGGCACCCGGCTGACAGTGCTCGAGGACCTGAAAAACGTGTTCCCACCCGAGG 319  Score
 TRBV15*00 341 a                                                                                341  1148
TRBJ2-3*00  26        gatacgcagtattttggcccaggcacccggctgacagtgctcg                               68   215
  TRBC2*00   0                                                   aggacctgaaaaacgtgttcccacccgagg 29   260


             V  A  V  F  E  P  S  D  S  Q _
 Quality     7777777777777777777777777766666
 Target0 320 TCGCTGTGTTTGAGCCATCAGATAGTCAATG 350  Score
TRBC2*00  30 tcgctgtgtttgagccatcaga          51   260

Now, the following pair of reads failed to align.

>>> Read ids: 1115



          _  N  P  V  G  L  R  C  Y  P  T  S  V  F  F  C  V  Y  L  Y  Q  Q  K  P  F  P  C
Quality   33553553353536366363363333633322336733363663633333633633733363222632222333655322
Target0 0 CAAACCCCGTGGGGCTGAGGTGCTACCCAACCTCTGTCTTTTTCTGTGTGTACTTGTACCAACAAAAACCCTTCCCCTGC 79  Score


            P  G  S  P  S  K  N  Y  Q  A  E  G  G  G  D  G  E  G  E  E  G  V  S  G  G  R  R
Quality    62222252552522333333633336333225522622222222252522525222222222222233522525222522
Target0 80 CCCGGGTCCCCCAGTAAGAATTATCAGGCCGAAGGGGGAGGAGACGGGGAGGGGGAAGAAGGAGTATCCGGGGGGCGGCG 159  Score

                                                                         FR3><CDR3
               V  V  L  K  K  K  L  N  L  S  S  L  E  L  V  D  S  A  L  Y  F  C  A  S  V  G
 Quality     22242225222672224452252522242225242626256626244262222424525222625452225222252225
 Target0 160 CGTTGTCTTAAAAAAAAAACTAAACCTGAGCTCTCTGGAGCTGGTGGACTCAGCTTTGTATTTCTGTGCCAGCGTCGGGT 239  Score
TRBV9*00 280                  aactaaacctgagctctctggagctggGggactcagctttgtatttctgtgccagc--AgCgt 340  200

             V><VP     VP>
             S   H   H  I
 Quality     242 22 252224
 Target0 240 CGC-AC-CACATA 250  Score
TRBV9*00 341 AgcTacGcTGCtG 353  200

             <CDR2        CDR2><FR3
              Y  H  K  G  E  E  R  A  K  G  N  I  L  E  R  F  S  A  Q  Q  F  P  D  F  L  F  F
 Quality     22224664272772777762664467626552222267767777765422666277777777766636665226665653
 Target1   0 TATCATAAAGGAGAAGAGAGAGCAAAAGGAAACATTCTTGAACGATTCTCCGCACAACAGTTCCCTGACTTTCTTTTTTT 79   Score
TRBV9*00 201 tatTataaTggagaagagagagcaaaaggaaacattcttgaacgattctccgcacaacagttccctgactt          271  327


             Q  A  E  D  G  I  R  H  R  S  R  H  S  C  *  T  A  L  P  I  *  T  P  V  E  L
Quality    27773753566265533552552226776565636377677777752777762626237777377772777667667733
Target1 80 TCAAGCAGAAGACGGCATACGACATCGGTCTCGGCATTCCTGCTGAACCGCTCTTCCGATTTGAACCCCTGTGGAGCTGA 159  Score


            R  C  N  Y  S  S  S  V  S  V  Y  L  F  *  Y  P  E  P  *  P  C  R  V  P  A  E  R
Quality     67777776777762277637766776776776667776676776636666776636366767337776636766636367
Target1 160 GGTGCAACTACTCATCGTCTGTTTCAGTGTATCTCTTCTGATATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGA 239  Score


             L  S  Q _
Quality     66776666366
Target1 240 CTTAGTCAGCC 250  Score

One can use BLAST and search for the not aligned parts of sequence in order to find out its origin.

Another quality report we should investigate is a chain abundance plot.

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

Most of the samples are more or less equally consist of TRA and TRB chains. Although we can also see a few samples (ex. CRC013_therapy-02) with no TRA chain wich might indicate some cDNA library reparation issues and needs further investigation.

Full-length clonotype assembly

iRepertoire protocol allows to recover a broader BCR receptor sequence then just CDR3 region. According to the protocol, forward primers are located in FR1 region, thus we can safely use an assembling feature that starts from CDR1 and be sure that no primers will affect the original sequence. The reverse primers are located in FR4 region very close to CDR3, thus there is not much left from to include in clone assembly.

Taking into account what is mentioned above, the longest possible assembling feature for this protocol is "{CDR1Begin:CDR3End}".

