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Built-in presets

MiXCR provides a comprehensive list of built-in presets for many of available commercial kits, data types and library preparation protocols.

Preset can be used to run the whole upstream analysis pipeline with analyze command. For example:

mixcr analyze milab-human-bcr-multiplex-cdr3 \
    --dont-split-clones-by C \
      sample_R1.fastq.gz \
      sample_R2.fastq.gz \
      sample_result
runs upstream analysis for samples obtained using Milaboratories Human BCR kit with additional optional config --dont-split-clones-by C.

Command exportPreset can help to understand structure of preset.

Bellow you one can find a variety of presets for different types of input data and commercially available kits. Most of these presets do not require any additional arguments.

Kits

MiLaboratories

Human Ig RNA Multiplex

milab-human-bcr-multiplex-full-length · milab-human-bcr-multiplex-cdr3 · Link · Code

Allows to obtain full length IG heavy and light chain repertoires with UMI-based accuracy. Discriminates all IGH isotypes including IgM, IgD, IgG3, IgG1, IgA1, IgG2, IgG4, IgE, and IgA2. -cdr3 preset may be used to reduce clonotype assembling feature from full V-D-J region to CDR3 only.

By default, separates clonotypes by isotype which may be changed using --dont-split-clones-by C mix-in option.

Example:

mixcr analyze milab-human-bcr-multiplex-full-length \
      input_R1.fastq.gz \
      input_R2.fastq.gz \
      result 

Human TCR RNA Multiplex

milab-human-tcr-rna-multiplex-cdr3 · Link · Code · Tutorial

Allows to obtain human TCR alpha and beta CDR3 repertoires for different types of available RNA material, with high sensitivity and UMI-based accuracy.

Example:

mixcr analyze milab-human-tcr-rna-multiplex-cdr3 \
      input_R1.fastq.gz \
      input_R2.fastq.gz \
      result 

Human TCR RNA

milab-human-tcr-rna-race-cdr3 · milab-human-tcr-rna-race-full-length · Link · Code

Allows to obtain unbiased TCR alpha and beta repertoires with UMI-based accuracy. -cdr3 preset may be used to reduce clonotype assembling feature from full V-D-J region to CDR3 only.

Example:

mixcr analyze milab-human-tcr-rna-race-cdr3 \
      input_R1.fastq.gz \
      input_R2.fastq.gz \
      result 

Human TCR DNA Multiplex

milab-human-tcr-dna-multiplex-cdr3 · Link · Code

Allows to obtain TCR alpha and beta repertoires for different types of available DNA material, with the highest possible sensitivity. Clones are assembled by CDR3 sequence.

Example:

mixcr analyze milab-human-tcr-dna-multiplex-cdr3 \
      input_R1.fastq.gz \
      input_R2.fastq.gz \
      result 

Mouse TCR RNA

milab-mouse-tcr-rna-race-cdr3 · milab-mouse-tcr-rna-race-full-length · Link · Code

The kit allows to obtain unbiased TCR alpha and beta repertoires with UMI-based accuracy. -cdr3 preset may be used to reduce clonotype assembling feature from full V-D-J region to CDR3 only.

Example:

mixcr analyze milab-mouse-tcr-rna-race-cdr3 \
      input_R1.fastq.gz \
      input_R2.fastq.gz \
      result 

Takara Bio

SMART-Seq Human BCR (with UMIs)

takara-human-bcr-cdr3 · takara-human-bcr-full-length · Link · Code

SMART-Seq Human BCR Kit (with UMIs) provides a sensitive and reproducible solution for generating high-quality NGS libraries for profiling the human BCR repertoire. The kit leverages SMART (Switching Mechanism at 5' end of RNA Template) full-length cDNA synthesis technology and pairs NGS with a 5’-RACE approach to capture the complete V(D)J variable regions of all human B-cell receptor (BCR) heavy (IgG/M/D/A/E) and light (IgK/L) chains. The -cdr3 preset may be used to reduce clonotype assembling feature from full V-D-J region to CDR3 only. Mix-in option --dont-split-clones-by C may be used to not separate clones by isotypes.

Example:

mixcr analyze takara-human-bcr-cdr3 \
      input_R1.fastq.gz \
      input_R2.fastq.gz \
      result 

SMARTer Human BCR IgG IgM H/K/L Profiling Kit

takara-human-bcr-cdr3 · takara-human-bcr-full-length · Link · Code · Tutorial

SMARTer Human BCR IgG IgM H/K/L Profiling Kit pairs 5' RACE with NGS technology to provide a sensitive, accurate, and optimized approach to BCR profiling from RNA input samples. The 5' RACE method reduces variability and allows for priming from the constant region of BCR heavy or light chains. This kit combines these benefits with gene-specific amplification to capture complete V(D)J variable regions of BCR transcripts and provide a highly sensitive and reproducible method for profiling B-cell repertoires. The -cdr3 preset may be used to reduce clonotype assembling feature from full V-D-J region to CDR3 only. Mix-in option--dont-split-clones-by C mix-in may be used to not separate clones by isotypes.

