A wide variety of temperate animals rely on length of day (photoperiodism) to anticipate and prepare for changing seasons by regulating the timing of development, reproduction, dormancy, and migration. Although the molecular basis of circadian rhythms regulating daily activities is well defined, the molecular basis for the photoperiodic regulation of seasonal activities is largely unknown. We use geographic variation in the photoperiodic control of diapause in the pitcher-plant mosquito Wyeomyia smithii to create the first QTL map of photoperiodism in any animal. For critical photoperiod (CPP), we detect QTL that are unique, a QTL that is sex linked, QTL that overlap with QTL for stage of diapause (SOD), and a QTL that interacts epistatically with the circadian rhythm gene, timeless. Results presented here confirm earlier studies concluding that CPP is under directional selection over the climatic gradient of North America and that the evolution of CPP is genetically correlated with SOD. Despite epistasis between timeless and a QTL for CPP, timeless is not located within any detectable QTL, indicating that it plays an ancillary role in the evolution of photoperiodism in W. smithii. Finally, we highlight one region of the genome that includes loci contributing to CPP, SOD, and hormonal regulation of development.T HE annual change in day length at temperate latitudes provides a highly reliable indicator of future seasonal events and a wide variety of organisms use day length (photoperiod) to time their development, reproduction, dormancy, and migration. Concordance between individual photoperiodic response and local climate is an essential component of fitness for animals living in the temperate zone (Bradshaw et al. 2004), and modification of photoperiodic response is an important adaptation of animals during range expansion (Danilevskii 1965;Cooke 1977;Tauber et al. 1986;Danks 1987;Lounibos et al. 2003) or when confronted with rapid climate change (Bradshaw and Holzapfel 2001a, 2006). Photoperiodism provides an ecologically relevant, highly heritable trait whose adaptive significance in a temperate seasonal environment is not questioned. Length of the favorable or growing season in North America decreases with increasing latitude (Bradshaw 1976). Consequently, the optimal time to enter diapause advances to an earlier day in the year and the day length used to switch from active development to diapause [hereafter, the critical photoperiod (CPP)] is positively correlated with latitude and altitude among a wide variety of temperate arthropods (Danilevskii 1965;Taylor and Spalding 1986;Danks 1987). The consistent genetic change in photoperiodic response over geographic and climatic gradients provides one of the most robust examples of repeated adaptive evolution in nature.Although much progress has been made in identifying the genetic components of the circadian clock regulating daily activities, the molecular basis of the photoperiodic timer regulating seasonal activities is conspicuously absent from the ...

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The gold standard of molecular pathogen detection is the quantitative polymerase chain reaction (qPCR). Modern qPCR instruments are capable of detecting 4−6 analytes in a single sample: one per optical detection channel. However, many clinical applications require multiplexing beyond this traditional single-well capacity, including the task of simultaneously testing for SARS-CoV-2 and other respiratory pathogens. This can be addressed by dividing a sample across multiple wells, or using technologies such as genomic sequencing and spatial arrays, but at the expense of significantly higher cost and lower throughput compared with single-well qPCR. These trade-offs represent unacceptable compromises in high-throughput screening scenarios such as SARS-CoV-2 testing. We demonstrate a novel method of detecting up to 20 targets per well with standard qPCR instrumentation: high-definition PCR (HDPCR). HDPCR combines TaqMan chemistry and familiar workflows with robust encoding to enable far higher levels of multiplexing on a traditional qPCR system without an increase in cost or reduction in throughput. We utilize HDPCR with a custom 20-Plex assay, an 8-Plex assay using unmodified predesigned single-plex assays from Integrated DNA Technologies and a 9-Plex pathogen panel inclusive of SARS-CoV-2 and other common respiratory viruses. All three assays were successful when tested on a variety of samples, with overall sample accuracies of 98.8, 98.3, and 100%, respectively. The HDPCR technology enables the large install base of qPCR instrumentation to perform mid-density multiplex diagnostics without modification to instrumentation or workflow, meeting the urgent need for increased diagnostic yield at an affordable price without sacrificing assay performance.

The real time polymerase chain reaction (rtPCR) is an essential method for detecting nucleic acids that has a wide range of clinical and research applications. Current multiplexed rtPCR is capable of detecting four to six nucleic acid targets in a single sample. However, advances in clinical medicine are driving the need to measure many more targets at once. We demonstrate a novel method which significantly increases the multiplexing capability of any existing rtPCR instrument without new hardware, software, or chemistry. The technique works by varying the relative TaqMan probe concentrations amongst targets that are measured in a single fluorometric channel. Our fluorescent amplitude modulation method generates a unique rtPCR signature for every combination of targets present in a reaction. We demonstrate this technique by measuring nine different targets across three color channels with TaqMan reporting probes, yielding a detection accuracy of 98.9% across all combinations of targets. In principle this method could be extended to measure 6 or more targets per color channel across any number of color channels without loss in specificity.

Digital PCR (dPCR) is the gold-standard analytical platform for rapid highprecision quantification of genomic fragments. However, current dPCR assays are generally limited to monitoring 1−2 analytes per sample, thereby limiting the platform's ability to address some clinical applications that require the simultaneous monitoring of 20−50 analytes per sample. Here, we present virtual-partition dPCR (VPdPCR), a novel analysis methodology enabling the detection of 10 or more target regions per color channel using conventional dPCR hardware and workflow. Furthermore, VPdPCR enables dPCR instruments to overcome upper quantitation limits caused by partitioning error. While traditional dPCR analysis establishes a single threshold to separate negative and positive partitions, VPdPCR establishes multiple thresholds to identify the number of unique targets present in each positive droplet based on fluorescence intensity. Each physical partition is then divided into a series of virtual partitions, and the resulting increase in partition count substantially decreases partitioning error. We present both a theoretical analysis of the advantages of VPdPCR and an experimental demonstration in the form of a 20-plex assay for noninvasive fetal aneuploidy testing. This demonstration assaytested on 432 samples contrived from sheared cell-line DNA at multiple input concentrations and simulated fractions of euploid or trisomy-21 "fetal" DNAis analyzed using both traditional dPCR thresholding and VPdPCR. VPdPCR analysis significantly lowers the variance of the chromosomal ratio across replicates and increases the accuracy of trisomy identification when compared to traditional dPCR, yielding > 98% single-well sensitivity and specificity. VPdPCR has substantial promise for increasing the utility of dPCR in applications requiring ultrahigh-precision quantitation.