5′ Rapid Amplification of cDNA Ends and Illumina MiSeq Reveals B Cell Receptor Features in Healthy Adults, Adults With Chronic HIV-1 Infection, Cord Blood, and Humanized Mice

Waltari, Eric; Jia, Manxue; Jiang, Caroline S.; Liang, Hong; Huang, Jing; Fernández, Carmen; Finzi, Andrés; Kaufmann, Daniel E.; Markowitz, Martin; Tsuji, Moriya; Wu, Xueling

doi:10.3389/fimmu.2018.00628

Cited by 17 publications

(15 citation statements)

References 81 publications

(77 reference statements)

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“…We enriched the targets of point mutations and HBV integrations with a method similar to rapid amplification of cDNA ends (RACE) using multiple primers covering the coding regions of TP53, CTNNB1, and AXIN1, the promoter region of TERT, and the HBV sequence (SI Appendix, Fig. S2) (7,13). The reads from next generation sequencing can be tracked to the original cfDNA molecule with the DNA barcode to filter false positive single nucleotide variants (SNVs) from sequencing/amplification errors (14).…”

Section: Clinical Parameters Of Participants At Baseline In the Four mentioning

confidence: 99%

Detection of early-stage hepatocellular carcinoma in asymptomatic HBsAg-seropositive individuals by liquid biopsy

Wang

et al. 2019

Proc. Natl. Acad. Sci. U.S.A.

139

129

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Liquid biopsies, based on cell free DNA (cfDNA) and proteins, have shown the potential to detect early stage cancers of diverse tissue types. However, most of these studies were retrospective, using individuals previously diagnosed with cancer as cases and healthy individuals as controls. Here, we developed a liquid biopsy assay, named the hepatocellular carcinoma screen (HCCscreen), to identify HCC from the surface antigen of hepatitis B virus (HBsAg) positive asymptomatic individuals in the community population. The training cohort consisted of individuals who had liver nodules and/or elevated serum α-fetoprotein (AFP) levels, and the assay robustly separated those with HCC from those who were non-HCC with a sensitivity of 85% and a specificity of 93%. We further applied this assay to 331 individuals with normal liver ultrasonography and serum AFP levels. A total of 24 positive cases were identified, and a clinical follow-up for 6–8 mo confirmed four had developed HCC. No HCC cases were diagnosed from the 307 test-negative individuals in the follow-up during the same timescale. Thus, the assay showed 100% sensitivity, 94% specificity, and 17% positive predictive value in the validation cohort. Notably, each of the four HCC cases was at the early stage (<3 cm) when diagnosed. Our study provides evidence that the use of combined detection of cfDNA alterations and protein markers is a feasible approach to identify early stage HCC from asymptomatic community populations with unknown HCC status.

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Section: Clinical Parameters Of Participants At Baseline In the Four mentioning

confidence: 99%

Detection of early-stage hepatocellular carcinoma in asymptomatic HBsAg-seropositive individuals by liquid biopsy

Wang

et al. 2019

Proc. Natl. Acad. Sci. U.S.A.

139

129

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“…Besides the lineage specific analyses, high-throughput NGS approaches have also been used to investigate the whole B cell receptor repertoire of HIV-1-infected individuals either from combinatorial libraries [162][163][164] or from PBMCs or purified B cells [75,154,[165][166][167][168]. A recent study by Waltari et al detected slight shifts in V gene family usage, higher degrees of somatic hypermutation, and longer CDRH3s for HIV-1-infected individuals [167]. However, other studies could not find any differences but report variation within healthy or HIV-1-infected individuals to be as large as between the different cohorts [75,166].…”

Section: Informing About Vaccination Strategies (Ii): B Cell Receptormentioning

confidence: 99%

Exploiting B Cell Receptor Analyses to Inform on HIV-1 Vaccination Strategies

et al. 2020

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The human antibody repertoire is generated by the recombination of different gene segments as well as by processes of somatic mutation. Together these mechanisms result in a tremendous diversity of antibodies that are able to combat various pathogens including viruses and bacteria, or malignant cells. In this review, we summarize the opportunities and challenges that are associated with the analyses of the B cell receptor repertoire and the antigen-specific B cell response. We will discuss how recent advances have increased our understanding of the antibody response and how repertoire analyses can be exploited to inform on vaccine strategies, particularly against HIV-1.Antibodies are composed of heavy and light chains, both of which are divided into a constant and a variable region ( Figure 1a). The different isotypes for the heavy chain constant regions mediate different effector functions and are grouped into the classes IgM, IgD, and IgE [3]. The variable regions of heavy and light chains form the paratope that contacts the epitope on a particular antigen (e.g., on a bacterial or viral surface protein). Two essential steps act during the lifespan of a B cell to generate B cell receptor diversity: (i) the V(D)J recombination process that builds the naïve (i.e., antigen-inexperienced) B cell repertoire, and (ii) somatic hypermutation (SHM) during the process of affinity maturation that generates high affinity B cell receptors and antibodies (i.e., the antigen-experienced repertoire).lifespan of a B cell to generate B cell receptor diversity: i.) the V(D)J recombination process that 52 builds the naïve (i.e., antigen-inexperienced) B cell repertoire, and ii.) somatic hypermutation (SHM) 53 during the process of affinity maturation that generates high affinity B cell receptors and antibodies 54 (i.e., the antigen-experienced repertoire). 55The initial diversity of the B cell repertoire results from the assembly of the B cell receptor 56 during early B cell development in the bone marrow. The recombination-activating gene (RAG) 1/2 57 enzymes recombine variable (V), diversity (D), and joining (J) gene segments of the immunoglobulin 58 heavy (IgH) chain locus to first assemble the heavy chain variable region, followed by V and J gene 59 segment recombination within the Ig kappa (IgK) and Ig lambda (IgL) loci [4]. Junctional diversity is 60 further increased by RAG1/2 and other enzymes through the generation of palindromic (P) 61 nucleotides, as well as by the terminal deoxynuclotidyl transferase (TdT) through the addition of 62 non-template (N) nucleotides [5] (Figure 1b). Heavy and light chain V genes exclusively encode for 63 two complementarity determining regions (CDR1 and CDR2) that are usually structurally exposed 64 at the tip of the antibody and contribute to antigen recognition. A third CDR (CDR3) is generated by 65 the V(D)J recombination process and is the most variable part within B cell receptors and antibodies. 66The CDRs are interspersed and flanked with framework regions (FWR) that mainly function a...

