Menu

Auto Tag: Targeted

July 7, 2019

ConcatSeq: A method for increasing throughput of single molecule sequencing by concatenating short DNA fragments.

Single molecule sequencing (SMS) platforms enable base sequences to be read directly from individual strands of DNA in real-time. Though capable of long read lengths, SMS platforms currently suffer from low throughput compared to competing short-read sequencing technologies. Here, we present a novel strategy for sequencing library preparation, dubbed ConcatSeq, which increases the throughput of SMS platforms by generating long concatenated templates from pools of short DNA molecules. We demonstrate adaptation of this technique to two target enrichment workflows, commonly used for oncology applications, and feasibility using PacBio single molecule real-time (SMRT) technology. Our approach is capable of increasing the sequencing throughput of the PacBio RSII platform by more than five-fold, while maintaining the ability to correctly call allele frequencies of known single nucleotide variants. ConcatSeq provides a versatile new sample preparation tool for long-read sequencing technologies.

Read more
July 7, 2019

Plasmid composition in Aeromonas salmonicida subsp. salmonicida 01-B526 unravels unsuspected type three secretion system loss patterns.

Aeromonas salmonicida subsp. salmonicida is a ubiquitous psychrophilic waterborne bacterium and a fish pathogen. The numerous mobile elements, especially insertion sequences (IS), in its genome promote rearrangements that impact its phenotype. One of the main virulence factors of this bacterium, its type three secretion system (TTSS), is affected by these rearrangements. In Aeromonas salmonicida subsp. salmonicida most of the TTSS genes are encoded in a single locus on a large plasmid called pAsa5, and may be lost when the bacterium is cultivated at a higher temperature (25 °C), producing non-virulent mutants. In a previous study, pAsa5-rearranged strains that lacked the TTSS locus on pAsa5 were produced using parental strains, including 01-B526. Some of the generated deletions were explained by homologous recombination between ISs found on pAsa5, whereas the others remained unresolved. To investigate those rearrangements, short- and long-read high-throughput sequencing technologies were used on the A. salmonicida subsp. salmonicida 01-B526 whole genome.Whole genome sequencing of the 01-B526 strain revealed that its pAsa5 has an additional IS copy, an ISAS5, compared to the reference strain (A449) sequence, which allowed for a previously unknown rearrangement to occur. It also appeared that 01-B526 bears a second large plasmid, named pAsa9, which shares 40 kbp of highly similar sequences with pAsa5. Following these discoveries, previously unexplained deletions were elucidated by genotyping. Furthermore, in one of the derived strains a fusion of pAsa5 and pAsa9, involving the newly discovered ISAS5 copy, was observed.The loss of TTSS and hence virulence is explained by one consistent mechanism: IS-driven homologous recombination. The similarities between pAsa9 and pAsa5 also provide another example of genetic diversity driven by ISs.

Read more
July 7, 2019

Evidence for contemporary switching of the O-antigen gene cluster between Shiga toxin-producing Escherichia coli strains colonizing cattle.

Shiga toxin-producing Escherichia coli (STEC) comprise a group of zoonotic enteric pathogens with ruminants, especially cattle, as the main reservoir. O-antigens are instrumental for host colonization and bacterial niche adaptation. They are highly immunogenic and, therefore, targeted by the adaptive immune system. The O-antigen is one of the most diverse bacterial cell constituents and variation not only exists between different bacterial species, but also between individual isolates/strains within a single species. We recently identified STEC persistently infecting cattle and belonging to the different serotypes O156:H25 (n = 21) and O182:H25 (n = 15) that were of the MLST sequence types ST300 or ST688. These STs differ by a single nucleotide in purA only. Fitness-, virulence-associated genome regions, and CRISPR/CAS (clustered regularly interspaced short palindromic repeats/CRISPR associated sequence) arrays of these STEC O156:H25 and O182:H25 isolates were highly similar, and identical genomic integration sites for the stx converting bacteriophages and the core LEE, identical Shiga toxin converting bacteriophage genes for stx1a, identical complete LEE loci, and identical sets of chemotaxis and flagellar genes were identified. In contrast to this genomic similarity, the nucleotide sequences of the O-antigen gene cluster (O-AGC) regions between galF and gnd and very few flanking genes differed fundamentally and were specific for the respective serotype. Sporadic aEPEC O156:H8 isolates (n = 5) were isolated in temporal and spatial proximity. While the O-AGC and the corresponding 5′ and 3′ flanking regions of these aEPEC isolates were identical to the respective region in the STEC O156:H25 isolates, the core genome, the virulence associated genome regions and the CRISPR/CAS elements differed profoundly. Our cumulative epidemiological and molecular data suggests a recent switch of the O-AGC between isolates with O156:H8 strains having served as DNA donors. Such O-antigen switches can affect the evaluation of a strain’s pathogenic and virulence potential, suggesting that NGS methods might lead to a more reliable risk assessment.

