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The occurrence of mutations in the BCR-ABL1 kinase domain (KD) can lead to treatment resistance in chronic myeloid leukaemia patients. Nowadays, next-generation sequencing (NGS) is an alternative method for the detection of kinase domain mutations, compared to routinely used Sanger sequencing, providing a higher sensitivity of mutation detection. However, in the protocols established so far multiple rounds of amplification limit reliable mutation detection to approximately 5% variant allele frequency. Here, we present a simplified, one-round amplification NGS protocol for the Illumina platform, which offers a robust early detection of BCR-ABL1 KD mutations with a reliable detection limit of 3% variant allele frequency.
- Klíčová slova
- BCR-ABL1, TKI resistance, illumina, kinase domain mutation, next generation sequencing,
- MeSH
- bcr-abl fúzové proteiny genetika MeSH
- chronická myeloidní leukemie genetika MeSH
- lidé MeSH
- mutace MeSH
- vysoce účinné nukleotidové sekvenování metody MeSH
- zdraví dobrovolníci pro lékařské studie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- bcr-abl fúzové proteiny MeSH
Understanding the complex Entamoeba communities in the mammalian intestine has been, to date, complicated by the lack of a suitable approach for molecular detection of multiple variants co-occurring in mixed infections. Here, we report on the application of a high throughput sequencing approach based on partial 18S rDNA using the Illumina MiSeq platform. We describe, to our knowledge, for the first time, the Entamoeba communities in humans, free-ranging western lowland gorillas and central chimpanzees living in the Dja Faunal Reserve in Cameroon. We detected 36 Entamoeba haplotypes belonging to six haplotype clusters, containing haplotypes possessing high and low host specificity. Most of the detected haplotypes belonged to commensal Entamoeba, however, the pathogenic species (Entamoeba histolytica and Entamoeba nuttalli) were also detected. We observed that some Entamoeba haplotypes are shared between humans and other hosts, indicating their zoonotic potential. The findings are important not only for understanding the epidemiology of amoebiasis in humans in rural African localities, but also in the context of wild great ape conservation.
- Klíčová slova
- Central chimpanzee, Diversity, Entamoeba, Entamoeba histolytica, Humans, Metabarcoding, Mixed infections, Western lowland gorilla,
- MeSH
- entamébóza epidemiologie parazitologie veterinární MeSH
- Entamoeba * MeSH
- Gorilla gorilla parazitologie MeSH
- lidé MeSH
- nemoci lidoopů epidemiologie parazitologie MeSH
- Pan troglodytes parazitologie MeSH
- parazitární nemoci střev epidemiologie parazitologie veterinární MeSH
- vysoce účinné nukleotidové sekvenování * MeSH
- zachování přírodních zdrojů MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- Afrika epidemiologie MeSH
SUMMARY: The Sequencing Read Archive is one of the largest and fastest-growing repositories of sequencing data, containing tens of petabytes of sequenced reads. Its data is used by a wide scientific community, often beyond the primary study that generated them. Such analyses rely on accurate metadata concerning the type of experiment and library, as well as the organism from which the sequenced reads were derived. These metadata are typically entered manually by contributors in an error-prone process, and are frequently incomplete. In addition, easy-to-use computational tools that verify the consistency and completeness of metadata describing the libraries to facilitate data reuse, are largely unavailable. Here we introduce HTSinfer, a Python-based tool to infer metadata directly and solely from bulk RNA-sequencing data generated on Illumina platforms. HTSinfer leverages genome sequence information and diagnostic genes to rapidly and accurately infer the library source and library type, as well as the relative read orientation, 3' adapter sequence and read length statistics. HTSinfer is written in a modular manner, published under a permissible free and open-source license and encourages contributions by the community, enabling easy addition of new functionalities, for example for the inference of additional metrics, or the support of different experiment types or sequencing platforms. AVAILABILITY AND IMPLEMENTATION: HTSinfer is released under the Apache License 2.0. Latest code is available via GitHub at https://github.com/zavolanlab/htsinfer, while releases are published on Bioconda. A snapshot of the HTSinfer version described in this article was deposited at Zenodo at 10.5281/zenodo.13985958.
- Publikační typ
- časopisecké články MeSH
Many studies correlate changes in human gut microbiome with the onset of various diseases, mostly by 16S rRNA gene sequencing. Setting up the optimal sampling and DNA isolation procedures is crucial for robustness and reproducibility of the results. We performed a systematic comparison of several sampling and DNA isolation kits, quantified their effect on bacterial gDNA quality and the bacterial composition estimates at all taxonomic levels. Sixteen volunteers tested three sampling kits. All samples were consequently processed by two DNA isolation kits. We found that the choice of both stool sampling and DNA isolation kits have an effect on bacterial composition with respect to Gram-positivity, however the isolation kit had a stronger effect than the sampling kit. The proportion of bacteria affected by isolation and sampling kits was larger at higher taxa levels compared to lower taxa levels. The PowerLyzer PowerSoil DNA Isolation Kit outperformed the QIAamp DNA Stool Mini Kit mainly due to better lysis of Gram-positive bacteria while keeping the values of all the other assessed parameters within a reasonable range. The presented effects need to be taken into account when comparing results across multiple studies or computing ratios between Gram-positive and Gram-negative bacteria.
