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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 26|37|68|31|42||110.0 ;;; nan nan nan nan nan nan nan nan nan nan TGTGCGAGAGATTCCGTCTTCGACTTATTAATTGACTACTGG 45 nan nan nan nan nan nan nan CARDSVFDLLIDYW nan :::::::::0:1:12:21:-18:-4:30:31:-6:42:::
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 nan nan nan nan nan nan TGTGCGACTGCTTTGGTGGTGACTGCTGCATACTTCTGG 45 nan nan nan nan nan nan nan CATALVVTAAYFW nan :::::::::0:-4:7:13:-9:-5:27:31:-9:39:::
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 nan nan nan nan nan nan TGTGCGTCCCTACGGTGGCTACGGGGGGCCTTTGACTACTGG 45 nan nan nan nan nan nan nan CASLRWLRGAFDYW nan :::::::::0:-5:6:9:-3:1:23:29:-4:42:::
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 nan nan nan nan nan nan TGTGCGAGAGGCGACCGGAATTATCATGACAGTGGGTGGTCCGGCTGGTTCTTCGGTCTCTGG 45 nan nan nan nan nan nan nan CARGDRNYHDSGWSGWFFGLW nan :::::::::0:1:12:29:-8:1:43:44:-3:63:::
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 nan nan nan nan nan nan TGTGCGAGAGGCAATATGATAGTAGTGGCCTTTAGTGCTTTTGATATCTGG 45 nan nan nan nan nan nan nan CARGNMIVVAFSAFDIW nan :::::::::0:1:12:14:-7:-10:28:35:-3:51:::
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 nan nan nan nan nan nan TGTGCGAGTGAGAACGTGGATACAGGTATGGTTCCCTGGTTCGACCCCTGG 45 nan nan nan nan nan nan nan CASENVDTGMVPWFDPW nan :::::::::0:-3:8:13:2:-2:33:35:-4:51:::
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 nan nan nan nan nan nan TGTGCGAGTGAGAACGTGGATACAGGAATGGTTCCCTGGTTCGACCCCTGG 45 nan nan nan nan nan nan nan CASENVDTGMVPWFDPW nan :::::::::0:-3:8:13:2:-2:33:35:-4:51:::
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 nan nan nan nan nan nan TGTGCACACAGACTCGGCTCGTGGTTAAAGGGACTTCAGACTCACCCTATGATAGACCCAAGCGGGGACTGGTTCGACCCCTGG 45 nan nan nan nan nan nan nan CAHRLGSWLKGLQTHPMIDPSGDWFDPW nan :::::::::0:0:13:46:-6:-16:55:67:-3:84:::
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 nan nan nan nan nan nan TGTGCGAGACGGCCCCCAGTAGTGGTTATTCCGTTTGACTACTGG 45 nan nan nan nan nan nan nan CARRPPVVVIPFDYW nan :::::::::0:-2:9:17:-13:-5:30:33:-5:45:::
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 nan nan nan nan nan nan TGTGCGAGGTGGGGGCCTACGGTGACTCCCTACTTTGACTACTGG 45 nan nan nan nan nan nan nan CARWGPTVTPYFDYW nan :::::::::0:-3:8:16:-3:-2:27:29👎45:::
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 39|52|83|17|30||130.0 ;;; nan nan nan nan nan nan nan nan nan nan TGTGCGAGAGCCAACCATATGGACGTCTGG 45 nan nan nan nan nan nan nan CARANHMDVW nan :::::::::0:-1:10:12:14:-40:17:17:-19:30:::
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 nan nan nan nan nan nan TGTGCGGGCCAATTTGTAGTAGTTCCAGCTGCTAGTTACTACTACTACGCTATGGACGTCTGG 45 nan nan nan nan nan nan nan CAGQFVVVPAASYYYYAMDVW nan :::::::::0:-5:6:13:-6:-4:34:36:-5:63:::
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 nan nan nan nan nan nan TGTGCGAAATCCTATAGTGGGAGCTACTCCGCCGCTTTTGATATCTGG 45 nan nan nan nan nan nan nan CAKSYSGSYSAAFDIW nan :::::::::0:-2:9:12:-2:1:31:33:-4:48:::
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 nan nan nan nan nan nan TGTGCGCGTGAGAACGTGGATACAGGAATGGTTCCCTGGTTCGACCCCTGG 45 nan nan nan nan nan nan nan CARENVDTGMVPWFDPW nan :::::::::0:-5:6:13:2:-2:33:35:-4:51:::
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 24|37|68|29|42||130.0 ;;; nan nan nan nan nan nan nan nan nan nan TGTGCGTCCCTAAGGTGGCTACGGGGGGCCTTTGACTACTGG 45 nan nan nan nan nan nan nan CASLRWLRGAFDYW nan :::::::::0:-5:6:14:-9:-5:23:29:-4:42:::
