Current methods for genome-wide analysis of gene expression require fragmentation of original transcripts into small fragments for short-read sequencing. In bacteria, the resulting fragmented ...information hides operon complexity. Additionally, in vivo processing of transcripts confounds the accurate identification of the 5' and 3' ends of operons. Here we develop a methodology called SMRT-Cappable-seq that combines the isolation of un-fragmented primary transcripts with single-molecule long read sequencing. Applied to E. coli, this technology results in an accurate definition of the transcriptome with 34% of known operons from RegulonDB being extended by at least one gene. Furthermore, 40% of transcription termination sites have read-through that alters the gene content of the operons. As a result, most of the bacterial genes are present in multiple operon variants reminiscent of eukaryotic splicing. By providing such granularity in the operon structure, this study represents an important resource for the study of prokaryotic gene network and regulation.
The human reference genome assembly plays a central role in nearly all aspects of today's basic and clinical research. GRCh38 is the first coordinate-changing assembly update since 2009; it reflects ...the resolution of roughly 1000 issues and encompasses modifications ranging from thousands of single base changes to megabase-scale path reorganizations, gap closures, and localization of previously orphaned sequences. We developed a new approach to sequence generation for targeted base updates and used data from new genome mapping technologies and single haplotype resources to identify and resolve larger assembly issues. For the first time, the reference assembly contains sequence-based representations for the centromeres. We also expanded the number of alternate loci to create a reference that provides a more robust representation of human population variation. We demonstrate that the updates render the reference an improved annotation substrate, alter read alignments in unchanged regions, and impact variant interpretation at clinically relevant loci. We additionally evaluated a collection of new de novo long-read haploid assemblies and conclude that although the new assemblies compare favorably to the reference with respect to continuity, error rate, and gene completeness, the reference still provides the best representation for complex genomic regions and coding sequences. We assert that the collected updates in GRCh38 make the newer assembly a more robust substrate for comprehensive analyses that will promote our understanding of human biology and advance our efforts to improve health.
The human genome is arguably the most complete mammalian reference assembly, yet more than 160 euchromatic gaps remain and aspects of its structural variation remain poorly understood ten years after ...its completion. To identify missing sequence and genetic variation, here we sequence and analyse a haploid human genome (CHM1) using single-molecule, real-time DNA sequencing. We close or extend 55% of the remaining interstitial gaps in the human GRCh37 reference genome--78% of which carried long runs of degenerate short tandem repeats, often several kilobases in length, embedded within (G+C)-rich genomic regions. We resolve the complete sequence of 26,079 euchromatic structural variants at the base-pair level, including inversions, complex insertions and long tracts of tandem repeats. Most have not been previously reported, with the greatest increases in sensitivity occurring for events less than 5 kilobases in size. Compared to the human reference, we find a significant insertional bias (3:1) in regions corresponding to complex insertions and long short tandem repeats. Our results suggest a greater complexity of the human genome in the form of variation of longer and more complex repetitive DNA that can now be largely resolved with the application of this longer-read sequencing technology.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, KISLJ, NUK, PILJ, PNG, SAZU, SBMB, SIK, UILJ, UKNU, UL, UM, UPUK
In an effort to more fully understand the full spectrum of human genetic variation, we generated deep single-molecule, real-time (SMRT) sequencing data from two haploid human genomes. By using an ...assembly-based approach (SMRT-SV), we systematically assessed each genome independently for structural variants (SVs) and indels resolving the sequence structure of 461,553 genetic variants from 2 bp to 28 kbp in length. We find that >89% of these variants have been missed as part of analysis of the 1000 Genomes Project even after adjusting for more common variants (MAF > 1%). We estimate that this theoretical human diploid differs by as much as ∼16 Mbp with respect to the human reference, with long-read sequencing data providing a fivefold increase in sensitivity for genetic variants ranging in size from 7 bp to 1 kbp compared with short-read sequence data. Although a large fraction of genetic variants were not detected by short-read approaches, once the alternate allele is sequence-resolved, we show that 61% of SVs can be genotyped in short-read sequence data sets with high accuracy. Uncoupling discovery from genotyping thus allows for the majority of this missed common variation to be genotyped in the human population. Interestingly, when we repeat SV detection on a pseudodiploid genome constructed in silico by merging the two haploids, we find that ∼59% of the heterozygous SVs are no longer detected by SMRT-SV. These results indicate that haploid resolution of long-read sequencing data will significantly increase sensitivity of SV detection.
DNA methylation is the most common form of DNA modification in prokaryotic and eukaryotic genomes. We have applied the method of single-molecule, real-time (SMRT®) DNA sequencing that is capable of ...direct detection of modified bases at single-nucleotide resolution to characterize the specificity of several bacterial DNA methyltransferases (MTases). In addition to previously described SMRT sequencing of N6-methyladenine and 5-methylcytosine, we show that N4-methylcytosine also has a specific kinetic signature and is therefore identifiable using this approach. We demonstrate for all three prokaryotic methylation types that SMRT sequencing confirms the identity and position of the methylated base in cases where the MTase specificity was previously established by other methods. We then applied the method to determine the sequence context and methylated base identity for three MTases with unknown specificities. In addition, we also find evidence of unanticipated MTase promiscuity with some enzymes apparently also modifying sequences that are related, but not identical, to the cognate site.