- MeSH
- DNA bakterií genetika MeSH
- dospělí MeSH
- feces mikrobiologie MeSH
- fylogeneze MeSH
- gramnegativní bakterie klasifikace genetika izolace a purifikace MeSH
- grampozitivní bakterie klasifikace genetika izolace a purifikace MeSH
- lidé středního věku MeSH
- lidé MeSH
- mladý dospělý MeSH
- reagenční diagnostické soupravy MeSH
- reprodukovatelnost výsledků MeSH
- RNA ribozomální 16S genetika MeSH
- sekvenční analýza DNA MeSH
- vysoce účinné nukleotidové sekvenování metody MeSH
- zdraví dobrovolníci pro lékařské studie MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mladý dospělý MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- srovnávací studie MeSH
- Názvy látek
- DNA bakterií MeSH
- reagenční diagnostické soupravy MeSH
- RNA ribozomální 16S MeSH
Researchers have assembled thousands of eukaryotic genomes using Illumina reads, but traditional mate-pair libraries cannot span all repetitive elements, resulting in highly fragmented assemblies. However, both chromosome conformation capture techniques, such as Hi-C and Dovetail Genomics Chicago libraries and long-read sequencing, such as Pacific Biosciences and Oxford Nanopore, help span and resolve repetitive regions and therefore improve genome assemblies. One important livestock species of arid regions that does not have a high-quality contiguous reference genome is the dromedary (Camelus dromedarius). Draft genomes exist but are highly fragmented, and a high-quality reference genome is needed to understand adaptation to desert environments and artificial selection during domestication. Dromedaries are among the last livestock species to have been domesticated, and together with wild and domestic Bactrian camels, they are the only representatives of the Camelini tribe, which highlights their evolutionary significance. Here we describe our efforts to improve the North African dromedary genome. We used Chicago and Hi-C sequencing libraries from Dovetail Genomics to resolve the order of previously assembled contigs, producing almost chromosome-level scaffolds. Remaining gaps were filled with Pacific Biosciences long reads, and then scaffolds were comparatively mapped to chromosomes. Long reads added 99.32 Mbp to the total length of the new assembly. Dovetail Chicago and Hi-C libraries increased the longest scaffold over 12-fold, from 9.71 Mbp to 124.99 Mbp and the scaffold N50 over 50-fold, from 1.48 Mbp to 75.02 Mbp. We demonstrate that Illumina de novo assemblies can be substantially upgraded by combining chromosome conformation capture and long-read sequencing.
- Klíčová slova
- chromosome conformation capture, chromosome mapping, dromedary, genome annotation, genome assembly, scaffolding,
- MeSH
- genom * MeSH
- genomika metody MeSH
- pouštní klima MeSH
- sekvenční analýza DNA metody MeSH
- velbloudi genetika MeSH
- výpočetní biologie metody MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Here, we present first draft genome sequence of the trypanosomatid Herpetomonas muscarum ingenoplastis. This parasite was isolated repeatedly in the black blowfly, Phormia regina, and it forms a phylogenetically distinct clade in the Trypanosomatidae family.
- Klíčová slova
- Trypanosomatidae, genome assembly, insect trypanosomatids, monoxenous trypanosomatids, whole genome,
- Publikační typ
- časopisecké články MeSH
The large size and complex structural rearrangements inherent in the mitochondrial genomes of land plants pose challenges for their sequencing. Originally, the assembly of these genomes required the cloning of mitochondrial DNA fragments followed by Sanger sequencing. Subsequently, the advent of next-generation sequencing significantly expedited the process. This review highlights examples of plant mitochondrial genome assembly employing various technologies, including 454 sequencing, Illumina short sequencing reads, and Pacific Biosciences or Oxford Nanopore Technology long sequencing reads. The combination of short and long reads in hybrid assembly has proven to be the most efficient approach for achieving reliable assemblies of land plant mitochondrial genomes.
- Klíčová slova
- Hybrid assembly, Unicycler, land plants, mitochondrial genome, next-generation sequencing, rearrangement, recombination,
- MeSH
- genom mitochondriální * MeSH
- genom rostlinný MeSH
- sekvenční analýza DNA metody MeSH
- vysoce účinné nukleotidové sekvenování metody MeSH
- vyšší rostliny * genetika MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
BACKGROUND: Silene latifolia is a dioecious [corrected] plant with well distinguished X and Y chromosomes that is used as a model to study sex determination and sex chromosome evolution in plants. However, efficient utilization of this species has been hampered by the lack of large-scale sequencing resources and detailed analysis of its genome composition, especially with respect to repetitive DNA, which makes up the majority of the genome. METHODOLOGY/PRINCIPAL FINDINGS: We performed low-pass 454 sequencing followed by similarity-based clustering of 454 reads in order to identify and characterize sequences of all major groups of S. latifolia repeats. Illumina sequencing data from male and female genomes were also generated and employed to quantify the genomic proportions of individual repeat families. The majority of identified repeats belonged to LTR-retrotransposons, constituting about 50% of genomic DNA, with Ty3/gypsy elements being more frequent than Ty1/copia. While there were differences between the male and female genome in the abundance of several repeat families, their overall repeat composition was highly similar. Specific localization patterns on sex chromosomes were found for several satellite repeats using in situ hybridization with probes based on k-mer frequency analysis of Illumina sequencing data. CONCLUSIONS/SIGNIFICANCE: This study provides comprehensive information about the sequence composition and abundance of repeats representing over 60% of the S. latifolia genome. The results revealed generally low divergence in repeat composition between the sex chromosomes, which is consistent with their relatively recent origin. In addition, the study generated various data resources that are available for future exploration of the S. latifolia genome.