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 nan nan nan nan nan nan TGTGCGAGAGATCAGGGATACAGCTATGGGCCATGGGGGGACTACTACGGTATGGACGTCTGG 45 nan nan nan nan nan nan nan CARDQGYSYGPWGDYYGMDVW nan :::::::::0:2:13:15:-2:-4:29:40:-9:63:::
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 nan nan nan nan nan nan TGTGCGACTGAGAACGTGGATACAGGCATGGTTCCCTGGTTCGACCCCTGG 45 nan nan nan nan nan nan nan CATENVDTGMVPWFDPW nan :::::::::0:-4:7:13:2:-2:33:35:-4:51:::
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 22|37|68|21|36||150.0 ;;; nan nan nan nan nan nan nan nan nan nan TGTGCGAGGACCCTTTACGATTACTTTGACTACTGG 45 nan nan nan nan nan nan nan CARTLYDYFDYW nan :::::::::0:-3:8:14:-3:-21:21:21:-2:36:::
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||130.0 ;;; nan nan nan nan nan nan nan nan nan nan TGTGCGTCCCTACGGTGGCTTCGGGGGGCCTTTGACTACTGG 45 nan nan nan nan nan nan nan CASLRWLRGAFDYW nan :::::::::0:-5:6:9:-3:-7:18:29:-4:42:::
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 nan nan nan nan nan nan TGTGCGAGAATCTCCGTCTATGATAGTAGTGGTTATTACTCTGACTACTGG 45 nan nan nan nan nan nan nan CARISVYDSSGYYSDYW nan :::::::::0:-2:9:17:-6:-9:33:33:1:51:::
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 nan nan nan nan nan nan TGTGCGAGGCCGTATAGTGGGAGCTACTTTTCTTACTTTGACTACTGG 45 nan nan nan nan nan nan nan CARPYSGSYFSYFDYW nan :::::::::0:-4:7:11👎-2:28:33:-2:48:::
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 29|37|68|43|51||80.0 ;;; nan nan nan nan nan nan nan nan nan nan TGTGCGAGAGATGACGGAAGCAGCGGCTGGTATGGACCGGGGAACTACTGG 45 nan nan nan nan nan nan nan CARDDGSSGWYGPGNYW nan :::::::::0:1:12:18:-6:5:38:43:-9:51:::
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 nan nan nan nan nan nan TGTGCGAGTGAGAACGTGGACACAGGTATGGTTCCCTGGTTCGACCCCTGG 45 nan nan nan nan nan nan nan CASENVDTGMVPWFDPW nan :::::::::0:-3:8:13:2:-2:33:35:-4:51:::
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 nan nan nan nan nan nan TGTGCGACCGGGATACAGTTACAGCTATGGTCTCACTACTTTGACTACTGG 45 nan nan nan nan nan nan nan CATGIQLQLWSHYFDYW nan :::::::::0:-4:7:19:-5:-3:31:34:0:51:::
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 nan nan nan nan nan nan TGTGCGACTGCTTTGGTGGTGACTGCTGCATACTTCTGG 45 nan nan nan nan nan nan nan CATALVVTAAYFW nan :::::::::0:-4:7:13:-9:-5:27:35:-17:39:::
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 nan nan nan nan nan nan TGTGCGACTGAGAACGTGGATACAGGTATGGTTCCCTGGTTCGACCCCTGG 45 nan nan nan nan nan nan nan CATENVDTGMVPWFDPW nan :::::::::0:-4:7:13:2:-2:33:35:-4:51:::
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 22|37|68|39|54|SC24TST27C|92.0 ;;; nan nan nan nan nan nan nan nan nan nan TGTGCACACAGGCGGGGAAGGGCCACATGGATGGCTGGTTATTTCGACTACTGG 45 nan nan nan nan nan nan nan CAHRRGRATWMAGYFDYW nan :::::::::0:1:14:31:-11:-2:39:39:-2:54:::
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 nan nan nan nan nan nan TGTGCGAGAGATGACGGAAGTACCGGCTGGTATGGACCGGGGAACTACTGG 45 nan nan nan nan nan nan nan CARDDGSTGWYGPGNYW nan :::::::::0:1:12:18:-13:-2:34:43:-9:51:::
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||30.0;37|40|71|36|39||30.0 ;;; nan nan nan nan nan nan nan nan nan nan TGTGCGAGAAGGGACTTCCAGAGGTCGTTCCTCTTTTGG 45 nan nan nan nan nan nan nan CARRDFQRSFLFW nan :::::::::0:-2:9:20:-14:8:31:36:-14:39:::
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 nan nan nan nan nan nan TGTGCGAGACGGCCGGCTACTATGATATTTCTGGCCCACACCAAGAAAGACTACTTTGACTACTGG 45 nan nan nan nan nan nan nan CARRPATMIFLAHTKKDYFDYW nan :::::::::0:-2:9:17:-4:-17:27:49:0:66:::
