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NEBNext® Immune Sequencing

Here we will discuss how to process BCR cDNA libraries obtained with NEBNext® Immune Sequencing Kit.

Data libraries

This tutorial uses the data from the following publication: Germinal centre-driven maturation of B cell response to mRNA vaccination Wooseob, et al, Nature volume 604, pages141–145 (2022) doi:10.1038/s41586-022-04527-1

The data includes samples taken from 8 patients before and several time points after receiving SARS-CoV-2 mRNA vaccine. Samples include: germinal center B cells from lymph node, plasmablasts from lymph node, plasma cells from bone marrow and memory B cells from blood. Sorted GC B cells and LNPCs from FNA, enriched BMPCs from bone marrow or enriched MBCs from PBMCs from blood were used for library preparation for bulk BCR sequecning. Circulating MBCs were magnetically isolated by first staining with IgD-PE and MojoSort anti-PE Nanobeads (BioLegend), and then processing with the EasySep Human B Cell Isolation Kit (StemCell Technologies) to negatively enrich IgDlo B cells. RNA was prepared from each sample using the RNeasy Plus Micro kit (Qiagen). Libraries were prepared using the NEBNext Immune Sequencing Kit for Human (New England Biolabs) according to the manufacturer’s instructions without modifications. Only heavy chain primers were used. High-throughput paired-end sequencing was performed on the Illumina MiSeq platform, 325 cycles for read 1 and 275 cycles for read 2.

On the scheme bellow you can see structure of cDNA library. UMI is located in the first 17 bp of R2, followed by 7 to 10 bp occurred in the process of template switching (TS).

The set of primers used in NEBNext® Immune Sequencing Kits. The list includes primers for human and mice bcr and tcr kits. 
>Human-IGHM
GAATTCTCACAGGAGACGAGG
>Human-IGHD
TGTCTGCACCCTGATATGATGG
>Human-IGHA
GGGTGCTGYMGAGGCTCAG
>Human-IGHE
TTGCAGCAGCGGGTCAAGG
>Human-IGHG
CCAGGGGGAAGACSGATG
>Human-IGK
GACAGATGGTGCAGCCACAG
>Human-IGL
AGGGYGGGAACAGAGTGAC
>Human-TRA
CACGGCAGGGTCAGGGTTC
>Human-TRB
CGACCTCGGGTGGGAACAC

>Mus-p5-IgGb
CCAGGGACCAAGGGATAGAC
>Mus-p5-IgGa
CCAGTGGATAGACHGATGGGG
>Mus-p5-IgE
GCTTTAAGGGGTAGAGCTGAG
>Mus-p5-IgD
CTCTGAGAGGAGGAACATGTCA
>Mus-p5-IgM
GGGAAGACATTTGGGAAGGAC
>Mus-p5-IgA
GAATCAGGCAGCCGATTATCAC
>Mus-p5-IgK
AGATGGATGCAGTTGGTGCA
>Mus-p5-IgL
TCCTCAGAGGAAGGTGGAAAC
>Mus-p5-TRAC
ATCTTTTAACTGGTACACAGCAGG
>Mus-p5-TRBC
CAAGGAGACCTTGGGTGGAG
>Mus-p5-TRGC
AAGGAAGAAAAATAGTGGGCTTGG
>Mus-p5-TRDC
CATGATGAAAACAGATGGTTTGGC

All data may be downloaded directly from SRA (PRJNA777934) 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/SRR177/032/SRR17709532/SRR17709532_1.fastq.gz
  out=raw/13_d60_LN_germCenterB_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR177/032/SRR17709532/SRR17709532_2.fastq.gz
  out=raw/13_d60_LN_germCenterB_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR177/033/SRR17709533/SRR17709533_1.fastq.gz
  out=raw/07_d110_LN_plasmablast_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR177/033/SRR17709533/SRR17709533_2.fastq.gz
  out=raw/07_d110_LN_plasmablast_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR177/034/SRR17709534/SRR17709534_1.fastq.gz
  out=raw/07_d60_LN_plasmablast_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR177/034/SRR17709534/SRR17709534_2.fastq.gz
  out=raw/07_d60_LN_plasmablast_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR177/036/SRR17709536/SRR17709536_1.fastq.gz
  out=raw/01a_d60_LN_germCenterB_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR177/036/SRR17709536/SRR17709536_2.fastq.gz
  out=raw/01a_d60_LN_germCenterB_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR177/031/SRR17709531/SRR17709531_1.fastq.gz
  out=raw/13_d110_LN_germCenterB_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR177/031/SRR17709531/SRR17709531_2.fastq.gz
  out=raw/13_d110_LN_germCenterB_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR177/035/SRR17709535/SRR17709535_1.fastq.gz
  out=raw/01a_d110_LN_germCenterB_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR177/035/SRR17709535/SRR17709535_2.fastq.gz
  out=raw/01a_d110_LN_germCenterB_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR177/030/SRR17709530/SRR17709530_1.fastq.gz
  out=raw/13_d60_LN_plasmablast_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR177/030/SRR17709530/SRR17709530_2.fastq.gz
  out=raw/13_d60_LN_plasmablast_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR177/029/SRR17709529/SRR17709529_1.fastq.gz
  out=raw/13_d110_LN_plasmablast_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR177/029/SRR17709529/SRR17709529_2.fastq.gz
  out=raw/13_d110_LN_plasmablast_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR177/027/SRR17709527/SRR17709527_1.fastq.gz
  out=raw/22_d110_LN_germCenterB_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR177/027/SRR17709527/SRR17709527_2.fastq.gz
  out=raw/22_d110_LN_germCenterB_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR177/028/SRR17709528/SRR17709528_1.fastq.gz
  out=raw/22_d60_LN_germCenterB_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR177/028/SRR17709528/SRR17709528_2.fastq.gz
  out=raw/22_d60_LN_germCenterB_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR177/026/SRR17709526/SRR17709526_1.fastq.gz
  out=raw/22_d60_LN_plasmablast_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR177/026/SRR17709526/SRR17709526_2.fastq.gz
  out=raw/22_d60_LN_plasmablast_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR177/025/SRR17709525/SRR17709525_1.fastq.gz
  out=raw/22_d110_LN_plasmablast_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR177/025/SRR17709525/SRR17709525_2.fastq.gz
  out=raw/22_d110_LN_plasmablast_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR177/024/SRR17709524/SRR17709524_1.fastq.gz
  out=raw/01a_d60_LN_plasmablast_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR177/024/SRR17709524/SRR17709524_2.fastq.gz
  out=raw/01a_d60_LN_plasmablast_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR177/022/SRR17709522/SRR17709522_1.fastq.gz
  out=raw/20_d110_LN_germCenterB_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR177/022/SRR17709522/SRR17709522_2.fastq.gz
  out=raw/20_d110_LN_germCenterB_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR177/023/SRR17709523/SRR17709523_1.fastq.gz
  out=raw/20_d60_LN_germCenterB_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR177/023/SRR17709523/SRR17709523_2.fastq.gz
  out=raw/20_d60_LN_germCenterB_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR177/021/SRR17709521/SRR17709521_1.fastq.gz
  out=raw/20_d60_LN_plasmablast_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR177/021/SRR17709521/SRR17709521_2.fastq.gz
  out=raw/20_d60_LN_plasmablast_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR177/020/SRR17709520/SRR17709520_1.fastq.gz
  out=raw/20_d110_LN_plasmablast_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR177/020/SRR17709520/SRR17709520_2.fastq.gz
  out=raw/20_d110_LN_plasmablast_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR177/019/SRR17709519/SRR17709519_1.fastq.gz
  out=raw/02a_d35_LN_germCenterB_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR177/019/SRR17709519/SRR17709519_2.fastq.gz
  out=raw/02a_d35_LN_germCenterB_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR177/018/SRR17709518/SRR17709518_1.fastq.gz
  out=raw/02a_d60_LN_germCenterB_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR177/018/SRR17709518/SRR17709518_2.fastq.gz
  out=raw/02a_d60_LN_germCenterB_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR177/017/SRR17709517/SRR17709517_1.fastq.gz
  out=raw/02a_d35_LN_plasmablast_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR177/017/SRR17709517/SRR17709517_2.fastq.gz
  out=raw/02a_d35_LN_plasmablast_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR177/015/SRR17709515/SRR17709515_1.fastq.gz
  out=raw/02a_d35_LN_germCenterB_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR177/015/SRR17709515/SRR17709515_2.fastq.gz
  out=raw/02a_d35_LN_germCenterB_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR177/016/SRR17709516/SRR17709516_1.fastq.gz
  out=raw/02a_d60_LN_plasmablast_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR177/016/SRR17709516/SRR17709516_2.fastq.gz
  out=raw/02a_d60_LN_plasmablast_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR177/014/SRR17709514/SRR17709514_1.fastq.gz
  out=raw/02a_d60_LN_germCenterB_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR177/014/SRR17709514/SRR17709514_2.fastq.gz
  out=raw/02a_d60_LN_germCenterB_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR177/013/SRR17709513/SRR17709513_1.fastq.gz
  out=raw/01a_d110_LN_plasmablast_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR177/013/SRR17709513/SRR17709513_2.fastq.gz
  out=raw/01a_d110_LN_plasmablast_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR177/012/SRR17709512/SRR17709512_1.fastq.gz
  out=raw/02a_d35_LN_plasmablast_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR177/012/SRR17709512/SRR17709512_2.fastq.gz
  out=raw/02a_d35_LN_plasmablast_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR177/011/SRR17709511/SRR17709511_1.fastq.gz
  out=raw/02a_d60_LN_plasmablast_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR177/011/SRR17709511/SRR17709511_2.fastq.gz
  out=raw/02a_d60_LN_plasmablast_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR177/009/SRR17709509/SRR17709509_1.fastq.gz
  out=raw/10_d60_LN_plasmablast_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR177/009/SRR17709509/SRR17709509_2.fastq.gz
  out=raw/10_d60_LN_plasmablast_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR177/010/SRR17709510/SRR17709510_1.fastq.gz
  out=raw/10_d60_LN_germCenterB_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR177/010/SRR17709510/SRR17709510_2.fastq.gz
  out=raw/10_d60_LN_germCenterB_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR177/008/SRR17709508/SRR17709508_1.fastq.gz
  out=raw/10_d110_LN_germCenterB_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR177/008/SRR17709508/SRR17709508_2.fastq.gz
  out=raw/10_d110_LN_germCenterB_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR177/007/SRR17709507/SRR17709507_1.fastq.gz
  out=raw/10_d110_LN_plasmablast_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR177/007/SRR17709507/SRR17709507_2.fastq.gz
  out=raw/10_d110_LN_plasmablast_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR177/006/SRR17709506/SRR17709506_1.fastq.gz
  out=raw/10_d60_LN_germCenterB_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR177/006/SRR17709506/SRR17709506_2.fastq.gz
  out=raw/10_d60_LN_germCenterB_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR177/005/SRR17709505/SRR17709505_1.fastq.gz
  out=raw/10_d60_LN_plasmablast_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR177/005/SRR17709505/SRR17709505_2.fastq.gz
  out=raw/10_d60_LN_plasmablast_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR177/003/SRR17709503/SRR17709503_1.fastq.gz
  out=raw/10_d110_LN_plasmablast_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR177/003/SRR17709503/SRR17709503_2.fastq.gz
  out=raw/10_d110_LN_plasmablast_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR177/004/SRR17709504/SRR17709504_1.fastq.gz
  out=raw/10_d110_LN_germCenterB_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR177/004/SRR17709504/SRR17709504_2.fastq.gz
  out=raw/10_d110_LN_germCenterB_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR177/002/SRR17709502/SRR17709502_1.fastq.gz
  out=raw/04_d28_LN_germCenterB_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR177/002/SRR17709502/SRR17709502_2.fastq.gz
  out=raw/04_d28_LN_germCenterB_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR177/001/SRR17709501/SRR17709501_1.fastq.gz
  out=raw/01a_d201_BM_plasmaCell_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR177/001/SRR17709501/SRR17709501_2.fastq.gz
  out=raw/01a_d201_BM_plasmaCell_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR177/000/SRR17709500/SRR17709500_1.fastq.gz
  out=raw/01a_d201_BM_plasmaCell_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR177/000/SRR17709500/SRR17709500_2.fastq.gz
  out=raw/01a_d201_BM_plasmaCell_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR177/099/SRR17709499/SRR17709499_1.fastq.gz
  out=raw/02a_d201_BM_plasmaCell_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR177/099/SRR17709499/SRR17709499_2.fastq.gz
  out=raw/02a_d201_BM_plasmaCell_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR177/098/SRR17709498/SRR17709498_1.fastq.gz
  out=raw/04_d201_BM_plasmaCell_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR177/098/SRR17709498/SRR17709498_2.fastq.gz
  out=raw/04_d201_BM_plasmaCell_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR177/096/SRR17709496/SRR17709496_1.fastq.gz
  out=raw/13_d201_BM_plasmaCell_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR177/096/SRR17709496/SRR17709496_2.fastq.gz
  out=raw/13_d201_BM_plasmaCell_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR177/097/SRR17709497/SRR17709497_1.fastq.gz
  out=raw/07_d201_BM_plasmaCell_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR177/097/SRR17709497/SRR17709497_2.fastq.gz
  out=raw/07_d201_BM_plasmaCell_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR177/095/SRR17709495/SRR17709495_1.fastq.gz
  out=raw/20_d201_BM_plasmaCell_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR177/095/SRR17709495/SRR17709495_2.fastq.gz
  out=raw/20_d201_BM_plasmaCell_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR177/094/SRR17709494/SRR17709494_1.fastq.gz
  out=raw/22_d201_BM_plasmaCell_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR177/094/SRR17709494/SRR17709494_2.fastq.gz
  out=raw/22_d201_BM_plasmaCell_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR177/092/SRR17709492/SRR17709492_1.fastq.gz
  out=raw/04_d201_d208_PBMC_Bmem_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR177/092/SRR17709492/SRR17709492_2.fastq.gz
  out=raw/04_d201_d208_PBMC_Bmem_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR177/093/SRR17709493/SRR17709493_1.fastq.gz
  out=raw/02a_d201_PBMC_Bmem_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR177/093/SRR17709493/SRR17709493_2.fastq.gz
  out=raw/02a_d201_PBMC_Bmem_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR177/091/SRR17709491/SRR17709491_1.fastq.gz
  out=raw/04_d60_LN_germCenterB_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR177/091/SRR17709491/SRR17709491_2.fastq.gz
  out=raw/04_d60_LN_germCenterB_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR177/089/SRR17709489/SRR17709489_1.fastq.gz
  out=raw/10_d201_PBMC_Bmem_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR177/089/SRR17709489/SRR17709489_2.fastq.gz
  out=raw/10_d201_PBMC_Bmem_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR177/090/SRR17709490/SRR17709490_1.fastq.gz
  out=raw/07_d201_PBMC_Bmem_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR177/090/SRR17709490/SRR17709490_2.fastq.gz
  out=raw/07_d201_PBMC_Bmem_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR177/088/SRR17709488/SRR17709488_1.fastq.gz
  out=raw/13_d201_PBMC_Bmem_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR177/088/SRR17709488/SRR17709488_2.fastq.gz
  out=raw/13_d201_PBMC_Bmem_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR177/086/SRR17709486/SRR17709486_1.fastq.gz
  out=raw/22_d201_PBMC_Bmem_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR177/086/SRR17709486/SRR17709486_2.fastq.gz
  out=raw/22_d201_PBMC_Bmem_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR177/087/SRR17709487/SRR17709487_1.fastq.gz
  out=raw/20_d201_PBMC_Bmem_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR177/087/SRR17709487/SRR17709487_2.fastq.gz
  out=raw/20_d201_PBMC_Bmem_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR177/085/SRR17709485/SRR17709485_1.fastq.gz
  out=raw/10_d280_BM_plasmaCell_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR177/085/SRR17709485/SRR17709485_2.fastq.gz
  out=raw/10_d280_BM_plasmaCell_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR177/084/SRR17709484/SRR17709484_1.fastq.gz
  out=raw/20_d280_BM_plasmaCell_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR177/084/SRR17709484/SRR17709484_2.fastq.gz
  out=raw/20_d280_BM_plasmaCell_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR177/083/SRR17709483/SRR17709483_1.fastq.gz
  out=raw/04_d28_LN_plasmablast_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR177/083/SRR17709483/SRR17709483_2.fastq.gz
  out=raw/04_d28_LN_plasmablast_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR177/082/SRR17709482/SRR17709482_1.fastq.gz
  out=raw/04_d60_LN_plasmablast_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR177/082/SRR17709482/SRR17709482_2.fastq.gz
  out=raw/04_d60_LN_plasmablast_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR177/080/SRR17709480/SRR17709480_1.fastq.gz
  out=raw/07_d110_LN_germCenterB_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR177/080/SRR17709480/SRR17709480_2.fastq.gz
  out=raw/07_d110_LN_germCenterB_R2.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR177/081/SRR17709481/SRR17709481_1.fastq.gz
  out=raw/07_d60_LN_germCenterB_R1.fastq.gz
ftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR177/081/SRR17709481/SRR17709481_2.fastq.gz
  out=raw/07_d60_LN_germCenterB_R2.fastq.gz

