June 1, 2021  |  

Allele-level sequencing and phasing of full-length HLA class I and II genes using SMRT Sequencing technology

The three classes of genes that comprise the MHC gene family are actively involved in determining donor-recipient compatibility for organ transplant, as well as susceptibility to autoimmune diseases via cross-reacting immunization. Specifically, Class I genes HLA-A, -B, -C, and class II genes HLA-DR, -DQ and -DP are considered medically important for genetic analysis to determine histocompatibility. They are highly polymorphic and have thousands of alleles implicated in disease resistance and susceptibility. The importance of full-length HLA gene sequencing for genotyping, detection of null alleles, and phasing is now widely acknowledged. While DNA-sequencing-based HLA genotyping has become routine, only 7% of the HLA genes have been characterized by allele-level sequencing, while 93% are still defined by partial sequences. The gold-standard Sanger sequencing technology is being quickly replaced by second-generation, high- throughput sequencing methods due to its inability to generate unambiguous phased reads from heterozygous alleles. However, although these short, high-throughput, clonal sequencing methods are better at heterozygous allele detection, they are inadequate at generating full-length haploid gene sequences. Thus, full-length gene sequencing from an enhancer-promoter region to a 3’UTR that includes phasing information without the need for imputation still remains a technological challenge. The best way to overcome these challenges is to sequence these genes with a technology that is clonal in nature and has the longest possible read lengths. We have employed Single Molecule Real-Time (SMRT) sequencing technology from Pacific Biosciences for sequencing full-length HLA class I and II genes.


June 1, 2021  |  

De novo PacBio long-read assembled avian genomes correct and add to genes important in neuroscience and conservation research

To test the impact of high-quality genome assemblies on biological research, we applied PacBio long-read sequencing in conjunction with the new, diploid-aware FALCON-Unzip assembler to a number of bird species. These included: the zebra finch, for which a consortium-generated, Sanger-based reference exists, to determine how the FALCON-Unzip assembly would compare to the current best references available; Anna’s hummingbird genome, which had been assembled with short-read sequencing methods as part of the Avian Phylogenomics phase I initiative; and two critically endangered bird species (kakapo and ‘alala) of high importance for conservations efforts, whose genomes had not previously been sequenced and assembled.


June 1, 2021  |  

SMRT-Cappable-seq reveals the complex operome of bacteria

SMRT-Cappable-seq combines the isolation of full-length prokaryotic primary transcripts with long read sequencing technology. It is the first experimental methodology to sequence entire prokaryotic transcripts. It identifies the transcription start site and termination site, thereby directly defines the operon structures genome-wide in prokaryotes. Applied to E.coli, SMRT-Cappable-seq identifies a total of ~2300 operons, among which ~900 are novel. Importantly, our result reveals a pervasive read-through of previous experimentally validated transcription termination sites. Termination read-through represents a powerful strategy to control gene expression. Taken together this data provides a first glance at the complexity of the ‘operome’ in bacteria and presents an invaluable resource for understanding gene regulation and function in bacteria.


June 1, 2021  |  

Allelic specificity of immunoglobulin heavy chain ([email protected]) translocation in B-cell acute lymphoblastic leukemia (B-ALL) unveiled by long-read sequencing

Oncogenic fusion of IGH-DUX4 has recently been reported as a hallmark that defines a B-ALL subtype present in up to 7% of adolescents and young adults B-ALL. The translocation of DUX4 into IGH results in aberrant activation of DUX4 by hijacking the intronic IGH enhancer (Eµ). How IGH-DUX4 translocation interplays with IGH allelic exclusion was never been explored. We investigated this in Nalm6 B-ALL cell line, using long-read (PacBio Iso-Seq method and 10X Chromium WGS), short-read (Illumina total stranded RNA and WGS), epigenome (H3K27ac ChIP-seq, ATAC-seq) and 3-D genome (Hi-C, H3K27ac HiChIP, Capture-C).


