We report the sequencing and assembly of a reference genome for the human GM12878 Utah/Ceph cell line using the MinION (Oxford Nanopore Technologies) nanopore sequencer. 91.2 Gb of sequence data, representing ~30× theoretical coverage, were produced. Reference-based alignment enabled detection of large structural variants and epigenetic modifications. De novo assembly of nanopore reads alone yielded a contiguous assembly (NG50 ~3 Mb). Next, we developed a protocol to generate ultra-long reads (N50 > 100kb, up to 882 kb). Incorporating an additional 5×-coverage of this data type more than doubled the assembly contiguity (NG50 ~6.4 Mb). The final assembled genome was 2,867 million bases in size, covering 85.8% of the reference. Assembly accuracy, after incorporating complementary short-read sequencing data, exceeded 99.8%. Ultra-long reads enabled assembly and phasing of the 4 Mb major histocompatibility complex (MHC) locus in its entirety, measurement of telomere repeat length and closure of gaps in the reference human genome assembly GRCh38.

The Oxford Nanopore MinION sequences DNA by sensing changes in electrical current flow in real-time as molecules traverse nanopores. Optionally, the voltage across specific nanopores can be reversed, ejecting the DNA molecule. This enables “Read Until”, the selection of specific DNA molecules for sequencing. We use dynamic time warping to match reads to reference, selecting regions of small genomes, individual amplicons, or normalization of the amplicon set. This first demonstration of direct selection of specific DNA molecules in real-time enables many novel future applications.

A key challenge in the production of second generation biofuels is the conversion of lignocellulosic substrates into fermentable sugars. Enzymes, particularly those from fungi, are a central part of this process, and many have been isolated and characterised. However, relatively little is known of how fungi respond to lignocellulose and produce the enzymes necessary for dis-assembly of plant biomass. We studied the physiological response of the fungus Aspergillus niger when exposed to wheat straw as a model lignocellulosic substrate. Using RNA sequencing we showed that, 24 hours after exposure to straw, gene expression of known and presumptive plant cell wall–degrading enzymes represents a huge investment for the cells (about 20% of the total mRNA). Our results also uncovered new esterases and surface interacting proteins that might form part of the fungal arsenal of enzymes for the degradation of plant biomass. Using transcription factor deletion mutants (xlnR and creA) to study the response to both lignocellulosic substrates and low carbon source concentrations, we showed that a subset of genes coding for degradative enzymes is induced by starvation. Our data support a model whereby this subset of enzymes plays a scouting role under starvation conditions, testing for available complex polysaccharides and liberating inducing sugars, that triggers the subsequent induction of the majority of hydrolases. We also showed that antisense transcripts are abundant and that their expression can be regulated by growth conditions.

DNA replication initiates at defined sites called origins, which serve as binding sites for initiator proteins that recruit the replicative machinery. Origins differ in number and structure across the three domains of life1 and their properties determine the dynamics of chromosome replication. Bacteria and some archaea replicate from single origins, whilst most archaea and all eukaryotes replicate using multiple origins. Initiation mechanisms that rely on homologous recombination operate in some viruses. Here we show that such mechanisms also operate in archaea. We have used deep sequencing to study replication in Haloferax volcanii. Four chromosomal origins of differing activity were identified. Deletion of individual origins resulted in perturbed replication dynamics and reduced growth. However, a strain lacking all origins has no apparent defects and grows significantly faster than wild-type. Origin-less cells initiate replication at dispersed sites rather than at discrete origins and have an absolute requirement for the recombinase RadA, unlike strains lacking individual origins. Our results demonstrate that homologous recombination alone can efficiently initiate the replication of an entire cellular genome. This raises the question of what purpose replication origins serve and why they have evolved.