The project contains 48 paired FASTQ files. Each file name encodes the information about donor, time-point relative to vaccination, tissue of origin and cell population. For example for the first file from the above listing:

13_d60_LN_germCenterB_R1.fastq.gz - 13 - donor id - d60 - 60 days after vaccination - LN - lymph node (BM-bone marrow) - germCenterB - germinal center B cell (cell population)

Upstream analysis

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

mixcr analyze neb-human-rna-xcr-umi-nebnext \
    raw/13_d60_LN_germCenterB_R1.fastq.gz \
    raw/13_d60_LN_germCenterB_R2.fastq.gz \
    results/13_d60_LN_germCenterB

Running the command above will generate the following files:

> ls result/

# human-readable reports 
13_d60_LN_germCenterB.report
# raw alignments (highly compressed binary file)
13_d60_LN_germCenterB.vdjca
# alignments with corrected UMI barcode sequences 
13_d60_LN_germCenterB.refined.vdjca
# IGH, IGK and IGL CDR3 clonotypes (highly compressed binary file)
13_d60_LN_germCenterB.clns
# IGH, IGK and IGL CDR3 clonotypes exported in tab-delimited txt
13_d60_LN_germCenterB.clones.IGH.tsv
13_d60_LN_germCenterB.clones.IGK.tsv
13_d60_LN_germCenterB.clones.IGL.tsv

In order to run the analysis for all samples in the project on Linux we can use GNU Parallel in the following way:

#!/usr/bin/env bash

mkdir -p results

ls /raw/*R1* |
    parallel -j 2 --line-buffer \
    "mixcr analyze neb-human-rna-xcr-umi-nebnext \
    {} \
    {=s:R1:R2:=} \
    {=s:.*/:results/:;s:_R.*::=}"