April 21, 2020  |  

Transcriptional initiation of a small RNA, not R-loop stability, dictates the frequency of pilin antigenic variation in Neisseria gonorrhoeae.

Neisseria gonorrhoeae, the sole causative agent of gonorrhea, constitutively undergoes diversification of the Type IV pilus. Gene conversion occurs between one of the several donor silent copies located in distinct loci and the recipient pilE gene, encoding the major pilin subunit of the pilus. A guanine quadruplex (G4) DNA structure and a cis-acting sRNA (G4-sRNA) are located upstream of the pilE gene and both are required for pilin antigenic variation (Av). We show that the reduced sRNA transcription lowers pilin Av frequencies. Extended transcriptional elongation is not required for Av, since limiting the transcript to 32 nt allows for normal Av frequencies. Using chromatin immunoprecipitation (ChIP) assays, we show that cellular G4s are less abundant when sRNA transcription is lower. In addition, using ChIP, we demonstrate that the G4-sRNA forms a stable RNA:DNA hybrid (R-loop) with its template strand. However, modulating R-loop levels by controlling RNase HI expression does not alter G4 abundance quantified through ChIP. Since pilin Av frequencies were not altered when modulating R-loop levels by controlling RNase HI expression, we conclude that transcription of the sRNA is necessary, but stable R-loops are not required to promote pilin Av. © 2019 John Wiley & Sons Ltd.


April 21, 2020  |  

A Novel Bacteriophage Exclusion (BREX) System Encoded by the pglX Gene in Lactobacillus casei Zhang.

The bacteriophage exclusion (BREX) system is a novel prokaryotic defense system against bacteriophages. To our knowledge, no study has systematically characterized the function of the BREX system in lactic acid bacteria. Lactobacillus casei Zhang is a probiotic bacterium originating from koumiss. By using single-molecule real-time sequencing, we previously identified N6-methyladenine (m6A) signatures in the genome of L. casei Zhang and a putative methyltransferase (MTase), namely, pglX This work further analyzed the genomic locus near the pglX gene and identified it as a component of the BREX system. To decipher the biological role of pglX, an L. casei Zhang pglX mutant (?pglX) was constructed. Interestingly, m6A methylation of the 5′-ACRCAG-3′ motif was eliminated in the ?pglX mutant. The wild-type and mutant strains exhibited no significant difference in morphology or growth performance in de Man-Rogosa-Sharpe (MRS) medium. A significantly higher plasmid acquisition capacity was observed for the ?pglX mutant than for the wild type if the transformed plasmids contained pglX recognition sites (i.e., 5′-ACRCAG-3′). In contrast, no significant difference was observed in plasmid transformation efficiency between the two strains when plasmids lacking pglX recognition sites were tested. Moreover, the ?pglX mutant had a lower capacity to retain the plasmids than the wild type, suggesting a decrease in genetic stability. Since the Rebase database predicted that the L. casei PglX protein was bifunctional, as both an MTase and a restriction endonuclease, the PglX protein was heterologously expressed and purified but failed to show restriction endonuclease activity. Taken together, the results show that the L. casei Zhang pglX gene is a functional adenine MTase that belongs to the BREX system.IMPORTANCELactobacillus casei Zhang is a probiotic that confers beneficial effects on the host, and it is thus increasingly used in the dairy industry. The possession of an effective bacterial immune system that can defend against invasion of phages and exogenous DNA is a desirable feature for industrial bacterial strains. The bacteriophage exclusion (BREX) system is a recently described phage resistance system in prokaryotes. This work confirmed the function of the BREX system in L. casei and that the methyltransferase (pglX) is an indispensable part of the system. Overall, our study characterizes a BREX system component gene in lactic acid bacteria. Copyright © 2019 American Society for Microbiology.


April 21, 2020  |  

Sensitivity to the two peptide bacteriocin plantaricin EF is dependent on CorC, a membrane-bound, magnesium/cobalt efflux protein.