Example:

mixcr analyze takara-human-bcr-cdr3 \
      input_R1.fastq.gz \
      input_R2.fastq.gz \
      result 

SMARTer Human TCR a/b Profiling Kit v2

takara-human-tcr-V2-cdr3 · takara-human-tcr-V2-full-length · Link · Code

The SMARTer Human TCR a/b Profiling Kit v2 (TCRv2) is powered by robust chemistry that provides unparalleled sensitivity and reproducibility. The kit leverages SMART (Switching Mechanism at 5' end of RNA Template) full-length cDNA synthesis technology and pairs NGS with a 5'-RACE approach to capture the complete V(D)J variable regions of TRA and TRB genes. The -cdr3 preset may be used to reduce clonotype assembling feature from full V-D-J region to CDR3 only.

Example:

mixcr analyze takara-human-tcr-V2-cdr3 \
      input_R1.fastq.gz \
      input_R2.fastq.gz \
      result 

SMARTer Human TCR a/b Profiling Kit

takara-human-tcr-V1-cdr3 · takara-human-tcr-V1-full-length · Link · Code

SMARTer Human TCR a/b Profiling Kit allows to obtain full-length sequences of TCR-alpha and TCR-beta V(D)J variable regions. The -cdr3 preset may be used to reduce clonotype assembling feature from full V-D-J region to CDR3 only.

Example:

mixcr analyze takara-human-tcr-V1-cdr3 \
      input_R1.fastq.gz \
      input_R2.fastq.gz \
      result 

SMARTer Mouse BCR IgG H/K/L Profiling Kit

takara-mouse-bcr-cdr3 · takara-mouse-bcr-full-length · Link · Code

The SMARTer Mouse BCR IgG H/K/L Profiling Kit pairs 5' RACE with NGS technology to provide a sensitive, accurate, and optimized approach to BCR profiling. The 5'-RACE method reduces variability and allows for priming from the constant region of BCR heavy or light chains. This kit combines these benefits with gene-specific amplification to capture complete V(D)J variable regions of BCR transcripts and provide a highly sensitive and reproducible method for profiling B-cell repertoires. The -cdr3 preset may be used to reduce clonotype assembling feature from full V-D-J region to CDR3 only.

Example:

mixcr analyze takara-mouse-bcr-cdr3 \
      input_R1.fastq.gz \
      input_R2.fastq.gz \
      result 

SMARTer Mouse TCR a/b Profiling Kit

takara-mouse-tcr-cdr3 · takara-mouse-tcr-full-length · Link · Code · Tutorial

The SMARTer Mouse TCR a/b Profiling Kit provides a powerful new solution for those seeking to perform T-cell receptor (TCR) repertoire analysis using NGS. The kit employs a 5'-RACE-based approach to capture complete V(D)J variable regions of TCR transcripts, starting from as little as 10 ng to 500 ng of total RNA obtained from mouse spleen, thymus, or PBMCs, or from 1,000 to 10,000 purified T cells. As the name suggests, the kit can be used to generate data for both TCR-alpha and TCR-beta chain diversity, either in the same experiment or separately. The -cdr3 preset may be used to reduce clonotype assembling feature from full V-D-J region to CDR3 only.

Example:

mixcr analyze takara-mouse-tcr-cdr3 \
      input_R1.fastq.gz \
      input_R2.fastq.gz \
      result 

10xGenomics

10x Genomics single cell VDJ

10x-vdj-tcr · 10x-vdj-bcr · Link · Code

Chromium Single Cell Immune Profiling provides a solution to your immunology questions. Analyze full-length V(D)J sequences for paired B-cell or T-cell receptors, all from a single cell. Notice that on the scheme bellow reads' length is shown according to the protocol recommendations, but the presets will work regardless of sequencing reads length.

The --species option is required.

Example:

mixcr analyze 10x-vdj-bcr \
     --species hsa \
     sample_R1.fastq.gz \
     sample_R2.fastq.gz \
     sample_result

10x Genomics single cell 5' gene expression

10x-5gex-cdr3 · 10x-5gex-full-length · Link · Code

These presets are specifically optimized to extract TCR and BCR repertoires from non-enriched single cell 5' RNA-seq cDNA libraries.

The --species option is required.

