Background: The state-of-the-art in nucleic acid based biodetection continues to be polymerase chain reaction (PCR), and many real-time PCR assays targeting biodefense pathogens for biosurveillance are in widespread use. These assays are predominantly singleplex; i.e. one assay tests for the presence of one target, found in a single organism, one sample at a time. Due to the intrinsic limitations of such tests, there exists a critical need for highthroughput multiplex assays to reduce the time and cost incurred when screening multiple targets, in multiple pathogens, and in multiple samples. Such assays allow users to make an actionable call while maximizing the utility of the small volumes of test samples. Unfortunately, current multiplex real-time PCR assays are limited in the number of targets that can be probed simultaneously due to the availability of fluorescence channels in real-time PCR instruments. Results: To address this gap, we developed a pipeline in which the amplicons produced by a 14-plex end-point PCR assay using spiked samples were subsequently sequenced using Nanopore technology. We used bar codes to sequence multiple samples simultaneously, leading to the generation and subsequent analysis of sequence data resulting from a short sequencing run time (< 10 min). We compared the limits of detection (LoD) of real-time PCR assays to Oxford Nanopore Technologies (ONT)-based amplicon sequencing and estimated the sample-to-answer time needed for this approach. Overall, LoDs determined from the first 10 min of sequencing data were at least one to two orders of magnitude lower than real-time PCR. Given enough time, the amplicon sequencing approach is approximately 100 times more sensitive than real-time PCR, with detection of amplicon specific reads even at the lowest tested spiking concentration (around 2.5-50 Colony Forming Units (CFU)/ml).

Setaria viridis (green foxtail) is an important model system for improving cereal crops due to its diploid genome, ease of cultivation, and use of C4 photosynthesis. The S. viridis accession ME034V is exceptionally transformable, but the lack of a sequenced genome for this accession has limited its utility. We present a 397 Mb highly contiguous de novo assembly of ME034V using ultra-long nanopore sequencing technology (read N50=41kb). We estimate that this genome is largely complete based on our updated k-mer based genome size estimate of 401 Mb for S. viridis. Genome annotation identified 37,908 protein-coding genes and >300k repetitive elements comprising 46% of the genome. We compared the ME034V assembly with two other previously sequenced Setaria genomes as well as to a diversity panel of 235 S. viridis accessions. We found the genome assemblies to be largely syntenic, but numerous unique polymorphic structural variants were discovered. Several ME034V deletions may be associated with recent retrotransposition of copia and gypsy LTR repeat families, as evidenced by their low genotype frequencies in the sampled population. Lastly, we performed a phylogenomic analysis to identify gene families that have expanded in Setaria, including those involved in specialized metabolism and plant defense response. The high continuity of the ME034V genome assembly validates the utility of ultra-long DNA sequencing to improve genetic resources for emerging model organisms. Structural variation present in Setaria illustrates the importance of obtaining the proper genome reference for genetic experiments. Thus, we anticipate that the ME034V genome will be of significant utility for the Setaria research community.

Reference genome fidelity is critically important for genome wide association studies, yet most vary widely from the study population. A typical whole genome sequencing approach implies short-read technologies resulting in fragmented assemblies with regions of ambiguity. Further information is lost by economic necessity when genotyping populations, as lower resolution technologies such as genotyping arrays are commonly used. Here, we present a phased reference genome for Canis lupus familiaris using high molecular weight DNA-sequencing technologies. We tested wet laboratory and bioinformatic approaches to demonstrate a minimum workflow to generate the 2.4 gigabase genome for a Labrador Retriever. The de novo assembly required eight Oxford Nanopore R9.4 flowcells (∼23X depth) and running a 10X Genomics library on the equivalent of one lane of an Illumina NovaSeq S1 flowcell (∼88X depth), bringing the cost of generating a nearly complete reference genome to less than $10K (USD). Mapping of short-read data from 10 Labrador Retrievers against this reference resulted in 1% more aligned reads versus the current reference (CanFam3.1, P < 0.001), and a 15% reduction of variant calls, increasing the chance of identifying true, low-effect size variants in a genome-wide association studies. We believe that by incorporating the cost to produce a full genome assembly into any large-scale genotyping project, an investigator can improve study power, decrease costs, and optimize the overall scientific value of their study.

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24Setaria viridis (green foxtail) is an important model system for improving cereal crops 25 due to its diploid genome, ease of cultivation, and use of C4 photosynthesis. The S. 26 viridis cultivar ME034V is exceptionally transformable, but the lack of a sequenced 27 genome for this cultivar has limited its utility. We present a 397 Mb highly contiguous de 28 novo assembly of ME034V using ultra-long nanopore sequencing technology (read 29 N50=41kb). We estimate that this genome is largely complete based on our updated k-30 mer based genome size estimate of 401 Mb for S. viridis. Genome annotation identified 31 37,908 protein-coding genes and >300k repetitive elements comprising 46% of the 32 genome. We compared the ME034V assembly with two other previously sequenced 33Setaria genomes as well as to a diversity panel of 235 S. viridis cultivars. We found the 34 genome assemblies to be largely syntenic, but numerous unique polymorphic structural 35 variants were discovered. Several ME034V deletions may be associated with recent 36 retrotransposition of copia and gypsy LTR repeat families, as evidenced by their low 37 genotype frequencies in the sampled population. Lastly, we performed a phylogenomic 38 analysis to identify gene families that have expanded in Setaria, including those 39 involved in specialized metabolism and plant defense response. The high continuity of 40 the ME034V genome assembly validates the utility of ultra-long DNA sequencing to 41 improve genetic resources for emerging model organisms. Structural variation present 42 in Setaria illustrates the importance of obtaining the proper genome reference for 43 genetic experiments. Thus, we anticipate that the ME034V genome will be of significant 44 utility for the Setaria research community. 45 47 Grasses of the genus Setaria represent diverse species, with phenotypes ranging from 48 the domesticated food crop foxtail millet, S. italica, to its weedy ancestral progenitor, 49 green foxtail, S. viridis (Li and Brutnell 2011). Simple growth requirements, small 50 stature, and short lifecycle make Setaria a tractable monocot model system for studying 51 C4 photosynthesis (Brutnell et al. 2010; Li and Brutnell 2011; Van Eck and Swartwood 52 2015). Furthermore, close phylogenetic relationships with agriculturally important crops 53 such as maize and sorghum promise to inform genetic and cell biology knowledge of 54 other food crops of global importance. Current genome resources for Setaria include a 55 reference genome for S. italica (Bennetzen et al. 2012; Zhang et al. 2012) based on the 56 cultivar Yugu1, a variety of foxtail millet widely grown as a food crop in China. 57 Additionally, a de novo assembly of S. viridis cultivar A10.1 (hereafter referred to as 58 A10) was recently made available alongside low coverage resequencing of more than 59 600 Setaria ecotypes (Huang et al. 2019). 60 61 Efficient genetic modification is a primary requirement for development of any model 62 organism. Approaches for Setaria protoplasting, particle bombardment, and 63...