Lactic acid bacteria produce a variety of antimicrobial peptides known as bacteriocins. Most bacteriocins are understood to kill sensitive bacteria through receptor-mediated disruptions. Here, we report on the identification of the Lactobacillus plantarum plantaricin EF (PlnEF) receptor. Spontaneous PlnEF-resistant mutants of the PlnEF-indicator strain L. plantarum NCIMB 700965 (LP965) were isolated and confirmed to maintain cellular ATP levels in the presence of PlnEF. Genome comparisons resulted in the identification of a single mutated gene annotated as the membrane-bound, magnesium/cobalt efflux protein CorC. All isolates contained a valine (V) at position 334 instead of a glycine (G) in a cysteine-ß-synthase domain at the C-terminal region of CorC. In silico template-based modeling of this domain indicated that the mutation resides in a loop between two ß-strands. The relationship between PlnEF, CorC, and metal homeostasis was supported by the finding that PlnEF-resistance was lost when PlnEF was applied together with high concentrations of Mg2+ , Co2+ , Zn2+ , or Cu2+ . Lastly, PlnEF sensitivity was increased upon heterologous expression of LP965 corC but not the G334V CorC mutant in the PlnEF-resistant strain Lactobacillus casei BL23. These results show that PlnEF kills sensitive bacteria by targeting CorC. © 2019 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.


April 21, 2020  |  

Genome-Wide Screening for Enteric Colonization Factors in Carbapenem-Resistant ST258 Klebsiella pneumoniae.

A diverse, antibiotic-naive microbiota prevents highly antibiotic-resistant microbes, including carbapenem-resistant Klebsiella pneumoniae (CR-Kp), from achieving dense colonization of the intestinal lumen. Antibiotic-mediated destruction of the microbiota leads to expansion of CR-Kp in the gut, markedly increasing the risk of bacteremia in vulnerable patients. While preventing dense colonization represents a rational approach to reduce intra- and interpatient dissemination of CR-Kp, little is known about pathogen-associated factors that enable dense growth and persistence in the intestinal lumen. To identify genetic factors essential for dense colonization of the gut by CR-Kp, we constructed a highly saturated transposon mutant library with >150,000 unique mutations in an ST258 strain of CR-Kp and screened for in vitro growth and in vivo intestinal colonization in antibiotic-treated mice. Stochastic and partially reversible fluctuations in the representation of different mutations during dense colonization revealed the dynamic nature of intestinal microbial populations. We identified genes that are crucial for early and late stages of dense gut colonization and confirmed their role by testing isogenic mutants in in vivo competition assays with wild-type CR-Kp Screening of the transposon library also identified mutations that enhanced in vivo CR-Kp growth. These newly identified colonization factors may provide novel therapeutic opportunities to reduce intestinal colonization by CR-KpIMPORTANCEKlebsiella pneumoniae is a common cause of bloodstream infections in immunocompromised and hospitalized patients, and over the last 2 decades, some strains have acquired resistance to nearly all available antibiotics, including broad-spectrum carbapenems. The U.S. Centers for Disease Control and Prevention has listed carbapenem-resistant K. pneumoniae (CR-Kp) as an urgent public health threat. Dense colonization of the intestine by CR-Kp and other antibiotic-resistant bacteria is associated with an increased risk of bacteremia. Reducing the density of gut colonization by CR-Kp is likely to reduce their transmission from patient to patient in health care facilities as well as systemic infections. How CR-Kp expands and persists in the gut lumen, however, is poorly understood. Herein, we generated a highly saturated mutant library in a multidrug-resistant K. pneumoniae strain and identified genetic factors that are associated with dense gut colonization by K. pneumoniae This study sheds light on host colonization by K. pneumoniae and identifies potential colonization factors that contribute to high-density persistence of K. pneumoniae in the intestine. Copyright © 2019 Jung et al.


