Single-Molecule Sequencing Revealing the Presence of Distinct JC Polyomavirus Populations in Patients With Progressive Multifocal Leukoencephalopathy

Background. Progressive multifocal leukoencephalopathy (PML) is a fatal disease caused by reactivation of JC polyomavirus (JCPyV) in immunosuppressed individuals and lytic infection by neurotropic JCPyV in glial cells. The exact content of neurotropic mutations within individual JCPyV strains has not been studied to our knowledge. Methods. We exploited the capacity of single-molecule real-time sequencing technology to determine the sequence of complete JCPyV genomes in single reads. The method was used to precisely characterize individual neurotropic JCPyV strains of 3 patients with PML without the bias caused by assembly of short sequence reads. Results. In the cerebrospinal fluid sample of a 73-year-old woman with rapid PML onset, 3 distinct JCPyV populations could be identified. All viral populations were characterized by rearrangements within the noncoding regulatory region (NCCR) and 1 point mutation, S267L in the VP1 gene, suggestive of neurotropic strains. One patient with PML had a single neurotropic strain with rearranged NCCR, and 1 patient had a single strain with small NCCR alterations. Conclusions. We report here, for the first time, full characterization of individual neurotropic JCPyV strains in the cerebrospinal fluid of patients with PML. It remains to be established whether PML pathogenesis is driven by one or several neurotropic strains in an individual.Peer reviewe

BK polyomavirus microRNA expression and sequence variation in polyomavirus-associated nephropathy

Background: BK polyomavirus (BKPyV) infection is a common asymptomatic viral infection in the general population. Severe complications are seen in immunocompromised individuals, such as polyomavirus-associated nephropathy (PyVAN) in renal transplant recipients. Information on BKPyV microRNA expressions is scarce, although polyomavirus-encoded microRNAs have been shown to control viral replication and assist in immune evasion. Whereas the pathogenic role of rearrangements in JC polyomavirus has been well established, little is known about BKPyV rearrangements in PyVAN. Objectives: To assess viral microRNA expression and transcriptional control region (TCR) sequence variation in PyVAN patients. Study design: bkv-miR-B1-3p and bkv-miR-B1-5p microRNA expression was quantified in 55 plasma samples from 9 PyVAN patients and 2 controls using specific miRNA assays. TCR architectures among the viral populations in each patient were characterized by massive parallel sequencing. Results: bkv-miR-B1-3p and bkv-miR-B1-5p miRNA expression was established in 85.5% and 98.2% of samples, respectively. On average, an 8.9-fold (bkv-miR-B1-3p) and 8.7-fold (bkv-miR-B1-5p) higher expression levels were detected in PyVAN patients as compared to controls. Rearranged BKPyV strains with duplications and deletions were detected in 7/9 PyVAN patients, but 77.6-99.9% of all sequence reads in all samples represented archetype strains. Conclusions: The frequent detection and increased expression of miRNAs suggest involvement in PyVAN pathogenesis. Despite the predominance of archetype BKPyV strains, the frequent detection of minor rearranged viral populations urges further study on their role in severe kidney disease. Our results suggest that miRNA expression is increased in PyVAN patients, as well as in the presence of rearranged viral strains.Peer reviewe

Microbial Communities of Cladonia Lichens and Their Biosynthetic Gene Clusters Potentially Encoding Natural Products

Lichens have been widely used in traditional medicine, especially by indigenous communities worldwide. However, their slow growth and difficulties in the isolation of lichen symbionts and associated microbes have hindered the pharmaceutical utilisation of lichen-produced compounds. Advances in high-throughput sequencing techniques now permit detailed investigations of the complex microbial communities formed by fungi, green algae, cyanobacteria, and other bacteria within the lichen thalli. Here, we used amplicon sequencing, shotgun metagenomics, and in silico metabolomics together with compound extractions to study reindeer lichens collected from Southern Finland. Our aim was to evaluate the potential of Cladonia species as sources of novel natural products. We compared the predicted biosynthetic pathways of lichen compounds from isolated genome-sequenced lichen fungi and our environmental samples. Potential biosynthetic genes could then be further used to produce secondary metabolites in more tractable hosts. Furthermore, we detected multiple compounds by metabolite analyses, which revealed connections between the identified biosynthetic gene clusters and their products. Taken together, our results contribute to metagenomic data studies from complex lichen-symbiotic communities and provide valuable new information for use in further biochemical and pharmacological studies

Microbial Communities of Cladonia Lichens and Their Biosynthetic Gene Clusters Potentially Encoding Natural Products

Lichens have been widely used in traditional medicine, especially by indigenous communities worldwide. However, their slow growth and difficulties in the isolation of lichen symbionts and associated microbes have hindered the pharmaceutical utilisation of lichen-produced compounds. Advances in high-throughput sequencing techniques now permit detailed investigations of the complex microbial communities formed by fungi, green algae, cyanobacteria, and other bacteria within the lichen thalli. Here, we used amplicon sequencing, shotgun metagenomics, and in silico metabolomics together with compound extractions to study reindeer lichens collected from Southern Finland. Our aim was to evaluate the potential of Cladonia species as sources of novel natural products. We compared the predicted biosynthetic pathways of lichen compounds from isolated genome-sequenced lichen fungi and our environmental samples. Potential biosynthetic genes could then be further used to produce secondary metabolites in more tractable hosts. Furthermore, we detected multiple compounds by metabolite analyses, which revealed connections between the identified biosynthetic gene clusters and their products. Taken together, our results contribute to metagenomic data studies from complex lichen-symbiotic communities and provide valuable new information for use in further biochemical and pharmacological studies

