Advances in genome sequencing and assembly technologies are generating many high-quality genome sequences, but assemblies of large, repeat-rich polyploid genomes, such as that of bread wheat, remain fragmented and incomplete. We have generated a new wheat whole-genome shotgun sequence assembly using a combination of optimized data types and an assembly algorithm designed to deal with large and complex genomes. The new assembly represents >78% of the genome with a scaffold N50 of 88.8 kb that has a high fidelity to the input data. Our new annotation combines strand-specific Illumina RNA-seq and Pacific Biosciences (PacBio) full-length cDNAs to identify 104,091 high-confidence protein-coding genes and 10,156 noncoding RNA genes. We confirmed three known and identified one novel genome rearrangements. Our approach enables the rapid and scalable assembly of wheat genomes, the identification of structural variants, and the definition of complete gene models, all powerful resources for trait analysis and breeding of this key global crop.

Accurate classification of a microbial mock community using MinION sequencing. We benchmarked MinION technology by profiling a bacterial mock community using R7.3 flow cells. Reads were analysed with NanoOK 18 and produced alignments to the 20 microbial reference sequences with 82-89% identity 19. Coverage ranged from almost 0 × (8 reads) of Actinomyces odontolyticus to 13 × (7,695 reads) of Streptococcus mutans, which is consistent with expected mock concentrations (Supplementary Table 1). Benchmarking to Illumina sequencing demonstrated high correlation with expected proportions (Fig. 1a, log-transformed Pearson's r = 0.94 for MinION and 0.97 for Illumina), and with each other (log-transformed Pearson's r = 0.98). Broadly similar abundance levels across both platforms were observed, with some differences in assignment to species versus genus/family (Fig. 1b). This is probable since the longer length Nanopore reads should provide

Advances in genome sequencing and assembly technologies are generating many high quality genome sequences, but assemblies of large, repeat-rich polyploid genomes, such as that of bread wheat, remain fragmented and incomplete. We have generated a new wheat whole-genome shotgun sequence assembly using a combination of optimised data types and an assembly algorithm designed to deal with large and complex genomes. The new assembly represents more than 78% of the genome with a scaffold N50 of 88.8kbp that has a high fidelity to the input data. Our new annotation combines strand-specific Illumina RNAseq and PacBio full-length cDNAs to identify 104,091 high confidence protein-coding genes and 10,156 non-coding RNA genes. We confirmed three known and identified one novel genome rearrangements. Our approach enables the rapid and scalable assembly of wheat genomes, the identification of structural variants, and the definition of complete gene models, all powerful resources for trait analysis and breeding of this key global crop. [Supplemental material is available for this article.]Running title: "An improved wheat genome assembly and annotation"

Background Riverine ecosystems are biogeochemical powerhouses driven largely by microbial communities that inhabit water columns and sediments. Because rivers are used extensively for anthropogenic purposes (drinking water, recreation, agriculture, and industry), it is essential to understand how these activities affect the composition of river microbial consortia. Recent studies have shown that river metagenomes vary considerably, suggesting that microbial community data should be included in broad-scale river ecosystem models. But such ecogenomic studies have not been applied on a broad “aquascape” scale, and few if any have applied the newest nanopore technology. Results We investigated the metagenomes of 11 rivers across 3 continents using MinION nanopore sequencing, a portable platform that could be useful for future global river monitoring. Up to 10 Gb of data per run were generated with average read lengths of 3.4 kb. Diversity and diagnosis of river function potential was accomplished with 0.5–1.0 ⋅ 106 long reads. Our observations for 7 of the 11 rivers conformed to other river-omic findings, and we exposed previously unrecognized microbial biodiversity in the other 4 rivers. Conclusions Deeper understanding that emerged is that river microbial consortia and the ecological functions they fulfil did not align with geographic location but instead implicated ecological responses of microbes to urban and other anthropogenic effects, and that changes in taxa manifested over a very short geographic space.