April 21, 2020  |  

FadR1, a pathway-specific activator of fidaxomicin biosynthesis in Actinoplanes deccanensis Yp-1.

Fidaxomicin, an 18-membered macrolide antibiotic, is highly active against Clostridium difficile, the most common cause of diarrhea in hospitalized patients. Though the biosynthetic mechanism of fidaxomicin has been well studied, little is known about its regulatory mechanism. Here, we reported that FadR1, a LAL family transcriptional regulator in the fidaxomicin cluster of Actinoplanes deccanensis Yp-1, acts as an activator for fidaxomicin biosynthesis. The disruption of fadR1 abolished the ability to synthesize fidaxomicin, and production could be restored by reintegrating a single copy of fadR1. Overexpression of fadR1 resulted in an approximately 400 % improvement in fidaxomicin production. Electrophoretic mobility shift assays indicated that fidaxomicin biosynthesis is under the control of FadR1 through its binding to the promoter regions of fadM, fadA1-fadP2, fadS2-fadC, and fadE-fadF, respectively. And the conserved binding sites of FadR1 within the four promoter regions were determined by footprinting experiment. All results indicated that fadR1 encodes a pathway-specific positive regulator of fidaxomicin biosynthesis and upregulates the transcription levels of most of genes by binding to the four above intergenic regions. In summary, we not only clearly elucidate the regulatory mechanism of FadR1 but also provide strategies for the construction of industrial high-yield strain of fidaxomicin.


April 21, 2020  |  

Potential of TLR-gene diversity in Czech indigenous cattle for resistance breeding as revealed by hybrid sequencing

A production herd of Czech Simmental cattle (Czech Red Pied, CRP), the conserved subpopulation of this breed, and the ancient local breed Czech Red cattle (CR) were screened for diversity in the antibacterial toll-like receptors (TLRs), which are members of the innate immune system. Polymerase chain reaction (PCR) amplicons of TLR1, TLR2, TLR4, TLR5, and TLR6 from pooled DNA samples were sequenced with PacBio technology, with 3–5×?coverage per gene per animal. To increase the reliability of variant detection, the gDNA pools were sequenced in parallel with the Illumina X-ten platform at low coverage (60× per gene). The diversity in conserved CRP and CR was similar to the diversity in conserved and modern CRP, representing 76.4?% and 70.9?% of its variants, respectively. Sixty-eight (54.4?%) polymorphisms in the five TLR genes were shared by the two breeds, whereas 38 (30.4?%) were specific to the production herd of CRP; 4 (3.2?%) were specific to the broad CRP population; 7 (5.6?%) were present in both conserved populations; 5 (4.0?%) were present solely for the conserved CRP; and 3 (2.4?%) were restricted to CR. Consequently, gene pool erosion related to intensive breeding did not occur in Czech Simmental cattle. Similarly, no considerable consequences were found from known bottlenecks in the history of Czech Red cattle. On the other hand, the distinctness of the conserved populations and their potential for resistance breeding were only moderate. This relationship might be transferable to other non-abundant historical cattle breeds that are conserved as genetic resources. The estimates of polymorphism impact using Variant Effect Predictor and SIFT software tools allowed for the identification of candidate single-nucleotide polymorphisms (SNPs) for association studies related to infection resistance and targeted breeding. Knowledge of TLR-gene diversity present in Czech Simmental populations may aid in the potential transfer of variant characteristics from other breeds.


April 21, 2020  |  

A systematic review of the Trypanosoma cruzi genetic heterogeneity, host immune response and genetic factors as plausible drivers of chronic chagasic cardiomyopathy.