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

See first 100 records from FebControl1.clones.IGH.tsv clonotype table
cloneId cloneCount uniqueTagCountUMI 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 4763 969 0.108675 TGTGCGAGAGATTCCGTCTTCGACTTATTAATTGACTACTGG NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN IGHV4-39*00(2146.9) IGHD3-1000(31),IGHD3-2200(30),IGHD3-3*00(30) IGHJ4*00(416.3) IGHG100(74.8),IGHG200(74.8),IGHG400(74.8),IGHGP00(74.8) 288 300 319 0 12 SC297G 91.0 49 58 93 21 30 SG51C 31.0;52 58 93 24 30 30.0;52 58 93 24 30 30.0
1 2392 486 0.054577 TGTGCGACTGCTTTGGTGGTGACTGCTGCATACTTCTGG NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN IGHV4-39*00(2317.7) IGHD2-21*00(56) IGHJ4*00(211.9) IGHG100(75.1),IGHG200(75),IGHG400(75),IGHGP00(75) 288 295 319 0 7 70.0 37 51 84 13 27 ST46C 56.0 29 37 68 31 39 SA32T 51.0 ;;; nan nan nan nan
2 2237 476 0.0510404 TGTGCGTCCCTACGGTGGCTACGGGGGGCCTTTGACTACTGG NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN IGHV4-39*00(2270.8) IGHD4-17*00(56) IGHJ4*00(429.3) IGHG100(75.2),IGHG200(75.2),IGHG400(75.2),IGHGP00(75.2) 288 294 319 0 6 60.0 19 33 48 9 23 SA27G 56.0 24 37 68 29 42 130.0 ;;; nan nan nan nan
3 1342 284 0.0306197 TGTGCGAGAGGCGACCGGAATTATCATGACAGTGGGTGGTCCGGCTGGTTCTTCGGTCTCTGG NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN IGHV4-34*00(1365.2) IGHD6-19*00(42) IGHJ2*00(293.3) IGHG100(74.8),IGHG200(74.8),IGHG400(74.8),IGHGP00(74.3) 282 294 313 0 12 120.0 29 43 63 29 43 SC35GSA40C 42.0 23 42 73 44 63 SA28TSA34G 132.0 ;;; nan nan nan nan
4 1135 255 0.0258967 TGTGCGAGAGGCAATATGATAGTAGTGGCCTTTAGTGCTTTTGATATCTGG NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN IGHV4-34*00(2388) IGHD3-22*00(70) IGHJ3*00(443.4) IGHG100(75.4),IGHG200(75.4),IGHG400(75.4),IGHGP00(75.4) 282 294 313 0 12 120.0 38 52 93 14 28 70.0 23 39 70 35 51 160.0 ;;; nan nan nan nan
5 904 187 0.0206261 TGTGCGAGTGAGAACGTGGATACAGGTATGGTTCCCTGGTTCGACCCCTGG NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN IGHV4-39*00(2148.7) IGHD5-5*00(86) IGHJ5*00(465.8) IGHG100(75),IGHG200(75),IGHG400(75),IGHGP00(75) 288 296 319 0 8 80.0 18 38 60 13 33 SC30G 86.0 24 40 71 35 51 160.0 ;;; nan nan nan nan
6 736 148 0.0167929 TGTGCGAGTGAGAACGTGGATACAGGAATGGTTCCCTGGTTCGACCCCTGG NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN IGHV4-39*00(2268.5) IGHD5-5*00(72) IGHJ5*00(465.4) IGHG100(75.5),IGHG200(75.5),IGHG400(75.5),IGHGP00(75.5) 288 296 319 0 8 80.0 18 38 60 13 33 SC30GST31A 72.0 24 40 71 35 51 160.0 ;;; nan nan nan nan
7 709 139 0.0161769 TGTGCACACAGACTCGGCTCGTGGTTAAAGGGACTTCAGACTCACCCTATGATAGACCCAAGCGGGGACTGGTTCGACCCCTGG NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN IGHV2-5*00(2446.5) IGHD3-22*00(45) IGHJ5*00(478.5) IGHG100(74.9),IGHG200(74.9),IGHG400(74.9),IGHGP00(74.9) 288 301 321 0 13 130.0 37 46 93 46 55 45.0 23 40 71 67 84 170.0 ;;; nan nan nan nan
8 704 143 0.0160628 TGTGCGAGACGGCCCCCAGTAGTGGTTATTCCGTTTGACTACTGG NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN IGHV4-34*00(2164) IGHD3-22*00(65) IGHJ4*00(427.9) IGHG100(75.8),IGHG200(75.8),IGHG400(75.8),IGHGP00(75.8) 282 291 313 0 9 90.0 44 57 93 17 30 65.0 25 37 68 33 45 120.0 ;;; nan nan nan nan
9 693 160 0.0158118 TGTGCGAGGTGGGGGCCTACGGTGACTCCCTACTTTGACTACTGG NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN IGHV3-53*00(2094.5) IGHD4-17*00(55) IGHJ4*00(465.4) IGHA100(144.4),IGHA200(144.4) 282 290 313 0 8 80.0 19 30 48 16 27 55.0 21 37 68 29 45 160.0 ; nan nan nan nan
10 679 155 0.0154924 TGTGCGAGAGCCAACCATATGGACGTCTGG NNNNNNNNNNNNNNNNNNNNNNNNNNNNNN IGHV3-30*00(2105.7) IGHD3-1000(25),IGHD3-2200(25),IGHD3-3*00(25) IGHJ6*00(435.8) IGHG100(75.3),IGHG200(75.3),IGHG400(75.3),IGHGP00(75.3) 285 295 316 0 10 SA292G 71.0 17 22 93 12 17 25.0;8 13 93 12 17 25.0;8 13 93 12 17 25.0
11 627 128 0.0143059 TGTGCGGGCCAATTTGTAGTAGTTCCAGCTGCTAGTTACTACTACTACGCTATGGACGTCTGG NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN IGHV4-39*00(2084.7) IGHD2-2*00(91) IGHJ6*00(546.8) IGHG100(75),IGHG200(75),IGHG400(75),IGHGP00(75) 288 294 319 0 6 60.0 37 58 93 13 34 SA47T 91.0 25 52 83 36 63 SG38C 241.0 ;;; nan nan nan nan
12 591 142 0.0134845 TGTGCGAAATCCTATAGTGGGAGCTACTCCGCCGCTTTTGATATCTGG NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN IGHV3-30*00(2218.3) IGHD1-26*00(81) IGHJ3*00(438.3) IGHG100(74.7),IGHG200(74.3),IGHG400(74.3),IGHGP00(74.3) 285 294 316 0 9 90.0 22 41 60 12 31 SA38C 81.0 24 39 70 33 48 150.0 ;;; nan nan nan nan
13 490 99 0.0111801 TGTGCGCGTGAGAACGTGGATACAGGAATGGTTCCCTGGTTCGACCCCTGG NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN IGHV4-39*00(2257.6) IGHD5-5*00(72) IGHJ5*00(466.4) IGHG100(74.6),IGHG200(74.6),IGHG400(74.6),IGHGP00(74.6) 288 294 319 0 6 60.0 18 38 60 13 33 SC30GST31A 72.0 24 40 71 35 51 160.0 ;;; nan nan nan nan
14 432 84 0.00985671 TGTGCGTCCCTAAGGTGGCTACGGGGGGCCTTTGACTACTGG NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN IGHV4-39*00(2280.2) IGHD5-1200(45),IGHD5-1800(45),IGHD4-17*00(42) IGHJ4*00(428.6) IGHG100(74.8),IGHG200(74.8),IGHG400(74.8),IGHGP00(74.8) 288 294 319 0 6 60.0 32 41 69 14 23 45.0;32 41 69 14 23 45.0;19 33 48 9 23 SC22ASA27G 42.0
15 419 102 0.0095601 TGTGCGAGAGATCAGGGATACAGCTATGGGCCATGGGGGGACTACTACGGTATGGACGTCTGG NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN IGHV3-21*00(2077.3) IGHD5-5*00(70) IGHJ6*00(537.1) IGHG100(74.8),IGHG200(74.8),IGHG400(74.8),IGHGP00(74.8) 285 298 316 0 13 130.0 22 36 60 15 29 70.0 29 52 83 40 63 230.0 ;;; nan nan nan nan
16 410 83 0.00935475 TGTGCGACTGAGAACGTGGATACAGGCATGGTTCCCTGGTTCGACCCCTGG NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN IGHV4-39*00(2221.2) IGHD5-5*00(72) IGHJ5*00(439.7) IGHG100(74.9),IGHG200(74.9),IGHG400(74.9),IGHGP00(74.9) 288 295 319 0 7 70.0 18 38 60 13 33 SC30GST31C 72.0 24 40 71 35 51 160.0 ;;; nan nan nan nan
17 387 88 0.00882997 TGTGCGAGGACCCTTTACGATTACTTTGACTACTGG NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN IGHV3-53*00(2307.8) IGHD3-300(35),IGHD3-900(35),IGHD5-12*00(30) IGHJ4*00(458.1) IGHG100(75),IGHG200(75),IGHG400(75),IGHGP00(75) 282 290 313 0 8 80.0 34 41 93 14 21 35.0;34 41 93 14 21 35.0;37 43 69 15 21 30.0
18 324 70 0.00739253 TGTGCGTCCCTACGGTGGCTTCGGGGGGCCTTTGACTACTGG NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN IGHV4-39*00(2207.9) IGHD4-2300(45),IGHD4-1700(42) IGHJ4*00(408.8) IGHG100(75),IGHG200(75),IGHG400(75),IGHGP00(75) 288 294 319 0 6 60.0 22 31 57 9 18 45.0;19 33 48 9 23 SA27GSA30T 42.0 24 37 68 29 42
19 319 73 0.00727845 TGTGCGAGAATCTCCGTCTATGATAGTAGTGGTTATTACTCTGACTACTGG NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN IGHV1-69D*00(2193.7) IGHD3-22*00(80) IGHJ4*00(426.6) IGHG100(74.7),IGHGP00(74.7),IGHG200(73.8),IGHG400(73.8) 285 294 316 0 9 90.0 37 53 93 17 33 80.0 19 37 68 33 51 SC21TST26C 122.0 ;;; nan nan nan nan
20 307 75 0.00700465 TGTGCGAGGCCGTATAGTGGGAGCTACTTTTCTTACTTTGACTACTGG NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN IGHV3-30*00(2040.4) IGHD1-26*00(85) IGHJ4*00(457.8) IGHG100(74.8),IGHG200(74.8),IGHG400(74.8),IGHGP00(74.8) 285 292 316 0 7 70.0 21 38 60 11 28 85.0 22 37 68 33 48 150.0 ;;; nan nan nan nan
21 301 69 0.00686776 TGTGCGAGAGATGACGGAAGCAGCGGCTGGTATGGACCGGGGAACTACTGG NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN IGHV1-69D*00(2140.9) IGHD6-1300(58),IGHD6-1900(58),IGHD6-25*00(50) IGHJ4*00(387.9) IGHG100(75.6),IGHG200(75.6),IGHG400(75.6),IGHGP00(75.6) 285 297 316 0 12 120.0 27 47 63 18 38 SA33GSC41TST43G 58.0;27 47 63 18 38 ST32CSC41TST43G 58.0;24 34 54 18 28 50.0
22 296 62 0.00675367 TGTGCGAGTGAGAACGTGGACACAGGTATGGTTCCCTGGTTCGACCCCTGG NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN IGHV4-39*00(2253.5) IGHD5-5*00(72) IGHJ5*00(463.9) IGHG100(74.8),IGHG200(74.8),IGHG400(74.8),IGHGP00(74.8) 288 296 319 0 8 80.0 18 38 60 13 33 ST25CSC30G 72.0 24 40 71 35 51 160.0 ;;; nan nan nan nan
23 284 62 0.00647988 TGTGCGACCGGGATACAGTTACAGCTATGGTCTCACTACTTTGACTACTGG NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN IGHV4-39*00(2432.8) IGHD5-5*00(60) IGHJ4*00(474.6) IGHA100(144.2),IGHA200(144.2) 288 295 319 0 7 70.0 25 37 60 19 31 60.0 20 37 68 34 51 170.0 ; nan nan nan nan
24 264 59 0.00602355 TGTGCGACTGCTTTGGTGGTGACTGCTGCATACTTCTGG NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN IGHV4-39*00(2487.2) IGHD2-21*00(56) IGHJ1*00(139.5) IGHG100(74.8),IGHG200(74.8),IGHG400(74.8),IGHGP00(74.8) 288 295 319 0 7 70.0 37 51 84 13 27 ST46C 56.0 37 41 72 35 39 40.0 ;;; nan nan nan nan
