Project description:This project carries out the pilot CRISPR/Cas9 screens in the K562 background. Its goals are to confirm that positive controls work, and to assess the effects of experimental parameters (listed below) on the sequencing-based fitness readout. We test 1) length of selection 2) biological replicates 3) sampling variation during bottlenecks 4) sampling variation during DNA preparation 5) sequencing depth to inform the setup for the next round of experiments. To do so, we propose to sequence 13 samples (6 timepoints, 2 biological replicates, 2 severe bottlenecks during growth, 2 bottlenecks during DNA preparation, and the screening library itself) on two lanes of HiSeq, using 19bp reads. The sequencing libraries are prepared in our lab.This data is part of a pre-publication release. For information on the proper use of pre-publication data shared by the Wellcome Trust Sanger Institute (including details of any publication moratoria), please see http://www.sanger.ac.uk/datasharing/
Project description:Electronic nicotine delivery systems (ENDS) are increasingly used worldwide, but their effects on respiratory microbial communities remain understudied. This pilot cross-sectional study compared the expectorated sputum microbiota of Lebanese adult ENDS users and non-ENDS users. Sputum samples were collected from 15 ENDS users and 15 non-ENDS users without chronic respiratory illness or recent antibiotic exposure. Samples were analyzed using 16S rRNA V3-V4 amplicon sequencing. Overall microbial richness, diversity, and evenness did not differ significantly between groups. In addition, presence/absence-based community composition showed no significant group-level separation. Both groups shared broadly similar dominant bacterial profiles with Bacillota, Pseudomonadota, and Bacteroidota among the most abundant phyla. Moreover, there was substantial overlap at the genus level. Despite these broad similarities, selective taxonomic differences were observed at finer resolutions including increased relative abundance of Nanosynbacter and decreased abundance of a putative Rothia species-level taxon among ENDS users after FDR correction. Actinomyces showed a nominal increase in ENDS users, but this did not remain significant after FDR correction. These findings suggest that ENDS use was not associated with broad sputum microbiota disruption in this cohort but may be associated with selective shifts in specific bacterial taxa.