Read more
July 7, 2019

Three novel species with peptidoglycan cell walls form the new genus Lacunisphaera gen. nov. in the family Opitutaceae of the verrucomicrobial subdivision 4.

The cell wall of free-living bacteria consists of peptidoglycan (PG) and is critical for maintenance of shape as dissolved solutes cause osmotic pressure and challenge cell integrity. Surprisingly, the subdivision 4 of the phylum Verrucomicrobia appears to be exceptional in this respect. Organisms of this subdivision are described to be devoid of muramic or diaminopimelic acid (DAP), usually found as components of PG in bacterial cell walls. Here we describe three novel bacterial strains from a freshwater lake, IG15(T), IG16b(T), and IG31(T), belonging to a new genus in the subdivision 4 of Verrucomicrobia which we found to possess PG as part of their cell walls. Biochemical analysis revealed the presence of DAP not only in these novel strains, but also in Opitutus terrae PB90-1(T), the closest described relative of strains IG15(T), IG16b(T), and IG31(T). Furthermore, we found that nearly all genes necessary for peptidoglycan synthesis are present in genomes of subdivision 4 members, as well as in the complete genome sequence of strain IG16b(T). In addition, we isolated and visualized PG-sacculi for strain IG16b(T). Thus, our results challenge the concept of peptidoglycan-less free-living bacteria. Our polyphasic taxonomy approach places the novel strains in a new genus within the family Opitutaceae, for which the name Lacunisphaera gen. nov. is proposed. Strain designations for IG15(T), IG16b(T) and IG31(T) are Lacunisphaera parvula sp. nov. (=DSM 26814 = LMG 29468), L. limnophila sp. nov. (=DSM 26815 = LMG 29469) and L. anatis sp. nov. (=DSM 103142 = LMG 29578) respectively, with L. limnophila IG16b(T) being the type species of the genus.

Read more
July 7, 2019

Comparative analysis of Ralstonia solanacearum methylomes.

Ralstonia solanacearum is an important soil-borne plant pathogen with broad geographical distribution and the ability to cause wilt disease in many agriculturally important crops. Genome sequencing of multiple R. solanacearum strains has identified both unique and shared genetic traits influencing their evolution and ability to colonize plant hosts. Previous research has shown that DNA methylation can drive speciation and modulate virulence in bacteria, but the impact of epigenetic modifications on the diversification and pathogenesis of R. solanacearum is unknown. Sequencing of R. solanacearum strains GMI1000 and UY031 using Single Molecule Real-Time technology allowed us to perform a comparative analysis of R. solanacearum methylomes. Our analysis identified a novel methylation motif associated with a DNA methylase that is conserved in all complete Ralstonia spp. genomes and across the Burkholderiaceae, as well as a methylation motif associated to a phage-borne methylase unique to R. solanacearum UY031. Comparative analysis of the conserved methylation motif revealed that it is most prevalent in gene promoter regions, where it displays a high degree of conservation detectable through phylogenetic footprinting. Analysis of hyper- and hypo-methylated loci identified several genes involved in global and virulence regulatory functions whose expression may be modulated by DNA methylation. Analysis of genome-wide modification patterns identified a significant correlation between DNA modification and transposase genes in R. solanacearum UY031, driven by the presence of a high copy number of ISrso3 insertion sequences in this genome and pointing to a novel mechanism for regulation of transposition. These results set a firm foundation for experimental investigations into the role of DNA methylation in R. solanacearum evolution and its adaptation to different plants.

Read more
July 7, 2019

Evolution of the wheat blast fungus through functional losses in a host specificity determinant.

Wheat blast first emerged in Brazil in the mid-1980s and has recently caused heavy crop losses in Asia. Here we show how this devastating pathogen evolved in Brazil. Genetic analysis of host species determinants in the blast fungus resulted in the cloning of avirulence genes PWT3 and PWT4, whose gene products elicit defense in wheat cultivars containing the corresponding resistance genes Rwt3 and Rwt4 Studies on avirulence and resistance gene distributions, together with historical data on wheat cultivation in Brazil, suggest that wheat blast emerged due to widespread deployment of rwt3 wheat (susceptible to Lolium isolates), followed by the loss of function of PWT3 This implies that the rwt3 wheat served as a springboard for the host jump to common wheat. Copyright © 2017, American Association for the Advancement of Science.

Read more
July 7, 2019

Hybrid assembly with long and short reads improves discovery of gene family expansions.