25 258 52 0.00588665 TGTGCGACTGAGAACGTGGATACAGGTATGGTTCCCTGGTTCGACCCCTGG NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN IGHV4-39*00(2360.1) IGHD5-5*00(86) IGHJ5*00(467) IGHG100(75),IGHG200(75),IGHG400(75),IGHGP00(75) 288 295 319 0 7 70.0 18 38 60 13 33 SC30G 86.0 24 40 71 35 51 160.0 ;;; nan nan nan nan
26 250 53 0.00570412 TGTGCACACAGGCGGGGAAGGGCCACATGGATGGCTGGTTATTTCGACTACTGG NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN IGHV2-5*00(2447.1) IGHD6-1900(40),IGHD5-1200(36),IGHD5-5*00(36) IGHJ4*00(400.5) IGHG100(75.2),IGHG200(75.2),IGHG400(75.2),IGHGP00(73.8) 288 302 321 0 14 SA299G 111.0 32 40 63 31 39 40.0;19 29 69 22 32 SG23A 36.0;16 26 60 22 32 SG20A 36.0
27 244 49 0.00556722 TGTGCGAGAGATGACGGAAGTACCGGCTGGTATGGACCGGGGAACTACTGG NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN IGHV1-69D*00(2165.1) IGHD2-2*00(52) IGHJ4*00(386) IGHG100(74.9),IGHG200(74.9),IGHG400(74.9),IGHGP00(74.9) 285 297 316 0 12 120.0 44 60 93 18 34 SA50GSC55G 52.0 29 37 68 43 51 80.0 ;;; nan nan nan nan
28 244 44 0.00556722 TGTGCGAGAAGGGACTTCCAGAGGTCGTTCCTCTTTTGG NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN IGHV4-4*00(2190.5) IGHD1-100(41),IGHD1-2000(41) IGHJ400(337.4),IGHJ500(337.4) IGHG100(76.5),IGHG200(76.5),IGHG400(76.5),IGHGP00(76.5) 285 294 316 0 9 90.0 31 42 51 20 31 SC33G 41.0;31 42 51 20 31 SC33G 41.0 34 37 68 36 39
29 238 55 0.00543032 TGTGCGAGACGGCCGGCTACTATGATATTTCTGGCCCACACCAAGAAAGACTACTTTGACTACTGG NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN IGHV4-34*00(2147.8) IGHD3-22*00(50) IGHJ4*00(478.2) IGHG100(74.7),IGHG200(74.7),IGHG400(74.7),IGHGP00(74.7) 282 291 313 0 9 90.0 35 45 93 17 27 50.0 20 37 68 49 66 170.0 ;;; nan nan nan nan
30 237 49 0.0054075 TGTGCCAGACGGCCCCCAGTAGTGGTTATTCCGTTTGACTACTGG NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN IGHV4-34*00(2228.4) IGHD3-22*00(65) IGHJ4*00(428.9) IGHG100(74.8),IGHG200(74.8),IGHG400(74.8),IGHGP00(74.8) 282 291 313 0 9 SG287C 61.0 44 57 93 17 30 65.0 25 37 68 33 45 120.0 ;;; nan nan nan nan
31 229 51 0.00522497 TGTGCGAGAGATGACGGAAGCAGCAGCTGGTATGGACCGGGGAACTACTGG NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN IGHV1-69D*00(2258) IGHD6-13*00(72) IGHJ4*00(389.5) IGHG100(75),IGHG200(75),IGHG400(75),IGHGP00(75) 285 297 316 0 12 120.0 27 47 63 18 38 SC41TST43G 72.0 29 37 68 43 51 80.0 ;;; nan nan nan nan
32 226 52 0.00515652 TGTGCGAGCAGTGGCTGGCACCGGGGGGCCTTTGACTACTGG NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN IGHV3-66*00(2370.4) IGHD6-19*00(51) IGHJ4*00(436.5) IGHG100(74.9),IGHG200(74.9),IGHG400(74.9),IGHGP00(74.9) 282 290 313 0 8 80.0 29 42 63 8 21 ST39C 51.0 24 37 68 29 42 130.0 ;;; nan nan nan nan
33 212 55 0.00483709 TGTGCGAGCGCTCCCCGCCCGTATTACTATGATAGTAGTGCTTATTACCCATTTGACTACTGG NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN IGHV3-48*00(2081.7) IGHD3-22*00(131) IGHJ4*00(428.4) IGHG100(75),IGHG200(75),IGHG400(75),IGHGP00(75) 285 293 316 0 8 80.0 30 59 93 19 48 SG51C 131.0 25 37 68 51 63 120.0 ;;; nan nan nan nan
34 196 37 0.00447203 TGTGCGAGAGGAGCTACGGGAGATGCTTTTGATATCTGG NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN IGHV4-39*00(2201.8) IGHD1-26*00(40) IGHJ3*00(438.3) IGHG100(74.1),IGHG200(74.1),IGHG400(74.1),IGHGP00(74.1) 288 297 319 0 9 90.0 29 37 60 9 17 40.0 21 39 70 21 39 180.0 ;;; nan nan nan nan
35 195 43 0.00444921 TGTGCGATTGAGAACGTGGATACAGGTATGGTTCCCTGGTTCGACCCCTGG NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN IGHV4-39*00(2276) IGHD5-5*00(86) IGHJ5*00(469.6) IGHG100(75.5),IGHG200(75.5),IGHG400(75.5),IGHGP00(75.5) 288 295 319 0 7 70.0 18 38 60 13 33 SC30G 86.0 24 40 71 35 51 160.0 ;;; nan nan nan nan
36 182 44 0.0041526 TGTGCGAGAGAACGGGATATTGTAGTAGTACCAGCGTCTTACTTTGACTACTGG NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN IGHV1-46*00(2381.6) IGHD2-2*00(105) IGHJ4*00(456.1) IGHG100(74.7),IGHG200(74.7),IGHG400(74.7),IGHGP00(74.7) 285 296 316 0 11 110.0 32 53 93 14 35 105.0 22 37 68 39 54 150.0 ;;; nan nan nan nan
37 173 38 0.00394725 TGTGCGAGAGAAAGTCCCGAGCGCATTGTAGTGGAGGTAGCTGCTACGACCAACTGGTTCGACCCCTGG NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN IGHV1-2*00(1962.1) IGHD2-15*00(101) IGHJ5*00(495) IGHG100(74.9),IGHG200(74.9),IGHG400(74.9),IGHGP00(74.9) 285 296 316 0 11 110.0 36 59 93 24 47 ST46A 101.0 21 40 71 50 69 190.0 ;;; nan nan nan nan
38 170 37 0.0038788 TGTGCGAAAGATCGGGGCGATGACTACGCCAGCTACTACTACGGTATGGACGTCTGG NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN IGHV1-69D*00(2161.3) IGHD4-1700(45),IGHD4-2300(45),IGHD4-11*00(40) IGHJ6*00(502.4) IGHG100(74.9),IGHG200(74.9),IGHG400(74.9),IGHGP00(74.9) 285 298 316 0 13 SG292A 101.0 15 24 48 19 28 45.0;18 27 57 19 28 45.0;15 23 48 19 27 40.0
39 167 45 0.00381035 TGTGCGAGAGTGAATGGCTACGGAAATGCTTTTGATATCTGG NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN IGHV3-23*00(2161.1) IGHD5-1200(40),IGHD5-1800(40),IGHD5-24*00(40) IGHJ3*00(398.4) IGHG100(74.9),IGHG200(74.9),IGHG400(74.9),IGHGP00(74.9) 285 295 316 0 10 SA292G 71.0 33 41 69 14 22 40.0;33 41 69 14 22 40.0;26 34 60 13 21 40.0
40 163 37 0.00371908 TGTGCGAGACCAGGGTGGCTACGAGGTGCTTTTGATATCTGG NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN IGHV4-39*00(2345) IGHD5-1200(50),IGHD5-1800(50) IGHJ3*00(462.2) IGHG100(74.8),IGHG200(74.8),IGHG400(74.8),IGHGP00(74.8) 288 298 319 0 10 100.0 32 42 69 14 24 50.0;32 42 69 14 24 50.0 23 39 70 26 42
41 155 36 0.00353655 TGTGCGAGACATCCCTCACCTTATTACTATGGTTCGGGGAGATATGAGTACGGTGTGGACGTCTGG NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN IGHV5-51*00(2241.9) IGHD3-10*00(92) IGHJ6*00(454.7) IGHG100(74.9),IGHG200(74.9),IGHG400(74.9),IGHGP00(74.9) 285 297 316 0 12 120.0 32 56 93 21 45 SA46GST52A 92.0 34 52 83 48 66 SA40G 151.0 ;;; nan nan nan nan
42 155 34 0.00353655 TGTGCGAGAGATCAGGGATACAGCTATGGGCCATGGGGGGACTACTACGGTATGGACGACTGG NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN IGHV3-21*00(2242.4) IGHD5-5*00(70) IGHJ6*00(475.5) IGHG100(74.9),IGHG200(74.9),IGHG400(74.9),IGHGP00(72.8) 285 298 316 0 13 130.0 22 36 60 15 29 70.0 29 52 83 40 63 ST47A 201.0 ;;; nan nan nan nan
43 155 41 0.00353655 TGTGCAAAAGATATAGGGTATAGCAGTGGTGGGACCGACTACTGG NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN IGHV3-9*00(2385.3) IGHD6-19*00(74) IGHJ4*00(396) IGHG100(74.9),IGHG200(74.9),IGHG400(74.9),IGHGP00(74.9) 285 300 318 0 15 150.0 21 42 63 15 35 DC35ST39G 74.0 28 37 68 36 45 90.0 ;;; nan nan nan nan
44 154 35 0.00351374 TGTGCGGGCCAATTTGTAGTAGTACCAGCTGCTAGTTACTACTACTACGCTATGGACGTCTGG NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN IGHV4-39*00(2289.6) IGHD2-2*00(105) IGHJ6*00(542.6) IGHG100(74.9),IGHG200(74.9),IGHG400(74.9),IGHGP00(74.9) 288 294 319 0 6 60.0 37 58 93 13 34 105.0 25 52 83 36 63 SG38C 241.0 ;;; nan nan nan nan
45 154 39 0.00351374 TGTGCGAGGTGTTTCGGTGGGAACTATTACGGGCCCTTTGACTACTGG NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN IGHV3-30*00(2123.5) IGHD1-2600(47),IGHD4-2300(41) IGHJ4*00(428.1) IGHG100(75),IGHG200(75),IGHG400(75),IGHGP00(75) 285 292 316 0 7 70.0 26 41 60 16 31 SG32ASC36T 47.0;25 36 57 14 25 ST31G 41.0 24 37 68 35 48
46 151 33 0.00344529 TGTGCGGGCCAATCTGTAGTAGTACCAGCTGCTAGTTACTACTACTACGCTATGGACGTCTGG NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN IGHV4-39*00(2130.8) IGHD2-2*00(100) IGHJ6*00(549.9) IGHG100(75),IGHG200(75),IGHG400(75),IGHGP00(75) 288 294 319 0 6 60.0 38 58 93 14 34 100.0 25 52 83 36 63 SG38C 241.0 ;;; nan nan nan nan
47 139 34 0.00317149 TGTGCGAAGTCCTATAGTGGGAGCTACTCCGCCGCTTTTGATATCTGG NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN IGHV3-30*00(2227.7) IGHD1-26*00(81) IGHJ3*00(372.4) IGHG100(75),IGHG200(75),IGHG400(75),IGHGP00(75) 285 293 316 0 8 80.0 22 41 60 12 31 SA38C 81.0 24 39 70 33 48 150.0 ;;; nan nan nan nan
48 136 37 0.00310304 TGTGCGAGCAGTGGCTGGCACCGGGGGGCCTTTGACTACTGG NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN IGHV3-53*00(2225.4) IGHD6-19*00(51) IGHJ4*00(438.2) IGHG100(75),IGHG200(75),IGHG400(75),IGHGP00(75) 282 290 313 0 8 80.0 29 42 63 8 21 ST39C 51.0 24 37 68 29 42 130.0 ;;; nan nan nan nan
49 130 28 0.00296614 TGTGCGACCGGGATACAGTTACACCTATGGTCTCACTACTTTGACTACTGG NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN IGHV4-39*00(2349.5) IGHD5-5*00(46) IGHJ4*00(475.6) IGHA100(144.5),IGHA200(144.5) 288 295 319 0 7 70.0 25 37 60 19 31 SG29C 46.0 20 37 68 34 51 170.0 ; nan nan nan nan