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 nan nan nan nan nan nan TGTGCCAGACGGCCCCCAGTAGTGGTTATTCCGTTTGACTACTGG 45 nan nan nan nan nan nan nan CARRPPVVVIPFDYW nan :::::::::0:-2:9:17:-13:-5:30:33:-5:45:::
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 nan nan nan nan nan nan TGTGCGAGAGATGACGGAAGCAGCAGCTGGTATGGACCGGGGAACTACTGG 45 nan nan nan nan nan nan nan CARDDGSSSWYGPGNYW nan :::::::::0:1:12:18:-6:5:38:43:-9:51:::
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 nan nan nan nan nan nan TGTGCGAGCAGTGGCTGGCACCGGGGGGCCTTTGACTACTGG 45 nan nan nan nan nan nan nan CASSGWHRGAFDYW nan :::::::::0:-3:8:8:-8:0:21:29:-4:42:::
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 nan nan nan nan nan nan TGTGCGAGCGCTCCCCGCCCGTATTACTATGATAGTAGTGCTTATTACCCATTTGACTACTGG 45 nan nan nan nan nan nan nan CASAPRPYYYDSSAYYPFDYW nan :::::::::0:-3:8:19:1:-3:48:51:-5:63:::
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 nan nan nan nan nan nan TGTGCGAGAGGAGCTACGGGAGATGCTTTTGATATCTGG 45 nan nan nan nan nan nan nan CARGATGDAFDIW nan :::::::::0:-2:9:9:-9:-3:17:21👎39:::
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 nan nan nan nan nan nan TGTGCGATTGAGAACGTGGATACAGGTATGGTTCCCTGGTTCGACCCCTGG 45 nan nan nan nan nan nan nan CAIENVDTGMVPWFDPW nan :::::::::0:-4:7:13:2:-2:33:35:-4:51:::
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 nan nan nan nan nan nan TGTGCGAGAGAACGGGATATTGTAGTAGTACCAGCGTCTTACTTTGACTACTGG 45 nan nan nan nan nan nan nan CARERDIVVVPASYFDYW nan :::::::::0:0:11:14👎-9:35:39:-2:54:::
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 nan nan nan nan nan nan TGTGCGAGAGAAAGTCCCGAGCGCATTGTAGTGGAGGTAGCTGCTACGACCAACTGGTTCGACCCCTGG 45 nan nan nan nan nan nan nan CARESPERIVVEVAATTNWFDPW nan :::::::::0:0:11:24:-5:-3:47:50👎69:::
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 27|52|83|32|57||250.0 ;;; nan nan nan nan nan nan nan nan nan nan TGTGCGAAAGATCGGGGCGATGACTACGCCAGCTACTACTACGGTATGGACGTCTGG 45 nan nan nan nan nan nan nan CAKDRGDDYASYYYGMDVW nan :::::::::0:2:13:19:1:-8:28:32:-7:57:::
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 22|39|70|25|42||170.0 ;;; nan nan nan nan nan nan nan nan nan nan TGTGCGAGAGTGAATGGCTACGGAAATGCTTTTGATATCTGG 45 nan nan nan nan nan nan nan CARVNGYGNAFDIW nan :::::::::0:-1:10:14:-10:-5:22:25:-2:42:::
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||160.0 ;;; nan nan nan nan nan nan nan nan nan nan TGTGCGAGACCAGGGTGGCTACGAGGTGCTTTTGATATCTGG 45 nan nan nan nan nan nan nan CARPGWLRGAFDIW nan :::::::::0:-1:10:14:-9:-4:24:26:-3:42:::
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 nan nan nan nan nan nan TGTGCGAGACATCCCTCACCTTATTACTATGGTTCGGGGAGATATGAGTACGGTGTGGACGTCTGG 45 nan nan nan nan nan nan nan CARHPSPYYYGSGRYEYGVDVW nan :::::::::0:1:12:21👎-6:45:48:-14:66:::
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 nan nan nan nan nan nan TGTGCGAGAGATCAGGGATACAGCTATGGGCCATGGGGGGACTACTACGGTATGGACGACTGG 45 nan nan nan nan nan nan nan CARDQGYSYGPWGDYYGMDDW nan :::::::::0:2:13:15:-2:-4:29:40:-9:63:::
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 nan nan nan nan nan nan TGTGCAAAAGATATAGGGTATAGCAGTGGTGGGACCGACTACTGG 45 nan nan nan nan nan nan nan CAKDIGYSSGGTDYW nan :::::::::0:2:15:15:0:0:35:36:-8:45:::
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 nan nan nan nan nan nan TGTGCGGGCCAATTTGTAGTAGTACCAGCTGCTAGTTACTACTACTACGCTATGGACGTCTGG 45 nan nan nan nan nan nan nan CAGQFVVVPAASYYYYAMDVW nan :::::::::0:-5:6:13:-6:-4:34:36:-5:63:::