Example:

mixcr analyze 10x-5gex-full-length \
     --species hsa \
     sample_R1.fastq.gz \
     sample_R2.fastq.gz \
     sample_result

Illumina

AmpliSeq for Illumina Immune Repertoire Plus, TCR beta Panel

ampliseq-tcrb-plus-cdr3 · ampliseq-tcrb-plus-full-length · Link · Code

AmpliSeq for Illumina Immune Repertoire Plus, TCR beta Panel is a highly multiplexed targeted resequencing panel to measure T cell diversity and clonal expansion by sequencing T cell receptor (TCR) beta chain rearrangements. RNA evaluation of TCRβ chain rearrangements, including CDR1, CDR2, and CDR3 (with up to 400 bp read-length amplicons). The -cdr3 preset may be used to reduce clonotype assembling feature from {CDR1:FR4} region to CDR3 only.

Example:

mixcr analyze ampliseq-tcrb-plus-cdr3 \
      input_R1.fastq.gz \
      input_R2.fastq.gz \
      result 

AmpliSeq™ for Illumina® TCR beta-SR Panel

ampliseq-tcrb-sr-cdr3 · Link · Code

Sequences TCR beta chain rearrangements, with up to 80 bp read-length amplicons for characterizing CDR3.

Example:

mixcr analyze ampliseq-tcrb-sr-cdr3 \
      input_R1.fastq.gz \
      input_R2.fastq.gz \
      result 

Qiagen

QIAseq™ Human TCR Panel Immune Repertoire RNA Library Kit

qiaseq-human-tcr-cdr3 · qiaseq-human-tcr-full-length · Link · Code · Tutorial

The QIAseq Human TCR Panel Immune Repertoire RNA Library Kit uses unique Molecular Indices (UMI) with gene-specific primers to target specific RNAs for NGS sequencing. The Human T-cell Receptors Panel is used for sequencing the V(D)J region of the alpha, beta, delta and gamma genes, including the CDR3 regions. The -cdr3 preset may be used to reduce clonotype assembling feature from full V-D-J region to CDR3 only.

Example:

mixcr analyze qiaseq-human-tcr-cdr3 \
      input_R1.fastq.gz \
      input_R2.fastq.gz \
      result 

QIAseq™ Mouse TCR Panel Immune Repertoire RNA Library Kit

qiaseq-mouse-tcr-cdr3 · qiaseq-mouse-tcr-full-length · Link · Code

The QIAseq Mouse TCR Panel Immune Repertoire RNA Library Kit uses unique Molecular Indices (UMI) with gene-specific primers to target specific RNAs for NGS sequencing. The Mouse T-cell Receptors Panel is used for sequencing the V(D)J region of the alpha, beta, delta and gamma genes, including the CDR3 regions. The -cdr3 preset may be used to reduce clonotype assembling feature from full V-D-J region to CDR3 only.

Example:

mixcr analyze qiaseq-mouse-tcr-cdr3 \
      input_R1.fastq.gz \
      input_R2.fastq.gz \
      result 

Cellecta

DriverMap Adaptive Immune Receptor (AIR) TCR-BCR Profiling

cellecta-air-human · Link · Code

Cellecta’s DriverMap™ AIR TCR-BCR assay is designed to specifically amplify only functional CDR3 RNA molecules' TCR and BCR cells, avoiding non-functional pseudogenes with similar structures. The assay simultaneously amplifies, in a single, multiplex RT-PCR reaction, all TCR and BCR CDR3 regions using a set of 300 experimentally validated PCR primers to yield Illumina-compatible, next-generation sequencing (NGS) libraries.

Example:

mixcr analyze cellecta-air-human \
      input_R1.fastq.gz \
      input_R2.fastq.gz \
      result 

iRepertoire

iRepertoire has multiple different primer systems that vary by the regions targeted, the desired read length, and the species.

Repertoire’s RepSeq service (formerly AMP2Seq) utilizes arm-PCR technology, which uses hundreds of VDJ-specific primers in one reaction to semi-quantitatively and inclusively amplify all the expressed V(D)Js in B or T cells from a single sample.

Human RepSeq RNA Reagent System

irepertoire-human-rna-tcr-sr · irepertoire-human-rna-tcr-lr · irepertoire-human-rna-bcr-sr · irepertoire-human-rna-bcr-lr · Link · Code

Short-read (SR) RNA-compatible human primers (presets that end with sr) cover from within Framework-3 (FR3) into the Constant Region (C). These SR primers are compatible with 100/150 paired end read (PER) sequencing. Long-read (LR) primer systems (presets that end with ls) cover from within FR1 and continue through to the C region. iRepertoire’s LR primers are compatible with 250 PER sequencing. Note that by default the clones are assembled by the regions not covered by the primers (CDR3 for sr and {CDR1Begin:FR4End} for lr).