50 130 30 0.00296614 TGTGCGAGACTTACCAGCAGTTTTCCTCCGTACTACTTTGACTACTGG NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN IGHV3-23*00(1988.5) IGHD6-1300(36),IGHD6-1900(36),IGHD2-2*00(35) IGHJ4*00(496.8) IGHA100(143.4),IGHA200(143.4) 285 292 316 0 7 70.0 40 50 63 8 18 SG42T 36.0;40 50 63 8 18 SG42T 36.0;46 53 93 11 18 35.0
51 128 29 0.00292051 TGTGCGAGAGATCTGGGGGGCTGGTTCGACCCCTGG NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN IGHV3-30*00(1972.5) IGHD3-16*00(25) IGHJ5*00(465.4) IGHG100(74.9),IGHG200(74.9),IGHG400(74.9),IGHGP00(74.9) 285 299 316 0 14 SA292G 111.0 54 59 111 14 19 25.0 24 40 71 20 36 160.0 ;;; nan nan nan nan
52 124 22 0.00282924 TGTGCGAGAAGGCAGATGCCCTACACCCTTGACTACTGG NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN IGHV4-4*00(2353.8) IGHD2-800(30),IGHD5-2400(29),IGHD6-25*00(26) IGHJ4*00(419.5) IGHG100(75),IGHG200(75),IGHG400(75),IGHGP00(75) 285 294 316 0 9 90.0 9 15 93 21 27 30.0;24 35 60 13 25 I29CSG29C 29.0;10 18 54 20 28 ST13C 26.0
53 123 30 0.00280643 TGTGCGAGAGATCAGGGATACAGCTATGGGCCATGGGGGGACTACTACGGTCTGGACGTCTGG NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN IGHV3-21*00(2122.7) IGHD5-5*00(70) IGHJ6*00(507.1) IGHG100(75),IGHG200(75),IGHG400(75),IGHGP00(75) 285 298 316 0 13 130.0 22 36 60 15 29 70.0 29 52 83 40 63 SA40C 201.0 ;;; nan nan nan nan
54 122 24 0.00278361 TGTGTGTCCCTACGGTGGCTACGGGGGGCCTTTGACTACTGG NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN IGHV4-39*00(2304.3) IGHD4-17*00(56) IGHJ4*00(438.4) IGHG100(75),IGHG200(75),IGHG400(75),IGHGP00(75) 288 292 319 0 4 40.0 19 33 48 9 23 SA27G 56.0 24 37 68 29 42 130.0 ;;; nan nan nan nan
55 122 25 0.00278361 TGTGCGTCCCTAGGGTGGCTACGGGGGGCCTTTGACTACTGG NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN IGHV4-39*00(2248.3) IGHD5-1200(45),IGHD5-1800(45),IGHD4-17*00(42) IGHJ4*00(427.1) IGHG100(75),IGHG200(75),IGHG400(75),IGHGP00(75) 288 294 319 0 6 60.0 32 41 69 14 23 45.0;32 41 69 14 23 45.0;19 33 48 9 23 SC22GSA27G 42.0
56 120 27 0.00273798 TGTGCGAGAGAAAGTCCCGAGCGGATTGTAGTGGTGCTAGCTGCTACGACCAACTGGTTCGACCCCTGG NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN IGHV1-2*00(2068.5) IGHD2-15*00(101) IGHJ5*00(495.3) IGHG100(74.1),IGHG200(74.1),IGHG400(74.1),IGHGP00(74.1) 285 296 316 0 11 110.0 36 59 93 24 47 SG48C 101.0 21 40 71 50 69 190.0 ;;; nan nan nan nan
57 119 23 0.00271516 TGTGCGGCAGATGACGGAAGCAGCGGCTGGTTTGGACCGGGGAACTACTGG NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN IGHV1-69D*00(2168.4) IGHD6-1300(51),IGHD6-1900(51),IGHD6-25*00(50) IGHJ4*00(386.6) IGHG100(74.9),IGHG200(74.9),IGHG400(74.9),IGHGP00(74.9) 285 297 316 0 12 SA291GSG292C 62.0 27 40 63 18 31 SA33G 51.0;27 40 63 18 31 ST32C 51.0;24 34 54 18 28 50.0
58 117 26 0.00266953 TGTGCGAGAGAAAGTCCCGAGCGGATTGTAGTGATGGTAGCTGCTACGACCAACTGGTTCGACCCCTGG NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN IGHV1-2*00(2073) IGHD2-15*00(101) IGHJ5*00(497.1) IGHG100(75),IGHG200(75),IGHG400(75),IGHGP00(75) 285 296 316 0 11 110.0 36 59 93 24 47 SG45A 101.0 21 40 71 50 69 190.0 ;;; nan nan nan nan
59 116 21 0.00264671 TGTGCGCGACTTTCCTACTACTACGGTATGGACGTCTGG NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN IGHV4-59*00(2214.1) nan IGHJ6*00(541.6) IGHG100(75),IGHG200(75),IGHG400(75),IGHGP00(75) 282 291 313 0 9 SA288C 61.0 nan 27 52 83 14 39 250.0 ;;; nan nan nan nan nan nan nan nan nan nan
60 115 25 0.00262389 TGTGCGAGACAACCCTTACGATATTTTGACTGGAACTGGCACTTCGATCTCTGG NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN IGHV4-59*00(2239.1) IGHD3-9*00(90) IGHJ2*00(473.6) IGHG100(74.9),IGHG200(74.9),IGHG400(74.9),IGHGP00(74.9) 282 291 313 0 9 90.0 34 52 93 15 33 90.0 22 42 73 34 54 ST27C 171.0 ;;; nan nan nan nan
61 115 20 0.00262389 TGTGCGAGAAGGGACTTCCAGACGTCGTTCCTCTTTTGG NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN IGHV4-4*00(2367.2) IGHD1-100(55),IGHD1-2000(55) IGHJ400(338.4),IGHJ500(338.4) IGHG100(75),IGHG200(75),IGHG400(75),IGHGP00(75) 285 294 316 0 9 90.0 31 42 51 20 31 55.0;31 42 51 20 31 55.0 34 37 68 36 39
62 113 30 0.00257826 TGTGCGAGAGATTACACCCCTCTCTCCTTCGGGGGGGCTCGCTACGGTATGGACGTCTGG NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN IGHV3-21*00(2323.2) IGHD2-800(30),IGHD3-1600(26),IGHD5-24*00(26) IGHJ6*00(486.2) IGHG100(75),IGHG200(75),IGHG400(75),IGHGP00(75) 285 297 316 0 12 120.0 9 15 93 12 18 30.0;51 59 111 27 35 ST53C 26.0;10 18 60 17 25 SA12C 26.0
63 112 19 0.00255544 TGTGCACAGAGCAGTGGCTGGTCGTACTACTTTGACTACTGG NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN IGHV2-5*00(2452.4) IGHD6-19*00(45) IGHJ4*00(490.5) IGHA100(143.9),IGHA200(143.9) 288 299 321 0 11 SC296G 81.0 29 38 63 11 20 45.0 15 37 68 20 42 SA17C 191.0 ; nan nan nan nan
64 110 24 0.00250981 TGTGCGAGGCTACTATGGTTCGGGGCATATGCTTTTGATATCTGG NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN IGHV4-39*00(2233.6) IGHD3-10*00(61) IGHJ3*00(475.7) IGHG100(75),IGHG200(75),IGHG400(75),IGHGP00(75) 288 296 319 0 8 80.0 35 50 93 10 25 SA46G 61.0 22 39 70 28 45 170.0 ;;; nan nan nan nan
65 109 28 0.00248699 TGTGCGAGCACCGATTGGGGTACTGGGAGATACTACAGTGCCTACTGG NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN IGHV3-53*00(2269) IGHD2-800(35),IGHD1-2600(31),IGHD2-2*00(30) IGHJ4*00(397.5) IGHG100(74.9),IGHG200(74.9),IGHG400(74.9),IGHGP00(74.9) 282 290 313 0 8 80.0 39 46 93 19 26 35.0;24 33 60 20 29 SG26C 31.0;12 18 93 18 24 30.0
66 106 25 0.00241855 TGTGCGTCTGTTTGGTGGCTGAGGGGGTACTTTGACTACTGG NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN IGHV3-48*00(2190.8) IGHD6-1900(35),IGHD2-2100(33),IGHD2-15*00(31) IGHJ4*00(459.1) IGHG100(75.6),IGHG200(75.6),IGHG400(75.6),IGHGP00(75.6) 285 291 316 0 6 60.0 31 38 63 14 21 35.0;37 46 84 12 22 I43C 33.0;46 55 93 12 21 SA50G 31.0
67 103 22 0.0023501 TGTGCGAGCCTCATGTATTTCTATGGTCCCCTTGACCACTGG NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN IGHV4-4*00(2333.4) IGHD3-10*00(51) IGHJ4*00(388.5) IGHG100(75),IGHG200(75),IGHG400(75),IGHGP00(75) 285 293 316 0 8 80.0 31 44 93 14 27 SA36T 51.0 26 37 68 31 42 ST31C 81.0 ;;; nan nan nan nan
68 103 26 0.0023501 TGTGCGAGAGGGTTACGATATTTTGACTGGTTATTTCACAGTCAAGTCAACGAACACAACTGGTTCGACCCCTGG NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN IGHV4-39*00(2097.1) IGHD3-9*00(115) IGHJ5*00(505.5) IGHG100(75),IGHG200(75),IGHG400(75),IGHGP00(75) 288 297 319 0 9 90.0 34 57 93 12 35 115.0 20 40 71 55 75 200.0 ;;; nan nan nan nan
69 102 22 0.00232728 TGTGCGACTGCTTTGGTGGTGACTGCTGCATACTTCTGG NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN IGHV4-39*00(2462.4) IGHD2-21*00(56) IGHJ6*00(135) IGHG100(75),IGHG200(75),IGHG400(75),IGHGP00(75) 288 295 319 0 7 70.0 37 51 84 13 27 ST46C 56.0 44 52 83 31 39 SG46T 51.0 ;;; nan nan nan nan
70 101 18 0.00230446 TGTGCACACACAGTGGATACGATTTTTGGAGTGGTTATTTTTGACTACTGG NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN IGHV2-5*00(2407.5) IGHD3-3*00(110) IGHJ4*00(416) IGHG100(75),IGHG200(75),IGHG400(75),IGHGP00(75) 288 298 321 0 10 100.0 35 57 93 17 39 110.0 25 37 68 39 51 120.0 ;;; nan nan nan nan
71 100 22 0.00228165 TGTGCTAGAGTGGGCGGCGCTTTTGATATCTGG NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN IGHV1-46*00(2335.5) IGHD6-25*00(25) IGHJ3*00(453.9) IGHG100(75),IGHG200(75),IGHG400(75),IGHGP00(75) 285 295 316 0 10 SG290T 71.0 28 33 54 13 18 25.0 24 39 70 18 33 150.0 ;;; nan nan nan nan
72 100 21 0.00228165 TGTGCGAGAGATCGGGGCGATGACTACGCCAGTTACTACTACGGTATGGACGTCTGG NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN IGHV1-69D*00(2349.7) IGHD4-1700(45),IGHD4-2300(45),IGHD4-11*00(40) IGHJ6*00(544.9) IGHG100(75),IGHG200(75),IGHG400(75),IGHGP00(75) 285 298 316 0 13 130.0 15 24 48 19 28 45.0;18 27 57 19 28 45.0;15 23 48 19 27 40.0
73 100 27 0.00228165 TGTGCGAGAGAAAGTCCCGAGCGGATTGTAGTGGTGATAGCTACTACGACCAACTGGTTTGACCCCTGG NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN IGHV1-2*00(2136.3) IGHD2-15*00(87) IGHJ5*00(467.9) IGHG100(75),IGHG200(75),IGHG400(75),IGHGP00(75) 285 296 316 0 11 110.0 36 59 93 24 47 SG48ASG54A 87.0 21 40 71 50 69 SC30T 161.0 ;;; nan nan nan nan
74 99 22 0.00225883 TGTGCGTCCCTACGGTGGCTACGGGGGGCCTTTGAGTACTGG NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN IGHV4-39*00(2273.1) IGHD4-17*00(56) IGHJ4*00(397.6) IGHG100(75),IGHG200(75),IGHG400(75),IGHGP00(75) 288 294 319 0 6 60.0 19 33 48 9 23 SA27G 56.0 24 37 68 29 42 SC30G 101.0 ;;; nan nan nan nan
