Menu

Auto Tag: Targeted

July 19, 2019

Improved maize reference genome with single-molecule technologies.

Complete and accurate reference genomes and annotations provide fundamental tools for characterization of genetic and functional variation. These resources facilitate the determination of biological processes and support translation of research findings into improved and sustainable agricultural technologies. Many reference genomes for crop plants have been generated over the past decade, but these genomes are often fragmented and missing complex repeat regions. Here we report the assembly and annotation of a reference genome of maize, a genetic and agricultural model species, using single-molecule real-time sequencing and high-resolution optical mapping. Relative to the previous reference genome, our assembly features a 52-fold increase in contig length and notable improvements in the assembly of intergenic spaces and centromeres. Characterization of the repetitive portion of the genome revealed more than 130,000 intact transposable elements, allowing us to identify transposable element lineage expansions that are unique to maize. Gene annotations were updated using 111,000 full-length transcripts obtained by single-molecule real-time sequencing. In addition, comparative optical mapping of two other inbred maize lines revealed a prevalence of deletions in regions of low gene density and maize lineage-specific genes.

Read more
July 19, 2019

Quality control of the traditional patent medicine Yimu Wan based on SMRT Sequencing and DNA barcoding.

Substandard traditional patent medicines may lead to global safety-related issues. Protecting consumers from the health risks associated with the integrity and authenticity of herbal preparations is of great concern. Of particular concern is quality control for traditional patent medicines. Here, we establish an effective approach for verifying the biological composition of traditional patent medicines based on single-molecule real-time (SMRT) sequencing and DNA barcoding. Yimu Wan (YMW), a classical herbal prescription recorded in the Chinese Pharmacopoeia, was chosen to test the method. Two reference YMW samples were used to establish a standard method for analysis, which was then applied to three different batches of commercial YMW samples. A total of 3703 and 4810 circular-consensus sequencing (CCS) reads from two reference and three commercial YMW samples were mapped to the ITS2 and psbA-trnH regions, respectively. Moreover, comparison of intraspecific genetic distances based on SMRT sequencing data with reference data from Sanger sequencing revealed an ITS2 and psbA-trnH intergenic spacer that exhibited high intraspecific divergence, with the sites of variation showing significant differences within species. Using the CCS strategy for SMRT sequencing analysis was adequate to guarantee the accuracy of identification. This study demonstrates the application of SMRT sequencing to detect the biological ingredients of herbal preparations. SMRT sequencing provides an affordable way to monitor the legality and safety of traditional patent medicines.

Read more
July 19, 2019

Detecting AGG interruptions in male and female FMR1 premutation carriers by single-molecule sequencing.

The FMR1 gene contains an unstable CGG repeat in its 5′ untranslated region. Premutation alleles range between 55 and 200 repeat units and confer a risk for developing fragile X-associated tremor/ataxia syndrome or fragile X-associated primary ovarian insufficiency. Furthermore, the premutation allele often expands to a full mutation during female germline transmission giving rise to the fragile X syndrome. The risk for a premutation to expand depends mainly on the number of CGG units and the presence of AGG interruptions in the CGG repeat. Unfortunately, the detection of AGG interruptions is hampered by technical difficulties. Here, we demonstrate that single-molecule sequencing enables the determination of not only the repeat size, but also the complete repeat sequence including AGG interruptions in male and female alleles with repeats ranging from 45 to 100 CGG units. We envision this method will facilitate research and diagnostic analysis of the FMR1 repeat expansion. © 2016 WILEY PERIODICALS, INC.

Read more
July 19, 2019

Iterative optimization of xylose catabolism in Saccharomyces cerevisiae using combinatorial expression tuning.