Long-read and short-read sequencing technologies offer competing advantages for eukaryotic genome sequencing projects. Combinations of both may be appropriate for surveys of within-species genomic variation.We developed a hybrid assembly pipeline called “Alpaca” that can operate on 20X long-read coverage plus about 50X short-insert and 50X long-insert short-read coverage. To preclude collapse of tandem repeats, Alpaca relies on base-call-corrected long reads for contig formation.Compared to two other assembly protocols, Alpaca demonstrated the most reference agreement and repeat capture on the rice genome. On three accessions of the model legume Medicago truncatula, Alpaca generated the most agreement to a conspecific reference and predicted tandemly repeated genes absent from the other assemblies.Our results suggest Alpaca is a useful tool for investigating structural and copy number variation within de novo assemblies of sampled populations.

Read more
July 7, 2019

CLOVE: classification of genomic fusions into structural variation events.

A precise understanding of structural variants (SVs) in DNA is important in the study of cancer and population diversity. Many methods have been designed to identify SVs from DNA sequencing data. However, the problem remains challenging because existing approaches suffer from low sensitivity, precision, and positional accuracy. Furthermore, many existing tools only identify breakpoints, and so not collect related breakpoints and classify them as a particular type of SV. Due to the rapidly increasing usage of high throughput sequencing technologies in this area, there is an urgent need for algorithms that can accurately classify complex genomic rearrangements (involving more than one breakpoint or fusion).We present CLOVE, an algorithm for integrating the results of multiple breakpoint or SV callers and classifying the results as a particular SV. CLOVE is based on a graph data structure that is created from the breakpoint information. The algorithm looks for patterns in the graph that are characteristic of more complex rearrangement types. CLOVE is able to integrate the results of multiple callers, producing a consensus call.We demonstrate using simulated and real data that re-classified SV calls produced by CLOVE improve on the raw call set of existing SV algorithms, particularly in terms of accuracy. CLOVE is freely available from http://www.github.com/PapenfussLab .

Read more
July 7, 2019

Evolutionary dynamics of pathoadaptation revealed by three independent acquisitions of the VirB/D4 type IV secretion system in Bartonella.

The a-proteobacterial genus Bartonella comprises a group of ubiquitous mammalian pathogens that are studied as a model for the evolution of bacterial pathogenesis. Vast abundance of two particular phylogenetic lineages of Bartonella had been linked to enhanced host adaptability enabled by lineage-specific acquisition of a VirB/D4 type IV secretion system (T4SS) and parallel evolution of complex effector repertoires. However, the limited availability of genome sequences from one of those lineages as well as other, remote branches of Bartonella has so far hampered comprehensive understanding of how the VirB/D4 T4SS and its effectors called Beps have shaped Bartonella evolution. Here, we report the discovery of a third repertoire of Beps associated with the VirB/D4 T4SS of B. ancashensis, a novel human pathogen that lacks any signs of host adaptability and is only distantly related to the two species-rich lineages encoding a VirB/D4 T4SS. Furthermore, sequencing of ten new Bartonella isolates from under-sampled lineages enabled combined in silico analyses and wet lab experiments that suggest several parallel layers of functional diversification during evolution of the three Bep repertoires from a single ancestral effector. Our analyses show that the Beps of B. ancashensis share many features with the two other repertoires, but may represent a more ancestral state that has not yet unleashed the adaptive potential of such an effector set. We anticipate that the effectors of B. ancashensis will enable future studies to dissect the evolutionary history of Bartonella effectors and help unraveling the evolutionary forces underlying bacterial host adaptation.© The Author(s) 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Read more
July 7, 2019

The MHC locus and genetic susceptibility to autoimmune and infectious diseases.

In the past 50 years, variants in the major histocompatibility complex (MHC) locus, also known as the human leukocyte antigen (HLA), have been reported as major risk factors for complex diseases. Recent advances, including large genetic screens, imputation, and analyses of non-additive and epistatic effects, have contributed to a better understanding of the shared and specific roles of MHC variants in different diseases. We review these advances and discuss the relationships between MHC variants involved in autoimmune and infectious diseases. Further work in this area will help to distinguish between alternative hypotheses for the role of pathogens in autoimmune disease development.

Read more
July 7, 2019

Complete genome sequence of the Bifidobacterium animalis subspecies lactis BL3, preventive probiotics for acute colitis and colon cancer.

We report the genome sequence of Bifidobacterium animalis subspecies lactis BL3, which has preventive properties on acute colitis and colon cancer. The genome of BL3, which was isolated from Korean faeces, consisted of a 1 944 323 bp size single chromosome, and its G+C content was 60.5%. Genome comparison against the closest Bifidobacterium animalis strain revealed that BL3 had particularly different regions of four areas encoding flavin-nucleotide-binding protein, transposase, multidrug ABC transporter and ATP binding protein.