Example:

mixcr analyze irepertoire-human-rna-tcr-sr \
      input_R1.fastq.gz \
      input_R2.fastq.gz \
      result 

Mouse RepSeq RNA Reagent System

irepertoire-mouse-rna-tcr-sr · irepertoire-mouse-rna-tcr-lr · irepertoire-mouse-rna-bcr-sr · irepertoire-mouse-rna-bcr-lr · Link · Code

Short-read (SR) RNA-compatible human primers (presets that end with sr) cover from within Framework-3 (FR3) into the Constant Region (C). These SR primers are compatible with 100/150 paired end read (PER) sequencing. Long-read (LR) primer systems (presets that end with ls) cover from within FR2 and continue through to the C region. iRepertoire’s LR primers are compatible with 250 PER sequencing. Note that by default the clones are assembled by the regions not covered by the primers (CDR3 for sr and {CDR2Begin:FR4End} for lr).

Example:

mixcr analyze irepertoire-mouse-rna-tcr-sr \
      input_R1.fastq.gz \
      input_R2.fastq.gz \
      result 

Human RepSeq+ Reagent System

irepertoire-human-rna-repseq-plus · Link · Code

iRepertoire’s RepSeq+ service utilizes dam-PCR technology, which allows for any combination of TCR and BCR chains (TCR-alpha, TCR-beta, TCR-delta, TCR-gamma, BCR-IgHeavy, and BCR-kappa/lambda) to be amplified within a single reaction.

Example:

mixcr analyze irepertoire-human-rna-repseq-plus \
      input_R1.fastq.gz \
      input_R2.fastq.gz \
      result 

Human gDNA based RepSeq Reagent System

irepertoire-human-dna-trb-sr · irepertoire-human-dna-trb-lr · irepertoire-human-dna-igh-sr · irepertoire-human-dna-igh-lr · Link · Code

SR gDNA compatible primers (SR-VJ) cover from within FR3 to the end of the J-gene. These have been LR gDNA compatible primers cover from within FR1 to the end of the J-gene. LR versions are available for both human TCR beta and human IgH. These can be sequenced as single end read on 300-cycle kits or for full amplicon coverage as 250 PER sequencing.

Example:

mixcr analyze irepertoire-human-dna-igh-lr \
      input_R1.fastq.gz \
      input_R2.fastq.gz \
      result 

Thermo Fisher

Oncomine™ TCR Beta‑LR Assay

oncomine-human-tcrb-lr-cdr3 · oncomine-human-tcrb-lr-full-length · Link · Code

The Oncomine™ TCR Beta‑LR Assay is a highly sensitive, RNA-based NGS assay that enables the characterization of the T-cell receptor β (TCRβ) sequences, including all complementarity-determining regions (CDR1, 2, and 3) of the variable gene. The assay accurately measures T‑cell repertoire diversity, clonal expansion, and allows for identification of allele-specific polymorphisms, in a wide array of sample types.

Example:

mixcr analyze oncomine-human-tcrb-lr-cdr3 \
      input.fastq.gz \
      result 

Oncomine™ TCR Beta‑SR Assay

oncomine-human-tcrb-sr-cdr3 · Link · Code

The Oncomine™ TCR Beta‑SR Assay is a highly sensitive RNA- or DNA-based NGS assay that enables the characterization of the T-cell receptor β (TCRβ) complementarity-determining region 3 (CDR3) sequences of the TCRβ chain. The assay accurately measures T‑cell repertoire diversity and clonal expansion in a wide array of sample types, including those derived from FFPE-preserved or degraded material.

Example:

mixcr analyze oncomine-human-tcrb-sr-cdr3 \
      input.fastq.gz \
      result 

Oncomine™ BCR IGH‑LR Assay Kit

oncomine-human-bcr-ihg-lr-cdr3 · oncomine-human-bcr-igh-lr-full-length · Link · Code

The Oncomine™ BCR IGH‑LR Assay is a highly sensitive, RNA-based NGS assay that enables the characterization of immunoglobulin heavy-chain sequences, including all complementarity-determining regions (CDR1, 2, and 3) and the CH1 domain of the constant gene. The assay accurately measures repertoire diversity, clonal expansion, allows for determination of B cell isotype (and subtype), and reports the level of somatic hypermutation, in a wide array of sample types

Example:

mixcr analyze oncomine-human-bcr-igh-lr-full-length \
      input.fastq.gz \
      result 

Oncomine™ BCR IGH‑SR Assay

oncomine-human-bcr-igh-sr-cdr3 · Link · Code

The Oncomine™ BCR IGH‑SR Assay is a highly sensitive RNA- or DNA-based NGS assay that enables the characterization of the immunoglobulin heavy chain complementarity-determining region 3 (CDR3) sequences. The assay accurately measures B cell repertoire diversity and clonal expansion in a wide array of sample types, including samples derived from FFPE-preserved or degraded material