75 99 24 0.00225883 TGTGCGAAGGAAGGGGCGTTCGGGGAGTTAGGGCTCGGCCTTGACTACTGG NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN IGHV3-30*00(1863.7) IGHD3-1000(51),IGHD3-1600(46) IGHJ4*00(416.5) IGHA100(144.5),IGHA200(144.5) 285 296 316 0 11 SA293G 81.0 42 55 93 17 30 SA46G 51.0;52 64 111 18 30 SG54C 46.0 26 37 68 40 51
76 99 23 0.00225883 TGTACGAGACTGCCGGATTACTATGATAGTAGTGACTACTGG NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN IGHV4-39*00(2195.4) IGHD3-22*00(81) IGHJ4*00(408.5) IGHG100(75),IGHG200(75),IGHG400(75),IGHGP00(75) 288 298 319 0 10 SG291A 71.0 30 49 93 13 32 ST32G 81.0 27 37 68 32 42 100.0 ;;; nan nan nan nan
77 98 21 0.00223601 TGTGCGGGCCAATTTGTAGTAGTGCCAGCCGCTAGTTACTACTATTACGCTATGGACGTCTGG NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN IGHV4-39*00(2242) IGHD2-2*00(77) IGHJ6*00(519.2) IGHG100(75),IGHG200(75),IGHG400(75),IGHGP00(75) 288 294 319 0 6 60.0 37 58 93 13 34 SA47GST53C 77.0 25 52 83 36 63 SC33TSG38C 212.0 ;;; nan nan nan nan
78 98 20 0.00223601 TGTGCGAGACTTTCCTACTACTACGGTATGGACGTCTGG NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN IGHV4-59*00(2199.1) nan IGHJ6*00(524.8) IGHG100(74.8),IGHG200(74.8),IGHG400(74.8),IGHGP00(74.8) 282 291 313 0 9 90.0 nan 27 52 83 14 39 250.0 ;;; nan nan nan nan nan nan nan nan nan nan
79 98 24 0.00223601 TGTGCGAGAGAAAGTCCCGAGCGGATTGTAGTGGTGTTAGCTGCTACGACCAACTGGTTCGACCCCTGG NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN IGHV1-2*00(2118) IGHD2-15*00(101) IGHJ5*00(497.8) IGHG1*00(74.6) 285 296 316 0 11 110.0 36 59 93 24 47 SG48T 101.0 21 40 71 50 69 190.0 nan nan nan nan nan
80 97 21 0.0022132 TGTGCGTCCCTTCGGTGGCTACGGGGGGCCTTTGACTACTGG NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN IGHV4-39*00(2375.9) IGHD5-1200(45),IGHD5-1800(45),IGHD4-17*00(42) IGHJ4*00(437.2) IGHG100(74.9),IGHG200(74.9),IGHG400(74.9),IGHGP00(74.9) 288 294 319 0 6 60.0 32 41 69 14 23 45.0;32 41 69 14 23 45.0;19 33 48 9 23 SA21TSA27G 42.0
81 94 21 0.00214475 TGTGCGAGACGTCCGGCCACTACGATAGTACTGGCCCACACCAAGAAAGACTACTTTGACTACTGG NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN IGHV4-34*00(2089.9) IGHD3-2200(52),IGHD4-1100(46),IGHD4-4*00(46) IGHJ4*00(475.8) IGHG100(75),IGHG200(75),IGHG400(75),IGHGP00(75) 282 291 313 0 9 90.0 36 52 93 18 34 ST40CSG48C 52.0;27 42 48 18 33 I33ADT36 46.0;27 42 48 18 33 I33ADT36 46.0
82 94 23 0.00214475 TGTGCGAAATCCTACAGTGGGAGCTACTCCGCCGCTTTTGATATCTGG NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN IGHV3-30*00(2374.9) IGHD1-26*00(67) IGHJ3*00(370.2) IGHG100(75),IGHG200(75),IGHG400(75),IGHGP00(75) 285 294 316 0 9 90.0 22 41 60 12 31 ST24CSA38C 67.0 24 39 70 33 48 150.0 ;;; nan nan nan nan
83 91 21 0.0020763 TGTGCGAGAGAAAGTCCCGAGCGGATTGTAGTGGTGGTAGCTGCTACGACCAACTGGTTCGACCCCTGG NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN IGHV1-2*00(1923.5) IGHD2-15*00(115) IGHJ5*00(495.9) IGHG100(75),IGHG200(75),IGHG400(75),IGHGP00(75) 285 296 316 0 11 110.0 36 59 93 24 47 115.0 21 40 71 50 69 190.0 ;;; nan nan nan nan
84 89 20 0.00203067 TGTGCGGGTGAGAACGTGGATACAGGAATGGTTCCCTGGTTCGACCCCTGG NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN IGHV4-39*00(2194.4) IGHD5-5*00(72) IGHJ5*00(450.8) IGHG100(74.9),IGHG200(74.9),IGHG400(74.9),IGHGP00(74.9) 288 294 319 0 6 60.0 18 38 60 13 33 SC30GST31A 72.0 24 40 71 35 51 160.0 ;;; nan nan nan nan
85 89 26 0.00203067 TGTGCGAGGTGTTTCGGTGGGAGCTACTACGGGCCCTTTGACTACTGG NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN IGHV3-30*00(2097.5) IGHD1-26*00(75) IGHJ4*00(438) IGHG100(75),IGHG200(75),IGHG400(75),IGHGP00(75) 285 292 316 0 7 70.0 26 41 60 16 31 75.0 24 37 68 35 48 130.0 ;;; nan nan nan nan
86 86 18 0.00196222 TGTGCACACAGACGGGGAAGGGCCACATGGATGGCTGGTTATTTCGACTACTGG NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN IGHV2-5*00(2398.7) IGHD6-1900(40),IGHD5-1200(36),IGHD5-5*00(36) IGHJ4*00(383.1) IGHG100(74.7),IGHG200(74.7),IGHG400(74.7),IGHGP00(74.7) 288 302 321 0 14 140.0 32 40 63 31 39 40.0;19 29 69 22 32 SG23A 36.0;16 26 60 22 32 SG20A 36.0
87 85 19 0.0019394 TGTGCGACCGGGATACAGTTACAGTTATGGTCTCACTACTTTGACTACTGG NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN IGHV4-39*00(2351.9) IGHD1-700(46),IGHD5-500(46),IGHD1-20*00(41) IGHJ4*00(477) IGHA100(144.6),IGHA200(144.6) 288 295 319 0 7 70.0 2 14 51 16 28 SC6A 46.0;25 37 60 19 31 SC30T 46.0;3 14 51 17 28 SC6A 41.0
88 85 22 0.0019394 TGTGCGAGGTGGGGGCCTACGGTGACTCCCTACTTTGACTACTGG NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN IGHV3-53*00(2380.4) IGHD4-17*00(55) IGHJ4*00(466.9) IGHG100(75),IGHG200(75),IGHG400(75),IGHGP00(75) 282 290 313 0 8 80.0 19 30 48 16 27 55.0 21 37 68 29 45 160.0 ;;; nan nan nan nan
89 83 17 0.00189377 TGTGCGAGACGGCCCCCACTAGTGGTTATTCCGTTTGACTACTGG NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN IGHV4-34*00(2173.2) IGHD3-2200(55),IGHD3-300(50) IGHJ4*00(427.2) IGHG100(75.9),IGHG200(75.9),IGHG400(75.9),IGHGP00(75.9) 282 291 313 0 9 90.0 46 57 93 19 30 55.0;47 57 93 20 30 50.0 25 37 68 33 45
90 83 18 0.00189377 TGTGCGAGAGACGAAATTCGGGGAATTAGATCCAGAACCTTTGACTACTGG NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN IGHV4-4*00(2455.5) IGHD3-1000(32),IGHD3-1600(32),IGHD1-7*00(31) IGHJ4*00(436.1) IGHG100(74.9),IGHG200(74.9),IGHG4*00(74.9) 285 296 316 0 11 110.0 43 55 93 16 28 SA46GSG51A 32.0;52 64 111 16 28 SG54CSG60A 32.0;1 10 51 26 35 ST4A 31.0
91 82 20 0.00187095 TGTGCACGGATACCGGGGGATTTTTGGAGTGGTTATTCCTACTTTGACTACTGG NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN IGHV2-70*00(2387.2) IGHD3-3*00(95) IGHJ4*00(465.1) IGHG100(75),IGHG200(75),IGHG400(75),IGHGP00(75) 288 301 321 0 13 130.0 38 57 93 18 37 95.0 21 37 68 38 54 160.0 ;;; nan nan nan nan
92 81 17 0.00184813 TGTGCGAGAGGCAATATGATAATAGTGGCCTTTAGTGCTTTTGATATCTGG NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN IGHV4-34*00(2438) IGHD3-22*00(56) IGHJ3*00(417.5) IGHG100(75),IGHG200(75),IGHG400(75),IGHGP00(75) 282 294 313 0 12 120.0 38 52 93 14 28 SG45A 56.0 23 39 70 35 51 160.0 ;;; nan nan nan nan
93 81 20 0.00184813 TGTGCGAGAGGAGGTGGATACAGCTATGGGGCCCTTGACTACTGG NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN IGHV4-34*00(2432.1) IGHD5-5*00(80) IGHJ4*00(418.7) IGHG100(74.8),IGHG200(74.8),IGHG400(74.8),IGHGP00(74.8) 282 293 313 0 11 110.0 20 36 60 13 29 80.0 26 37 68 34 45 110.0 ;;; nan nan nan nan
94 80 18 0.00182532 TGTGCGCGAGATCTCACTACGGTGTCGATCTGG NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN IGHV3-48*00(2152.3) IGHD4-1700(45),IGHD4-2300(45) IGHJ3*00(362.3) IGHG100(74.7),IGHG200(74.7),IGHG400(74.7),IGHGP00(74.7) 285 300 316 0 15 SA291C 121.0 18 27 48 15 24 45.0;21 30 57 15 24 45.0 33 39 70 27 33
95 80 22 0.00182532 TGTGCGAGCGCTCCCCGCCCGTATTACTATGATAGTAGTGGTTATTACCCATTTGACTACTGG NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN IGHV3-48*00(2155.1) IGHD3-22*00(145) IGHJ4*00(426.6) IGHG100(74.6),IGHG200(74.6),IGHG400(74.6),IGHGP00(74.6) 285 293 316 0 8 80.0 30 59 93 19 48 145.0 25 37 68 51 63 120.0 ;;; nan nan nan nan
96 78 15 0.00177968 TGTGCGACTGAGAACGTGGATACAGGAATGGTTCCCTGGTTCGACCCCTGG NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN IGHV4-39*00(2367.5) IGHD5-5*00(72) IGHJ5*00(467.5) IGHG100(75),IGHG200(75),IGHG400(75),IGHGP00(75) 288 295 319 0 7 70.0 18 38 60 13 33 SC30GST31A 72.0 24 40 71 35 51 160.0 ;;; nan nan nan nan
97 78 21 0.00177968 TGTGCGAACAGTGGCTGGCACCGGGGGGCCTTTGACTACTGG NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN IGHV3-53*00(2155.6) IGHD6-19*00(51) IGHJ4*00(435.5) IGHG100(75),IGHG200(75),IGHG400(75),IGHGP00(75) 282 289 313 0 7 70.0 29 42 63 8 21 ST39C 51.0 24 37 68 29 42 130.0 ;;; nan nan nan nan
98 76 18 0.00173405 TGTGCGAGACGGCCGGCTACTCTGATAGTAATGGCCCACACCAAGAATGACTACTTTGACTACTGG NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN IGHV4-34*00(2074.4) IGHD3-22*00(57) IGHJ4*00(452.7) IGHG100(74.8),IGHG200(74.8),IGHG400(74.8),IGHGP00(74.8) 282 291 313 0 9 90.0 35 52 93 17 34 SA39CSG48A 57.0 20 37 68 49 66 170.0 ;;; nan nan nan nan
99 74 15 0.00168842 TGTGCGAGAGACGAAATTCGGGGAGTTAGATCCAGAATGTTTGACTACTGG NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN IGHV4-4*00(2253.9) IGHD3-1000(46),IGHD3-1600(46) IGHJ4*00(396.7) IGHG100(75),IGHG200(75),IGHG400(75),IGHGP00(75) 285 296 316 0 11 110.0 43 55 93 16 28 SA46G 46.0;52 64 111 16 28 SG54C 46.0 25 37 68 39 51