MiXCR has a specific preset to obtain full-length BCR clones with Biomed2 protocol:

mixcr analyze irepertoire-human-rna-xcr-repseq-lr \
    raw/CRC016_preTherapy_R1.fastq.gz \
    raw/CRC016_preTherapy_R2.fastq.gz \
    results/CRC016_preTherapy

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

mixcr assemble \
    -OassemblingFeatures="{CDR1Begin:CDR3End}" \
    -OseparateByJ=true \
    --report results/SRR8365468_HIP2_male.report \
    --json-report results/SRR8365468_HIP2_male.json \
    results/CRC016_preTherapy.vdjca \
    results/CRC016_preTherapy.clns

-OassemblingFeatures="{CDR1Begin:CDR3End}": sets the assembling feature to the region which starts from CDR1Begin and ends at the end of CDR3.

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/CRC016_preTherapy.clns \
    figs/CRC016_preTherapy.report.txt

mixcr exportReports \
    --json \
    results/CRC016_preTherapy.clns \
    figs/CRC016_preTherapy.report.json

Show report file

.txt.json

============== Align Report ==============
Input file(s): /raw/iRepertoire/CRC016_preTherapy_R1.fastq.gz,/raw/iRepertoire/CRC016_preTherapy_R2.fastq.gz
Output file(s): results-trimmed/CRC016_preTherapy.vdjca
Version: 4.0.0-331-protocols; built=Thu Oct 06 19:33:27 CEST 2022; rev=83ef6ba9c4; lib=repseqio.v2.0
Command line arguments: --report results-trimmed/CRC016_preTherapy.align.report.txt --json-report results-trimmed/CRC016_preTherapy.align.report.json --preset local:irepertoire-human-tcr-lr-cdr3 /raw/iRepertoire/CRC016_preTherapy_R1.fastq.gz /raw/iRepertoire/CRC016_preTherapy_R2.fastq.gz results-trimmed/CRC016_preTherapy.vdjca
Analysis time: 0ns
Total sequencing reads: 409090
Successfully aligned reads: 386658 (94.52%)
Paired-end alignment conflicts eliminated: 3486 (0.85%)
Alignment failed, no hits (not TCR/IG?): 4045 (0.99%)
Alignment failed because of absence of V hits: 29 (0.01%)
Alignment failed because of absence of J hits: 17953 (4.39%)
No target with both V and J alignments: 203 (0.05%)
Alignment failed because of low total score: 202 (0.05%)
Overlapped: 392724 (96%)
Overlapped and aligned: 371487 (90.81%)
Alignment-aided overlaps: 722 (0.19%)
Overlapped and not aligned: 21237 (5.19%)
No CDR3 parts alignments, percent of successfully aligned: 94 (0.02%)
Partial aligned reads, percent of successfully aligned: 575 (0.15%)
V gene chimeras: 481 (0.12%)
TRA chains: 370 (0.1%)
TRA non-functional: 57 (15.41%)
TRB chains: 386282 (99.9%)
TRB non-functional: 8075 (2.09%)
TRD chains: 5 (0%)
TRD non-functional: 0 (0%)
IGH chains: 1 (0%)
IGH non-functional: 0 (0%)
Realigned with forced non-floating bound: 34176 (8.35%)
Realigned with forced non-floating right bound in left read: 9768 (2.39%)
Realigned with forced non-floating left bound in right read: 9768 (2.39%)
============== Assemble Report ==============
Input file(s): results-trimmed/CRC016_preTherapy.vdjca
Output file(s): results-trimmed/CRC016_preTherapy.clns
Version: 4.0.0-331-protocols; built=Thu Oct 06 19:33:27 CEST 2022; rev=83ef6ba9c4; lib=repseqio.v2.0
Command line arguments: --report results-trimmed/CRC016_preTherapy.assemble.report.txt --json-report results-trimmed/CRC016_preTherapy.assemble.report.json results-trimmed/CRC016_preTherapy.vdjca results-trimmed/CRC016_preTherapy.clns
Analysis time: 0ns
Final clonotype count: 24390
Average number of reads per clonotype: 14.7
Reads used in clonotypes, percent of total: 358492 (87.63%)
Reads used in clonotypes before clustering, percent of total: 379951 (92.88%)
Number of reads used as a core, percent of used: 377014 (99.23%)
Mapped low quality reads, percent of used: 2937 (0.77%)
Reads clustered in PCR error correction, percent of used: 21459 (5.65%)
Reads pre-clustered due to the similar VJC-lists, percent of used: 76 (0.02%)
Reads dropped due to the lack of a clone sequence, percent of total: 2924 (0.71%)
Reads dropped due to a too short clonal sequence, percent of total: 143 (0.03%)
Reads dropped due to low quality, percent of total: 1 (0%)
Reads dropped due to failed mapping, percent of total: 3571 (0.87%)
Reads dropped with low quality clones, percent of total: 1 (0%)
Clonotypes eliminated by PCR error correction: 9372
Clonotypes dropped as low quality: 1
Clonotypes pre-clustered due to the similar VJC-lists: 69
TRB chains: 24390 (100%)
TRB non-functional: 1117 (4.58%)