A common challenge in metabolic engineering is rapidly identifying rate-controlling enzymes in heterologous pathways for subsequent production improvement. We demonstrate a workflow to address this challenge and apply it to improving xylose utilization in Saccharomyces cerevisiae. For eight reactions required for conversion of xylose to ethanol, we screened enzymes for functional expression in S. cerevisiae, followed by a combinatorial expression analysis to achieve pathway flux balancing and identification of limiting enzymatic activities. In the next round of strain engineering, we increased the copy number of these limiting enzymes and again tested the eight-enzyme combinatorial expression library in this new background. This workflow yielded a strain that has a ~70% increase in biomass yield and ~240% increase in xylose utilization. Finally, we chromosomally integrated the expression library. This library enriched for strains with multiple integrations of the pathway, which likely were the result of tandem integrations mediated by promoter homology. Biotechnol. Bioeng. 2017;114: 1301-1309. © 2017 Wiley Periodicals, Inc.© 2017 Wiley Periodicals, Inc.

Read more
July 19, 2019

Diversity of the TLR4 immunity receptor in Czech native cattle breeds revealed using the Pacific Biosciences sequencing platform.

The allelic variants of immunity genes in historical breeds likely reflect local infection pressure and therefore represent a reservoir for breeding. Screening to determine the diversity of the Toll-like receptor gene TLR4 was conducted in two conserved cattle breeds: Czech Red and Czech Red Pied. High-throughput sequencing of pooled PCR amplicons using the PacBio platform revealed polymorphisms, which were subsequently confirmed via genotyping techniques. Eight SNPs found in coding and adjacent regions were grouped into 18 haplotypes, representing a significant portion of the known diversity in the global breed panel and presumably exceeding diversity in production populations. Notably, the ancient Czech Red breed appeared to possess greater haplotype diversity than the Czech Red Pied breed, a Simmental variant, although the haplotype frequencies might have been distorted by significant crossbreeding and bottlenecks in the history of Czech Red cattle. The differences in haplotype frequencies validated the phenotypic distinctness of the local breeds. Due to the availability of Czech Red Pied production herds, the effect of intensive breeding on TLR diversity can be evaluated in this model. The advantages of the Pacific Biosciences technology for the resequencing of long PCR fragments with subsequent direct phasing were independently validated.

Read more
July 19, 2019

Sequencing the CYP2D6 gene: from variant allele discovery to clinical pharmacogenetic testing.

CYP2D6 is one of the most studied enzymes in the field of pharmacogenetics. The CYP2D6 gene is highly polymorphic with over 100 catalogued star (*) alleles, and clinical CYP2D6 testing is increasingly accessible and supported by practice guidelines. However, the degree of variation at the CYP2D6 locus and homology with its pseudogenes make interrogating CYP2D6 by short-read sequencing challenging. Moreover, accurate prediction of CYP2D6 metabolizer status necessitates analysis of duplicated alleles when an increased copy number is detected. These challenges have recently been overcome by long-read CYP2D6 sequencing; however, such platforms are not widely available. This review highlights the genomic complexities of CYP2D6, current sequencing methods and the evolution of CYP2D6 from allele discovery to clinical pharmacogenetic testing.

Read more
July 19, 2019

SMRT Gate: A method for validation of synthetic constructs on Pacific Biosciences sequencing platforms.

Current DNA assembly methods are prone to sequence errors, requiring rigorous quality control (QC) to identify incorrect assemblies or synthesized constructs. Such errors can lead to misinterpretation of phenotypes. Because of this intrinsic problem, routine QC analysis is generally performed on three or more clones using a combination of restriction endonuclease assays, colony PCR, and Sanger sequencing. However, as new automation methods emerge that enable high-throughput assembly, QC using these techniques has become a major bottleneck. Here, we describe a quick and affordable methodology for the QC of synthetic constructs. Our method involves a one-pot digestion-ligation DNA assembly reaction, based on the Golden Gate assembly methodology, that is coupled with Pacific Biosciences’ Single Molecule, Real-Time (PacBio SMRT) sequencing technology.

Read more
July 19, 2019

Defective HIV-1 proviruses are expressed and can be recognized by cytotoxic T lymphocytes, which shape the proviral landscape.

