Genes of the major histocompatibility complex (MHC) code for cell surface proteins essential for adaptive immunity. They show the most outstanding genetic diversity in vertebrates, which has been connected with various fitness traits and thus with the long-term persistence of populations. In this study, polymorphism of the MHC class II DRB locus was investigated in chamois with Single-Strand Conformation Polymorphism (SSCP)/Sanger genotyping and Ion Torrent S5 next-generation sequencing (NGS). From eight identified DRB variants in 28 individuals, five had already been described, and three were new, undescribed alleles. With conventional SSCP/Sanger sequencing, we were able to detect seven alleles, all of which were also detected with NGS. We found inconsistencies in the individual genotypes between the two methods, which were mainly caused by allelic dropout in the SSCP/Sanger method. Six out of 28 individuals were falsely classified as homozygous with SSCP/Sanger analysis. Overall, 25% of the individuals were identified as genotyping discrepancies between the two methods. Our results show that NGS technologies are better performing in sequencing highly variable regions such as the MHC, and they also have a higher detection capacity, thus allowing a more accurate description of the genetic composition, which is crucial for evolutionary and population genetic studies.

• Klíčová slova
• Ion Torrent, Rupicapra rupicapra, major histocompatibility complex, next-generation sequencing,
• Publikační typ
• časopisecké články MeSH

One of main steps in a study of microbial communities is resolving their composition, diversity and function. In the past, these issues were mostly addressed by the use of amplicon sequencing of a target gene because of reasonable price and easier computational postprocessing of the bioinformatic data. With the advancement of sequencing techniques, the main focus shifted to the whole metagenome shotgun sequencing, which allows much more detailed analysis of the metagenomic data, including reconstruction of novel microbial genomes and to gain knowledge about genetic potential and metabolic capacities of whole environments. On the other hand, the output of whole metagenomic shotgun sequencing is mixture of short DNA fragments belonging to various genomes, therefore this approach requires more sophisticated computational algorithms for clustering of related sequences, commonly referred to as sequence binning. There are currently two types of binning methods: taxonomy dependent and taxonomy independent. The first type classifies the DNA fragments by performing a standard homology inference against a reference database, while the latter performs the reference-free binning by applying clustering techniques on features extracted from the sequences. In this review, we describe the strategies within the second approach. Although these strategies do not require prior knowledge, they have higher demands on the length of sequences. Besides their basic principle, an overview of particular methods and tools is provided. Furthermore, the review covers the utilization of the methods in context with the length of sequences and discusses the needs for metagenomic data preprocessing in form of initial assembly prior to binning.

• Klíčová slova
• Abundance, Genomic signature, Metagenomics, Sequence binning, Taxonomy independent, Visualization,
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

Pre-mRNA splicing represents an important regulatory layer of eukaryotic gene expression. In the simple budding yeast Saccharomyces cerevisiae, about one-third of all mRNA molecules undergo splicing, and splicing efficiency is tightly regulated, for example, during meiotic differentiation. S. cerevisiae features a streamlined, evolutionarily highly conserved splicing machinery and serves as a favourite model for studies of various aspects of splicing. RNA-seq represents a robust, versatile, and affordable technique for transcriptome interrogation, which can also be used to study splicing efficiency. However, convenient bioinformatics tools for the analysis of splicing efficiency from yeast RNA-seq data are lacking. We present a complete workflow for the calculation of genome-wide splicing efficiency in S. cerevisiae using strand-specific RNA-seq data. Our pipeline takes sequencing reads in the FASTQ format and provides splicing efficiency values for the 5' and 3' splice junctions of each intron. The pipeline is based on up-to-date open-source software tools and requires very limited input from the user. We provide all relevant scripts in a ready-to-use form. We demonstrate the functionality of the workflow using RNA-seq datasets from three spliceosome mutants. The workflow should prove useful for studies of yeast splicing mutants or of regulated splicing, for example, under specific growth conditions.

• MeSH
• databáze nukleových kyselin MeSH
• mutace genetika   MeSH
• prekurzory RNA genetika   MeSH
• průběh práce * MeSH
• Saccharomyces cerevisiae genetika   MeSH
• sekvenční analýza RNA metody   MeSH
• sestřih RNA genetika   MeSH
• spliceozomy genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• prekurzory RNA MeSH