Example:

mixcr analyze oncomine-human-bcr-igh-sr-cdr3 \
      input.fastq.gz \
      result 

Oncomine™ BCR Pan-Clonality Assay

oncomine-human-bcr-pan-clonality-cdr3 · Link · Code

The Oncomine™ BCR Pan-Clonality Assay is a highly sensitive, single reaction, DNA-based NGS assay that enables the characterization of B cell heavy chain (IGH) and light chain (IgK and IgL) receptor sequences, including each complementarity-determining region 3 (CDR3). The assay accurately measures B cell repertoire metrics such as repertoire diversity and clonality in multiple receptors in a single reaction.

Example:

mixcr analyze oncomine-human-bcr-pan-clonality-cdr3 \
      input.fastq.gz \
      result 

Oncomine™ IGH FR3(d)-J Assay

oncomine-human-igh-fr3-j · Link · Code

The Oncomine™ IGH FR3(d)-J Assay is a highly sensitive, DNA-based NGS assays that enable the characterization of B cell heavy chain (IGH) sequences, including complementarity-determining region 3 (CDR3). The assays accurately measure B cell repertoire metrics such as repertoire diversity and clonality.

Example:

mixcr analyze oncomine-human-igh-fr3-j \
      input.fastq.gz \
      result 

Oncomine™ IGH FR2-J Assay

oncomine-human-igh-fr2-j · Link · Code

Oncomine™ IGH FR2-J Assay is a highly sensitive, DNA-based NGS assays that enable the characterization of B cell heavy chain (IGH) sequences, including complementarity-determining region 3 (CDR3). The assays accurately measure B cell repertoire metrics such as repertoire diversity and clonality. The Oncomine™ IGH FR2-J Assay uses multiplex Ion AmpliSeq™ primers to target the FR2 region of the variable gene and the joining gene segment of IGH rearrangements in genomic DNA producing 200–250 bp amplicons. Note, that clonotypes in this preset are assembled by {CDR2Begin:CDR3End} feature, because primers are located in FR2 and FR4.

Example:

mixcr analyze oncomine-human-igh-fr2-j \
      input.fastq.gz \
      result 

Oncomine™ IGH FR1-J Assay

oncomine-human-igh-fr1-j · Link · Code

The Oncomine IGH FR1-J Assay is a targeted next-generation sequencing (NGS) assay for detection of somatic hypermutation (SHM) in the variable region of the immunoglobulin heavy chain of B-cell receptors. SHM increases the affinity of the B-cell receptor for antigen, post-VDJ recombination, and is frequently a region of interest in CLL research. Primers have been specifically designed to target framework 1 and joining gene regions of the immunoglobulin heavy chain, resulting in an amplicon ∼300 bp in length. Note, that clonotypes in this preset are assembled by {CDR1Begin:CDR3End} feature, because primers are located in FR1 and FR4.

Example:

mixcr analyze oncomine-human-igh-fr1-j \
      input.fastq.gz \
      result 

Oncomine™ IGHV Leader-J Assay

oncomine-human-igh-leader-j · Link · Code

The Oncomine BCR IGHV Leader-J Assay is a targeted next-generation sequencing (NGS) assay for detection of somatic hypermutation (SHM) in the variable region of the immunoglobulin heavy chain of B-cell receptors. SHM increases the affinity of the B-cell receptor for antigen, post-VDJ recombination, and is used as a biomarker in chronic lymphocytic leukemia (CLL). Primers have been specifically designed to target the leader and joining regions of the immunoglobulin heavy chain, resulting in an amplicon ∼480 bp in length. Note, that clonotypes in this preset are assembled by {FR1Begin:CDR3End} feature, because primers are located in the leader sequence and FR4.

Example:

mixcr analyze oncomine-human-igh-leader-j \
      input.fastq.gz \
      result 

Oncomine™ TCR Pan-Clonality Assay

oncomine-human-tcr-pan-clonality-cdr3 · Link · Code

The Oncomine TCR Pan-Clonality Assay is a targeted next-generation sequencing (NGS) assay for detection of T-cell diversity to measure clonal expansion/contraction with accuracy and for detection of rare clones with high sensitivity. Primers have been specifically designed to sequence the FR3-J regions of the TCR beta chain and TCR gamma chain. Through the use of Ion AmpliSeq technology, sequencing of the CDR3 region of the beta and gamma T-cell receptor chains occurs in a single reaction. When combined with Ion Reporter Software, data can be easily analyzed and reports created within a short period of time.

