Characterization of the naive murine antibody repertoire using unamplified high-throughput sequencing

Rettig, Trisha A.; Ward, Claire; Bye, Bailey A.; Pecaut, Michael J.; Chapes, Stephen K.

doi:10.1371/journal.pone.0190982

Cited by 44 publications

(48 citation statements)

References 59 publications

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“…The majority of raw reads were downloaded from the European Nucleotide Archive (30) and the National Center for Biotechnology Information websites (31). In a small number of cases, another public Ig-seq repository was specified [e.g (14,(32)(33)(34)]. Metadata were manually extracted from the deposited datasets and arranged in a reproducible format.…”

Section: Methodsmentioning

confidence: 99%

Observed Antibody Space: A Resource for Data Mining Next-Generation Sequencing of Antibody Repertoires

Kovaltsuk

Leem

Kelm

et al. 2018

The Journal of Immunology

209

235

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Abs are immune system proteins that recognize noxious molecules for elimination. Their sequence diversity and binding versatility have made Abs the primary class of biopharmaceuticals. Recently, it has become possible to query their immense natural diversity using next-generation sequencing of Ig gene repertoires (Ig-seq). However, Ig-seq outputs are currently fragmented across repositories and tend to be presented as raw nucleotide reads, which means nontrivial effort is required to reuse the data for analysis. To address this issue, we have collected Ig-seq outputs from 55 studies, covering more than half a billion Ab sequences across diverse immune states, organisms (primarily human and mouse), and individuals. We have sorted, cleaned, annotated, translated, and numbered these sequences and make the data available via our Observed Antibody Space (OAS) resource at http://antibodymap.org The data within OAS will be regularly updated with newly released Ig-seq datasets. We believe OAS will facilitate data mining of immune repertoires for improved understanding of the immune system and development of better biotherapeutics.

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Section: Methodsmentioning

confidence: 99%

Observed Antibody Space: A Resource for Data Mining Next-Generation Sequencing of Antibody Repertoires

Kovaltsuk

Leem

Kelm

et al. 2018

The Journal of Immunology

209

235

View full text Add to dashboard Cite

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“…The 37 amino acid CDR H3 of VRC26UCA is far above the average CDR H3 length of mouse antibodies, and the mouse SLC may not be adapted to accommodating such extraordinarily long CDR H3s. By comparison, antibodies with long CDR H3s are more common in human than in mouse repertoires (34,35,62). The difference may explain more efficient pairing of VRC26UCA HC with human SLC than with mouse SLC.…”

Section: Discussionmentioning

confidence: 99%

Conditional Antibody Expression to Avoid Central B Cell Deletion in a Humanized HIV-1 Vaccine Mouse Models

Tian¹,

McGovern²,

Cheng³

et al. 2020

Preprint

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HIV-1 vaccine development aims to elicit broadly neutralizing antibodies (bnAbs) against diverse viral strains. In some HIV-1 infected individuals, bnAbs evolve from precursor antibodies through affinity maturation. To induce bnAbs, a vaccine must mediate a similar process of antibody maturation. One way to test vaccination strategies is to immunize mouse models that express human bnAb precursors. Such immunization experiments can assess whether the vaccine can convert precursor antibody into bnAb. A major problem with such mouse models is that bnAb expression often hinders B cell development in the bone marrow. Such developmental blocks may be attributed to unusual properties of bnAb variable regions, such as poly-reactivity and long antigen-binding loops, which are often under negative selection during primary B cell development. To address this problem, we devised a method to circumvent B cell developmental block by expressing bnAbs conditionally in mature B cells. We validated this method by expressing the unmutated common ancestor (UCA) of the human VRC26 bnAb in transgenic mice. Constitutive expression of combined immunoglobulin heavy and light chains of VRC26UCA led to developmental arrest of B cell progenitors in the bone marrow; poly-reactivity of VRC26UCA and poor pairing of VRC26UCA IgH chain with mouse surrogate light chain may contribute to the phenotype. The conditional expression strategy circumvented this developmental impediment, allowing the VRC26UCA to be expressed in mature peripheral B cells. This method should be generally applicable for expressing other antibodies that are under negative selection during B cell development.Receptor editing can delete rearranged VJ exons in the Igk locus, leading to the rearrangement of the other Igk allele or the Igl locus (19)(20)(21)(22). This process likely accounted for

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“…We assessed all functional V‐gene segments as identified by IMGT. We also include three putative functional genes (V5S21, V1S100, and V3S7) which were detected in rearranged transcripts (containing a CDR3 C‐xx‐W motif or class switched) in our previous analysis of the normal C57BL/6 repertoire . IMGT's High‐V Quest occasionally assigned multiple potential V‐gene segments to a single sequence, likely due to incomplete capture of the entire V‐gene sequence or high homology between gene segments.…”

Section: Methodsmentioning

confidence: 99%

A comparison of unamplified and massively multiplexed PCR amplification for murine antibody repertoire sequencing

2018

Self Cite

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Sequencing antibody repertoires has steadily become cheaper and easier. Sequencing methods usually rely on some form of amplification, often a massively multiplexed PCR prior to sequencing. To eliminate potential biases and create a data set that could be used for other studies, our laboratory compared unamplified sequencing results from the splenic heavy‐chain repertoire in the mouse to those processed through two commercial applications. We also compared the use of mRNA vs total RNA, reverse transcriptase, and primer usage for cDNA synthesis and submission. The use of mRNA for cDNA synthesis resulted in higher read counts but reverse transcriptase and primer usage had no statistical effects on read count. Although most of the amplified data sets contained more antibody reads than the unamplified data set, we detected more unique variable (V)‐gene segments in the unamplified data set. Although unique CDR3 detection was much lower in the unamplified data set, RNASeq detected 98% of the high‐frequency CDR3s. We have shown that unamplified profiling of the antibody repertoire is possible, detects more V‐gene segments, and detects high‐frequency clones in the repertoire.

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Characterization of the naive murine antibody repertoire using unamplified high-throughput sequencing

Cited by 44 publications

References 59 publications

Observed Antibody Space: A Resource for Data Mining Next-Generation Sequencing of Antibody Repertoires

Observed Antibody Space: A Resource for Data Mining Next-Generation Sequencing of Antibody Repertoires

Conditional Antibody Expression to Avoid Central B Cell Deletion in a Humanized HIV-1 Vaccine Mouse Models

A comparison of unamplified and massively multiplexed PCR amplification for murine antibody repertoire sequencing

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