No abstract

The detection of minority variants in mixed samples demands methods for enrichment and accurate sequencing of small genomic intervals. We describe an efficient approach based on sequential rounds of hybridization with biotinylated oligonucleotides, enabling more than one-million fold enrichment of genomic regions of interest. In conjunction with error correcting double-stranded molecular tags, our approach enables the quantification of mutations in individual DNA molecules.

Cockayne syndrome (CS) and xeroderma pigmentosum (XP) are human photosensitive diseases with mutations in the nucleotide excision repair (NER) pathway, which repairs DNA damage from UV exposure. CS is mutated in the transcription-coupled repair (TCR) branch of the NER pathway and exhibits developmental and neurological pathologies. The XP-C group of XP patients have mutations in the global genome repair (GGR) branch of the NER pathway and have a very high incidence of UV-induced skin cancer. Cultured cells from both diseases have similar sensitivity to UV-induced cytotoxicity, but CS patients have never been reported to develop cancer, although they often exhibit photosensitivity. Because cancers are associated with increased mutations, especially when initiated by DNA damage, we examined UV-induced mutagenesis in both XP-C and CS cells, using duplex sequencing for high-sensitivity mutation detection. Duplex sequencing detects rare mutagenic events, independent of selection and in multiple loci, enabling examination of all mutations rather than just those that confer major changes to a specific protein. We found telomerase-positive normal and CS-B cells had increased background mutation frequencies that decreased upon irradiation, purging the population of subclonal variants. Primary XP-C cells had increased UV-induced mutation frequencies compared with normal cells, consistent with their GGR deficiency. CS cells, in contrast, had normal levels of mutagenesis despite their TCR deficiency. The lack of elevated UV-induced mutagenesis in CS cells reveals that their TCR deficiency, although increasing cytotoxicity, is not mutagenic. Therefore the absence of cancer in CS patients results from the absence of UV-induced mutagenesis rather than from enhanced lethality.

Transcriptional mutagenesis (TM) due to misincorporation during RNA transcription can result in mutant RNAs, or epimutations, that generate proteins with altered properties. TM has long been hypothesized to play a role in aging, cancer, and viral and bacterial evolution. However, inadequate methodologies have limited progress in elucidating a causal association. We present a high-throughput, highly accurate RNA sequencing method to measure epimutations with single-molecule sensitivity. Accurate RNA consensus sequencing (ARCseq) uniquely combines RNA barcoding and generation of multiple cDNA copies per RNA molecule to eliminate errors introduced during cDNA synthesis, PCR, and sequencing. The stringency of ARC-seq can be scaled to accommodate the quality of input RNAs. We apply ARCseq to directly assess transcriptome-wide epimutations resulting from RNA polymerase mutants and oxidative stress.transcriptional mutagenesis | epimutations | RNA mutations | molecular misreading | RNAseq I nfidelity during RNA transcription, termed transcriptional mutagenesis (TM), has long been hypothesized to contribute to aging (1) and age-associated diseases, including cancer (2, 3) and neurodegeneration (4, 5). RNA mutations resulting from TM, termed epimutations, have also been implicated in bacterial and viral evolution and resistance (6-8). Studies on RNA polymerases have revealed the fidelity of in vitro transcription by multiple RNA polymerases to be on the order of 10 −5 epimutations per nucleotide (9-13). This rate can dramatically increase during transcription of damaged templates and certain sequence contexts, such as repetitive DNA (14). Additionally, in vivo assays have revealed that TM can result in phenotypic changes in nondividing (15) and dividing cells (16)(17)(18)(19), with the potential for TM-induced phenotypic changes to be heritable (20, 21), indicating that a single mutant transcript has the potential to have profound effects on cellular function.The bulk of the evidence for TM has been generated using in vitro fidelity assays and highly expressed reporter genes that encompass a small number of sequence contexts, are limited in the spectrum of mutations that can be monitored, and are subject to translational errors convoluting the results (9, 22). Consequently, the results of these studies cannot be easily extrapolated to understand the extent of epimutations in cells, where transcription factors, repair enzymes, chromatin, and gene expression levels modulate transcriptional fidelity. Thus, to elucidate the roles of TM-induced epimutations in physiology, disease, and evolution, it is necessary to study individual RNA molecules transcribed in vivo in a high-throughput manner.De novo epimutations remain a challenging target for highthroughput RNA sequencing (RNAseq). While in vitro studies estimate RNA polymerase infidelity to be on the order of one in 100,000 epimutations per nucleotide, reverse transcriptase used to generate cDNA from RNA makes approximately one error per 10,000 bases (23). Additionally, Illu...