Example:

mixcr analyze oncomine-human-tcr-pan-clonality-cdr3 \
      input.fastq.gz \
      result 

AbHelix

BCR data

abhelix-human-bcr-cdr3 · abhelix-human-bcr-full-length · Link · Code · Tutorial

This kit allows identification of IgG1,IgG1,IgG1,IgG1,IgGM,IgA isotypes. Isotypes are separated prior to the final PCR reaction, in a way that resulting sequences don't cover C region enough. Thus, this preset does not separate clones by C-gene, implying that different isotypes have been already separated into different samples. The -cdr3 preset may be used to reduce clonotype assembling feature from full V-D-J region to CDR3 only.

Example:

mixcr analyze abhelix-human-bcr-cdr3 \
      input_R1.fastq.gz \
      input_R2.fastq.gz \
      result 

TCR data

abhelix-human-tcr-cdr3 · abhelix-human-tcr-full-length · Link · Code

The kit allows to obtain full-length sequences of TCR-alpha and TCR-beta V(D)J variable regions. The -cdr3 preset may be used to reduce clonotype assembling feature from full V-D-J region to CDR3 only.

Example:

mixcr analyze abhelix-human-tcr-cdr3 \
      input_R1.fastq.gz \
      input_R2.fastq.gz \
      result 

BD Rhapsody

BD Rhapsody™ VDJ CDR3 Protocol with V1 beads

bd-rhapsody-human-tcr-v1 · bd-rhapsody-human-bcr-v1 · bd-rhapsody-mouse-tcr-v1 · bd-rhapsody-mouse-bcr-v1 · Link · Code

The BD Rhapsody™ VDJ CDR3 Protocol utilizes the existing BD Rhapsody™ Targeted Kits and the human/mouse immune response primer panel and is designed to work alongside the BD® AbSeq Assay and BD® Single-Cell Multiplexing Kit (SMK). Details about the individual reagents needed to run the assay are included in the respective protocols. A dedicated bioinformatics pipeline is also available for you to analyze sequencing data generated using the CDR3 protocol.

Example:

mixcr analyze bd-rhapsody-mouse-tcr-v1 \
     sample_R1.fastq.gz \
     sample_R2.fastq.gz \
     sample_result

BD Rhapsody™ VDJ CDR3 Protocol with Enhanced Bead (introduced in 2022)

bd-rhapsody-human-tcr-v2 · bd-rhapsody-human-bcr-v2 · bd-rhapsody-mouse-tcr-v2 · bd-rhapsody-mouse-bcr-v2

Link · Code

In 2022, BD introduced a new version of beads , called Enhanced Beads, which have slightly different oilgo design. Use these presets for the new updated protocol.

Example:

mixcr analyze bd-rhapsody-mouse-bcr-v2 \
     sample_R1.fastq.gz \
     sample_R2.fastq.gz \
     sample_result

BD Rhapsody™ VDJ Full Length Protocol

bd-rhapsody-human-bcr-full-length · bd-rhapsody-mouse-bcr-full-length · bd-rhapsody-human-tcr-full-length · bd-rhapsody-mouse-tcr-full-length

Link · Code

The BD Rhapsody™ TCR/BCR Multiomic Assay helps you assemble the full length VDJ sequence for the T cell and B cell receptor.

Example:

mixcr analyze bd-rhapsody-mouse-tcr-full-length \
     sample_R1.fastq.gz \
     sample_R2.fastq.gz \
     sample_result

Oxford Nanopore Technologies

Full-length RNA-seq

ont-rna-seq-vdj-full-length · Link · Code

The preset is designed to handle long reads RNA-seq data obtained with Oxford Nanopore Technologies sequencer.

The --species option is required.

Example:

mixcr analyze ont-rna-seq-vdj-full-length \
     --species hsa \
     sample_R1.fastq.gz \
     sample_R2.fastq.gz \
     sample_result

Parse Biosciences

Parse Evercode™ single-cell

parse-bio-vdj-3gex · Link · Code

Conventional droplet-based single-cell technologies struggle as cell or experiment sizes change. Parse makes it easy to scale your experiments regardless of cell size or sample type. The Evercode™ Whole Transcriptome technology, originally based on the split-pool combinatorial barcoding method published in Science and known widely as SPLiT-Seq, is accessible to any standard biology lab. Please note that as a 3'end RNA-seq based protocol it was not originally optimized for immune repertoire analysis, thus TCR/BCR yield might be low.

The --species option is required.

Example:

mixcr analyze parsebio-vdj-3gex \
     --species hsa \
     sample_R1.fastq.gz \
     sample_R2.fastq.gz \
     sample_result

Singleron

GEXSCOPE Single Cell D(D)J Kit

singleron-2.0.1-vdj-cdr3 · Link · Code

The GEXSCOPE® Single Cell V(D)J Kit enables simultaneous detection of T-cell or B-cell receptor variable region (CDR3) together with the whole transcriptome expression at single-cell level.

