Towards error-free profiling of immune repertoires

Shugay, Mikhail; Britanova, Olga V.; Merzlyak, Ekaterina M.; Turchaninova, Maria A.; Mamedov, Ilgar Z.; Tuganbaev, Timur; Bolotin, Dmitriy A.; Staroverov, Dmitry B; Putintseva, Ekaterina V.; Plevová, Karla; Linnemann, Carsten; Shagin, Dmitry A.; Pospı́šilová, Šárka; Lukyanov, Sergey; Schumacher, Ton N.; Chudakov, Dmitriy M.

doi:10.1038/nmeth.2960

Cited by 397 publications

(432 citation statements)

References 23 publications

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“…For example, if one UMI of 12 nt length is amplified and sequenced 10 4 times, it may routinely produce ∼10-20 erroneous UMI subvariants generated during PCR amplification, which may be represented altogether by .100-200 sequencing reads, according to our experience with spike-in CDR3 sequence controls in BCR and TCR libraries (28,29,36). In this situation, straightforward data interpretation could be that there were 11-21 TCR or BCR cDNA molecules at the start, whereas there was only 1.…”

Section: Revising Umi-based Analysismentioning

confidence: 99%

“…For each library, preliminary MiTCR (29) analysis without using UMI information identified from 61,860 to 702,195 CDR3-containing sequencing reads (Supplemental Table IIa). In MIGEC analysis (36), reads that carried the same molecular identifier and thus covered the same starting cDNA molecule were grouped. Erroneous UMI subvariants were filtered according to the logic described above.…”

Section: Experiments 1: Eight Replicas Of 4000 Pbmcsmentioning

confidence: 99%

“…This approach minimizes quantitative biases associated with uneven amplification efficiency for different TCR and BCR genes (3,32). Additionally, we employed barcoding of each cDNA molecule using unique molecular identifiers (UMIs) (5,(33)(34)(35)(36), which provides several important advantages.…”

mentioning

confidence: 99%

“…This information is averaged, which efficiently eliminates most PCR and sequencing errors. As an additional step, MIGEC filters the hot-spot errors, which is critical to achieve deep error-free sequencing (36). In contrast to the frequency-based grouping of homologous reads (27,28), selective grouping of reads basing on UMI information does not result in the loss of true sample diversity.…”

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confidence: 99%

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Quantitative Profiling of Immune Repertoires for Minor Lymphocyte Counts Using Unique Molecular Identifiers

Egorov

Merzlyak

Shelenkov

et al. 2015

The Journal of Immunology

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Emerging high-throughput sequencing methods for the analyses of complex structure of TCR and BCR repertoires give a powerful impulse to adaptive immunity studies. However, there are still essential technical obstacles for performing a truly quantitative analysis. Specifically, it remains challenging to obtain comprehensive information on the clonal composition of small lymphocyte populations, such as Ag-specific, functional, or tissue-resident cell subsets isolated by sorting, microdissection, or fine needle aspirates. In this study, we report a robust approach based on unique molecular identifiers that allows profiling Ag receptors for several hundred to thousand lymphocytes while preserving qualitative and quantitative information on clonal composition of the sample. We also describe several general features regarding the data analysis with unique molecular identifiers that are critical for accurate counting of starting molecules in high-throughput sequencing applications.

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Section: Revising Umi-based Analysismentioning

confidence: 99%

Section: Experiments 1: Eight Replicas Of 4000 Pbmcsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Quantitative Profiling of Immune Repertoires for Minor Lymphocyte Counts Using Unique Molecular Identifiers

Egorov

Merzlyak

Shelenkov

et al. 2015

The Journal of Immunology

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“…A further step forward came with the use of template switch enzymes and 5′ RACE, as has been frequently used in T cell biology 59, 60, 61, 62, 63, 64, 65. The 5′ RACE method has an advantage over consensus immunoglobulin PCR because it only requires priming in the constant region and adds a primer landing site in the 5′ with the addition of a template switch oligo (TSO).…”

Section: Repertoire Analysis Approachesmentioning

confidence: 99%

Immunoglobulin gene analysis as a tool for investigating human immune responses

Dunn-Walters

Townsend

Sinclair

et al. 2018

Immunological Reviews

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SummaryThe human immunoglobulin repertoire is a hugely diverse set of sequences that are formed by processes of gene rearrangement, heavy and light chain gene assortment, class switching and somatic hypermutation. Early B cell development produces diverse IgM and IgD B cell receptors on the B cell surface, resulting in a repertoire that can bind many foreign antigens but which has had self‐reactive B cells removed. Later antigen‐dependent development processes adjust the antigen affinity of the receptor by somatic hypermutation. The effector mechanism of the antibody is also adjusted, by switching the class of the antibody from IgM to one of seven other classes depending on the required function. There are many instances in human biology where positive and negative selection forces can act to shape the immunoglobulin repertoire and therefore repertoire analysis can provide useful information on infection control, vaccination efficacy, autoimmune diseases, and cancer. It can also be used to identify antigen‐specific sequences that may be of use in therapeutics. The juxtaposition of lymphocyte development and numerical evaluation of immune repertoires has resulted in the growth of a new sub‐speciality in immunology where immunologists and computer scientists/physicists collaborate to assess immune repertoires and develop models of immune action.

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High‐throughput sequencing of immune repertoires in multiple sclerosis

Lossius

Johansen

Vartdal

et al. 2016

Ann Clin Transl Neurol

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T cells and B cells are crucial in the initiation and maintenance of multiple sclerosis (MS), and the activation of these cells is believed to be mediated through specific recognition of antigens by the T‐ and B‐cell receptors. The antigen receptors are highly polymorphic due to recombination (T‐ and B‐cell receptors) and mutation (B‐cell receptors) of the encoding genes, which can therefore be used as fingerprints to track individual T‐ and B‐cell clones. Such studies can shed light on mechanisms driving the immune responses and provide new insights into the pathogenesis. Here, we summarize studies that have explored the T‐ and B‐cell receptor repertoires using earlier methodological approaches, and we focus on how high‐throughput sequencing has provided new knowledge by surveying the immune repertoires in MS in even greater detail and with unprecedented depth.

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Towards error-free profiling of immune repertoires

Cited by 397 publications

References 23 publications

Quantitative Profiling of Immune Repertoires for Minor Lymphocyte Counts Using Unique Molecular Identifiers

Quantitative Profiling of Immune Repertoires for Minor Lymphocyte Counts Using Unique Molecular Identifiers

Immunoglobulin gene analysis as a tool for investigating human immune responses

High‐throughput sequencing of immune repertoires in multiple sclerosis

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