No abstract

Long read sequencing technologies now allow routine highly contiguous assembly of bacterial genomes. However, because of the lower accuracy of some long read data, it is often combined with short read data (e.g. Illumina), to improve assembly quality. There are a number of methods available for producing such hybrid assemblies. Here we use Illumina and Oxford Nanopore (ONT) data from 49 natural isolates of Escherichia coli to characterise differences in assembly accuracy for five assembly methods (Canu, Unicycler, Raven, Flye, and Redbean). We evaluate assembly accuracy using five metrics designed to measure structural accuracy and sequence accuracy (indel and substitution frequency). We assess structural accuracy by quantifying (1) the contiguity of chromosomes and plasmids; (2) the fraction of concordantly mapped Illumina reads withheld from the assembly; and (3) whether rRNA operons are correctly oriented. We assess indel and substitution frequency by quantifying (1) the fraction of open reading frames that appear truncated and (2) the number of variants that are called using Illumina reads only. Applying these assembly metrics to a large number of E. coli strains, we find that different assembly methods offer different advantages. In particular, we find that Unicycler assemblies have the highest sequence accuracy in non-repetitive regions, while Flye and Raven tend to be the most structurally accurate. In addition, we find that there are unidentified strain-specific characteristics that affect ONT consensus accuracy, despite individual reads having similar levels of accuracy. The differences in consensus accuracy of the ONT reads can preclude accurate assembly regardless of assembly method. These results provide quantitative insight into the best approaches for hybrid assembly of bacterial genomes and the expected levels of structural and sequence accuracy. They also show that there are intrinsic idiosyncratic strain-level differences that inhibit accurate long read bacterial genome assembly. However, we also show it is possible to diagnose problematic assemblies, even in the absence of ground truth, by comparing long-read first and short-read first assemblies.Author NotesAll supporting data, code and protocols have been provided within the article or through supplementary data files. The supporting code is available from the GitHub repository https://github.com/GeorgiaBreckell/assembly_pipeline. nine supplementary figures and three supplementary tables are available with the online version of this article.Data summarySequence data and genome assemblies for the natural isolates are available at https://www.ebi.ac.uk/ena/browser/view/PRJEB36951. Genome assemblies for additional E. coli strains used here are available from NCBI: (MG1655, SE11, REL606, CFT073, W, IA136, O157:H7-EDL933)

Escherichia coli is commonly considered a host-associated bacterium. However, there is evidence that some strains occupy environmental (non-host-associated) niches. Here, we report the complete genomes of 47 Escherichia coli environmental isolates. These will be useful for understanding the dynamics of plasmids, phages, and other repetitive genetic elements.

DNA methylation in bacteria frequently serves as a simple immune system, allowing recognition of DNA from foreign sources, such as phages or selfish genetic elements. However, DNA methylation also affects other cell phenotypes in a heritable manner (i.e. epigenetically). While there are several examples of methylation affecting transcription in an epigenetic manner in highly localised contexts, it is not well established how frequently methylation serves a more general epigenetic function over larger genomic scales. To address this question, here we use Oxford Nanopore sequencing to profile DNA modification marks in three natural isolates of E. coli. We first identify the DNA sequence motifs targeted by the methyltransferases in each strain. We then quantify the frequency of methylation at each of these motifs across the entire genome in different growth conditions. We find that motifs in specific regions of the genome consistently exhibit high or low levels of methylation. Furthermore, we show that there are replicable and consistent differences in methylated regions across different growth conditions. This suggests that during growth, E. coli transiently differentiates into distinct methylation states that depend on the growth state, raising the possibility that measuring DNA methylation alone can be used to infer bacterial growth states without additional information such as transcriptome or proteome data. These results show the utility of using Oxford Nanopore sequencing as an economic means to infer DNA methylation status. They also provide new insights into the dynamics of methylation during bacterial growth, and provide evidence of differentiated cell states, a transient analogue to what is observed in the differentiation of cell types in multicellular organisms.

14Introduced species of mammals in New Zealand have had catastrophic effects on 15 populations of diverse native species. Quantifying the diets of these omnivorous and 16 predatory species is critical for understanding which native species are most impacted, 17 and to prioritize which mammal species and locations should be targeted with control 2009). These metabarcoding methods require a PCR step using primers that bind to 24 highly conserved genomic regions (e.g. mitochondrial COI) to amplify specific regions 25 for sequencing. This step introduces significant bias, primarily due to the lack of a 26 universal primer set (King et al. 2008). Here we show that direct metagenomic 27 sequencing using the Oxford Nanopore Minion allows rapid quantification of rat diets. 28Using a sample of rats collected from within 100km of Auckland, NZ, we show that 29 these rats consume a wide variety of plant, invertebrate, vertebrate, and fungal taxa, 30 with substantial differences in diet content between locales. We then show that, based 31 on diet content alone, it is possible to pinpoint the sampling location of an individual rat 32 within tens of kilometres. We expect that the rapidly increasing accuracy and 33 throughput of nanopore-based sequencing, as well as increases in the species 34 diversity of genomic databases, will soon allow rapid and unbiased assessments of 35 animal diets in field settings.