In many plant species, somatic cell differentiation is accompanied by endoreduplication, a process during which cells undergo one or more rounds of DNA replication cycles in the absence of mitosis, resulting in nuclei with multiples of 2C DNA amounts (4C, 8C, 16C, etc.). In some orchids, a disproportionate increase in nuclear DNA contents has been observed, where successive endoreduplication cycles result in DNA amounts 2C + P, 2C + 3P, 2C + 7P, etc., where P is the DNA content of the replicated part of the 2C nuclear genome. This unique phenomenon was termed "progressively partial endoreplication" (PPE). We investigated processes behind the PPE in Ludisia discolor using flow cytometry (FCM) and Illumina sequencing. In particular, we wanted to determine whether chromatin elimination or incomplete genome duplication was involved, and to identify types of DNA sequences that were affected. Cell cycle analysis of root tip cell nuclei pulse-labeled with EdU revealed two cell cycles, one ending above the population of nuclei with 2C + P content, and the other with a typical "horseshoe" pattern of S-phase nuclei ranging from 2C to 4C DNA contents. The process leading to nuclei with 2C + P amounts therefore involves incomplete genome replication. Subsequent Illumina sequencing of flow-sorted 2C and 2C + P nuclei showed that all types of repetitive DNA sequences were affected during PPE; a complete elimination of any specific type of repetitive DNA was not observed. We hypothesize that PPE is part of a highly controlled transition mechanism from proliferation phase to differentiation phase of plant tissue development.
- Klíčová slova
- DNA replication, EdU, Ludisia discolor, cell cycle, endoreduplication, orchids,
- MeSH
- buněčné jádro genetika MeSH
- endoreduplikace genetika MeSH
- genom rostlinný MeSH
- listy rostlin genetika MeSH
- mitóza genetika MeSH
- Orchidaceae genetika MeSH
- polyploidie MeSH
- průtoková cytometrie metody MeSH
- replikace DNA genetika MeSH
- vysoce účinné nukleotidové sekvenování metody MeSH
- Publikační typ
- časopisecké články MeSH
Complete mitochondrial genomes and nuclear rRNA operons of eight geographically distinct isolates of the Asian fish tapeworm Schyzocotyle acheilognathi (syn. Bothriocephalus acheilognathi), representing the parasite's global diversity spanning four continents, were fully characterised using an Illumina sequencing platform. This cestode species represents an extreme example of a highly invasive, globally distributed pathogen of veterinary importance with exceptionally low host specificity unseen elsewhere within the parasitic flatworms. In addition to eight specimens of S. acheilognathi, we fully characterised its closest known relative and the only congeneric species, Schyzocotyle nayarensis, from cyprinids in the Indian subcontinent. Since previous nucleotide sequence data on the Asian fish tapeworm were restricted to a single molecular locus of questionable phylogenetic utility-the nuclear rRNA genes-separating internal transcribed spacers-the mitogenomic data presented here offer a unique opportunity to gain the first detailed insights into both the intraspecific phylogenetic relationships and population genetic structure of the parasite, providing key baseline information for future research in the field. Additionally, we identify a previously unnoticed source of error and demonstrate the limited utility of the nuclear rRNA sequences, including the internal transcribed spacers that has likely misled most of the previous molecular phylogenetic and population genetic estimates on the Asian fish tapeworm.
- Klíčová slova
- Asian fish tapeworm, Bothriocephalus acheilognathi, Illumina sequencing, Invasive parasite, Mitochondrial genome, Phylogeny, Ribosomal RNA, Schyzocotyle acheilognathi,
- MeSH
- biodiverzita MeSH
- Cestoda klasifikace genetika MeSH
- cestodózy parazitologie veterinární MeSH
- Cyprinidae parazitologie MeSH
- DNA helmintů genetika MeSH
- druhová specificita MeSH
- fylogeneze MeSH
- fylogeografie MeSH
- geny rRNA * MeSH
- nemoci ryb parazitologie MeSH
- operon MeSH
- ribozomální proteiny genetika MeSH
- vysoce účinné nukleotidové sekvenování metody MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- DNA helmintů MeSH
- ribozomální proteiny MeSH
