Shaipanich, T. | Search bioRxiv

bioRxiv2023

Single-cell RNA-sequencing of bronchoscopy specimens: development of a rapid, minimal-handling protocol

Single-cell RNA-sequencing (scRNA-seq) is an important tool for understanding disease pathophysiology, including airways diseases. Currently, the majority of scRNA-seq studies in airways diseases have used invasive methods (airway biopsy, surgical resection) which carry inherent risks and thus present a major limitation to scRNA-seq investigation of airway pathology. Bronchial brushing, where the airway mucosa is sampled using a cytological brush, is a viable, less invasive method of obtaining airway cells for scRNA-seq. Here we are describing the development of a rapid and minimal-handling protocol for preparing single cell suspensions from bronchial brush specimens for scRNA-seq. Our optimized protocol maximises cell recovery and cell quality, and may facilitate large-scale profiling of the airway transcriptome at single cell resolution. Lay abstractSingle-cell RNA-sequencing (scRNA-seq) measures the gene expression of individual cells, and may be useful for understanding disease processes. scRNA-seq may be used to investigate lung diseases, but using invasive methods such as biopsy or surgery limits our ability to conduct large research studies. Bronchial brushing, where a soft brush is used to collect cells from inside the lungs, is a safer method but we need a better way to isolate individual cells from the brush specimens. We developed a method that is faster and involves less handling of the specimens compared to other published methods. Our method may therefore be useful for conducting large scRNA-seq studies in lung diseases.

genomics↗

bioRxiv2022

HIV proviral genetic diversity, compartmentalization and inferred dynamics in lung and blood during long-term suppressive antiretroviral therapy

The lung is an understudied site of HIV persistence. We isolated 882 subgenomic proviral sequences by single-genome approaches from blood and lung from nine individuals on long-term suppressive antiretroviral therapy (ART), and characterized genetic diversity and compartmentalization using formal tests. Consistent with clonal expansion as a driver of HIV persistence, identical sequences comprised between 9% to 86% of within-host datasets, though their location (blood vs. lung) followed no consistent pattern. The majority (77%) of participants harbored at least one sequence shared across blood and lung, supporting the migration of clonally-expanded cells between sites. No participant exhibited genetic compartmentalization so obvious that it was visually apparent in a phylogeny. When formal tests were applied however, two (22%) participants showed modest yet significant support for compartmentalization when analysis was restricted to distinct proviruses per site. This increased to four participants (44%) when considering all within-host sequences. Thus, while a minority of individuals harbor somewhat distinctive proviral populations in blood and lung, these can simply be due to unequal distributions of clonally-expanded sequences. Importantly, the extent of lung proviral diversity on ART strongly reflected that in blood (Spearman {rho} = 0.98, p < 0.0001), confirming blood as a strong indicator of total body proviral diversity. Analysis of on-ART proviral diversity in context of pre-therapy viral diversity in two participants revealed marked differences in proviral longevity and dynamics. Whereas one participants proviral pool was rich in ancestral sequences that recapitulated more of HIVs within-host evolutionary history, the others largely comprised more contemporary sequences, including ones that re-seeded the reservoir during a three-year treatment interruption. Results highlight the genetic complexity of proviruses persisting in lung and blood during ART, and the uniqueness of each individuals proviral composition. Individualized HIV remission and cure strategies may be needed to overcome these challenges. Author SummaryHIV persists in the body despite long-term suppressive antiretroviral therapy. Much of our knowledge about the HIV reservoir comes from studying proviruses in blood, but a fundamental question is whether these are distinct from those in tissues. The lung could theoretically engender genetically distinctive HIV populations, but this remains understudied. Our analysis of nearly 900 proviral sequences from blood and lung of nine individuals with HIV receiving long-term therapy revealed substantial within-host heterogeneity, yet some common patterns. Identical sequences (consistent with clonal expansion of infected cells) were observed in everyone, though at different (9-86%) frequencies. Recovery of shared sequences across blood and lung was common (77% of participants). Fewer than half of participants exhibited blood-lung genetic compartmentalization, and only modestly so. Moreover, when present, compartmentalization was often attributable to differential distribution of identical sequences, not the presence of genetically distinctive populations, across sites. Results also revealed marked inter-individual differences in proviral dynamics, as evidenced by the proportion of persisting proviruses that represented ancestral versus more recently-circulating viral sequences. Critically, the extent of within-host proviral diversity in blood correlated strongly with that in lung, indicating that despite inter-individual heterogeneity, blood is a strong indicator of proviral diversity elsewhere in the body.

molecular biology↗