RESUMO
This communication describes our experience in large-scale G group-level high resolution HLA typing using three different DNA sequencing platforms - ABI 3730 xl, Illumina MiSeq and PacBio RS II. Recent advances in DNA sequencing technologies, so-called next generation sequencing (NGS), have brought breakthroughs in deciphering the genetic information in all living species at a large scale and at an affordable level. The NGS DNA indexing system allows sequencing multiple genes for large number of individuals in a single run. Our laboratory has adopted and used these technologies for HLA molecular testing services. We found that each sequencing technology has its own strengths and weaknesses, and their sequencing performances complement each other. HLA genes are highly complex and genotyping them is quite challenging. Using these three sequencing platforms, we were able to meet all requirements for G group-level high resolution and high volume HLA typing.
Assuntos
Antígenos HLA/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Teste de Histocompatibilidade , Alelos , Biologia Computacional/métodos , Sequenciamento de Nucleotídeos em Larga Escala/normas , Humanos , Reação em Cadeia da Polimerase , Reprodutibilidade dos Testes , Análise de Sequência de DNARESUMO
We present an electronic format for exchanging data for HLA and KIR genotyping with extensions for next-generation sequencing (NGS). This format addresses NGS data exchange by refining the Histoimmunogenetics Markup Language (HML) to conform to the proposed Minimum Information for Reporting Immunogenomic NGS Genotyping (MIRING) reporting guidelines (miring.immunogenomics.org). Our refinements of HML include two major additions. First, NGS is supported by new XML structures to capture additional NGS data and metadata required to produce a genotyping result, including analysis-dependent (dynamic) and method-dependent (static) components. A full genotype, consensus sequence, and the surrounding metadata are included directly, while the raw sequence reads and platform documentation are externally referenced. Second, genotype ambiguity is fully represented by integrating Genotype List Strings, which use a hierarchical set of delimiters to represent allele and genotype ambiguity in a complete and accurate fashion. HML also continues to enable the transmission of legacy methods (e.g. site-specific oligonucleotide, sequence-specific priming, and Sequence Based Typing (SBT)), adding features such as allowing multiple group-specific sequencing primers, and fully leveraging techniques that combine multiple methods to obtain a single result, such as SBT integrated with NGS.
Assuntos
Técnicas de Genotipagem , Antígenos HLA/genética , Sequenciamento de Nucleotídeos em Larga Escala , Teste de Histocompatibilidade , Canais de Potássio Corretores do Fluxo de Internalização/genética , Relatório de Pesquisa/normas , Software , Alelos , Biologia Computacional/métodos , Biologia Computacional/normas , Sistemas de Gerenciamento de Base de Dados , Genótipo , Humanos , Reprodutibilidade dos Testes , Análise de Sequência de DNARESUMO
The cucumber mosaic virus (CMV) replication complex has previously been shown to associate with cellular membranes. However, it remains unknown whether any host factors participate in this process. In this study, five groups of Arabidopsis tonoplast intrinsic protein (TIP) genes were isolated and the proteins they encoded were evaluated with regard to their interactions with CMV proteins. TIP1 and TIP2 were found to interact with the CMV 1a protein in the Sos recruitment system, whereas no interactions with the other three TIP subgroups were observed in this assay. The interaction of CMV 1a with the TIP1 and TIP2 proteins was confirmed via co-immunoprecipitation assays. Additionally, CMV 1a co-localized with TIP1 and TIP2 in transfected Arabidopsis protoplasts. The findings of this study suggest that members of two TIP subfamilies might affect CMV replication via interaction with CMV 1a in the tonoplasts.
Assuntos
Aquaporinas/metabolismo , Proteínas de Arabidopsis/metabolismo , Cucumovirus/fisiologia , Metiltransferases/metabolismo , Proteínas Virais/metabolismo , Aquaporinas/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Metiltransferases/genética , Protoplastos/metabolismo , Transfecção , Técnicas do Sistema de Duplo-Híbrido , Proteínas Virais/genética , Replicação ViralRESUMO
Using a yeast two-hybrid system, we identified a plant cellular factor that interacts with the proteins of the Cucumber mosaic virus (CMV). Initially 14 candidate genes were isolated from Nicotiana tabacum, using a full-length CMV 1a gene as bait. Among the candidate genes, two were encoding thaumatin-like proteins (TLP), and were designated as Nicotiana tabacum thaumatin-like protein 1 (NtTLP1). Consistent with this observation, recombinant GST-NtTLP1 protein, which was expressed and purified in E. coli, bound tightly to CMV 1a in vitro. In planta interaction was also verified via co-immunoprecipitation. Additionally, NtTLP1 specifically interacted with the CMV movement-related proteins, movement protein and coat protein, in yeast. Real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis showed that the expression of NtTLP1 increased as the result of CMV inoculation.