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||130.0 ;;; nan nan nan nan nan nan nan nan nan nan TGTGCGAGGTGTTTCGGTGGGAACTATTACGGGCCCTTTGACTACTGG 45 nan nan nan nan nan nan nan CARCFGGNYYGPFDYW nan :::::::::0:-4:7:16:-6:1:31:35:-4:48:::
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 nan nan nan nan nan nan TGTGCGGGCCAATCTGTAGTAGTACCAGCTGCTAGTTACTACTACTACGCTATGGACGTCTGG 45 nan nan nan nan nan nan nan CAGQSVVVPAASYYYYAMDVW nan :::::::::0:-5:6:14:-7:-4:34:36:-5:63:::
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 nan nan nan nan nan nan TGTGCGAAGTCCTATAGTGGGAGCTACTCCGCCGCTTTTGATATCTGG 45 nan nan nan nan nan nan nan CAKSYSGSYSAAFDIW nan :::::::::0:-3:8:12:-2:1:31:33:-4:48:::
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 nan nan nan nan nan nan TGTGCGAGCAGTGGCTGGCACCGGGGGGCCTTTGACTACTGG 45 nan nan nan nan nan nan nan CASSGWHRGAFDYW nan :::::::::0:-3:8:8:-8:0:21:29:-4:42:::
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 nan nan nan nan nan nan TGTGCGACCGGGATACAGTTACACCTATGGTCTCACTACTTTGACTACTGG 45 nan nan nan nan nan nan nan CATGIQLHLWSHYFDYW nan :::::::::0:-4:7:19:-5:-3:31:34:0:51:::
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 18|37|68|29|48||190.0 ; nan nan nan nan nan nan nan nan nan nan TGTGCGAGACTTACCAGCAGTTTTCCTCCGTACTACTTTGACTACTGG 45 nan nan nan nan nan nan nan CARLTSSFPPYYFDYW nan :::::::::0:-4:7:8:-19:8:18:29:2:48:::
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 nan nan nan nan nan nan TGTGCGAGAGATCTGGGGGGCTGGTTCGACCCCTGG 45 nan nan nan nan nan nan nan CARDLGGWFDPW nan :::::::::0:3:14:14:-17:-15:19:20:-4:36:::
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 26|37|68|28|39||110.0 ;;; nan nan nan nan nan nan nan nan nan nan TGTGCGAGAAGGCAGATGCCCTACACCCTTGACTACTGG 45 nan nan nan nan nan nan nan CARRQMPYTLDYW nan :::::::::0:-2:9:21:22:-47:27:28:-6:39:::
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 nan nan nan nan nan nan TGTGCGAGAGATCAGGGATACAGCTATGGGCCATGGGGGGACTACTACGGTCTGGACGTCTGG 45 nan nan nan nan nan nan nan CARDQGYSYGPWGDYYGLDVW nan :::::::::0:2:13:15:-2:-4:29:40:-9:63:::
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 nan nan nan nan nan nan TGTGTGTCCCTACGGTGGCTACGGGGGGCCTTTGACTACTGG 45 nan nan nan nan nan nan nan CVSLRWLRGAFDYW nan :::::::::0:-7:4:9:-3:1:23:29:-4:42:::
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 24|37|68|29|42||130.0 ;;; nan nan nan nan nan nan nan nan nan nan TGTGCGTCCCTAGGGTGGCTACGGGGGGCCTTTGACTACTGG 45 nan nan nan nan nan nan nan CASLGWLRGAFDYW nan :::::::::0:-5:6:14:-9:-5:23:29:-4:42:::
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 nan nan nan nan nan nan TGTGCGAGAGAAAGTCCCGAGCGGATTGTAGTGGTGCTAGCTGCTACGACCAACTGGTTCGACCCCTGG 45 nan nan nan nan nan nan nan CARESPERIVVVLAATTNWFDPW nan :::::::::0:0:11:24:-5:-3:47:50👎69:::
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 29|37|68|43|51||80.0 ;;; nan nan nan nan nan nan nan nan nan nan TGTGCGGCAGATGACGGAAGCAGCGGCTGGTTTGGACCGGGGAACTACTGG 45 nan nan nan nan nan nan nan CAADDGSSGWFGPGNYW nan :::::::::0:1:12:18:-6:-2:31:43:-9:51:::
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 nan nan nan nan nan nan TGTGCGAGAGAAAGTCCCGAGCGGATTGTAGTGATGGTAGCTGCTACGACCAACTGGTTCGACCCCTGG 45 nan nan nan nan nan nan nan CARESPERIVVMVAATTNWFDPW nan :::::::::0:0:11:24:-5:-3:47:50👎69:::
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 TGTGCGCGACTTTCCTACTACTACGGTATGGACGTCTGG 45 nan nan nan nan nan nan nan CARLSYYYGMDVW nan :::::::::0:-2:9:::::14:-7:39:::
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 nan nan nan nan nan nan TGTGCGAGACAACCCTTACGATATTTTGACTGGAACTGGCACTTCGATCTCTGG 45 nan nan nan nan nan nan nan CARQPLRYFDWNWHFDLW nan :::::::::0:-2:9:15:-3:-10:33:34:-2:54:::
