Project description:Long-read RNA sequencing (RNA-seq) is a powerful technology for transcriptome analysis, but the relatively low throughput of current long-read sequencing platforms limits transcript coverage. We present TEQUILA-seq, a versatile, easy-to-implement, and low-cost method for targeted long-read RNA-seq. TEQUILA-seq can be broadly used for targeted sequencing of full-length transcripts in diverse biomedical research settings.
Project description:Revertant mosaicism (RM) is a phenomenon in which inherited mutations are spontaneously corrected in somatic cells. RM occurs in some congenital skin diseases, although genetic validation of RM in clinically revertant skin has been challenging, especially when homologous recombination (HR) is responsible for RM. We introduced Nanopore Cas9-targeted sequencing (nCATS) for identifying HR in clinically revertant skin.
Project description:These experiments use a barcoded pool of reporter transcripts, each of which encode the same mScarlet-PPIG_LCD fusion protein, but using different degrees of GA-multivalency via codon bias, and containing a different number of constitutive introns. In order to be able to perform experiments using this pool, it was necessary to perform long-read sequencing of the plasmid pool to relate the barcodes in the 3' ends of the reporter to their gene structure. Therefore, we performed long-read sequencing of the plasmid pool (both the original pool used for transfection and the ePB plasmid used for PiggyBac integration). Furthermore, to determine the splicing patterns of the reporter genes, we transfected the plasmid pool into HeLa cells for 16 hours, then performed targeted long-read sequencing of the reporter plasmids via RT-PCR. Note: the Nanopore adapter ligation strategy means that reads can come in either orientation. To determine the gene architectures and barcodes, we used fuzzy string matching. First we matched to various fixed sequences throughout the reporter transcripts to determine the orientation of the read and that the read spanned the full length of the transcript. Then we used the same string matching strategy to detect the presence of the different intronic or exonic sequences - the gene architecture. Then we extracted the associated unique plasmid barcode associated with that gene architecture. Example reporter sequences can be found here: https://benchling.com/faraway/f_/kXCfddtQ-public-reporter-plasmid-maps/ or alternatively, in Supplemental Table 2 of the bioRxiv submission here: https://www.biorxiv.org/content/10.1101/2023.08.21.554177v1.supplementary-material
Project description:Evaluation of short-read-only, long-read-only, and hybrid assembly approaches on metagenomic samples demonstrating how they affect gene and protein prediction which is relevant for downstream functional analyses. For a human gut microbiome sample, we use complementary metatranscriptomic, and metaproteomic data to evaluate the metagenomic-based protein predictions.
