The accuracy of DNA replication, gene transcription, and protein translation is critical to maintain the integrity of the genetic code. Mistakes that occur during transcription can cause genomic instability and human diseases. Here, we employed Circular Sequencing (Cir-seq) to investigate the mutational profiles and landscape of the yeast Saccharomyces Cerevisiae W303-1A-Rad5^R535 strain that carries a variant of Rad5 gene that may impairs transcription-coupled DNA repair or promote transcription-replication conflicts. Three batches of Cir-seq are prepared with error rates of 1.99 × 10^-6, 4.66 × 10^-6 and 5.27× 10^-6. Dominant G to U mutations are found in two batches with a high coverage number. G to U errors are clustered when the focal base of G is flanked by GU and UC. A list o...[ Read more ]

The accuracy of DNA replication, gene transcription, and protein translation is critical to maintain the integrity of the genetic code. Mistakes that occur during transcription can cause genomic instability and human diseases. Here, we employed Circular Sequencing (Cir-seq) to investigate the mutational profiles and landscape of the yeast Saccharomyces Cerevisiae W303-1A-Rad5^R535 strain that carries a variant of Rad5 gene that may impairs transcription-coupled DNA repair or promote transcription-replication conflicts. Three batches of Cir-seq are prepared with error rates of 1.99 × 10^-6, 4.66 × 10^-6 and 5.27× 10^-6. Dominant G to U mutations are found in two batches with a high coverage number. G to U errors are clustered when the focal base of G is flanked by GU and UC. A list of hotspots is obtained for an error rate range of 10^-3 to 10^-6 with all types of mutations, most of which are related to the ribosomal gene, which may impact proteostasis.

Background error model-coupled precision nuclear run-on circular-sequencing (EmPC-seq) is our newly developed method for accurate sequencing of nascent RNA produced by RNA polymerase I. We present the initial development of EmPC-seq and identification of error hotspots and the features of the RNA polymerase I transcribed region by introducing a background baseline. Mismatch rates are greatly reduced from 1.43±0.11×10^-3 (PRO-seq) to 3.79±0.81×10^-5 (EmPC-seq), implying that EmPC-seq can successfully remove noise generated mainly during reverse transcription. One feature of RNA polymerase I is that errors tend to cluster at the 3’ end. EmPC-seq can be further extended to study different transcriptional error spectra of pre-mRNA (RNA polymerase II transcribed), siRNAs, and regulatory RNA between normal and disease cells.

Following the development of EmPC-seq, we were inspired to develop nucleotide resolution sequencing to obtain the influenza A polymerase (FluA pol) distribution and mutation spectrum by utilizing the PRO-seq and NET-seq principle. This method is temporarily named Pre-terminated RNA Elongation with Circular Influenza-A-transcript Sequencing Effort, PRECISE. We demonstrated that the incorporation of ß-D-2’-deoxy-2’-α-fluoro guanosine triphosphate (2’FdGTP) can stall influenza A polymerase upon elongation, terminating the transcript with a 2’FdGTP end, which resists RNase R degradation. FluA pol also demonstrated the ability to protect RNA (at least 14 nt) in the active site from various kinds of nuclease in permeabilized extracts.