SummaryAccurate measurement of clonal genotypes, mutational processes, and replication states from individual tumor-cell genomes will facilitate improved understanding of tumor evolution. We have developed DLP+, a scalable single-cell whole-genome sequencing platform implemented using commodity instruments, image-based object recognition, and open source computational methods. Using DLP+, we have generated a resource of 51,926 single-cell genomes and matched cell images from diverse cell types including cell lines, xenografts, and diagnostic samples with limited material. From this resource we have defined variation in mitotic mis-segregation rates across tissue types and genotypes. Analysis of matched genomic and image measurements revealed correlations between cellular morphology and genome ploidy states. Aggregation of cells sharing copy number profiles allowed for calculation of single-nucleotide resolution clonal genotypes and inference of clonal phylogenies and avoided the limitations of bulk deconvolution. Finally, joint analysis over the above features defined clone-specific chromosomal aneuploidy in polyclonal populations.

Essential features of cancer tissue cellular heterogeneity such as negatively selected genome topologies, sub-clonal mutation patterns and genome replication states can only effectively be studied by sequencing single-cell genomes at scale and high fidelity. Using an amplification-free single-cell genome sequencing approach implemented on commodity hardware (DLP+) coupled with a cloud-based computational platform, we define a resource of 40,000 single-cell genomes characterized by their genome states, across a wide range of tissue types and conditions. We show that shallow sequencing across thousands of genomes permits reconstruction of clonal genomes to single nucleotide resolution through aggregation analysis of cells sharing higher order genome structure. From large-scale population analysis over thousands of cells, we identify rare cells exhibiting mitotic mis-segregation of whole chromosomes. We observe that tissue derived scWGS libraries exhibit lower rates of whole chromosome anueploidy than cell lines, and loss of p53 results in a shift in event type, but not overall prevalence in breast epithelium. Finally, we demonstrate that the replication states of genomes can be identified, allowing the number and proportion of replicating cells, as well as the chromosomal pattern of replication to be unambiguously identified in single-cell genome sequencing experiments.The combined annotated resource and approach provide a re-implementable large scale platform for studying lineages and tissue heterogeneity. Biological and physical determinants of high quality DLP+ library constructionWe initially applied the same reaction conditions from microfluidic DLP (Zahn et al., 2017) to establish amplificationfree single cell WGS in open arrays (DLP+). However this resulted in many poor quality libraries measured by a high proportion of: i) alignments for which interpretable, integer state copy number profiles could not be inferred and ii) failed libraries where coverage was low or absent (Figure 2a, c ii: 1 nL G2 buffer). We therefore sought to quantitatively establish the physical reaction determinants of high quality libraries (e.g. Figure S4b), based on the computed quality score from the classifier. We systematically varied and evaluated several factors: cell lysis volume and buffer type; transposase (Tn5) concentration; post-indexing PCR cycles; cell lysis/DNA solubilisation time; and cell viability state.We observed the following properties as determinants of high quality libraries. Cell lysis volume and buffer type exhibited a combinatorial effect, whereby increased volumes to avoid meniscus effects and evaporation required specific buffers that could be diluted in one pot reactions without impacting subsequent reactions (Figure 2c ii).As expected, increasing transposase (Tn5) concentrations increased library success (Figure 2c iii), but with a tradeoff of increasing bias in sequence GC representation (Figure 2e) and consequently genome coverage (Figure 2f).Similarly, we observed that an increase in post-indexing PCR...

a b s t r a c tGully erosion is one of the main causes of soil loss in drylands. Understanding the dominant mechanisms of erosion is important to achieve effective erosion control, thus in this study our main objective was to quantify the mechanisms involved in gully bank retreat as a result of three processes, falling of entire soil aggregates, transport of soil material by splash and by water running along gully banks (runoff), during rainfall events. The study was conducted in the sloping lands of the KwaZulu-Natal province, a region that is highly affected by gully erosion. Artificial rain was applied at 60 mm h −1 for 45 min at the vertical wall of a gully bank typical to the area. The splash material was collected by using a network of 0.045 m 2 buckets. The sediments in the running water were assessed by sampling the runoff collected from a microplot inserted within the base of the bank, and collecting the fallen aggregates after the rainfall simulation was complete. Results indicated that the overall erosion for the simulation was 721 g m −2 h −1 . Runoff erosion proved to be the dominant mechanism and amounted to 450 g m −2 h −1 , followed by splash and fall down of aggregates (about 170 g m −2 h −1 ). Gully bank retreat occurred at a rate of 0.55 mm h −1 and assuming that the soil bulk density is 1.3 g cm −3 , this corresponds to a retreat of 8.8 mm y −1 . Extrapolations to the watershed level, where about 500 m 2 of gully bank are observed per hectare, would lead to an erosion rate of 4.8 t ha −1 y −1 . These limited results based on a simulated storm show that the three main mechanisms (runoff, splash and fall down of aggregates) are responsible for the retreat of gully banks and that to mitigate gully erosion, appropriate measures are required to control all three mechanisms. Further research studies are needed to confirm and to scale up, both in time and space, as these data are obtained at one location and from a single artificial storm.

Detection of Leifsonia xyli subsp. xyli (Lxx) on a large scale is based on serological assays such as evaporative-binding enzyme-linked immunoassay (EB-EIA). These methods are time consuming and require well-equipped laboratories. This study presents the development of a loop-mediated isothermal amplification (LAMP) assay which allows detection of Lxx in 30 min at 65°C, using xylem sap as the template. The assay requires minimal laboratory equipment and could be used at near farm conditions, thus saving time and money required to transfer samples from remote areas to diagnostic laboratories. The LAMP method shows potential as an alternative detection method for RSD.

Ecological studies of microbial communities often use profiling methods but the true community diversity can be underestimated in methods that separate amplicons based on sequence length using performance optimized polymer 4. Taxonomically, unrelated organisms can produce the same length amplicon even though the amplicons have different sequences. F-108 polymer has previously been shown to resolve same length amplicons by sequence polymorphisms. In this study, we showed F-108 polymer, using the ABI Prism 310 Genetic Analyzer and CE, resolved four bacteria that produced the same length amplicon for the 16S rRNA domain V3 but have variable nucleotide content. Second, a microbial mat community profile was resolved and supported by NextGen sequencing where the number of peaks in the F-108 profile was in concordance with the confirmed species numbers in the mat. Third, equine DNA was analyzed for SNPs. The F-108 polymer was able to distinguish heterozygous and homozygous individuals for the melanocortin 1 receptor coat color gene. The method proved to be rapid, inexpensive, reproducible, and uses common CE instruments. The potential for F-108 to resolve DNA mixtures or SNPs can be applied to various sample types-from SNPs to forensic mixtures to ecological communities.