Age-related changes can significantly alter the state of adaptive immune system and often lead to attenuated response to novel pathogens and vaccination. In present study we employed 5'RACE UMI-based full length and nearly error-free immunoglobulin profiling to compare plasma cell antibody repertoires in young (19-26 years) and middle-age (45-58 years) individuals vaccinated with a live yellow fever vaccine, modeling a newly encountered pathogen. Our analysis has revealed age-related differences in the responding antibody repertoire ranging from distinct IGH CDR3 repertoire properties to differences in somatic hypermutation intensity and efficiency and antibody lineage tree structure. Overall, our findings suggest that younger individuals respond with a more diverse antibody repertoire and employ a more efficient somatic hypermutation process than elder individuals in response to a newly encountered pathogen.

• Keywords
• age, immunoglobulin repertoire, plasma cell, vaccination, yellow fever,
• MeSH
• Immunity, Active * genetics   MeSH
• B-Lymphocytes immunology   metabolism   MeSH
• Adult MeSH
• Immunoglobulin Constant Regions genetics   MeSH
• Middle Aged MeSH
• Humans MeSH
• Young Adult MeSH
• Antibodies, Viral immunology   MeSH
• Receptors, Antigen, B-Cell genetics   metabolism   MeSH
• Somatic Hypermutation, Immunoglobulin MeSH
• Immunoglobulin Heavy Chains genetics   MeSH
• Yellow Fever Vaccine immunology   MeSH
• Vaccination MeSH
• Yellow Fever prevention & control   MeSH
• Animals MeSH
• Check Tag
• Adult MeSH
• Middle Aged MeSH
• Humans MeSH
• Young Adult MeSH
• Male MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Immunoglobulin Constant Regions MeSH
• Antibodies, Viral MeSH
• Receptors, Antigen, B-Cell MeSH
• Immunoglobulin Heavy Chains MeSH
• Yellow Fever Vaccine MeSH

BACKGROUND: Recently we proposed efficient method to exclude undesirable primers at any stage of amplification reaction, here termed NOPE (NOnsense-mediated Primer Exclusion). According to this method, added oligonucleotide overlapping with the 3'-end of unwanted amplification primer (NOPE oligo) simultaneously provides a template for its elongation. This elongation disrupts specificity of unwanted primer, preventing its further participation in PCR. The suggested approach allows to rationally manage the course of PCR reactions in order to facilitate analysis of complex DNA mixtures as well as to perform multistage PCR bypassing intermediate purification steps. RESULTS: Here we apply NOPE method to DNA library preparation for the high-throughput sequencing (HTS) with the PCR-based introduction of unique molecular identifiers (UMI). We show that NOPE oligo efficiently neutralizes UMI-containing oligonucleotides after introduction of UMI into sample DNA molecules, thus allowing to proceed with further amplification steps without purification and associated loss of starting material. At the same time, NOPE oligo does not affect the efficiency of target PCR amplification. CONCLUSION: We describe a simple, robust and cheap modification of UMI-labeled HTS libraries preparation procedure, that allows to bypass purification step and thus to preserve starting material which may be limited, e.g. circulating tumor DNA, circulating fetal DNA, or small amounts of isolated cells of interest. Furthermore, demonstrated simplicity and robustness of NOPE method should make it popular in various PCR protocols.

• Keywords
• High-throughput sequencing, PCR, Targeted resequencing, Unique molecular identifiers,
• MeSH
• DNA Primers genetics   MeSH
• ErbB Receptors genetics   MeSH
• Gene Library * MeSH
• Polymerase Chain Reaction methods   MeSH
• Sequence Analysis, DNA MeSH
• High-Throughput Nucleotide Sequencing MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• DNA Primers MeSH
• ErbB Receptors MeSH

The accuracy with which DNA polymerase can replicate a template DNA sequence is an extremely important property that can vary by an order of magnitude from one enzyme to another. The rate of nucleotide misincorporation is shaped by multiple factors, including PCR conditions and proofreading capabilities, and proper assessment of polymerase error rate is essential for a wide range of sensitive PCR-based assays. In this paper, we describe a method for studying polymerase errors with exceptional resolution, which combines unique molecular identifier tagging and high-throughput sequencing. Our protocol is less laborious than commonly-used methods, and is also scalable, robust and accurate. In a series of nine PCR assays, we have measured a range of polymerase accuracies that is in line with previous observations. However, we were also able to comprehensively describe individual errors introduced by each polymerase after either 20 PCR cycles or a linear amplification, revealing specific substitution preferences and the diversity of PCR error frequency profiles. We also demonstrate that the detected high-frequency PCR errors are highly recurrent and that the position in the template sequence and polymerase-specific substitution preferences are among the major factors influencing the observed PCR error rate.

• MeSH
• Alleles MeSH
• Analysis of Variance MeSH
• Gene Library MeSH
• Polymorphism, Single Nucleotide MeSH
• Polymerase Chain Reaction methods   standards   MeSH
• Sequence Analysis, DNA MeSH
• High-Throughput Nucleotide Sequencing * methods   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH