NLM DIR Seminar Schedule

Abstract:

Capturing the single nuclear and single cellular transcriptomics using snRNA-seq and scRNA-seq have advantages of their own. In snRNA-seq the message made by a cell at a certain time is captured whereas in scRNA-seq, the message stored for a longer period is captured as well. Similarly, RNA-seq profiles (sn or sc) captured from different stages of the life-cycle can reveal differences in the transcriptome across life stages. However, co-analyzing such diverse datasets together to gain biological insights poses significant challenges as the datasets could have batch effects eclipsing the biological differences, or even have significantly different RNA-features. Here we use an alternate approach where we first analyze the datasets separately utilizing the individual nuances and then contrast the analyses by a post-analysis alignment procedure. Specifically, we align the trajectory of germ cells from different data sets and set out to identify if there are any differences in the germ cell developmental stages revealed by the nuclear and whole cell transcriptomics at either life-stages.

We analyzed three datasets from different labs: 1) snRNA-seq on adult testis from Fly Single Cell Atlas, 2) scRNA-seq on adult testis and 3) scRNA-seq on larval testis. The snRNA-seq germline has 21,061 nuclei whereas the adult and the larval scRNA-seq have 6,438 and 9,044 cells respectively. The trajectory analyses using monocle3 on these datasets separately arrange the germ cells that progress from spermatogonia to spermatids via different developmental stages. However, to contrast these individual trajectories for uncovering differences in biology, we adapted Dynamic Time Warping (DTW). We were able to align the three different trajectories on a common warped pseudotime scale. The alignment of germline adult and larval scRNA-seq pseudotime revealed cell states in adults (elongating spermatids) that are absent in the larvae confirming the legitimate alignment. Buoyed by the accuracy of the alignment by DTW, we next focussed on the difference between snRNA-seq and scRNA-seq of adult germ cells and identified a set of genes for which the cell stops producing the RNAs in the nucleus while differentiating from spermatocyte to spermatid but shows perdurance of transcript in the cytoplasm. While some of these genes are known in literature, some are our novel findings that need to be validated experimentally.