Chagas disease is a complex tropical pathology caused by the kinetoplastid Trypanosoma cruzi. This parasite displays massive genetic diversity and has been classified by international consensus in at least six Discrete Typing Units (DTUs) that are broadly distributed in the American continent. The main clinical manifestation of the disease is the chronic chagasic cardiomyopathy (CCC) that is lethal in the infected individuals. However, one intriguing feature is that only 30-40% of the infected individuals will develop CCC. Some authors have suggested that the immune response, host genetic factors, virulence factors and even the massive genetic heterogeneity of T. cruzi are responsible of this clinical pattern. To date, no conclusive data support the reason why a few percentages of the infected individuals will develop CCC. Therefore, we decided to conduct a systematic review analysing the host genetic factors, immune response, cytokine production, virulence factors and the plausible association of the parasite DTUs and CCC. The epidemiological and clinical implications are herein discussed.


April 21, 2020  |  

Long-read sequence capture of the haemoglobin gene clusters across codfish species.

Combining high-throughput sequencing with targeted sequence capture has become an attractive tool to study specific genomic regions of interest. Most studies have so far focused on the exome using short-read technology. These approaches are not designed to capture intergenic regions needed to reconstruct genomic organization, including regulatory regions and gene synteny. Here, we demonstrate the power of combining targeted sequence capture with long-read sequencing technology for comparative genomic analyses of the haemoglobin (Hb) gene clusters across eight species separated by up to 70 million years. Guided by the reference genome assembly of the Atlantic cod (Gadus morhua) together with genome information from draft assemblies of selected codfishes, we designed probes covering the two Hb gene clusters. Use of custom-made barcodes combined with PacBio RSII sequencing led to highly continuous assemblies of the LA (~100 kb) and MN (~200 kb) clusters, which include syntenic regions of coding and intergenic sequences. Our results revealed an overall conserved genomic organization of the Hb genes within this lineage, yet with several, lineage-specific gene duplications. Moreover, for some of the species examined, we identified amino acid substitutions at two sites in the Hbb1 gene as well as length polymorphisms in its regulatory region, which has previously been linked to temperature adaptation in Atlantic cod populations. This study highlights the use of targeted long-read capture as a versatile approach for comparative genomic studies by generation of a cross-species genomic resource elucidating the evolutionary history of the Hb gene family across the highly divergent group of codfishes. © 2018 The Authors. Molecular Ecology Resources Published by John Wiley & Sons Ltd.


April 21, 2020  |  

Deciphering bacterial epigenomes using modern sequencing technologies.

Prokaryotic DNA contains three types of methylation: N6-methyladenine, N4-methylcytosine and 5-methylcytosine. The lack of tools to analyse the frequency and distribution of methylated residues in bacterial genomes has prevented a full understanding of their functions. Now, advances in DNA sequencing technology, including single-molecule, real-time sequencing and nanopore-based sequencing, have provided new opportunities for systematic detection of all three forms of methylated DNA at a genome-wide scale and offer unprecedented opportunities for achieving a more complete understanding of bacterial epigenomes. Indeed, as the number of mapped bacterial methylomes approaches 2,000, increasing evidence supports roles for methylation in regulation of gene expression, virulence and pathogen-host interactions.


April 21, 2020  |  

Genome assembly of a tropical maize inbred line provides insights into structural variation and crop improvement.

Maize is one of the most important crops globally, and it shows remarkable genetic diversity. Knowledge of this diversity could help in crop improvement; however, gold-standard genomes have been elucidated only for modern temperate varieties. Here, we present a high-quality reference genome (contig N50 of 15.78?megabases) of the maize small-kernel inbred line, which is derived from a tropical landrace. Using haplotype maps derived from B73, Mo17 and SK, we identified 80,614 polymorphic structural variants across 521 diverse lines. Approximately 22% of these variants could not be detected by traditional single-nucleotide-polymorphism-based approaches, and some of them could affect gene expression and trait performance. To illustrate the utility of the diverse SK line, we used it to perform map-based cloning of a major effect quantitative trait locus controlling kernel weight-a key trait selected during maize improvement. The underlying candidate gene ZmBARELY ANY MERISTEM1d provides a target for increasing crop yields.


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