Despite antiretroviral therapy, HIV-1 persists in memory CD4(+) T cells, creating a barrier to cure. The majority of HIV-1 proviruses are defective and considered clinically irrelevant. Using cells from HIV-1-infected individuals and reconstructed patient-derived defective proviruses, we show that defective proviruses can be transcribed into RNAs that are spliced and translated. Proviruses with defective major splice donors (MSDs) can activate novel splice sites to produce HIV-1 transcripts, and cells with these proviruses can be recognized by HIV-1-specific cytotoxic T lymphocytes (CTLs). Further, cells with proviruses containing lethal mutations upstream of CTL epitopes can also be recognized by CTLs, potentially through aberrant translation. Thus, CTLs may change the landscape of HIV-1 proviruses by preferentially targeting cells with specific types of defective proviruses. Additionally, the expression of defective proviruses will need to be considered in the measurement of HIV-1 latency reversal. Copyright © 2017 Elsevier Inc. All rights reserved.

Read more
July 19, 2019

Evolutionary restoration of fertility in an interspecies hybrid yeast, by whole-genome duplication after a failed mating-type switch.

Many interspecies hybrids have been discovered in yeasts, but most of these hybrids are asexual and can replicate only mitotically. Whole-genome duplication has been proposed as a mechanism by which interspecies hybrids can regain fertility, restoring their ability to perform meiosis and sporulate. Here, we show that this process occurred naturally during the evolution of Zygosaccharomyces parabailii, an interspecies hybrid that was formed by mating between 2 parents that differed by 7% in genome sequence and by many interchromosomal rearrangements. Surprisingly, Z. parabailii has a full sexual cycle and is genetically haploid. It goes through mating-type switching and autodiploidization, followed by immediate sporulation. We identified the key evolutionary event that enabled Z. parabailii to regain fertility, which was breakage of 1 of the 2 homeologous copies of the mating-type (MAT) locus in the hybrid, resulting in a chromosomal rearrangement and irreparable damage to 1 MAT locus. This rearrangement was caused by HO endonuclease, which normally functions in mating-type switching. With 1 copy of MAT inactivated, the interspecies hybrid now behaves as a haploid. Our results provide the first demonstration that MAT locus damage is a naturally occurring evolutionary mechanism for whole-genome duplication and restoration of fertility to interspecies hybrids. The events that occurred in Z. parabailii strongly resemble those postulated to have caused ancient whole-genome duplication in an ancestor of Saccharomyces cerevisiae.

Read more
July 19, 2019

Discovery and biosynthesis of gladiolin: A Burkholderia gladioli antibiotic with promising activity against Mycobacterium tuberculosis.

An antimicrobial activity screen of Burkholderia gladioli BCC0238, a clinical isolate from a cystic fibrosis patient, led to the discovery of gladiolin, a novel macrolide antibiotic with potent activity against Mycobacterium tuberculosis H37Rv. Gladiolin is structurally related to etnangien, a highly unstable antibiotic from Sorangium cellulosum that is also active against Mycobacteria. Like etnangien, gladiolin was found to inhibit RNA polymerase, a validated drug target in M. tuberculosis. However, gladiolin lacks the highly labile hexaene moiety of etnangien and was thus found to possess significantly increased chemical stability. Moreover, gladiolin displayed low mammalian cytotoxicity and good activity against several M. tuberculosis clinical isolates, including four that are resistant to isoniazid and one that is resistant to both isoniazid and rifampicin. Overall, these data suggest that gladiolin may represent a useful starting point for the development of novel drugs to tackle multidrug-resistant tuberculosis. The B. gladioli BCC0238 genome was sequenced using Single Molecule Real Time (SMRT) technology. This resulted in four contiguous sequences: two large circular chromosomes and two smaller putative plasmids. Analysis of the chromosome sequences identified 49 putative specialized metabolite biosynthetic gene clusters. One such gene cluster, located on the smaller of the two chromosomes, encodes a trans-acyltransferase (trans-AT) polyketide synthase (PKS) multienzyme that was hypothesized to assemble gladiolin. Insertional inactivation of a gene in this cluster encoding one of the PKS subunits abrogated gladiolin production, confirming that the gene cluster is responsible for biosynthesis of the antibiotic. Comparison of the PKSs responsible for the assembly of gladiolin and etnangien showed that they possess a remarkably similar architecture, obfuscating the biosynthetic mechanisms responsible for most of the structural differences between the two metabolites.