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“…[ 16 ] This eliminates the biases that occur in DNA‐based assays, which necessitate the use of degenerate V primer sets required to amplify from all V H families (Figure 2). [ 17,18 ] Moreover, in the step of reverse transcription converting RNA to cDNA, unique molecular identifier (UMI) tags can be added to correct sequencing errors, [ 19 ] and indexing barcodes can be introduced for pooled high‐throughput sequencing, thus multiple samples can be sequenced in a lane to reduce costs and eliminate sequencing errors of different batches. [ 20 ] To provide a better understanding of antibody repertoires in the context of specific B cell populations, there has been increased application of single B cell sequencing, a commercially available RNA‐based sequencing technology that uses the polyA tail of mRNA to capture BCR genes.…”

Section: Overview Of Ig‐seq Pipelinementioning

confidence: 99%

Deep Mining of Human Antibody Repertoires: Concepts, Methodologies, and Applications

Tian

et al. 2020

Small Methods

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of V-J genes in the immunoglobulin light chains variable regions (V L), during the early stages of B cell maturation in the bone marrow; [1] 2) junctional diversity resulting from exonuclease trimmings and the random addition of palindrome (P) or non-template encoded (N) nucleotides in the process of rearrangement; [2] and 3) B cell proliferation after antigen stimulation in order to produce high-affinity antibodies and eliminate the antigen through subsequent class switch recombination (CSR) and somatic hypermutation (SHM). [3,4] It is estimated that there are over 10 13 BCRs in the human antibody repertoire. [5] This astronomical size prevents a global analysis of the antibody repertoire using conventional DNA sequencing. However, advancements in highthroughput sequencing (HTS) for Ig-Seq have improved the ability to characterize the antibody repertoire on a large scale. [6] Over a decade of development, a variety of new technologies and approaches have improved Ig-Seq methods, resulting in improved understanding of B-cell immunology and accumulation of massive antibody sequence data. In this review, an overview of Ig-Seq and advanced methodologies applied to HTS of antibody repertoires will be discussed. We will describe the wide applications of Ig-Seq in basic and clinical immunology, and strategies for functional antibody discovery from antibody repertoires. Finally, we will provide our perspective on the future directions and challenges in human antibody repertoire deep mining. 2. Overview of Ig-Seq Pipeline Although multiple strategies have been used for studying antibody repertoires, each strategy follows a similar workflow including sample collection, library preparation and sequencing, data cleaning, sequence alignment, and high-level processing (Figure 1). For sample collection, peripheral blood mononuclear cells (PBMCs) and total B cells have been generally used because of their accessibility. [9-11] However, because antibody repertoire properties are divergent between antigen-specific B cells and plasma cells, [12] specific B cell populations or B cells located in targeted tissues should be enriched for more precise studies. Considering that as few as 2% of total B cells are circulating in The ability of the human adaptive immune system to respond to antigens relies upon the tremendous diversity of T cell receptors (TCR) and B cell receptors (BCR). The entirety of an individual's BCRs, often referred to as an antibody repertoire, shapes the humoral immune system. Therefore, technologies to identify and characterize antibody repertoires are critical for understanding fundamental aspects of the development and maintenance of the humoral immune system. Recently, innovative methodologies and technologies devoted to high-throughput sequencing of antibody repertoires (Ig-Seq) have broadened the understanding of humoral immunity. This review provides an overview of the Ig-Seq pipeline from sample collection, library preparation, and sequencing, to data cleaning, sequence alignment, and highlevel processing....

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5′ Rapid Amplification of cDNA Ends and Illumina MiSeq Reveals B Cell Receptor Features in Healthy Adults, Adults With Chronic HIV-1 Infection, Cord Blood, and Humanized Mice

Cited by 17 publications

References 81 publications

Detection of early-stage hepatocellular carcinoma in asymptomatic HBsAg-seropositive individuals by liquid biopsy

Detection of early-stage hepatocellular carcinoma in asymptomatic HBsAg-seropositive individuals by liquid biopsy

Exploiting B Cell Receptor Analyses to Inform on HIV-1 Vaccination Strategies

Deep Mining of Human Antibody Repertoires: Concepts, Methodologies, and Applications

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