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Bachmann, J. A.

Publications and source records attributed to Bachmann, J. A..

3 recordsLinked to original sources

Adapter dilution and input optimisation for Qiagen QIAseq miRNA Library kit

MicroRNAs (miRNAs) are small, non-coding RNA that play a critical role in regulating gene expressions that are important for a multitude of biological processes. In the preparation of miRNA sequencing libraries by using QIAseq miRNA library kit, adapter dimers might occur inadvertently and compromise the sequencing performance. In this technical note, we tested different adapter dilution strategies to minimise or diminish adapter dimers without compromising data quality. In our experiments, we found that RNA input amount plays a larger role in adapter dimer formation rather than the availability of the adapters itself. We suggest preparing the sequencing library with 10 times more RNA than the recommended input amount of the chosen protocol, e.g. following the adapter dilution and PCR cycle number of the standard 1 ng protocol with 10 ng RNA input. We also suggest analysing the data with deduplication by using unique molecular identifiers (UMIs) as it helps remove unusable reads.

genomics↗

Single-cell omics-based characterization of human basophils reveals two transcriptionally distinct populations

BackgroundBasophils are implicated in various diseases including allergies, but a comprehensive characterization of human basophils has yet to be performed at the single-cell level. ObjectiveWe aimed to generate a single-cell omics-based resource of circulating human basophils, to be made accessible to the research community. We also sought to investigate basophil heterogeneity at the transcriptional and surface epitope levels. MethodsCirculating basophils were analyzed using short- and long-read single-cell RNA-sequencing in combination with large-scale immunoprofiling of more than 100 cell surface markers. ResultsWe used a cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) based cell barcoding system to accurately track side scatterlow lineage- CCR3+ Fc{varepsilon}RI+ basophils through the single-cell omics analysis. The approach allowed for accurate transcriptional profiling of basophils among low-density peripheral blood leukocytes. The generation of an additional dataset with only basophils revealed two transcriptionally distinct populations. Single-cell transcriptomic analysis of samples from additional donors was performed and coupled with a large-scale immunoprofiling screen, producing a third CITE-seq dataset containing basophils. The analysis verified the existence of the two transcriptionally distinct populations and revealed that these populations have similar immunophenotypes with regard to the investigated surface markers. Long-read single-cell RNA-sequencing analysis provided further insights into the gene expression dynamics of the circulating basophils and confirmed the transcriptionally defined basophil states. ConclusionsThe single-cell omics analysis revealed two transcriptionally distinct basophil populations. Our single-cell omics resource constitutes a reference for charting human basophils at the cellular and molecular levels in health and disease. Capsule summaryShort- and long-read single-cell RNA-sequencing coupled with large-scale immunoprofiling characterize human basophils in peripheral blood and reveal two transcriptionally distinct populations.

immunology↗

Genetic basis and timing of a major mating system shift in Capsella

Shifts from outcrossing to self-fertilisation have occurred repeatedly in many different lineages of flowering plants, and often involve the breakdown of genetic outcrossing mechanisms. In the Brassicaceae, self-incompatibility (SI) allows plants to ensure outcrossing by recognition and rejection of self-pollen on the stigma. This occurs through the interaction of female and male specificity components, consisting of a pistil based receptor and a pollen-coat protein, both of which are encoded by tightly linked genes at the S-locus. When benefits of selfing are higher than costs of inbreeding, theory predicts that loss-of-function mutations in the male (pollen) SI component should be favoured, especially if they are dominant. However, it remains unclear whether mutations in the male component of SI are predominantly responsible for shifts to self-compatibility, and testing this prediction has been difficult due to the challenges of sequencing the highly polymorphic and repetitive ~100 kbp S-locus. The crucifer genus Capsella offers an excellent opportunity to study multiple transitions from outcrossing to self-fertilization, but so far, little is known about the genetic basis and timing of loss of SI in the self-fertilizing diploid Capsella orientalis. Here, we show that loss of SI in C. orientalis occurred within the past 2.6 Mya and maps as a dominant trait to the S-locus. Using targeted long-read sequencing of multiple complete S-haplotypes, we identify a frameshift deletion in the male specificity gene SCR that is fixed in C. orientalis, and we confirm loss of male SI specificity. We further analyze RNA sequencing data to identify a conserved, S-linked small RNA (sRNA) that is predicted to cause dominance of self-compatibility. Our results suggest that degeneration of pollen SI specificity in dominant S-alleles is important for shifts to self-fertilization in the Brassicaceae.\n\nAuthor SummaryAlready Darwin was fascinated by the widely varying modes of plant reproduction. The shift from outcrossing to self-fertilization is considered one of the most frequent evolutionary transitions in flowering plants, yet we still know little about the genetic basis of these shifts. In the Brassicaceae, outcrossing is enforced by a self-incompatibility (SI) system that enables the recognition and rejection of self pollen. This occurs through the action of two tightly linked genes at the S-locus, that encode a receptor protein located on the stigma (female component) and a pollen ligand protein (male component), respectively. Nevertheless, SI has frequently been lost, and theory predicts that mutations in the male component should have an advantage during the loss of SI, especially if they are dominant. To test this hypothesis, we mapped the loss of SI in a selfing species from the genus Capsella, a model system for evolutionary genomics. We found that loss of SI mapped to the S-locus, which harbored a dominant loss-of-function mutation in the male SI protein, and as expected, we found that male specificity was indeed lost in C. orientalis. Our results suggest that transitions to selfing often involve parallel genetic changes.

evolutionary biology↗