{
  "type": "alignerReport",
  "commandLine": "--report results-trimmed/CRC016_preTherapy.align.report.txt --json-report results-trimmed/CRC016_preTherapy.align.report.json --preset local:irepertoire-human-tcr-lr-cdr3 /raw/iRepertoire/CRC016_preTherapy_R1.fastq.gz /raw/iRepertoire/CRC016_preTherapy_R2.fastq.gz results-trimmed/CRC016_preTherapy.vdjca",
  "inputFiles": [
    "/raw/iRepertoire/CRC016_preTherapy_R1.fastq.gz",
    "/raw/iRepertoire/CRC016_preTherapy_R2.fastq.gz"
  ],
  "outputFiles": [
    "results-trimmed/CRC016_preTherapy.vdjca"
  ],
  "version": "4.0.0-331-protocols; built=Thu Oct 06 19:33:27 CEST 2022; rev=83ef6ba9c4; lib=repseqio.v2.0",
  "trimmingReport": null,
  "totalReadsProcessed": 409090,
  "aligned": 386658,
  "notAligned": 22432,
  "notAlignedReasons": {
    "NoCDR3Parts": 0,
    "NoBarcode": 0,
    "LowTotalScore": 202,
    "NoHits": 4045,
    "VAndJOnDifferentTargets": 203,
    "NoVHits": 29,
    "NoJHits": 17953
  },
  "chimeras": 0,
  "overlapped": 392724,
  "alignmentAidedOverlaps": 722,
  "overlappedAligned": 371487,
  "overlappedNotAligned": 21237,
  "pairedEndAlignmentConflicts": 3486,
  "vChimeras": 481,
  "jChimeras": 0,
  "chainUsage": {
    "type": "chainUsage",
    "chimeras": 0,
    "total": 386658,
    "chains": {
      "TRA": {
        "total": 370,
        "nonFunctional": 57,
        "isOOF": 41,
        "hasStops": 16
      },
      "TRB": {
        "total": 386282,
        "nonFunctional": 8075,
        "isOOF": 6763,
        "hasStops": 1312
      },
      "TRD": {
        "total": 5,
        "nonFunctional": 0,
        "isOOF": 0,
        "hasStops": 0
      },
      "IGH": {
        "total": 1,
        "nonFunctional": 0,
        "isOOF": 0,
        "hasStops": 0
      }
    }
  },
  "realignedWithForcedNonFloatingBound": 34176,
  "realignedWithForcedNonFloatingRightBoundInLeftRead": 9768,
  "realignedWithForcedNonFloatingLeftBoundInRightRead": 9768,
  "noCDR3PartsAlignments": 94,
  "partialAlignments": 575,
  "tagReport": null
}
{
  "type": "assemblerReport",
  "commandLine": "--report results-trimmed/CRC016_preTherapy.assemble.report.txt --json-report results-trimmed/CRC016_preTherapy.assemble.report.json results-trimmed/CRC016_preTherapy.vdjca results-trimmed/CRC016_preTherapy.clns",
  "inputFiles": [
    "results-trimmed/CRC016_preTherapy.vdjca"
  ],
  "outputFiles": [
    "results-trimmed/CRC016_preTherapy.clns"
  ],
  "version": "4.0.0-331-protocols; built=Thu Oct 06 19:33:27 CEST 2022; rev=83ef6ba9c4; lib=repseqio.v2.0",
  "preCloneAssemblerReport": null,
  "totalReadsProcessed": 409090,
  "initialClonesCreated": 33832,
  "readsDroppedNoTargetSequence": 2924,
  "readsDroppedTooShortClonalSequence": 143,
  "readsDroppedLowQuality": 68,
  "coreReads": 377014,
  "readsDroppedFailedMapping": 3571,
  "lowQualityRescued": 2937,
  "clonesClustered": 9372,
  "readsClustered": 21459,
  "clones": 24390,
  "clonesDroppedAsLowQuality": 1,
  "clonesPreClustered": 69,
  "readsPreClustered": 76,
  "readsInClones": 358492,
  "readsInClonesBeforeClustering": 379951,
  "readsDroppedWithLowQualityClones": 1,
  "clonalChainUsage": {
    "type": "chainUsage",
    "chimeras": 0,
    "total": 24390,
    "chains": {
      "TRB": {
        "total": 24390,
        "nonFunctional": 1117,
        "isOOF": 905,
        "hasStops": 212
      }
    }
  }
}