Under the hood pipeline:

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

align

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

mixcr align \
    --species hsa \
    -p generic-amplicon-with-umi \
    -OvParameters.geneFeatureToAlign="VTranscriptWithout5UTRWithP" \
    -OvParameters.parameters.floatingLeftBound=false \
    -OjParameters.parameters.floatingRightBound=false \
    -OcParameters.parameters.floatingRightBound=false \
    --tagPattern "^N{22}(R1:*) \ ^(UMI:N{17})(R2:*)" \
    --report results/13_d60_LN_germCenterB.report.txt \
    --json-report results/13_d60_LN_germCenterB.report.json \
     raw/13_d60_LN_germCenterB_R1.fastq.gz \
     raw/13_d60_LN_germCenterB_R2.fastq.gz \
     results/13_d60_LN_germCenterB.vdjca

Option --report is specified here explicitly.

--species hsa
determines the organism species (hsa for Homo Sapiens).
-p eneric-amplicon-with-umi
generic preset of MiXCR parameters for amplicon data with UMIs.
-OvParameters.geneFeatureToAlign="VTranscriptWithout5UTRWithP"
Sets a V gene feature to align. Check gene features for more info.
-OvParameters.parameters.floatingLeftBound=false
Results in a global alignment algorithm for V gene left bound. We use it because we don't have any primers covering V gene coding sequence.
-OjParameters.parameters.floatingRightBound=false -OcParameters.parameters.floatingRightBound=false
Results in a global alignment algorithm for J and C gene right bound, because primer sequences have been trimmed by --tagPattern.
--tagPattern "^N{22}(R1:*) \ ^(UMI:N{17})(R2:*)"
tag pattern specifies the location of the UMI barcode and also trims C primer sequences located in the beginning of R1 (22 b.p. is the length of the longest primer).

refineTagsAndSort

Corrects sequencing and PCR errors inside barcode sequences. This step does extremely important job by correcting artificial diversity caused by errors in barcodes. In the considered example project it corrects only sequences of UMIs.

mixcr refineTagsAndSort \
    --report results/13_d60_LN_germCenterB.report.txt \
    --json-report results/13_d60_LN_germCenterB.report.json \
    results/13_d60_LN_germCenterB.vdjca \
    results/13_d60_LN_germCenterB.refined.vdjca

assemble

Assembles alignments into clonotypes and applies several layers of errors correction:

  • quality-dependent correction for sequencing errors
  • PCR-error correction by clustering
  • UMI-based error correction)

Check mixcr assemble for more information.

mixcr assemble \
    -OassemblingFeatures="CDR3" \
    -OseparateByJ=true \
    -OseparateByV=true \
    -OseparateByC=true \
    --report results/13_d60_LN_germCenterB.report.txt \
    --json-report results/13_d60_LN_germCenterB.report.json \
    results/13_d60_LN_germCenterB.refined.vdjca \
    results/13_d60_LN_germCenterB.clns

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

-OassemblingFeatures=CDR3
By default nebnext-mouse-bcr-cdr3 preset assembles clones by CDR3 sequence.
-separateByJ: true
Separate clones with the same assembling feature, but different V-genes.
-separateByV: true
Separate clones with the same assembling feature, but different J-genes.
-separateByC: true
Separate clones with the same assembling feature, but different C-genes, which is essential for isotype identification.

export

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

mixcr exportClones \
    -c IGH \
    -uniqueTagCount UMI \
    results/13_d60_LN_germCenterB.clns \
    results/13_d60_LN_germCenterB.clones.IGH.txt
-с IGH
defines a specific chain to be exported.
-uniqueTagCount UMI
adds a column with the number of UMIs for each clone.

Quality control

Now when we have all files processed lets perform Quality Control. That can be easily done using mixcr exportQc function.

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

align QC

Most of the samples have a high successful alignment score. But samples that come from blood memory B cells clearly have a lower percentage of aligned reads, and the major reason for that is the lack of immune receptor sequences. That might be due to some issues during sample preparation. It is recommended to realign one of these samples and save not aligned reads (see mixcr align) into separate file for manual inspection. That can be done with the following command:

mkdir -p debug

mixcr align \
    --species hsa \
    -p kAligner2_4.0 \
    -OvParameters.geneFeatureToAlign="VTranscriptWithout5UTRWithP" \
    -OvParameters.parameters.floatingLeftBound=false \
    -OjParameters.parameters.floatingRightBound=false \
    -OcParameters.parameters.floatingRightBound=false \
    --tag-pattern "^N{22}(R1:*) \ ^(UMI:N{17})(R2:*)" \
    --report debug/13_d60_LN_germCenterB.report.txt \
    --json-report debug/13_d60_LN_germCenterB.report.json \
    --not-aligned-R1 debug/13_d60_LN_germCenterB_notAligned_R1.fastq \
    --not-aligned-R2 debug/13_d60_LN_germCenterB_notAligned_R2.fastq \
     raw/13_d60_LN_germCenterB_R1.fastq.gz \
     raw/13_d60_LN_germCenterB_R2.fastq.gz \
     debug/13_d60_LN_germCenterB.vdjca

Resulting 13_d60_LN_germCenterB_notAligned_R1.fastq and 13_d60_LN_germCenterB_notAligned_R2.fastq files can be manually inspected. A brief BLAST search revealed most not aligned sequences come from DNA contamination (they align within sequence between segments) and with immunoglobulin like genes (e.g.IGLL5).

Now Lets look at the chain distribution in every sample.

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

If we look at the chain usage plot, we see, as expected, that the samples are almost entirely consist of IGH chains, since only heavy BCR chains we sequenced.

chainUsage.svg

Full-length clonotype assembly

In the example above, we have assembled clones by a default CDR3 feature. But NEBNext® Immune Sequencing Kit covers the whole sequence of V and J genes, because it is a 5'RACE based protocol. And also C primers allow to capture enough sequence for isotyping.

Taking into account what is mentioned above, the longest possible assembling feature for this protocol is VDJRegion.