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||30.0;37|40|71|36|39||30.0 ;;; nan nan nan nan nan nan nan nan nan nan TGTGCGAGAAGGGACTTCCAGACGTCGTTCCTCTTTTGG 45 nan nan nan nan nan nan nan CARRDFQTSFLFW nan :::::::::0:-2:9:20:-14:8:31:36:-14:39:::
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 33|52|83|41|60||190.0 ;;; nan nan nan nan nan nan nan nan nan nan TGTGCGAGAGATTACACCCCTCTCTCCTTCGGGGGGGCTCGCTACGGTATGGACGTCTGG 45 nan nan nan nan nan nan nan CARDYTPLSFGGARYGMDVW nan :::::::::0:1:12:12:22:-47:18:41:-13:60:::
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 nan nan nan nan nan nan TGTGCACAGAGCAGTGGCTGGTCGTACTACTTTGACTACTGG 45 nan nan nan nan nan nan nan CAQSSGWSYYFDYW nan :::::::::0:-2:11:11:-8:-4:20:20:5:42:::
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 nan nan nan nan nan nan TGTGCGAGGCTACTATGGTTCGGGGCATATGCTTTTGATATCTGG 45 nan nan nan nan nan nan nan CARLLWFGAYAFDIW nan :::::::::0:-3:8:10:-4:-12:25:28:-2:45:::
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 19|37|68|30|48|ST25AST26GSA29C|93.0 ;;; nan nan nan nan nan nan nan nan nan nan TGTGCGAGCACCGATTGGGGTACTGGGAGATACTACAGTGCCTACTGG 45 nan nan nan nan nan nan nan CASTDWGTGRYYSAYW nan :::::::::0:-3:8:19:-8:-16:26:30:1:48:::
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 22|37|68|27|42||150.0 ;;; nan nan nan nan nan nan nan nan nan nan TGTGCGTCTGTTTGGTGGCTGAGGGGGTACTTTGACTACTGG 45 nan nan nan nan nan nan nan CASVWWLRGYFDYW nan :::::::::0:-5:6:14:-10:-4:21:27:-2:42:::
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 nan nan nan nan nan nan TGTGCGAGCCTCATGTATTTCTATGGTCCCCTTGACCACTGG 45 nan nan nan nan nan nan nan CASLMYFYGPLDHW nan :::::::::0:-3:8:14:0:-18:27:31:-6:42:::
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 nan nan nan nan nan nan TGTGCGAGAGGGTTACGATATTTTGACTGGTTATTTCACAGTCAAGTCAACGAACACAACTGGTTCGACCCCTGG 45 nan nan nan nan nan nan nan CARGLRYFDWLFHSQVNEHNWFDPW nan :::::::::0:-2:9:12:-3:-5:35:55:0:75:::
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 nan nan nan nan nan nan TGTGCGACTGCTTTGGTGGTGACTGCTGCATACTTCTGG 45 nan nan nan nan nan nan nan CATALVVTAAYFW nan :::::::::0:-4:7:13:-9:-5:27:31:-24:39:::
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 nan nan nan nan nan nan TGTGCACACACAGTGGATACGATTTTTGGAGTGGTTATTTTTGACTACTGG 45 nan nan nan nan nan nan nan CAHTVDTIFGVVIFDYW nan :::::::::0:-3:10:17:-4:-5:39:39:-5:51:::
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 nan nan nan nan nan nan TGTGCTAGAGTGGGCGGCGCTTTTGATATCTGG 45 nan nan nan nan nan nan nan CARVGGAFDIW nan :::::::::0:-1:10:13:-10:-3:18:18:-4:33:::
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 28|52|83|33|57||240.0 ;;; nan nan nan nan nan nan nan nan nan nan TGTGCGAGAGATCGGGGCGATGACTACGCCAGTTACTACTACGGTATGGACGTCTGG 45 nan nan nan nan nan nan nan CARDRGDDYASYYYGMDVW nan :::::::::0:2:13:19:1:-8:28:33:-8:57:::
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 nan nan nan nan nan nan TGTGCGAGAGAAAGTCCCGAGCGGATTGTAGTGGTGATAGCTACTACGACCAACTGGTTTGACCCCTGG 45 nan nan nan nan nan nan nan CARESPERIVVVIATTTNWFDPW nan :::::::::0:0:11:24:-5:-3:47:50👎69:::
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 nan nan nan nan nan nan TGTGCGTCCCTACGGTGGCTACGGGGGGCCTTTGAGTACTGG 45 nan nan nan nan nan nan nan CASLRWLRGAFEYW nan :::::::::0:-5:6:9:-3:1:23:29:-4:42:::
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||110.0 ; nan nan nan nan nan nan nan nan nan nan TGTGCGAAGGAAGGGGCGTTCGGGGAGTTAGGGCTCGGCCTTGACTACTGG 45 nan nan nan nan nan nan nan CAKEGAFGELGLGLDYW nan :::::::::0:0:11:17:-11:-7:30:40:-6:51:::