Example:

mixcr analyze singleron-2.0.1-vdj-cdr3 \
      input_R1.fastq.gz \
      input_R2.fastq.gz \
      result

New England BioLabs

NEBNext® Immune Sequencing Kit (Human) BCR & TCR

nebnext-human-bcr-cdr3 · nebnext-human-bcr-full-length · nebnext-human-tcr-cdr3 · nebnext-human-tcr-full-length · Link · Code · Tutorial

With the NEBNext® Immune Sequencing Kit (Human), sequence the full-length immune gene repertoires of B cells and T cells. Profile somatic mutations across all relevant contexts (e.g., V, D, and J segments and isotypes IgM, IgD, IgG, IgA, and IgE) with improved sequence accuracy. Characterize BCR light, BCR heavy, TCRα and TCRβ chains. This kit includes UMIs for source-molecule identification. The -cdr3 preset may be used to reduce clonotype assembling feature from full V-D-J region to CDR3 only. Mix-in option --dont-split-clones-by C may be used for BCR data to not separate clones by isotypes

Example:

mixcr analyze nebnext-human-bcr-cdr3 \
      input_R1.fastq.gz \
      input_R2.fastq.gz \
      result 

NEBNext® Immune Sequencing Kit (Mouse) BCR & TCR

nebnext-mouse-bcr-cdr3 · nebnext-mouse-bcr-full-length · nebnext-mouse-tcr-cdr3 · nebnext-mouse-tcr-full-length · Link · Code

With the NEBNext® Immune Sequencing Kit (Mouse), sequence the full-length immune gene repertoires of B cells and T cells. Profile somatic mutations across all relevant contexts (e.g., V, D, and J segments and isotypes IgM, IgD, IgG, IgA, and IgE) with improved sequence accuracy. Characterize BCR light, BCR heavy, TCRα, TCRβ, TCRγ and TCRδ chains. This kit includes UMIs for source-molecule identification. The -cdr3 preset may be used to reduce clonotype assembling feature from full V-D-J region to CDR3 only. Mix-in option --dont-split-clones-by C may be used for BCR data to not separate clones by isotypes

Example:

mixcr analyze nebnext-mouse-bcr-cdr3 \
      --dont-split-clones-by C \
      input_R1.fastq.gz \
      input_R2.fastq.gz \
      result 

Public protocols

Biomed2

biomed2-human-bcr-cdr3 · biomed2-human-bcr-full-length · Publication · Code · Tutorial

Biomed2 FR1-FR4 human multiplex BCR primer set. The -cdr3 presets used to extract CDR3 clonotypes while -full-length full VDJ region, starting from FR1.

Example:

mixcr analyze biomed2-human-bcr-full-length \
      input_R1.fastq.gz \
      input_R2.fastq.gz \
      result

Smart-Seq2

smartseq2-vdj-full-length · Publication · Code

For Smart-Seq2, single cells are lysed in a buffer that contains free dNTPs and oligo(dT)-tailed oligonucleotides with a universal 5'-anchor sequence. RT is performed, which adds 2–5 untemplated nucleotides to the cDNA 3′ end. A template-switching oligo (TSO) is added, carrying 2 riboguanosines and a modified guanosine to produce a LNA as the last base at the 3′ end. After the first-strand reaction, the cDNA is amplified using a limited number of cycles. Next, tagmentation is used to construct sequencing libraries quickly and efficiently from the amplified cDNA.

Note that Smart-Seq2 protocol implies that sequences are separated by cells in different FASTQ file pairs according to PCR plate row and column. In the example bellow we show how to provide cell id (row and column) information using filename patterns that have illumina indices in it. However, inplace of indices could be any text that is unique for column-row pair.

Example:

> ls

161014_SN163_0729_AH3VW7BCXY_L1_TAAGGCGA-CTCTCTAT_R1.fastq.gz
161014_SN163_0729_AH3VW7BCXY_L1_TAAGGCGA-CTCTCTAT_R2.fastq.gz
161014_SN163_0729_AH3VW7BCXY_L1_TAAGCTTA-GTGTTAAG_R1.fastq.gz
161014_SN163_0729_AH3VW7BCXY_L1_TAAGCTTA-GTGTTAAG_R2.fastq.gz

mixcr analyze smartseq2-vdj-full-length \
    -s hsa \
    161014_SN163_0729_AH3VW7BCXY_L1_{{CELL0ROW:a}}-{{CELL0COL:a}}_R1.fastq.gz \
    161014_SN163_0729_AH3VW7BCXY_L1_{{CELL0ROW:a}}-{{CELL0COL:a}}_R2.fastq.gz \
    result

SPLiT-seq

split-seq-vdj-3gex · Publication · Code

The SPLiT-seq uses the combinatorial indexing to identify single cells without single cell isolation. Multi-level indexing can be performed by ligation. Please note that as a 3'end RNA-seq based protocol it was not originally optimized for immune repertoire analysis, thus tcr/bcr yield might be low.

