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Method Article
NGS-based analysis of the mouse B-cell receptor repertoire
Nadja Zaborsky
Salzburg Cancer Research Institute - Laboratory for Immunological and Molecular Cancer Research (SCRI-LIMCR); Cancer Cluster Salzburg, Austria
Corresponding Author
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This work is licensed under a CC BY 4.0 License. Read Full License
The B cell receptor (BCR), especially the complementarity-determining region 3 (CDR3) encoded on the variable region of the immunoglobulin heavy chain, is the most important region that determines antigen binding and specificity. Furthermore, sequence analysis of the CDR3 region is important for understanding the clonal distribution and dynamics in the clonal evolution of leukemic cells.
Here we present a sensitive method for fast and precise analysis of the BCR repertoire of mouse chronic lymphocytic leukemia cells by amplification and high-throughput DNA sequencing of the genomic rearranged VDJ locus. Tagging and indexing of the PCR amplified VDJ locus enables multiplexing and Illumina sequencing of up to 3 x 96 samples (depending on the requested coverage) in parallel on a MiSeq system, with a total coverage of 25 million reads for a high output run. The protocol includes a suggestion for fast bioinformatics analysis using the MiXCR software.
Keywords
Mouse, PCR, BCR, High-throughput sequencing
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This is a preprint. It has not completed peer review.
Method Article
NGS-based analysis of the mouse B-cell receptor repertoire
Nadja Zaborsky
Salzburg Cancer Research Institute - Laboratory for Immunological and Molecular Cancer Research (SCRI-LIMCR); Cancer Cluster Salzburg, Austria
Corresponding Author
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
History
CURRENT STATUS: Posted
Version 1
Posted 16 Jan, 2019
No community comments so far
Metrics
Comments: 0
HTML Views: ...
License:
This work is licensed under a CC BY 4.0 License. Read Full License
The B cell receptor (BCR), especially the complementarity-determining region 3 (CDR3) encoded on the variable region of the immunoglobulin heavy chain, is the most important region that determines antigen binding and specificity. Furthermore, sequence analysis of the CDR3 region is important for understanding the clonal distribution and dynamics in the clonal evolution of leukemic cells.
Here we present a sensitive method for fast and precise analysis of the BCR repertoire of mouse chronic lymphocytic leukemia cells by amplification and high-throughput DNA sequencing of the genomic rearranged VDJ locus. Tagging and indexing of the PCR amplified VDJ locus enables multiplexing and Illumina sequencing of up to 3 x 96 samples (depending on the requested coverage) in parallel on a MiSeq system, with a total coverage of 25 million reads for a high output run. The protocol includes a suggestion for fast bioinformatics analysis using the MiXCR software.
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
Figure 8
Figure 9
Figure 10
Figure 11
Figure 12
Figure 13
Figure 14