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 nan nan nan nan nan nan TGTACGAGACTGCCGGATTACTATGATAGTAGTGACTACTGG 45 nan nan nan nan nan nan nan CTRLPDYYDSSDYW nan :::::::::0:-1:10:13:1:-13:32:32:-7:42:::
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 nan nan nan nan nan nan TGTGCGGGCCAATTTGTAGTAGTGCCAGCCGCTAGTTACTACTATTACGCTATGGACGTCTGG 45 nan nan nan nan nan nan nan CAGQFVVVPAASYYYYAMDVW nan :::::::::0:-5:6:13:-6:-4:34:36:-5:63:::
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 TGTGCGAGACTTTCCTACTACTACGGTATGGACGTCTGG 45 nan nan nan nan nan nan nan CARLSYYYGMDVW nan :::::::::0:-2:9:::::14:-7:39:::
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 nan nan nan nan nan nan TGTGCGAGAGAAAGTCCCGAGCGGATTGTAGTGGTGTTAGCTGCTACGACCAACTGGTTCGACCCCTGG 45 nan nan nan nan nan nan nan CARESPERIVVVLAATTNWFDPW nan :::::::::0:0:11:24:-5:-3:47:50👎69:::
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 24|37|68|29|42||130.0 ;;; nan nan nan nan nan nan nan nan nan nan TGTGCGTCCCTTCGGTGGCTACGGGGGGCCTTTGACTACTGG 45 nan nan nan nan nan nan nan CASLRWLRGAFDYW nan :::::::::0:-5:6:14:-9:-5:23:29:-4:42:::
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 20|37|68|49|66||170.0 ;;; nan nan nan nan nan nan nan nan nan nan TGTGCGAGACGTCCGGCCACTACGATAGTACTGGCCCACACCAAGAAAGACTACTTTGACTACTGG 45 nan nan nan nan nan nan nan CARRPATTIVLAHTKKDYFDYW nan :::::::::0:-2:9:18:-5:-10:34:49:0:66:::
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 nan nan nan nan nan nan TGTGCGAAATCCTACAGTGGGAGCTACTCCGCCGCTTTTGATATCTGG 45 nan nan nan nan nan nan nan CAKSYSGSYSAAFDIW nan :::::::::0:-2:9:12:-2:1:31:33:-4:48:::
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 nan nan nan nan nan nan TGTGCGAGAGAAAGTCCCGAGCGGATTGTAGTGGTGGTAGCTGCTACGACCAACTGGTTCGACCCCTGG 45 nan nan nan nan nan nan nan CARESPERIVVVVAATTNWFDPW nan :::::::::0:0:11:24:-5:-3:47:50👎69:::
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 nan nan nan nan nan nan TGTGCGGGTGAGAACGTGGATACAGGAATGGTTCCCTGGTTCGACCCCTGG 45 nan nan nan nan nan nan nan CAGENVDTGMVPWFDPW nan :::::::::0:-5:6:13:2:-2:33:35:-4:51:::
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 nan nan nan nan nan nan TGTGCGAGGTGTTTCGGTGGGAGCTACTACGGGCCCTTTGACTACTGG 45 nan nan nan nan nan nan nan CARCFGGSYYGPFDYW nan :::::::::0:-4:7:16:-6:1:31:35:-4:48:::
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 22|37|68|39|54|SC24TST27C|92.0 ;;; nan nan nan nan nan nan nan nan nan nan TGTGCACACAGACGGGGAAGGGCCACATGGATGGCTGGTTATTTCGACTACTGG 45 nan nan nan nan nan nan nan CAHRRGRATWMAGYFDYW nan :::::::::0:1:14:31:-11:-2:39:39:-2:54:::
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 20|37|68|34|51||170.0 ; nan nan nan nan nan nan nan nan nan nan TGTGCGACCGGGATACAGTTACAGTTATGGTCTCACTACTTTGACTACTGG 45 nan nan nan nan nan nan nan CATGIQLQLWSHYFDYW nan :::::::::0:-4:7:16:15:-20:28:34:0:51:::
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 nan nan nan nan nan nan TGTGCGAGGTGGGGGCCTACGGTGACTCCCTACTTTGACTACTGG 45 nan nan nan nan nan nan nan CARWGPTVTPYFDYW nan :::::::::0:-3:8:16:-3:-2:27:29👎45:::
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||120.0 ;;; nan nan nan nan nan nan nan nan nan nan TGTGCGAGACGGCCCCCACTAGTGGTTATTCCGTTTGACTACTGG 45 nan nan nan nan nan nan nan CARRPPLVVIPFDYW nan :::::::::0:-2:9:19:-15:-5:30:33:-5:45:::
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 24|37|68|38|51||130.0 ;; nan nan nan nan nan nan nan nan nan nan TGTGCGAGAGACGAAATTCGGGGAATTAGATCCAGAACCTTTGACTACTGG 45 nan nan nan nan nan nan nan CARDEIRGIRSRTFDYW nan :::::::::0:0:11:16:-12:-7:28:38:-4:51:::
