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BackgroundAn increasing number of studies have demonstrated the potential use of circulating cell-free DNA (cfDNA) for diagnosis, prognosis, disease progression, and treatment monitoring. However, many of these studies use assays covering a limited set of genes and therefore miss biologically and clinically relevant genetic alterations involving immuno-modulatory pathways which confer treatment resistance, and leading to changes in neoantigen status. To address this, we developed a whole-exome scale cfDNA platform, NeXT Liquid Biopsy™, that enables sensitive detection and tracking of mutations in approximately 20000 genes.MethodsTo enable sensitive detection across the exome, our enhanced exome assay and chemistry augments hard-to-sequence genomic regions, such as regions of high GC content, to enable more uniform coverage across the exome. We achieved a high mean sequencing depth of approximately 2000X exome-wide, with additionally boosted depth for 248 clinically relevant oncogenic and tumor suppressor genes to further enhance sensitivity. We developed a computational pipeline for our NeXT Liquid Biopsy assay optimized to lower the noise floor for variant detection, providing sensitive monitoring and de novo detection of variants over multiple time points.ResultsWe evaluated the sensitivity of our NeXT Liquid Biopsy platform in three ways. First, we evaluated the sensitivity within the coverage boosted regions using the SeraCare reference materials at multiple allele frequency (AF) dilutions. Our platform identified all 8 and 25 Horizon and SeraCare SNV events at 1% AF and above, respectively, and detected 24 out of 25 events at 0.5% for the SeraCare samples. Additionally, to enable sensitivity analysis at the whole-exome scale, we then developed a cell culture media system that models the shedding of tumor DNA fragments seen in human plasma samples and created tumor/normal dilution series in vitro. We achieved >95% sensitivity for variants with AF≥2%, and between 85% to 92% for mutations with AF of 1%-2%. Second, we evaluated false-positive rates on 12 cancer patients using digital droplet PCR. Third, we demonstrated our ability to longitudinally monitor treatment response using a clinical cancer cohort on checkpoint therapy, profiling putative tumor evolution while on therapy.ConclusionsIn conclusion, we have developed a whole-exome scale liquid biopsy platform, NeXT Liquid Biopsy, that enables sensitive monitoring and detection of somatic SNVs from cfDNA across ~20000 genes. The platform enables broader monitoring of changes in response to cancer therapy, acquired mechanisms of resistance, and intra- and inter-tumor heterogeneity.

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23A goal of speciation genetics is to understand how the genetic components underlying 24 interspecific reproductive barriers originate within species. Unilateral incompatibility (UI) is a 25 postmating prezygotic barrier in which pollen rejection in the female reproductive tract (style) 26 occurs in only one direction of an interspecific cross. Natural variation in the strength of UI has 27 been observed among populations within species in the wild tomato clade. In some cases, 28 molecular loci underlying self-incompatibility (SI) are associated with this variation in UI, but the 29 mechanistic connection between these intra-and inter-specific pollen rejection behaviors is 30 poorly understood in most instances. We generated an F 2 population between SI and SC 31 genotypes of a single species, Solanum pennellii, to examine the genetic basis of intraspecific 32 variation in the strength of UI against other species, and to determine whether loci underlying 33 SI are genetically associated with this variation. We found that F 2 individuals vary in the rate at 34 which UI rejection occurs. One large effect QTL detected for this trait co-localized with the SI-35 determining S-locus. Moreover, individuals that expressed S-RNase-the S-locus protein 36 involved in SI pollen rejection-in their styles had much more rapid UI responses compared to 37 those without S-RNase protein. Our analysis shows that intraspecific variation at mate choice 38 loci-in this case at loci that prevent self-fertilization-can contribute to variation in the 39 strength of interspecific isolation, including postmating prezygotic barriers. Understanding the 40 nature of such standing variation can provide insight into the accumulation of these barriers 41 between diverging lineages. 42 43 49reproducing organisms-reproductive isolation, among diverging lineages. Accordingly, loci that 50 contribute to this cumulative process between species must first arise within an individual 51 population prior to spreading to other conspecific populations within their own lineage. During 52 this process, populations of a single species are expected to show varying strengths of 53 reproductive isolation against other lineages; that is, there will be intraspecific genetic variation 54 for the magnitude of interspecific reproductive isolation from other lineages. Intraspecific 55 phenotypic variation in the strength of hybrid incompatibility has been observed in many 56 systems including mammals (Good, Handel and Nachman, 2007; Vyskočilová, Pražanová and 57 60 Sweigart, Mason and Willis, 2007). Understanding the nature, origin, and accumulation of this 61 variation, including the underlying molecular genetic variants responsible, can provide insight 62 into the evolutionary dynamics of lineage divergence (Cutter, 2012), including the order in 63 which alleles contributing to interspecific reproductive isolation arise and fix within diverging 64 lineages. 66The genetic basis of intraspecific variation for interspecific barriers has been 67 in...