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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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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

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

alignQc.svg

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 
============== 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
      }
    }
  }
}