Biology subjects

Hauenstein, J.

Publications and source records attributed to Hauenstein, J..

3 recordsLinked to original sources

bioRxiv2022

T-RHEX-RNAseq - A tagmentation-based, rRNA blocked, random hexamer primed RNAseq method for generating stranded RNAseq libraries directly from very low numbers of lysed cells.

BackgroundRNA sequencing has become the mainstay for studies of gene expression. Still, analysis of rare cells with random hexamer priming - to allow analysis of a broader range of transcripts - remains challenging. ResultsWe here describe a tagmentation-based, rRNA blocked, random hexamer primed RNAseq approach (T-RHEX-RNAseq) for generating stranded RNAseq libraries from very low numbers of FACS sorted cells without RNA purification steps. ConclusionT-RHEX-RNAseq provides an easy-to-use, time efficient and automation compatible method for generating stranded RNAseq libraries from rare cells.

molecular biology↗

bioRxiv2022

FOXO dictates initiation of B cell development and myeloid restriction in common lymphoid progenitors

The development of B cells relies on an intricate network of transcription factors critical for developmental progression and lineage commitment. In the B cell developmental trajectory, a temporal switch from predominant Foxo3 to Foxo1 expression occurs at the CLP stage. Utilizing VAV-iCre mediated conditional deletion, we found that the loss of FOXO3 impaired B cell development from LMPP down to B cell precursors, while the loss of FOXO1 impaired B cell commitment and resulted in a complete developmental block at the CD25 negative proB cell stage. Strikingly, the combined loss of FOXO1 and FOXO3 resulted in the failure to restrict the myeloid potential of CLPs and the complete loss of the B cell lineage. This is underpinned by the failure to enforce the early B-lineage gene regulatory circuitry upon a predominantly pre-established open chromatin landscape. Altogether, this demonstrates that FOXO3 and FOXO1 cooperatively govern early lineage restriction and initiation of B-lineage commitment in CLPs. SUMMARYCommon lymphoid progenitors co-express the transcription factors FOXO1 and FOXO3. Removing FOXO1 and FOXO3 at this developmental stage results in regained myeloid potential, failed establishment of the early B cell gene regulatory program, and the complete loss of the B cell lineage.

immunology↗

bioRxiv2021

Linked-read whole-genome sequencing resolves common and private structural variants in multiple myeloma

Multiple myeloma (MM) is an incurable and aggressive plasma cell malignancy characterized by a complex karyotype with multiple structural variants (SVs) and copy number variations (CNVs). Linked-read whole-genome sequencing (lrWGS) allows for refined detection and reconstruction of SVs by providing long-range genetic information from standard short-read sequencing. This makes lrWGS an attractive solution for capturing the full genomic complexity of MM. Here we show that high-quality lrWGS data can be generated from low numbers of FACS sorted cells without DNA purification. Using this protocol, we analyzed FACS sorted MM cells from 37 MM patients with lrWGS. We found high concordance between lrWGS and FISH for the detection of recurrent translocations and CNVs. Outside of the regions investigated by FISH, we identified >150 additional SVs and CNVs across the cohort. Analysis of the lrWGS data allowed for resolving the structure of diverse SVs affecting the MYC and t(11;14) loci causing the duplication of genes and gene regulatory elements. In addition, we identified private SVs causing the dysregulation of genes recurrently involved in translocations with the IGH locus and show that these can alter the molecular classification of the MM. Overall, we conclude that lrWGS allows for the detection of aberrations critical for MM prognostics and provides a feasible route for providing comprehensive genetics. Implementing lrWGS could provide more accurate clinical prognostics, facilitate genomic medicine initiatives, and greatly improve the stratification of patients included in clinical trials. KEY POINTS- Linked-read WGS can be performed without DNA purification and allows for resolving the diverse structural variants found in multiple myeloma. - Linked-read WGS can, as a stand-alone assay, provide comprehensive genetics in myeloma and other diseases with complex genomes.

genetics↗