Highlights d Ovarian cancer can be detected by ultra-accurate sequencing of uterine lavage DNA d However, low-frequency TP53 mutations also exist in normal tissue of healthy women d TP53 mutations are increasingly selected for with age, revealing somatic evolution d Age-associated, cancer-like mutations challenge specificity for cancer detection

Next-generation sequencing methods suffer from low recovery, uneven coverage, and false mutations. DNA fragmentation by sonication is a major contributor to these problems because it produces randomly sized fragments, PCR amplification bias, and end artifacts. In addition, oligonucleotide-based hybridization capture, a common target enrichment method, has limited efficiency for small genomic regions, contributing to low recovery. This becomes a critical problem in clinical applications, which value cost-effective approaches focused on the sequencing of small gene panels. To address these issues, we developed a targeted genome fragmentation approach based on CRISPR/Cas9 digestion that produces DNA fragments of similar length. These fragments can be enriched by a simple size selection, resulting in targeted enrichment of up to approximately 49,000-fold. Additionally, homogenous length fragments significantly reduce PCR amplification bias and maximize read usability. We combined this novel target enrichment approach with Duplex Sequencing, which uses double-strand molecular tagging to correct for sequencing errors. The approach, termed CRISPR-DS, enables efficient target enrichment of small genomic regions, even coverage, ultra-accurate sequencing, and reduced DNA input. As proof of principle, we applied CRISPR-DS to the sequencing of the exonic regions of and performed side-by-side comparisons with standard Duplex Sequencing. CRISPR-DS detected previously reported pathogenic mutations present as low as 0.1% in peritoneal fluid of women with ovarian cancer, while using 10- to 100-fold less DNA than standard Duplex Sequencing. Whether used as standalone enrichment or coupled with high-accuracy sequencing methods, CRISPR-based fragmentation offers a simple solution for fast and efficient small target enrichment.

Human colorectal cancers (CRCs) contain both clonal and subclonal mutations. Clonal driver mutations are positively selected, present in most cells, and drive malignant progression. Subclonal mutations are randomly dispersed throughout the genome, providing a vast reservoir of mutant cells that can expand, repopulate the tumor, and result in the rapid emergence of resistance, as well as being a major contributor to tumor heterogeneity. Here, we apply duplex sequencing (DS) methodology to quantify subclonal mutations in CRC tumor with unprecedented depth (104) and accuracy (<10−7). We measured mutation frequencies in genes encoding replicative DNA polymerases and in genes frequently mutated in CRC, and found an unexpectedly high effective mutation rate, 7.1 × 10−7. The curve of subclonal mutation accumulation as a function of sequencing depth, using DNA obtained from 5 different tumors, is in accord with a neutral model of tumor evolution. We present a theoretical approach to model neutral evolution independent of the infinite-sites assumption (which states that a particular mutation arises only in one tumor cell at any given time). Our analysis indicates that the infinite-sites assumption is not applicable once the number of tumor cells exceeds the reciprocal of the mutation rate, a circumstance relevant to even the smallest clinically diagnosable tumor. Our methods allow accurate estimation of the total mutation burden in clinical cancers. Our results indicate that no DNA locus is wild type in every malignant cell within a tumor at the time of diagnosis (probability of all cells being wild type, 10−308).

Highlights d wnt-3 is upregulated early in posterior wounds and is required for tail regeneration d wnt-3 is expressed in stem cells and is needed for stem cell proliferation at wound sites d A general wound response factor, egr, activates wnt-3 during regeneration d Pre-existing patterning information is required for wnt-3 upregulation

11Whole-body regeneration requires the re-establishment of body axes for appropriate patterning 12 of new and old tissue. Wnt signaling has been utilized to correctly regenerate tissues along the 13 primary axis in many animals. However, the causal molecular mechanisms that first launch Wnt 14 signaling during regeneration are poorly characterized. We used the acoel worm Hofstenia 15 miamia to identify processes that initiate Wnt signaling. Transcriptome profiling, in situ 16 hybridization, and functional studies revealed a Wnt ligand, wnt-3, as an early wound-induced 17 gene specifically activated in posterior-facing wound sites and was required for establishing 18 posterior identity during regeneration. wnt-3 was upregulated upon amputation in stem cells, 19 and its inhibition affected stem cell proliferation. Ectopic expression of anterior markers in wnt-3 20 RNAi head fragments was stem cell dependent. Chromatin accessibility data revealed that wnt-21 3 activation during regeneration required input from the general wound response. Additionally, 22 the expression of a different Wnt ligand, wnt-1, prior to amputation was required for activation of 23 wound-induced wnt-3 expression. Our study establishes a gene regulatory network for initiating 24 Wnt signaling in posterior tissues in a bilaterian. 25 26 Keywords 27 Regeneration, axial polarity, acoel, Wnt signaling, wound response, posterior identity 28 29 Although it has been hypothesized that transient suppression of the repressor must occur to 63 enable Wnt3 activation at oral-facing wound sites(Nakamura et al., 2011), and expression 64 analysis shows a corresponding absence of Sp5 prior to Wnt3 activation(Vogg et al., 2019), the 65 mechanisms leading to transcription of the Wnt3 locus upon amputation are not known in 66 cnidarians. 67 It is also unknown which transcriptional programs induce Wnt ligand expression upon 68 wounding in bilaterians. In planarian regeneration, mechanisms for inhibition of Wnt signaling pathways for initiation of Wnt 72 ligand expression, which occurs at both anterior-and posterior-facing wound sites, are yet to be 73 identified. The control of Wnt signaling during regeneration has not yet been investigated in 74 acoels. Here, we sought to assess the dynamics of Wnt pathway expression during 75 regeneration and to identify mechanisms that drive its activation upon amputation in Hofstenia. 76 Our analysis of the regeneration transcriptome of Hofstenia revealed that Wnt ligands 77 and other posterior markers are expressed at posterior-facing wound sites within six hours 78 following amputation. To find candidate genes for the initiation of this expression, we focused on 79 the earliest asymmetries between anterior-and posterior-facing wound sites during 80 regeneration. We found that a combination of a generic wound response factor and a pre-81 existing patterning gradient activates a Wnt ligand specifically at posterior-facing wound sites 82 within three hours upon wounding. Specific establishment of Wnt signaling at posterior-faci...