Bolotin, D.A. et al. Next generation sequencing for TCR repertoire profiling: platform-specific features and correction algorithms. Eur. J. Immunol. 42, 3073-3083

Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry RAS, Moscow, Russia.
European Journal of Immunology (Impact Factor: 4.03). 11/2012; 42(11). DOI: 10.1002/eji.201242517
Source: PubMed


The TCR repertoire is a mirror of the human immune system that reflects processes caused by infections, cancer, autoimmunity, and aging. Next generation sequencing (NGS) is becoming a powerful tool for deep TCR profiling; yet, questions abound regarding the methodological approaches for sample preparation and correct data interpretation. Accumulated PCR and sequencing errors along with library preparation bottlenecks and uneven PCR efficiencies lead to information loss, biased quantification, and generation of huge artificial TCR diversity. Here, we compare Illumina, 454, and Ion Torrent platforms for individual TCR profiling, evaluate the rate and character of errors, and propose advanced platform-specific algorithms to correct massive sequencing data. These developments are applicable to a wide variety of next generation sequencing applications. We demonstrate that advanced correction allows the removal of the majority of artificial TCR diversity with concomitant rescue of most of the sequencing information. Thus, this correction enhances the accuracy of clonotype identification and quantification as well as overall TCR diversity measurements.

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Available from: Ilgar Z Mamedov, Oct 06, 2015
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    • "NGS, in particular, has become a powerful tool for deep sequencing analyses of highly variable genomic populations, such as those arising during an infection with rapidly mutating pathogens, or metagenomics [2]. Similarly, in immunology, NGS is increasingly utilized for sequencing of highly polymorphic protein encoding regions, which are key elements of pathogen recognition, including HLA genes, T-cell receptor [3–5], and the B cell receptors [6]. NGS technology has been also utilized to study the evolution of complex viral populations evolving during an infection within host, such as the detection of rare viral variants in hepatitis C virus (HCV) [7], or in HIV [8]. "
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    ABSTRACT: Rapidly mutating viruses, such as hepatitis C virus (HCV) and HIV, have adopted evolutionary strategies that allow escape from the host immune response via genomic mutations. Recent advances in high-throughput sequencing are reshaping the field of immuno-virology of viral infections, as these allow fast and cheap generation of genomic data. However, due to the large volumes of data generated, a thorough understanding of the biological and immunological significance of such information is often difficult. This paper proposes a pipeline that allows visualization and statistical analysis of viral mutations that are associated with immune escape. Taking next generation sequencing data from longitudinal analysis of HCV viral genomes during a single HCV infection, along with antigen specific T-cell responses detected from the same subject, we demonstrate the applicability of these tools in the context of primary HCV infection. We provide a statistical and visual explanation of the relationship between cooccurring mutations on the viral genome and the parallel adaptive immune response against HCV.
    BioMed Research International 06/2014; 2014:264519. DOI:10.1155/2014/264519 · 2.71 Impact Factor
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    • "Also, Ion Torrent's sequencing chips match the common manufacturing standards used for commercial microchips, meaning that it can be used for other technologies at equal low cost [9]. Moreover, Ion Torrent requires as little as 10 ng/ul of DNA to generate accurate results for challenging samples [10] whereas the traditional method of Sanger sequencing requires higher amount of DNA. "
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    ABSTRACT: Hemoglobinopathies, such as sickle cell disease (SCD) and beta-thalassemia major (TM), are severe diseases and the most common autosomal recessive condition worldwide and in particular in Oman. Early screening and diagnosis of carriers are the key for primary prevention. Once a country-wide population screening program is mandated by law, a sequencing technology that can rapidly confirm or identify disease-causing mutations for a large number of patients in a short period of time will be necessary. While Sanger sequencing is the standard protocol for molecular diagnosis, next generation sequencing starts to become available to reference laboratories. Using the Ion Torrent PGM sequencer, we have analyzed a cohort of 297 unrelated Omani cases and reliably identified mutations in the beta-globin (HBB) gene. Our model study has shown that Ion Torrent PGM can rapidly sequence such a small gene in a large number of samples using a barcoded uni-directional or bi-directional sequence methodology, reducing cost, workload and providing accurate diagnosis. Based on our results we believe that the Ion Torrent PGM sequencing platform, able to analyze hundreds of patients simultaneously for a single disease gene can be a valid molecular screening alternative to ABI sequencing in the diagnosis of hemoglobinopathies and other genetic disorders in the near future.
    Blood Cells Molecules and Diseases 05/2014; 53(3). DOI:10.1016/j.bcmd.2014.05.002 · 2.65 Impact Factor
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    • "cDNA-based library preparation was performed essentially as described previously (9, 12, 16, 18, 21, 22). Briefly, we used the Mint kit (Evrogen, Russia) for first-strand cDNA synthesis. "
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    ABSTRACT: The relationship between maternal and child immunity has been actively studied in the context of complications during pregnancy, autoimmune diseases, and haploidentical transplantation of hematopoietic stem cells and solid organs. Here, we have for the first time used high-throughput Illumina HiSeq sequencing to perform deep quantitative profiling of T cell receptor (TCR) repertoires for peripheral blood samples of three mothers and their six children. Advanced technology allowed accurate identification of 5 × 10(5) to 2 × 10(6) TCR beta clonotypes per individual. We performed comparative analysis of these TCR repertoires with the aim of revealing characteristic features that distinguish related mother-child pairs, such as relative TCR beta variable segment usage frequency and relative overlap of TCR beta complementarity-determining region 3 (CDR3) repertoires. We show that thymic selection essentially and similarly shapes the initial output of the TCR recombination machinery in both related and unrelated pairs, with minor effect from inherited differences. The achieved depth of TCR profiling also allowed us to test the hypothesis that mature T cells transferred across the placenta during pregnancy can expand and persist as functional microchimeric clones in their new host, using characteristic TCR beta CDR3 variants as clonal identifiers.
    Frontiers in Immunology 12/2013; 4:463. DOI:10.3389/fimmu.2013.00463
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