The ability to identify and quantify the constituent plant species that make up a mixed‐species sample of pollen has important applications in ecology, conservation, and agriculture. Recently, metabarcoding protocols have been developed for pollen that can identify constituent plant species, but there are strong reasons to doubt that metabarcoding can accurately quantify their relative abundances. A PCR‐free, shotgun metagenomics approach has greater potential for accurately quantifying species relative abundances, but applying metagenomics to eukaryotes is challenging due to low numbers of reference genomes. We have developed a pipeline, RevMet (Reverse Metagenomics) that allows reliable and semi‐quantitative characterization of the species composition of mixed‐species eukaryote samples, such as bee‐collected pollen, without requiring reference genomes. Instead, reference species are represented only by ‘genome skims’: low‐cost, low‐coverage, short‐read sequence datasets. The skims are mapped to individual long reads sequenced from mixed‐species samples using the MinION, a portable nanopore sequencing device, and each long read is uniquely assigned to a plant species. We genome‐skimmed 49 wild UK plant species, validated our pipeline with mock DNA mixtures of known composition, and then applied RevMet to pollen loads collected from wild bees. We demonstrate that RevMet can identify plant species present in mixed‐species samples at proportions of DNA ≥ 1%, with few false positives and false negatives, and reliably differentiate species represented by high versus low amounts of DNA in a sample. RevMet could readily be adapted to generate semi‐quantitative datasets for a wide range of mixed eukaryote samples. Our per‐sample costs were £90 per genome skim and £60 per pollen sample, and new versions of sequencers available now will further reduce these costs.

The separation of germ cell populations from the soma is part of the evolutionary transition to multicellularity. Only genetic information present in the germ cells will be inherited by future generations, and any molecular processes affecting the germline genome are therefore likely to be passed on. Despite its prevalence across taxonomic kingdoms, we are only starting to understand details of the underlying micro-evolutionary processes occurring at the germline genome level. These include segregation, recombination, mutation and selection and can occur at any stage during germline differentiation and mitotic germline proliferation to meiosis and post-meiotic gamete maturation. Selection acting on germ cells at any stage from the diploid germ cell to the haploid gametes may cause significant deviations from Mendelian inheritance and may be more widespread than previously assumed. The mechanisms that affect and potentially alter the genomic sequence and allele frequencies in the germline are pivotal to our understanding of heritability. With the rise of new sequencing technologies, we are now able to address some of these unanswered questions. In this review, we comment on the most recent developments in this field and identify current gaps in our knowledge.

1. The ability to identify and quantify the constituent plant species that make up a mixed-species sample of pollen has important applications in ecology, conservation, and agriculture. Recently, metabarcoding protocols have been developed for pollen that can identify constituent plant species, but there are strong reasons to doubt that metabarcoding can accurately quantify their relative abundances. A PCR-free, shotgun metagenomics approach has greater potential for accurately quantifying species relative abundances, but applying metagenomics to eukaryotes is challenging due to low numbers of reference genomes.2. We have developed a pipeline, RevMet (Reverse Metagenomics) that allows reliable and semi-quantitative characterization of the species composition of mixed-species eukaryote samples, such as bee-collected pollen, without requiring reference genomes. Instead, reference species are represented only by 'genome skims': low-cost, low-coverage, short-read sequence datasets. The skims are mapped to individual long reads sequenced from mixed-species samples using the MinION, a portable nanopore sequencing device, and each long read is uniquely assigned to a plant species.3. We genome-skimmed 49 wild UK plant species, validated our pipeline with mock DNA mixtures of known composition, and then applied RevMet to pollen loads collected from wild bees. We demonstrate that RevMet can identify plant species present in mixed-species samples at proportions of DNA ≥ 1%, with few false positives and false negatives, and reliably differentiate species represented by high versus low amounts of DNA in a sample.4. RevMet could readily be adapted to generate semi-quantitative datasets for a wide range of mixed eukaryote samples. Our per-sample costs were £90 per genome skim and £60 per pollen sample, and new versions of sequencers available now will further reduce these costs. K E Y W O R D Sbees, diet analysis, genome skim, metabarcoding, metagenomics, MinION, pollen, quantitative | 1691Methods in Ecology and Evoluঞon PEEL Et aL.

Background Miscanthus is a commercial lignocellulosic biomass crop owing to its high biomass productivity and low chemical input requirements. Within an interspecific Miscanthus cross, progeny with high biomass yield were shown to have low concentrations of starch and sucrose but high concentrations of fructose. We performed a transcriptional RNA-seq analysis between selected Miscanthus hybrids with contrasting values for these phenotypes to clarify how these phenotypes are genetically controlled. Results We observed that genes directly involved in the synthesis and degradation of starch and sucrose were down-regulated in high-yielding Miscanthus hybrids. At the same time, glycolysis and export of triose phosphates were up-regulated in high-yielding Miscanthus hybrids. These differentially expressed genes and biological functions were regulated by a well-connected network of less than 25 co-regulated transcription factors. Conclusions Our results evidence a direct relationship between high expression of essential enzymatic genes in the starch and sucrose pathways and co-expression with their transcriptional regulators, with high starch concentrations and lower biomass production. The strong interconnectivity between gene expression and regulators, chemotype and agronomic traits opens the door to use the expression of well-characterised genes associated with carbohydrate metabolism, particularly in the starch and sucrose pathway, for the early selection of high biomass-yielding genotypes from large Miscanthus populations.