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[This corrects the article DOI: 10.1371/journal.pone.0166126.].
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Minimal residual disease (MRD) is known to be an independent prognostic factor in patients with acute lymphoblastic leukemia (ALL). High-throughput sequencing (HTS) is currently used in routine practice for the diagnosis and follow-up of patients with hematological neoplasms. In this retrospective study, we examined the role of immunoglobulin/T-cell receptor-based MRD in patients with ALL by HTS analysis of immunoglobulin H and/or T-cell receptor gamma chain loci in bone marrow samples from 11 patients with ALL, at diagnosis and during follow-up. We assessed the clinical feasibility of using combined HTS and bioinformatics analysis with interactive visualization using Vidjil software. We discuss the advantages and drawbacks of HTS for monitoring MRD. HTS gives a more complete insight of the leukemic population than conventional real-time quantitative PCR (qPCR), and allows identification of new emerging clones at each time point of the monitoring. Thus, HTS monitoring of Ig/TR based MRD is expected to improve the management of patients with ALL.
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Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Neoplasia Residual/diagnóstico , Leucemia-Linfoma Linfoblástico de Células Precursoras/diagnóstico , Médula Ósea , Células Clonales/patología , Estudios de Seguimiento , Genes Codificadores de la Cadena gamma de los Receptores de Linfocito T , Humanos , Cadenas Pesadas de Inmunoglobulina/genética , Monitorización Inmunológica , Neoplasia Residual/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Estudios Retrospectivos , Programas InformáticosRESUMEN
BACKGROUND: The B and T lymphocytes are white blood cells playing a key role in the adaptive immunity. A part of their DNA, called the V(D)J recombinations, is specific to each lymphocyte, and enables recognition of specific antigenes. Today, with new sequencing techniques, one can get billions of DNA sequences from these regions. With dedicated Repertoire Sequencing (RepSeq) methods, it is now possible to picture population of lymphocytes, and to monitor more accurately the immune response as well as pathologies such as leukemia. METHODS AND RESULTS: Vidjil is an open-source platform for the interactive analysis of high-throughput sequencing data from lymphocyte recombinations. It contains an algorithm gathering reads into clonotypes according to their V(D)J junctions, a web application made of a sample, experiment and patient database and a visualization for the analysis of clonotypes along the time. Vidjil is implemented in C++, Python and Javascript and licensed under the GPLv3 open-source license. Source code, binaries and a public web server are available at http://www.vidjil.org and at http://bioinfo.lille.inria.fr/vidjil. Using the Vidjil web application consists of four steps: 1. uploading a raw sequence file (typically a FASTQ); 2. running RepSeq analysis software; 3. visualizing the results; 4. annotating the results and saving them for future use. For the end-user, the Vidjil web application needs no specific installation and just requires a connection and a modern web browser. Vidjil is used by labs in hematology or immunology for research and clinical applications.
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Biología Computacional/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Recombinación V(D)J/genética , Navegador Web , Algoritmos , Secuencia de Bases , Humanos , Internet , Linfocitos/inmunología , Linfocitos/metabolismo , Reproducibilidad de los Resultados , Homología de Secuencia de Ácido NucleicoRESUMEN
High-throughput sequencing (HTS) is considered a technical revolution that has improved our knowledge of lymphoid and autoimmune diseases, changing our approach to leukaemia both at diagnosis and during follow-up. As part of an immunoglobulin/T cell receptor-based minimal residual disease (MRD) assessment of acute lymphoblastic leukaemia patients, we assessed the performance and feasibility of the replacement of the first steps of the approach based on DNA isolation and Sanger sequencing, using a HTS protocol combined with bioinformatics analysis and visualization using the Vidjil software. We prospectively analysed the diagnostic and relapse samples of 34 paediatric patients, thus identifying 125 leukaemic clones with recombinations on multiple loci (TRG, TRD, IGH and IGK), including Dd2/Dd3 and Intron/KDE rearrangements. Sequencing failures were halved (14% vs. 34%, P = 0.0007), enabling more patients to be monitored. Furthermore, more markers per patient could be monitored, reducing the probability of false negative MRD results. The whole analysis, from sample receipt to clinical validation, was shorter than our current diagnostic protocol, with equal resources. V(D)J recombination was successfully assigned by the software, even for unusual recombinations. This study emphasizes the progress that HTS with adapted bioinformatics tools can bring to the diagnosis of leukaemia patients.
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Biología Computacional/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Leucemia-Linfoma Linfoblástico de Células Precursoras/diagnóstico , Adolescente , Adulto , Niño , Preescolar , Células Clonales , Errores Diagnósticos/prevención & control , Reordenamiento Génico de Linfocito T , Secuenciación de Nucleótidos de Alto Rendimiento/normas , Humanos , Lactante , Recién Nacido , Neoplasia Residual/diagnóstico , Estudios Prospectivos , Programas Informáticos , Recombinación V(D)J/genética , Adulto JovenRESUMEN
BACKGROUND: V(D)J recombinations in lymphocytes are essential for immunological diversity. They are also useful markers of pathologies. In leukemia, they are used to quantify the minimal residual disease during patient follow-up. However, the full breadth of lymphocyte diversity is not fully understood. RESULTS: We propose new algorithms that process high-throughput sequencing (HTS) data to extract unnamed V(D)J junctions and gather them into clones for quantification. This analysis is based on a seed heuristic and is fast and scalable because in the first phase, no alignment is performed with germline database sequences. The algorithms were applied to TR γ HTS data from a patient with acute lymphoblastic leukemia, and also on data simulating hypermutations. Our methods identified the main clone, as well as additional clones that were not identified with standard protocols. CONCLUSIONS: The proposed algorithms provide new insight into the analysis of high-throughput sequencing data for leukemia, and also to the quantitative assessment of any immunological profile. The methods described here are implemented in a C++ open-source program called Vidjil.