Complete genome sequence of Leuconostoc gelidum subsp. gasicomitatum KG16-1, isolated from vacuum-packaged vegetable sausages

Leuconostoc gelidum subsp. gasicomitatum is a predominant lactic acid bacterium (LAB) in spoilage microbial communities of different kinds of modified-atmosphere packaged (MAP) food products. So far, only one genome sequence of a poultry-originating type strain of this bacterium (LMG 18811T) has been available. In the current study, we present the completely sequenced and functionally annotated genome of strain KG16-1 isolated from a vegetable-based product. In addition, six other vegetable-associated strains were sequenced to study possible “niche” specificity suggested by recent multilocus sequence typing. The genome of strain KG16-1 consisted of one circular chromosome and three plasmids, which together contained 2,035 CDSs. The chromosome carried at least three prophage regions and one of the plasmids encoded a galactan degradation cluster, which might provide a survival advantage in plant-related environments. The genome comparison with LMG 18811T and six other vegetable strains suggests no major differences between the meat- and vegetable-associated strains that would explain their “niche” specificity. Finally, the comparison with the genomes of other leuconostocs highlights the distribution of functionally interesting genes across the L. gelidum strains and the genus Leuconostoc.Peer reviewe

Complete genome sequence of Leuconostoc gelidum subsp. gasicomitatum KG16-1, isolated from vacuum-packaged vegetable sausages

Leuconostoc gelidum subsp. gasicomitatum is a predominant lactic acid bacterium (LAB) in spoilage microbial communities of different kinds of modified-atmosphere packaged (MAP) food products. So far, only one genome sequence of a poultry-originating type strain of this bacterium (LMG 18811T) has been available. In the current study, we present the completely sequenced and functionally annotated genome of strain KG16-1 isolated from a vegetable-based product. In addition, six other vegetable-associated strains were sequenced to study possible “niche” specificity suggested by recent multilocus sequence typing. The genome of strain KG16-1 consisted of one circular chromosome and three plasmids, which together contained 2,035 CDSs. The chromosome carried at least three prophage regions and one of the plasmids encoded a galactan degradation cluster, which might provide a survival advantage in plant-related environments. The genome comparison with LMG 18811T and six other vegetable strains suggests no major differences between the meat- and vegetable-associated strains that would explain their “niche” specificity. Finally, the comparison with the genomes of other leuconostocs highlights the distribution of functionally interesting genes across the L. gelidum strains and the genus Leuconostoc.Peer reviewe

Complete genome sequence of Leuconostoc gelidum subsp. gasicomitatum KG16-1, isolated from vacuum-packaged vegetable sausages

Leuconostoc gelidum subsp. gasicomitatum is a predominant lactic acid bacterium (LAB) in spoilage microbial communities of different kinds of modified-atmosphere packaged (MAP) food products. So far, only one genome sequence of a poultry-originating type strain of this bacterium (LMG 18811T) has been available. In the current study, we present the completely sequenced and functionally annotated genome of strain KG16-1 isolated from a vegetable-based product. In addition, six other vegetable-associated strains were sequenced to study possible “niche” specificity suggested by recent multilocus sequence typing. The genome of strain KG16-1 consisted of one circular chromosome and three plasmids, which together contained 2,035 CDSs. The chromosome carried at least three prophage regions and one of the plasmids encoded a galactan degradation cluster, which might provide a survival advantage in plant-related environments. The genome comparison with LMG 18811T and six other vegetable strains suggests no major differences between the meat- and vegetable-associated strains that would explain their “niche” specificity. Finally, the comparison with the genomes of other leuconostocs highlights the distribution of functionally interesting genes across the L. gelidum strains and the genus Leuconostoc.Peer reviewe

Complete Genome Sequences and Methylome Analyses of Cutibacterium acnes subsp. acnes Strains DSM 16379 and DSM 1897T

Cutibacterium acnes is a member of the normal human skin micro-biome. However, it is also associated with skin disorders and persistent infections of orthopedic implants. Here, we announce complete genome sequences and methyl-omes of the C. acnes subsp. acnes strains DSM 1897(T) and DSM 16379 together with their active restriction-modification systems.Peer reviewe

gapFinisher: A reliable gap filling pipeline for SSPACE-LongRead scaffolder output

Unknown sequences, or gaps, are present in many published genomes across public databases. Gap filling is an important finishing step in de novo genome assembly, especially in large genomes. The gap filling problem is nontrivial and while there are many computational tools partially solving the problem, several have shortcomings as to the reliability and correctness of the output, i.e. the gap filled draft genome. SSPACE-LongRead is a scaffolding tool that utilizes long reads from multiple third-generation sequencing platforms in finding links between contigs and combining them. The long reads potentially contain sequence information to fill the gaps created in the scaffolding, but SSPACE-LongRead currently lacks this functionality. We present an automated pipeline called gapFinisher to process SSPACE-LongRead output to fill gaps after the scaffolding. gapFinisher is based on the controlled use of a previously published gap filling tool FGAP and works on all standard Linux/UNIX command lines. We compare the performance of gapFinisher against two other published gap filling tools PBJelly and GMcloser. We conclude that gapFinisher can fill gaps in draft genomes quickly and reliably. In addition, the serial design of gapFinisher makes it scale well from prokaryote genomes to larger genomes with no increase in the computational footprint.Peer reviewe