Infants are widely thought to have weaker responses than toddlers to vaccines because of their developing immune systems 17. Although the antibody repertoire in fetuses 14, cord blood 15, young adults 6, and the elderly 6, 16 has been studied, infants and toddlers are among the most vulnerable age groups to many pathogenic challenges, yet their immune repertoires are not well understood. Given the wide use of IR-seq in basic research as well as clinical settings, we believe the method outlined here will serve as an important guideline for future IR-seq experimental designs.Īs a proof of principle, we use MIDCIRS to examine the antibody repertoire diversification in infants (<12 months old) and toddlers (12–47 months old) from a malaria endemic region in Mali before and during acute Plasmodium falciparum infection. Using naive B cells, we demonstrate that MIDCIRS has a high coverage of the diversity estimate, or different types of antibody sequences, that is consistent with the input cell number and a large dynamic range of three orders of magnitude compared to other MID-based immune repertoire-sequencing methods 10, 11. Here we report the development of MID clustering-based IR-seq (MIDCIRS) that further separates different RNA molecules tagged with the same MID. Despite these advancements, the large amount of input RNA required and low diversity coverage make it challenging to analyze small numbers of cells, such as memory B cells from dissected tissues or blood draws from young children, using IR-seq because these samples require many PCR cycles to generate enough material to make sequencing libraries, thus exacerbating PCR bias and errors. They are short stretches of nucleotide sequence tags composed of randomized nucleotides that are usually tagged to cDNA during reverse transcription to identify sequencing reads that originated from the same mRNA transcript. MIDs serve as barcodes to track genes of interest through amplification and sequencing. Although we and others have developed methods to control for artifacts from high amplification bias and sequencing error rates through data analysis 3, 5– 9, obtaining accurate sequencing information has now been made possible by the use of molecular identifiers (MID) 10– 13. Somatic hypermutation (SHM) further diversifies the antibody repertoire, which makes it impossible to quantify this diversity with nucleotide resolution until the development of high-throughput sequencing-based immune repertoire sequencing (IR-seq) 1– 4. V(D)J recombination creates hundreds of billions of antibodies and T cell receptors that collectively serve as the immune repertoire to protect the host from pathogens. These results highlight the potential of antibody repertoire diversification in infants and toddlers. Antibody clonal lineage analysis reveals that somatic hypermutation levels are increased in both infants and toddlers upon infection, and memory B cells isolated from individuals who previously experienced malaria continue to induce somatic hypermutations upon malaria rechallenge. Unexpectedly, we discover high levels of somatic hypermutation in infants as young as 3 months old. Here, we show this accurate and high-coverage repertoire-sequencing method can use as few as 1000 naive B cells. We develop molecular identifier clustering-based immune repertoire sequencing (MIDCIRS) and use it to study age-related antibody repertoire development and diversification before and during acute malaria in infants (< 12 months old) and toddlers (12–47 months old) with 4−8 ml of blood.
Accurately measuring antibody repertoire sequence composition in a small amount of blood is challenging yet important for understanding repertoire responses to infection and vaccination.
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