A primer set for comprehensive amplification of V-genes from rhesus macaque origin based on repertoire sequencing

Rosenfeld, Ronit; Zvi, Anat; Winter, Eitan; Hope, Ronen; Israeli, Ofir; Mazor, Ohad; Yaari, Gur

doi:10.1016/j.jim.2018.11.011

Cited by 10 publications

(24 citation statements)

References 12 publications

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“…The collection of unbiased full-length Ig and TCR transcript sequences also provided a unique opportunity for exploring the sequence diversity across the entire variable region, the target of current repertoire amplification efforts. We assessed the efficiencies of three commonly used rhesus-specific Ig MPCR strategies [27, 28, 31], by examining the sites targeted by their primers in the context of their respective Ig V genes. We selected the three most abundant IGHV genes represented among the unique Ig transcripts: IGHV4-2 (1,383 sequences), IGHV1-1 (163 sequences), and IGHV3-9 (128 sequences), and generated consensus profiles for each.…”

Section: Resultsmentioning

confidence: 99%

“…The IGHV4-2 consensus was constructed from 1,383 sequences, while IGHV1-1 was from 163 sequences and IGHV3-9 was from 128 sequences. The regions targeted by primers from (S)undling et al [27], (W)iehe et al [28], and (R)osenfeld et al [31] respectively have either a yellow, red, or green bar above.…”

Section: Resultsmentioning

confidence: 99%

“…Common rhesus-specific MPCR assays for amplifying IGH, IGK, and IGL sequences [27, 28, 31] and TCRA and TCRB sequences [63, 64] were tested via in silico PCR analysis using the UCSC isPcr tool [65]. For those primer sets with a nested design [27, 28], only the inner primers were used for the analysis.…”

Section: Methodsmentioning

confidence: 99%

“…The standard inner and outer reverse primers used for the 10x B cell and T cell V(D)J assays were also assessed via in silico PCR analysis [65] where a dummy adapter sequence was prepended to the 5’ end of sequences to enable analysis with the isPcr tool and only test reverse primer efficiencies. The efficiencies of rhesus-specific MPCR assays [27, 28, 31] in amplifying the variable region (forward primer) and constant region (reverse primer) were similarly tested by either appending and prepending a dummy adapter sequence, respectively. In all uses of the isPcr tool, default parameters were used, requiring a perfect match for the first 15 nucleotides on the 3’ end of the primer.…”

Section: Methodsmentioning

confidence: 99%

“…Typically, such PCR-based approaches are designed for individually sorted B or T cells [27] and facilitate cloning efforts [30]. A more recent rhesus-specific MPCR design aimed to expand coverage of the Ig repertoire [31]. There were also attempts to improve rhesus V and J germline gene annotation using 5’ RACE sequencing (RACE-seq) [17, 32].…”

Section: Introductionmentioning

confidence: 99%

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Systematic profiling of full-length immunoglobulin and T-cell receptor repertoire diversity in rhesus macaque through long read transcriptome sequencing

Brochu

Tseng

Smith

et al. 2019

Preprint

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The diversity of immunoglobulin (Ig) and T-cell receptor (TCR) repertoires is a focal point of immunological studies. Rhesus macaques are key for modeling human immune responses, placing critical importance on the accurate annotation and quantification of their Ig and TCR repertoires. However, due to incomplete reference resources, the coverage and accuracy of the traditional targeted amplification strategies for profiling rhesus Ig and TCR repertoires are largely unknown. Here, using long read sequencing, we sequenced four Indian-origin rhesus macaque tissues and obtained high quality, full-length sequences for over 6,000 unique Ig and TCR transcripts, without the need for sequence assembly. We constructed the first complete reference set for the constant regions of all known isotypes and chain types of rhesus Ig and TCR repertoires. We show that sequence diversity exists across the entire variable regions of rhesus Ig and TCR transcripts. Consequently, existing strategies using targeted amplification of rearranged variable regions comprised of V(D)J gene segments miss a significant fraction (27% to 53% and 42% to 49%) of rhesus Ig/TCR diversity. To overcome these limitations, we designed new rhesus-specific assays that remove the need for primers conventionally targeting variable regions and allow single cell-level Ig and TCR repertoire analysis. Our improved approach will enable future studies to fully capture rhesus Ig and TCR repertoire diversity and is applicable for improving annotations in any model organism.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Systematic profiling of full-length immunoglobulin and T-cell receptor repertoire diversity in rhesus macaque through long read transcriptome sequencing

Brochu

Tseng

Smith

et al. 2019

Preprint

View full text Add to dashboard Cite

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Rhesus and cynomolgus macaque immunoglobulin heavy-chain genotyping yields comprehensive databases of germline VDJ alleles

et al. 2021

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Full-length single-cell BCR sequencing paired with RNA sequencing reveals convergent responses to pneumococcal vaccination

Morgan,

Zhang,

Kim

et al. 2024

Commun Biol

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Single-cell RNA sequencing (scRNA-seq) can resolve transcriptional features from individual cells, but scRNA-seq techniques capable of resolving the variable regions of B cell receptors (BCRs) remain limited, especially from widely-used 3′-barcoded libraries. Here, we report a method that can recover paired, full-length variable region sequences of BCRs from 3′-barcoded scRNA-seq libraries. We first verify this method (B3E-seq) can produce accurate, full-length BCR sequences. We then apply this method to profile B cell responses elicited against the capsular polysaccharide of Streptococcus pneumoniae serotype 3 (ST3) by glycoconjugate vaccines in five infant rhesus macaques. We identify BCR features associated with specificity for the ST3 antigen which are present in multiple vaccinated monkeys, indicating a convergent response to vaccination. These results demonstrate the utility of our method to resolve key features of the B cell repertoire and profile antigen-specific responses elicited by vaccination.

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A primer set for comprehensive amplification of V-genes from rhesus macaque origin based on repertoire sequencing

Cited by 10 publications

References 12 publications

Systematic profiling of full-length immunoglobulin and T-cell receptor repertoire diversity in rhesus macaque through long read transcriptome sequencing

Systematic profiling of full-length immunoglobulin and T-cell receptor repertoire diversity in rhesus macaque through long read transcriptome sequencing

Rhesus and cynomolgus macaque immunoglobulin heavy-chain genotyping yields comprehensive databases of germline VDJ alleles

Full-length single-cell BCR sequencing paired with RNA sequencing reveals convergent responses to pneumococcal vaccination

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