RESUMEN
At present, low-pass whole-genome sequencing (WGS) is frequently used in clinical research and in the screening of copy number variations (CNVs). However, there are still some challenges in the detection of triploids. Restriction site-associated DNA sequencing (RAD-Seq) technology is a reduced-representation genome sequencing technology developed based on next-generation sequencing. Here, we verified whether RAD-Seq could be employed to detect CNVs and triploids. In this study, genomic DNA of 11 samples was extracted employing a routine method and used to build libraries. Five cell lines of known karyotypes and 6 triploid abortion tissue samples were included for RAD-Seq testing. The triploid samples were confirmed by STR analysis and also tested by low-pass WGS. The accuracy and efficiency of detecting CNVs and triploids by RAD-Seq were then assessed, compared with low-pass WGS. In our results, RAD-Seq detected 11 out of 11 (100%) chromosomal abnormalities, including 4 deletions and 1 aneuploidy in the purchased cell lines and all triploid samples. By contrast, these triploids were missed by low-pass WGS. Furthermore, RAD-Seq showed a higher resolution and more accurate allele frequency in the detection of triploids than low-pass WGS. Our study shows that, compared with low-pass WGS, RAD-Seq has relatively higher accuracy in CNV detection at a similar cost and is capable of identifying triploids. Therefore, the application of this technique in medical genetics has a significant potential value.
Asunto(s)
Variaciones en el Número de Copia de ADN/genética , Mapeo Restrictivo , Análisis de Secuencia de ADN/métodos , Triploidía , Línea Celular , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Secuenciación Completa del GenomaRESUMEN
CD4+ T cells play an essential role in protection against Mycobacterium tuberculosis (MTB) infection. We identified three HLA-DRB1*09:01-restricted CD4+ T-cell epitopes derived from the dominant secreted MTB antigens 38 kDa (Rv3804c) and Ag85A (Rv0934). The antigens were screened for epitopes by in silico prediction programs and analysis of IFN-γ induction in the peripheral blood mononuclear cells (PBMCs) from TB patients. In response to three of the high-affinity predicted epitopes derived from 38 kDa and Ag85A, CD4+ T cells from HLA-DRB1*09:01 TB patients were stimulated to produce IFN-γ and Tumor Necrosis Factor (TNF)-α. The three epitopes were also found to induce the proliferation of CD4+ T cells by carboxyfluorescein succinimidyl ester-diluted assays. These HLA-DRB1*09:01-restricted CD4+ T-cell epitopes facilitate analysis of the role of 38 kDa- and Ag85A-specific T cells in MTB infection and pave way for the design of vaccines against tuberculosis.
Asunto(s)
Antígenos Bacterianos/inmunología , Linfocitos T CD4-Positivos/efectos de los fármacos , Epítopos de Linfocito T/inmunología , Cadenas HLA-DRB1/inmunología , Mycobacterium tuberculosis/inmunología , Péptidos/farmacología , Tuberculosis Pulmonar/inmunología , Alelos , Secuencia de Aminoácidos , Antígenos Bacterianos/química , Bioensayo , Linfocitos T CD4-Positivos/inmunología , Proliferación Celular , Epítopos de Linfocito T/química , Expresión Génica , Frecuencia de los Genes , Cadenas HLA-DRB1/genética , Humanos , Interferón-alfa/biosíntesis , Interferón-alfa/metabolismo , Interferón gamma/biosíntesis , Interferón gamma/metabolismo , Interleucina-17/biosíntesis , Interleucina-17/metabolismo , Interleucina-4/biosíntesis , Interleucina-4/metabolismo , Mycobacterium tuberculosis/química , Mycobacterium tuberculosis/crecimiento & desarrollo , Péptidos/síntesis química , Cultivo Primario de Células , Tuberculosis Pulmonar/microbiología , Factor de Necrosis Tumoral alfa/biosíntesis , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
New vaccines are needed to combat Mycobacterium tuberculosis (MTB) infections. The currently employed Bacillus Calmette-Guérin vaccine is becoming ineffective, due in part to the emergence of multidrug-resistant tuberculosis (MDR-TB) strains and the reduced immune capacity in cases of HIV coinfection. CD8(+) T cells play an important role in the protective immunity against MTB infections, and the identification of immunogenic CD8(+) T cell epitopes specific for MTB is essential for the design of peptide-based vaccines. To identify CD8(+) T cell epitopes of MTB proteins, we screened a set of 94 MTB antigens for HLA class I A*11:01-binding motifs. HLA-A*11:01 is one of the most prevalent HLA molecules in Southeast Asians, and definition of T cell epitopes it can restrict would provide significant coverage for the Asian population. Peptides that bound with high affinity to purified HLA molecules were subsequently evaluated in functional assays to detect interferon-γ release and CD8(+) T cell proliferation in active pulmonary TB patients. We identified six novel epitopes, each derived from a unique MTB antigen, which were recognized by CD8(+) T cells from active pulmonary TB patients. In addition, a significant level of epitope-specific T cells could be detected ex vivo in peripheral blood mononuclear cells from active TB patients by an HLA-A*11:01 dextramer carrying the peptide Rv3130c194-204 (from the MTB triacylglycerol synthase Tgs1), which was the most frequently recognized epitope in our peptide library. In conclusion, this study identified six dominant CD8(+) T cell epitopes that may be considered potential targets for subunit vaccines or diagnostic strategies against TB.