Antibody repertoire3/23/2023 ![]() We quickly test whether each sequence roughly aligns to a reference germline V gene. The reads are the product of several rounds of PCR with antibody locus-specific primers, however, a subset of sequences may be to off-target loci. In our Reptor™ and Alicanto® workflows, we also apply a crude test of ‘antibody-ness’. Specifically, we remove consensus reads that are too short or too long, which can be signs that the read is truncated or was unstitchable. Once read pairs have been stitched into a single consensus read, additional filtering is applied. We construct a stitched read by identifying the overlap and constructing a consensus sequence in the overlapping region. This results in considerable overlap between reads. In our workflows, we use the Illumina MiSeq system to generate 2×300 nucleotide paired reads. In order to recover the full 330+ nucleotides of the variable region, paired-end reads or long single-end reads are needed. It’s important to recognize the unique challenges of antibody repertoire sequence and analysis, which make them ill-suited for standard RNA-seq or target-enrichment workflows. ![]() For a bit about that process, read our previous post on immunosequencing.ĭue to high sequence diversity, antibody repertoire sequencing and assembly of an individual repertoire is complex and requires specialized tools. Before repertoire construction and analysis can begin, the B-cell receptor transcripts must be sequenced. In this post, we discuss the approach to immune repertoire construction that we employ in our Reptor™ and Alicanto® services. The antibody repertoire is dynamic, with sequence diversity and composition of the repertoire changing dramatically over time. The total number of B cells and plasma cells that encode full-length immunoglobulins in an adult human is estimated to be 10¹º-10¹¹, which puts an upper bound on the total size of the B-cell receptor, or antibody, repertoire. At Digital Proteomics we focus primarily on the immunoglobulin, or B-cell receptor repertoires of humans and other mammals. The immune repertoire is the collection of unique immunoglobulin and T-cell receptor sequences present in an individual at a particular time.
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