Example:

mixcr analyze split-seq-3gex-vdj \
      input_R1.fastq.gz \
      input_R2.fastq.gz \
      result

Mikelov et al, 2021

mikelov-et-al-2021 · Publication · Code

Vergani et al, 2017

vergani-et-al-2017-cdr3 · vergani-et-al-2017-full-length · Publication · Code

Example:

mixcr analyze vergani-et-al-2017-full-length \
      input_R1.fastq.gz \
      input_R2.fastq.gz \
      result

Han et al, 2014

han-et-al-2014-tcr · han-et-al-2014-bcr · Publication · Code

These presets are optimized for a single-cell use of the protocol when each plate well contains a single cell.

Example:

mixcr analyze han-et-al-2014-bcr \
      --species hsa \
      input_R1.fastq.gz \
      input_R2.fastq.gz \
      result

Generic data

RNA-Seq data

rnaseq-cdr3 · rnaseq-full-length · Publication · Code

Non-enriched fragmented (shotgun) RNA-Seq data. Preset rnaseq-cdr3 is used to assemble CDR3 clonotypes, while rnaseq-full-length additionally runs consensus contig assembly to reconstruct all available parts of V-D-J-C receptor rearrangement sequence.

The --species option is required.

Example:

mixcr analyze rnaseq-full-length \
    --species hsa \
      input_R1.fastq.gz \
      input_R2.fastq.gz \
      result

Exome data

exome-cdr3 · exome-full-length · Code

Non-enriched fragmented (shotgun) Exome-Seq data. Preset exome-cdr3 is used to assemble CDR3 clonotypes, while exome-full-length additionally runs consensus contig assembly to reconstruct all available parts of V-D-J receptor rearrangement sequence.

The --species option is required.

Example:

mixcr analyze exome-full-length \
    --species hsa \
      input_R1.fastq.gz \
      input_R2.fastq.gz \
      result

Generic TCR amplicon

generic-tcr-amplicon · generic-tcr-amplicon-umi · Code · Tutorial

Generic TCR amplicon library with (-umi) or without UMIs. Required configs that must be specified with corresponding mix-in options:

Species;

Material type;

Tag pattern (for generic-tcr-amplicon-umi);

Left alignment boundary (5'-end);

Right alignment boundary (3'-end).

The following example runs upstream analysis for some bulk mouse 5'RACE TCR RNA library with 3'-end primers located on C-gene:

mixcr analyze generic-tcr-amplicon \
    --species mmu \
    --rna \
    --rigid-left-alignment-boundary \
    --floating-right-alignment-boundary C \
      input_R1.fastq.gz \
      input_R2.fastq.gz \
      result
The following mix-in options are used:

--species mmu
specify Mus Musculus species
--rna
set RNA as starting material (exon regions only will be used for alignments)
--rigid-left-alignment-boundary
use global left alignment boundary (5'RACE)
--floating-right-alignment-boundary C
use local right alignment boundary on C-segment as C-primers are used

Generic BCR amplicon

generic-bcr-amplicon · generic-bcr-amplicon-umi · Code · Tutorial · Tutorial

Generic BCR amplicon library with (-umi) or without UMIs. Required configs that must be specified with corresponding mix-in options:

Species;

Material type;

Tag pattern (for generic-bcr-amplicon-umi);

Left alignment boundary (5'-end);

Right alignment boundary (3'-end).

The following example runs upstream analysis for some full-length human BCR RNA multiplex library with 3'-end primers located on C-gene and UMIs spanning first 12 letters of 5'-end, followed by 4 letters of a fixed linker sequence:

mixcr analyze generic-bcr-amplicon-umi \
    --species hsa \
    --rna \
    --tag-pattern "^(R1:*) \ ^(UMI:N{12})GTAC(R2:*)" \
    --rigid-left-alignment-boundary \
    --floating-right-alignment-boundary C \
      input_R1.fastq.gz \
      input_R2.fastq.gz \
      result
The following mix-in options are used:

--species hsa
specify Homo Sapiens species
--rna
set RNA as starting material (exon regions only will be used for alignments)
--tag-pattern "^(R1:*) \ ^(UMI:N{12})GTAC(R2:*)"
UMI barcode pattern
--rigid-left-alignment-boundary
use global left alignment boundary as our tag pattern removes UMIs and linked sequensed from 5'-end of reads
--floating-right-alignment-boundary C
use local right alignment boundary on C-segment as C-primers are used