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 nan nan nan nan nan nan TGTGCACGGATACCGGGGGATTTTTGGAGTGGTTATTCCTACTTTGACTACTGG 45 nan nan nan nan nan nan nan CARIPGDFWSGYSYFDYW nan :::::::::0:0:13:18:-7:-5:37:38👎54:::
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 nan nan nan nan nan nan TGTGCGAGAGGCAATATGATAATAGTGGCCTTTAGTGCTTTTGATATCTGG 45 nan nan nan nan nan nan nan CARGNMIIVAFSAFDIW nan :::::::::0:1:12:14:-7:-10:28:35:-3:51:::
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 nan nan nan nan nan nan TGTGCGAGAGGAGGTGGATACAGCTATGGGGCCCTTGACTACTGG 45 nan nan nan nan nan nan nan CARGGGYSYGALDYW nan :::::::::0:0:11:13:0:-4:29:34:-6:45:::
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||60.0 ;;; nan nan nan nan nan nan nan nan nan nan TGTGCGCGAGATCTCACTACGGTGTCGATCTGG 45 nan nan nan nan nan nan nan CARDLTTVSIW nan :::::::::0:4:15:15:-2:-5:24:27:-13:33:::
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 nan nan nan nan nan nan TGTGCGAGCGCTCCCCGCCCGTATTACTATGATAGTAGTGGTTATTACCCATTTGACTACTGG 45 nan nan nan nan nan nan nan CASAPRPYYYDSSGYYPFDYW nan :::::::::0:-3:8:19:1:-3:48:51:-5:63:::
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 nan nan nan nan nan nan TGTGCGACTGAGAACGTGGATACAGGAATGGTTCCCTGGTTCGACCCCTGG 45 nan nan nan nan nan nan nan CATENVDTGMVPWFDPW nan :::::::::0:-4:7:13:2:-2:33:35:-4:51:::
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 nan nan nan nan nan nan TGTGCGAACAGTGGCTGGCACCGGGGGGCCTTTGACTACTGG 45 nan nan nan nan nan nan nan CANSGWHRGAFDYW nan :::::::::0:-4:7:8:-8:0:21:29:-4:42:::
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 nan nan nan nan nan nan TGTGCGAGACGGCCGGCTACTCTGATAGTAATGGCCCACACCAAGAATGACTACTTTGACTACTGG 45 nan nan nan nan nan nan nan CARRPATLIVMAHTKNDYFDYW nan :::::::::0:-2:9:17:-4:-10:34:49:0:66:::
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||120.0 ;;; nan nan nan nan nan nan nan nan nan nan TGTGCGAGAGACGAAATTCGGGGAGTTAGATCCAGAATGTTTGACTACTGG 45 nan nan nan nan nan nan nan CARDEIRGVRSRMFDYW nan :::::::::0:0:11:16:-12:-7:28:39:-5:51:::

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",
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  "trimmingReport": null,
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  "notAligned": 11405,
  "notAlignedReasons": {
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    "NoCDR3Parts": 0,
    "NoBarcode": 0,
    "NoHits": 3851,
    "LowTotalScore": 0,
    "NoVHits": 3260
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  "chimeras": 0,
  "overlapped": 49087,
  "alignmentAidedOverlaps": 2058,
  "overlappedAligned": 44737,
  "overlappedNotAligned": 4350,
  "pairedEndAlignmentConflicts": 30693,
  "vChimeras": 2068,
  "jChimeras": 5,
  "chainUsage": {
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    "chimeras": 0,
    "total": 88595,
    "chains": {
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        "nonFunctional": 2924,
        "isOOF": 516,
        "hasStops": 2408
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      "IGK": {
        "total": 253,
        "nonFunctional": 10,
        "isOOF": 7,
        "hasStops": 3
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      "IGL": {
        "total": 54,
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  "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",
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  "version": "; built=Mon Sep 26 10:55:18 CEST 2022; rev=8c998df1ab; lib=repseqio.v2.0",
