Abstract
Efficient and accurate high-throughput DNA sequencing of the adaptive immune receptor repertoire (AIRR) is necessary to study immune diversity in healthy subjects and disease-related conditions. The high complexity and diversity of the AIRR coupled with the limited amount of starting material, which can compromise identification of the full biological diversity makes such sequencing particularly challenging. AIRR sequencing protocols often fail to fully capture the sampled AIRR diversity, especially for samples containing restricted numbers of B lymphocytes. Here, we describe a library preparation method for immunoglobulin sequencing that results in an exhaustive full-length repertoire where virtually every sampled B-cell is sequenced. This maximizes the likelihood of identifying and quantifying the entire IGHV-D-J repertoire of a sample, including the detection of rearrangements present in only one cell in the starting population. The methodology establishes the importance of circumventing genetic material dilution in the preamplification phases and incorporates the use of certain described concepts: (1) balancing the starting material amount and depth of sequencing, (2) avoiding IGHV gene-specific amplification, and (3) using Unique Molecular Identifier. Together, this methodology is highly efficient, in particular for detecting rare rearrangements in the sampled population and when only a limited amount of starting material is available.
Highlights
The diversity of the adaptive immune system is the key to its ability to respond to a wide variety of antigens
B lymphocytes originate in the bone marrow where precursors pass through a series of highly regulated processes to generate a functional B-cell receptor that is necessary for the survival of mature B cells [1]
A defined number of B lymphocytes (
Summary
The diversity of the adaptive immune system is the key to its ability to respond to a wide variety of antigens. Recent advances in generation sequencing allow in-depth studies of AIRR of B (Ig-seq) and T lymphocytes. Its variability is increased as a consequence of imperfect joining with random nucleotide insertion and deletions occurring during the recombination process. This yields an antigen-inexperienced B cell with a virtually unique IGHV-D-J rearrangement, without a germline reference and, a characteristic antigen-binding site. The diversity and complexity of the AIRR obtained by recombination is further increased in secondary lymphoid tissues by another biological process termed somatic hypermutation, whereby the enzyme activation-induced deaminase introduces mutations in the rearranged IGHV-D-J
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