Streptococcus pneumoniae (the pneumococcus) is the world's foremost bacterial pathogen in both morbidity and mortality. Switching between phenotypic forms (or 'phases') that favour asymptomatic ...carriage or invasive disease was first reported in 1933. Here, we show that the underlying mechanism for such phase variation consists of genetic rearrangements in a Type I restriction-modification system (SpnD39III). The rearrangements generate six alternative specificities with distinct methylation patterns, as defined by single-molecule, real-time (SMRT) methylomics. The SpnD39III variants have distinct gene expression profiles. We demonstrate distinct virulence in experimental infection and in vivo selection for switching between SpnD39III variants. SpnD39III is ubiquitous in pneumococci, indicating an essential role in its biology. Future studies must recognize the potential for switching between these heretofore undetectable, differentiated pneumococcal subpopulations in vitro and in vivo. Similar systems exist in other bacterial genera, indicating the potential for broad exploitation of epigenetic gene regulation.
Public health officials have raised concerns that plasmid transfer between Enterobacteriaceae species may spread resistance to carbapenems, an antibiotic class of last resort, thereby rendering ...common health care-associated infections nearly impossible to treat. To determine the diversity of carbapenemase-encoding plasmids and assess their mobility among bacterial species, we performed comprehensive surveillance and genomic sequencing of carbapenem-resistant Enterobacteriaceae in the National Institutes of Health (NIH) Clinical Center patient population and hospital environment. We isolated a repertoire of carbapenemase-encoding Enterobacteriaceae, including multiple strains of Klebsiella pneumoniae, Klebsiella oxytoca, Escherichia coli, Enterobacter cloacae, Citrobacter freundii, and Pantoea species. Long-read genome sequencing with full end-to-end assembly revealed that these organisms carry the carbapenem resistance genes on a wide array of plasmids. K. pneumoniae and E. cloacae isolated simultaneously from a single patient harbored two different carbapenemase-encoding plasmids, indicating that plasmid transfer between organisms was unlikely within this patient. We did, however, find evidence of horizontal transfer of carbapenemase-encoding plasmids between K. pneumoniae, E. cloacae, and C. freundii in the hospital environment. Our data, including full plasmid identification, challenge assumptions about horizontal gene transfer events within patients and identify possible connections between patients and the hospital environment. In addition, we identified a new carbapenemase-encoding plasmid of potentially high clinical impact carried by K. pneumoniae, E. coli, E. cloacae, and Pantoea species, in unrelated patients and in the hospital environment.
Bacterial phosphorothioate (PT) DNA modifications are incorporated by Dnd proteins A-E and often function with DndF-H as a restriction-modification (R-M) system, as in Escherichia coli B7A. However, ...bacteria such as Vibrio cyclitrophicus FF75 lack dndF-H, which points to other PT functions. Here we report two novel, orthogonal technologies to map PTs across the genomes of B7A and FF75 with >90% agreement: single molecule, real-time sequencing and deep sequencing of iodine-induced cleavage at PT (ICDS). In B7A, we detect PT on both strands of GpsAAC/GpsTTC motifs, but with only 12% of 40,701 possible sites modified. In contrast, PT in FF75 occurs as a single-strand modification at CpsCA, again with only 14% of 160,541 sites modified. Single-molecule analysis indicates that modification could be partial at any particular genomic site even with active restriction by DndF-H, with direct interaction of modification proteins with GAAC/GTTC sites demonstrated with oligonucleotides. These results point to highly unusual target selection by PT-modification proteins and rule out known R-M mechanisms.
The Caulobacter DNA methyltransferase CcrM is one of five master cell-cycle regulators. CcrM is transiently present near the end of DNA replication when it rapidly methylates the adenine in ...hemimethylated GANTC sequences. The timing of transcription of two master regulator genes and two cell division genes is controlled by the methylation state of GANTC sites in their promoters. To explore the global extent of this regulatory mechanism, we determined the methylation state of the entire chromosome at every base pair at five time points in the cell cycle using single-molecule, real-time sequencing. The methylation state of 4,515 GANTC sites, preferentially positioned in intergenic regions, changed progressively from full to hemimethylation as the replication forks advanced. However, 27 GANTC sites remained unmethylated throughout the cell cycle, suggesting that these protected sites could participate in epigenetic regulatory functions. An analysis of the time of activation of every cell-cycle regulatory transcription start site, coupled to both the position of a GANTC site in their promoter regions and the time in the cell cycle when the GANTC site transitions from full to hemimethylation, allowed the identification of 59 genes as candidates for epigenetic regulation. In addition, we identified two previously unidentified N ⁶-methyladenine motifs and showed that they maintained a constant methylation state throughout the cell cycle. The cognate methyltransferase was identified for one of these motifs as well as for one of two 5-methylcytosine motifs.
The whole-genome sequence of a carbapenem-resistant Klebsiella pneumoniae strain, PittNDM01, which coproduces NDM-1 and OXA-232 carbapenemases, was determined in this study. The use of ...single-molecule, real-time (SMRT) sequencing provided a closed genome in a single sequencing run. K. pneumoniae PittNDM01 has a single chromosome of 5,348,284 bp and four plasmids: pPKPN1 (283,371 bp), pPKPN2 (103,694 bp), pPKPN3 (70,814 bp), and pPKPN4 (6,141 bp). The contents of the chromosome were similar to that of the K. pneumoniae reference genome strain MGH 78578, with the exception of a large inversion spanning 23.3% of the chromosome. In contrast, three of the four plasmids are unique. The plasmid pPKPN1, an IncHI1B-like plasmid, carries the blaNDM-1, armA, and qnrB1 genes, along with tellurium and mercury resistance operons. blaNDM-1 is carried on a unique structure in which Tn125 is further bracketed by IS26 downstream of a class 1 integron. The IncFIA-like plasmid pPKPN3 also carries an array of resistance elements, including blaCTX-M-15 and a mercury resistance operon. The ColE-type plasmid pPKPN4 carrying blaOXA-232 is identical to a plasmid previously reported from France. SMRT sequencing was useful in resolving the complex bacterial genomic structures in the de novo assemblies.
PDF