  "correctionReport": {
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    "outputRecords": 52064,
    "steps": [
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        "tagName": "UMI",
        "inputGroups": 1,
        "inputDiversity": 44135,
        "inputCount": 88595,
        "coreDiversity": 23770,
        "coreCount": 64187,
        "directlyCorrectedDiversity": 5918,
        "directlyCorrectedCount": 6351,
        "filteredDiversity": 14636,
        "filteredCount": 18247,
        "recursivelyCorrected": 6,
        "diversityFilteredByWhitelist": 0,
        "outputDiversity": 23581,
        "outputCount": 70348
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    "filterReport": {
      "type": "filter_groups_report",
      "groupingKeys": [
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      "numberOfGroups": 23581,
      "numberOfGroupsAccepted": 11348,
      "totalWeight": 70348,
      "totalWeightAccepted": 52064,
      "operatorReports": [
        {
          "type": "generic_hist_report",
          "threshold": 3
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      ],
      "metricHists": [
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          "metric": {
            "type": "group_metric_sum_weight",
            "reportHist": {
              "log": true,
              "binNumber": 0,
              "minBinWidth": 0.2
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          "hist": {
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              "minBinWidth": 0.2
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{
  "type": "assemblerReport",
  "commandLine": "--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",
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  "version": "; built=Mon Sep 26 10:55:18 CEST 2022; rev=8c998df1ab; lib=repseqio.v2.0",
  "preCloneAssemblerReport": {
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    "inputGroups": 11348,
    "inputAlignments": 52064,
    "clonotypes": 10644,
    "clonotypesPerGroup": {
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      "1": 10644
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    "coreAlignments": 47804,
    "discardedCoreAlignments": 1661,
    "empiricallyAssignedAlignments": 1695,
    "vjEmpiricallyAssignedAlignments": 0,
    "umiEmpiricallyAssignedAlignments": 0,
    "gatEmpiricallyAssignedAlignments": 1695,
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    "unassignedAlignments": 2430,
    "umiConflicts": 0,
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    "geneConflicts": {},
    "coreClonotypesDroppedByTagSuffix": 0,
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  "totalReadsProcessed": 100000,
  "initialClonesCreated": 1424,
  "readsDroppedNoTargetSequence": 2599,
  "readsDroppedTooShortClonalSequence": 0,
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  "coreReads": 49353,
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  "clonesClustered": 223,
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  "readsInClones": 47080,
  "readsInClonesBeforeClustering": 49371,
  "readsDroppedWithLowQualityClones": 57,
  "clonalChainUsage": {
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    "chimeras": 0,
    "total": 1180,
    "chains": {
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      "IGK": {
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      "IGL": {
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