MiXCR has a specific preset to obtain full-length BCR clones with NEBNext® Immune Sequencing Kit:

mixcr analyze neb-human-rna-xcr-umi-nebnext \
    raw/13_d60_LN_germCenterB_R1.fastq.gz \
    raw/13_d60_LN_germCenterB_R2.fastq.gz \
    results/13_d60_LN_germCenterB

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

mixcr assemble \
    -OassemblingFeatures="VDJRegion" \
    -OseparateByC=true \
    --report results/13_d60_LN_germCenterB.report.txt \
    --json-report results/13_d60_LN_germCenterB.report.json \
    results/13_d60_LN_germCenterB.refined.vdjca \
    results/13_d60_LN_germCenterB.clns
-OassemblingFeatures="VDJRegion"
sets the assembling feature to the region which starts from FR1Begin and ends at the end of FR4.

Notice we omit -OseparateByV=true and -OseparateByJ=true in this case because assembling feature already covers full V and J genes sequences, thus in case if clones have identical CDR3 they will still be separated. We still use -OseparateByC=true option for isotype identification.

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/13_d60_LN_germCenterB.clns \
    results/13_d60_LN_germCenterB.report.txt

mixcr exportReports \
    --json \
    results/13_d60_LN_germCenterB.clns \
    results/13_d60_LN_germCenterB.report.json
Show report file 
============== Align Report ==============
Input file(s): /raw/PRJNA777934_NEB_Immune_sequencing/13_d60_LN_germCenterB_R1.fastq.gz,/raw/PRJNA777934_NEB_Immune_sequencing/13_d60_LN_germCenterB_R2.fastq.gz
Output file(s): results/13_d60_LN_germCenterB.vdjca
Version: ; built=Mon Sep 26 10:55:18 CEST 2022; rev=8c998df1ab; lib=repseqio.v2.0
Command line arguments: --report results/13_d60_LN_germCenterB.align.report.txt --json-report results/13_d60_LN_germCenterB.align.report.json --preset local:nebnext-human-bcr-cdr3 +limitInput 100000 /raw/PRJNA777934_NEB_Immune_sequencing/13_d60_LN_germCenterB_R1.fastq.gz /raw/PRJNA777934_NEB_Immune_sequencing/13_d60_LN_germCenterB_R2.fastq.gz results/13_d60_LN_germCenterB.vdjca
Analysis time: 0ns
Total sequencing reads: 100000
Successfully aligned reads: 88595 (88.59%)
Paired-end alignment conflicts eliminated: 30693 (30.69%)
Alignment failed, no hits (not TCR/IG?): 3851 (3.85%)
Alignment failed because of absence of V hits: 3260 (3.26%)
Alignment failed because of absence of J hits: 2521 (2.52%)
No target with both V and J alignments: 1773 (1.77%)
Overlapped: 49087 (49.09%)
Overlapped and aligned: 44737 (44.74%)
Alignment-aided overlaps: 2058 (4.6%)
Overlapped and not aligned: 4350 (4.35%)
No CDR3 parts alignments, percent of successfully aligned: 44 (0.05%)
Partial aligned reads, percent of successfully aligned: 1970 (2.22%)
V gene chimeras: 2068 (2.07%)
J gene chimeras: 5 (0.01%)
IGH chains: 88288 (99.65%)
IGH non-functional: 2924 (3.31%)
IGK chains: 253 (0.29%)
IGK non-functional: 10 (3.95%)
IGL chains: 54 (0.06%)
IGL non-functional: 5 (9.26%)
Realigned with forced non-floating bound: 105942 (105.94%)
Realigned with forced non-floating right bound in left read: 25837 (25.84%)
Realigned with forced non-floating left bound in right read: 25837 (25.84%)
============== RefineTagsAndSort Report ==============
Input file(s): results/13_d60_LN_germCenterB.vdjca
Output file(s): results/13_d60_LN_germCenterB.refined.vdjca
Version: ; built=Mon Sep 26 10:55:18 CEST 2022; rev=8c998df1ab; lib=repseqio.v2.0
Command line arguments: --report results/13_d60_LN_germCenterB.refine.report.txt --json-report results/13_d60_LN_germCenterB.refine.report.json results/13_d60_LN_germCenterB.vdjca results/13_d60_LN_germCenterB.refined.vdjca
Analysis time: 0ns
Time spent in correction: 0ns
UMI input diversity: 44135
UMI output diversity: 23581 (53.43%)
UMI input reads: 88595
UMI output count: 70348 (79.4%)
UMI mean reads per tag: 2.01
UMI input core diversity: 23770 (53.86%)
UMI input core reads: 64187 (72.45%)
UMI directly corrected diversity: 5918 (13.41%)
UMI directly corrected reads: 6351 (7.17%)
UMI diversity filtered by tag quality: 14636 (33.16%)
UMI reads filtered by tag quality: 18247 (20.6%)
UMI diversity filtered by whitelist: 0 (0%)
UMI recursively corrected: 6
Number of output records: 52064 (58.77%)
Filter report:
  Number of groups: 23581
  Number of groups accepted: 11348 (48.12%)
  Total records weight: 70348.0
  Records weight accepted: 52064.0 (74.01%)
  Operator #0:
    Effective threshold: 3.0
============== Assemble Report ==============
Input file(s): results/13_d60_LN_germCenterB.refined.vdjca
Output file(s): results/13_d60_LN_germCenterB.clns
Version: ; built=Mon Sep 26 10:55:18 CEST 2022; rev=8c998df1ab; lib=repseqio.v2.0
Command line arguments: --report results/13_d60_LN_germCenterB.assemble.report.txt --json-report results/13_d60_LN_germCenterB.assemble.report.json results/13_d60_LN_germCenterB.refined.vdjca results/13_d60_LN_germCenterB.clns
Analysis time: 0ns
Number of input groups: 11348
Number of input alignments: 52064
Number of output pre-clonotypes: 10644
Number of clonotypes per group:
  0: + 674 (5.96%) = 674 (5.96%)
  1: + 10644 (94.04%) = 11318 (100%)
Number of core alignments: 47804 (91.82%)
Discarded core alignments: 1661 (3.47%)
Empirically assigned alignments: 1695 (3.26%)
Empirical assignment conflicts: 0 (0%)
UMI+VJ-gene empirically assigned alignments: 1695 (3.26%)
VJ-gene empirically assigned alignments: 0 (0%)
UMI empirically assigned alignments: 0 (0%)
Number of ambiguous UMIs: 0
Number of ambiguous UMI+V/J-gene combinations: 0
Unassigned alignments: 2430 (4.67%)
Final clonotype count: 1180
Average number of reads per clonotype: 39.9
Reads used in clonotypes, percent of total: 47080 (47.08%)
Reads used in clonotypes before clustering, percent of total: 49371 (49.37%)
Number of reads used as a core, percent of used: 49353 (99.96%)
Mapped low quality reads, percent of used: 18 (0.04%)
Reads clustered in PCR error correction, percent of used: 2291 (4.64%)
Reads pre-clustered due to the similar VJC-lists, percent of used: 108 (0.23%)
Reads dropped due to the lack of a clone sequence, percent of total: 2599 (2.6%)
Reads dropped due to a too short clonal sequence, percent of total: 0 (0%)
Reads dropped due to low quality, percent of total: 9 (0.01%)
Reads dropped due to failed mapping, percent of total: 71 (0.07%)
Reads dropped with low quality clones, percent of total: 57 (0.06%)
Clonotypes eliminated by PCR error correction: 223
Clonotypes dropped as low quality: 9
Clonotypes pre-clustered due to the similar VJC-lists: 12
IGH chains: 1173 (99.41%)
IGH non-functional: 31 (2.64%)
IGK chains: 5 (0.42%)
IGK non-functional: 0 (0%)
IGL chains: 2 (0.17%)
IGL non-functional: 0 (0%)

{
  "type": "alignerReport",
  "commandLine": "--report results/13_d60_LN_germCenterB.align.report.txt --json-report results/13_d60_LN_germCenterB.align.report.json --preset local:nebnext-human-bcr-cdr3 +limitInput 100000 /raw/PRJNA777934_NEB_Immune_sequencing/13_d60_LN_germCenterB_R1.fastq.gz /raw/PRJNA777934_NEB_Immune_sequencing/13_d60_LN_germCenterB_R2.fastq.gz results/13_d60_LN_germCenterB.vdjca",
  "inputFiles": [
    "/raw/PRJNA777934_NEB_Immune_sequencing/13_d60_LN_germCenterB_R1.fastq.gz",
    "/raw/PRJNA777934_NEB_Immune_sequencing/13_d60_LN_germCenterB_R2.fastq.gz"
  ],
  "outputFiles": [
    "results/13_d60_LN_germCenterB.vdjca"
  ],
  "version": "; built=Mon Sep 26 10:55:18 CEST 2022; rev=8c998df1ab; lib=repseqio.v2.0",
  "trimmingReport": null,
  "totalReadsProcessed": 100000,
  "aligned": 88595,
  "notAligned": 11405,
  "notAlignedReasons": {
    "NoJHits": 2521,
    "VAndJOnDifferentTargets": 1773,
    "NoCDR3Parts": 0,
    "NoBarcode": 0,
    "NoHits": 3851,
    "LowTotalScore": 0,
    "NoVHits": 3260
  },
  "chimeras": 0,
  "overlapped": 49087,
  "alignmentAidedOverlaps": 2058,
  "overlappedAligned": 44737,
  "overlappedNotAligned": 4350,
  "pairedEndAlignmentConflicts": 30693,
  "vChimeras": 2068,
  "jChimeras": 5,
  "chainUsage": {
    "type": "chainUsage",
    "chimeras": 0,
    "total": 88595,
    "chains": {
      "IGH": {
        "total": 88288,
        "nonFunctional": 2924,
        "isOOF": 516,
        "hasStops": 2408
      },
      "IGK": {
        "total": 253,
        "nonFunctional": 10,
        "isOOF": 7,
        "hasStops": 3
      },
      "IGL": {
        "total": 54,
        "nonFunctional": 5,
        "isOOF": 5,
        "hasStops": 0
      }
    }
  },
  "realignedWithForcedNonFloatingBound": 105942,
  "realignedWithForcedNonFloatingRightBoundInLeftRead": 25837,
  "realignedWithForcedNonFloatingLeftBoundInRightRead": 25837,
  "noCDR3PartsAlignments": 44,
  "partialAlignments": 1970,
  "tagReport": {
    "type": "tagReport"
  }
}
{
  "type": "refineTagsAndSort",
  "commandLine": "--report results/13_d60_LN_germCenterB.refine.report.txt --json-report results/13_d60_LN_germCenterB.refine.report.json results/13_d60_LN_germCenterB.vdjca results/13_d60_LN_germCenterB.refined.vdjca",
  "inputFiles": [
    "results/13_d60_LN_germCenterB.vdjca"
  ],
  "outputFiles": [
    "results/13_d60_LN_germCenterB.refined.vdjca"
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