Read more
July 7, 2019

Tandem duplications lead to novel expression patterns through exon shuffling in Drosophila yakuba.

One common hypothesis to explain the impacts of tandem duplications is that whole gene duplications commonly produce additive changes in gene expression due to copy number changes. Here, we use genome wide RNA-seq data from a population sample of Drosophila yakuba to test this ‘gene dosage’ hypothesis. We observe little evidence of expression changes in response to whole transcript duplication capturing 5′ and 3′ UTRs. Among whole gene duplications, we observe evidence that dosage sharing across copies is likely to be common. The lack of expression changes after whole gene duplication suggests that the majority of genes are subject to tight regulatory control and therefore not sensitive to changes in gene copy number. Rather, we observe changes in expression level due to both shuffling of regulatory elements and the creation of chimeric structures via tandem duplication. Additionally, we observe 30 de novo gene structures arising from tandem duplications, 23 of which form with expression in the testes. Thus, the value of tandem duplications is likely to be more intricate than simple changes in gene dosage. The common regulatory effects from chimeric gene formation after tandem duplication may explain their contribution to genome evolution.

Read more
July 7, 2019

Whole genome sequencing predicts novel human disease models in rhesus macaques.

Rhesus macaques are an important pre-clinical model of human disease. To advance our understanding of genomic variation that may influence disease, we surveyed genome-wide variation in 21 rhesus macaques. We employed best-practice variant calling, validated with Mendelian inheritance. Next, we used alignment data from our cohort to detect genomic regions likely to produce inaccurate genotypes, potentially due to either gene duplication or structural variation between individuals. We generated a final dataset of >16 million high confidence variants, including 13 million in Chinese-origin rhesus macaques, an increasingly important disease model. We detected an average of 131 mutations predicted to severely alter protein coding per animal, and identified 45 such variants that coincide with known pathogenic human variants. These data suggest that expanded screening of existing breeding colonies will identify novel models of human disease, and that increased genomic characterization can help inform research studies in macaques. Copyright © 2017 Elsevier Inc. All rights reserved.

Read more
July 7, 2019

Targeted disruption of the polyketide synthase gene pks15 affects virulence against insects and phagocytic survival in the fungus Beauveria bassiana.

The reducing clade III polyketide synthase genes, including pks15, are highly conserved among entomopathogenic fungi. To examine the function of pks15, we used targeted disruption to investigate the impact of Beauveria bassiana pks15 on insect pathogenesis. Southern analysis verified that the ?pks15 mutant was disrupted by a single integration of the transformation cassette at the pks15 locus. The ?pks15 mutant had a slight reduction in radial growth, and it produced fewer spores. Our insect bioassays indicated the ?pks15 mutant to be significantly reduced in virulence against beet armyworms compared to wild type (WT), which could be partially accounted for by its markedly decreased ability to survive phagocytosis. Total haemocyte count decreased sharply by 50-fold from days 1-3 post-inoculation in insects infected with WT, compared to a 5-fold decrease in the ?pks15 mutant. The mutant also produced fewer hemolymph hyphal bodies than WT by 3-fold. In co-culture studies with amoebae that have phagocytic ability similar to that of insect haemocytes, at 48 h the mortality rate of amoebae engulfing ?pks15 decreased by 72 %, and ?pks15 CFU decreased by 83 % compared to co-culture with WT. Thus, the ?pks15 mutant had a reduced ability to cope with phagocytosis and highly reduced virulence in an insect host. These data elucidate a mechanism of insect pathogenesis associated with polyketide biosynthesis. Copyright © 2017 British Mycological Society. Published by Elsevier Ltd. All rights reserved.

Read more
July 7, 2019

A large gene family in fission yeast encodes spore killers that subvert Mendel’s law.

Spore killers in fungi are selfish genetic elements that distort Mendelian segregation in their favor. It remains unclear how many species harbor them and how diverse their mechanisms are. Here, we discover two spore killers from a natural isolate of the fission yeast Schizosaccharomyces pombe. Both killers belong to the previously uncharacterized wtf gene family with 25 members in the reference genome. These two killers act in strain-background-independent and genome-location-independent manners to perturb the maturation of spores not inheriting them. Spores carrying one killer are protected from its killing effect but not that of the other killer. The killing and protecting activities can be uncoupled by mutation. The numbers and sequences of wtf genes vary considerably between S. pombe isolates, indicating rapid divergence. We propose that wtf genes contribute to the extensive intraspecific reproductive isolation in S. pombe, and represent ideal models for understanding how segregation-distorting elements act and evolve.

Read more

Subscribe for blog updates:

Talk with an expert

If you have a question, need to check the status of an order, or are interested in purchasing an instrument, we're here to help.