We present the draft genome sequences of six strains of Escherichia coli isolated from blood cultures collected from patients with sepsis. The strains were collected from two patient sets, those with a high severity of illness, and those with a low severity of illness. Each genome was sequenced by both Illumina and PacBio for comparison. Copyright © 2014 Dunitz et al.
Herein we present the applicability of single-molecule (PacBio RS) and second-generation sequencing technology (Illumina) to the characterization of large genomic deletions. By testing samples previously characterized using a Sanger approach, our methods determined that both next-generation sequencing platforms were able to identify the position of deletion breakpoints. Our results point out various advantages of next-generation sequencing platforms when characterizing genomic deletions; however, special attention must be dedicated to identical sequences flanking the breakpoints, such as poly(N) motifs.
HIV-1 coreceptor tropism assays are required to rule out the presence of CXCR4-tropic (non-R5) viruses prior treatment with CCR5 antagonists. Phenotypic (e.g., Trofile™, Monogram Biosciences) and genotypic (e.g., population sequencing linked to bioinformatic algorithms) assays are the most widely used. Although several next-generation sequencing (NGS) platforms are available, to date all published deep sequencing HIV-1 tropism studies have used the 454™ Life Sciences/Roche platform. In this study, HIV-1 co-receptor usage was predicted for twelve patients scheduled to start a maraviroc-based antiretroviral regimen. The V3 region of the HIV-1 env gene was sequenced using four NGS platforms: 454™, PacBio® RS (Pacific Biosciences), Illumina®, and Ion Torrent™ (Life Technologies). Cross-platform variation was evaluated, including number of reads, read length and error rates. HIV-1 tropism was inferred using Geno2Pheno, Web PSSM, and the 11/24/25 rule and compared with Trofile™ and virologic response to antiretroviral therapy. Error rates related to insertions/deletions (indels) and nucleotide substitutions introduced by the four NGS platforms were low compared to the actual HIV-1 sequence variation. Each platform detected all major virus variants within the HIV-1 population with similar frequencies. Identification of non-R5 viruses was comparable among the four platforms, with minor differences attributable to the algorithms used to infer HIV-1 tropism. All NGS platforms showed similar concordance with virologic response to the maraviroc-based regimen (75% to 80% range depending on the algorithm used), compared to Trofile (80%) and population sequencing (70%). In conclusion, all four NGS platforms were able to detect minority non-R5 variants at comparable levels suggesting that any NGS-based method can be used to predict HIV-1 coreceptor usage.
Borrelia (B.) burgdorferi sensu lato, including the tick-transmitted agents of human Lyme borreliosis, have particularly complex genomes, consisting of a linear main chromosome and numerous linear and circular plasmids. The number and structure of plasmids is variable even in strains within a single genospecies. Genes on these plasmids are known to play essential roles in virulence and pathogenicity as well as host and vector associations. For this reason, it is essential to explore methods for rapid and reliable characterisation of molecular level changes on plasmids. In this study we used three strains: a low passage isolate of B. burgdorferi sensu stricto strain B31(-NRZ) and two closely related strains (PAli and PAbe) that were isolated from human patients. Sequences of these strains were compared to the previously sequenced reference strain B31 (available in GenBank) to obtain proof-of-principle information on the suitability of next generation sequencing (NGS) library construction and sequencing methods on the assembly of bacterial plasmids. We tested the effectiveness of different short read assemblers on Illumina sequences, and of long read generation methods on sequence data from Pacific Bioscience single-molecule real-time (SMRT) and nanopore (Oxford Nanopore Technologies) sequencing technology.Inclusion of mate pair library reads improved the assembly in some plasmids as did prior enrichment of plasmids. While cp32 plasmids remained refractory to assembly using only short reads they were effectively assembled by long read sequencing methods. The long read SMRT and nanopore sequences came, however, at the cost of indels (insertions or deletions) appearing in an unpredictable manner. Using long and short read technologies together allowed us to show that the three B. burgdorferi s.s. strains investigated here, whilst having similar plasmid structures to each other (apart from fusion of cp32 plasmids), differed significantly from the reference strain B31-GB, especially in the case of cp32 plasmids.Short read methods are sufficient to assemble the main chromosome and many of the plasmids in B. burgdorferi. However, a combination of short and long read sequencing methods is essential for proper assembly of all plasmids including cp32 and thus, for gaining an understanding of host- or vector adaptations. An important conclusion from our work is that the evolution of Borrelia plasmids appears to be dynamic. This has important implications for the development of useful research strategies to monitor the risk of Lyme disease occurrence and how to medically manage it.
Long-read sequencing technologies such as Pacific Biosciences and Oxford Nanopore MinION are capable of producing long sequencing reads with average fragment lengths of over 10,000 base-pairs and maximum lengths reaching 100,000 base- pairs. Compared with short reads, the assemblies obtained from long-read sequencing platforms have much higher contig continuity and genome completeness as long fragments are able to extend paths into problematic or repetitive regions. Many successful assembly applications of the Pacific Biosciences technology have been reported ranging from small bacterial genomes to large plant and animal genomes. Recently, genome assemblies using Oxford Nanopore MinION data have attracted much attention due to the portability and low cost of this novel sequencing instrument. In this paper, we re-sequenced a well characterized genome, the Saccharomyces cerevisiae S288C strain using three different platforms: MinION, PacBio and MiSeq. We present a comprehensive metric comparison of assemblies generated by various pipelines and discuss how the platform associated data characteristics affect the assembly quality. With a given read depth of 31X, the assemblies from both Pacific Biosciences and Oxford Nanopore MinION show excellent continuity and completeness for the 16 nuclear chromosomes, but not for the mitochondrial genome, whose reconstruction still represents a significant challenge.
True flies are insects of the order Diptera and encompass one of the most diverse groups of animals on Earth. Within dipterans, Schizophora represents a recent radiation of insects that was used as a model to develop a pipeline for generating complete mitogenomes using various sequencing platforms and strategies. 91 mitogenomes from 32 different species were sequenced and assembled with high fidelity, using amplicon, whole genome shotgun or single molecule sequencing approaches. Based on the novel mitogenomes, we estimate the origin of Schizophora within the Cretaceous-Paleogene (K-Pg) boundary, about 68.3?Ma. Detailed analyses of the blowfly family (Calliphoridae) place its origin at 22?Ma, concomitant with the radiation of grazing mammals. The emergence of ectoparasitism within calliphorids was dated 6.95?Ma for the screwworm fly and 2.3?Ma for the Australian sheep blowfly. Varying population histories were observed for the blowfly Chrysomya megacephala and the housefly Musca domestica samples in our dataset. Whereas blowflies (n?=?50) appear to have undergone selective sweeps and/or severe bottlenecks in the New World, houseflies (n?=?14) display variation among populations from different zoogeographical zones and low levels of gene flow. The reported high-throughput mitogenomics approach for insects enables new insights into schizophoran diversity and population history of flies.
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