Read more
July 19, 2019

A case study into microbial genome assembly gap sequences and finishing strategies.

This study characterized regions of DNA which remained unassembled by either PacBio and Illumina sequencing technologies for seven bacterial genomes. Two genomes were manually finished using bioinformatics and PCR/Sanger sequencing approaches and regions not assembled by automated software were analyzed. Gaps present within Illumina assemblies mostly correspond to repetitive DNA regions such as multiple rRNA operon sequences. PacBio gap sequences were evaluated for several properties such as GC content, read coverage, gap length, ability to form strong secondary structures, and corresponding annotations. Our hypothesis that strong secondary DNA structures blocked DNA polymerases and contributed to gap sequences was not accepted. PacBio assemblies had few limitations overall and gaps were explained as cumulative effect of lower than average sequence coverage and repetitive sequences at contig termini. An important aspect of the present study is the compilation of biological features that interfered with assembly and included active transposons, multiple plasmid sequences, phage DNA integration, and large sequence duplication. Our targeted genome finishing approach and systematic evaluation of the unassembled DNA will be useful for others looking to close, finish, and polish microbial genome sequences.

Read more
July 19, 2019

A new method for sequencing the hypervariable Plasmodium falciparum gene var2csa from clinical samples.

VAR2CSA, a member of the Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) family, mediates the binding of P. falciparum-infected erythrocytes to chondroitin sulfate A, a surface-associated molecule expressed in placental cells, and plays a central role in the pathogenesis of placental malaria. VAR2CSA is a target of naturally acquired immunity and, as such, is a leading vaccine candidate against placental malaria. This protein is very polymorphic and technically challenging to sequence. Published var2csa sequences, mostly limited to specific domains, have been generated through the sequencing of cloned PCR amplicons using capillary electrophoresis, a method that is both time consuming and costly, and that performs poorly when applied to clinical samples that are commonly polyclonal. A next-generation sequencing platform, Pacific Biosciences (PacBio), offers an alternative approach to overcome these issues.PCR primers were designed that target a 5 kb segment in the 5′ end of var2csa and the resulting amplicons were sequenced using PacBio sequencing. The primers were optimized using two laboratory strains and were validated on DNA from 43 clinical samples, extracted from dried blood spots on filter paper or from cryopreserved P. falciparum-infected erythrocytes. Sequence reads were assembled using the SMRT-analysis ConsensusTools module.Here, a PacBio sequencing-based approach for recovering a segment encoding the majority of VAR2CSA’s extracellular region is described; this segment includes the totality of the first four domains in the 5′ end of var2csa (~5 kb), from clinical malaria samples. The feasibility of the method is demonstrated, showing a high success rate from cryopreserved samples and more limited success from dried blood spots stored at room temperature, and characterized the genetic variation of the var2csa locus.This method will facilitate a detailed analysis of var2csa genetic variation and can be adapted to sequence other hypervariable P. falciparum genes.

Read more
July 19, 2019

A mobile pathogenicity chromosome in Fusarium oxysporum for infection of multiple cucurbit species.

The genome of Fusarium oxysporum (Fo) consists of a set of eleven ‘core’ chromosomes, shared by most strains and responsible for housekeeping, and one or several accessory chromosomes. We sequenced a strain of Fo f.sp. radicis-cucumerinum (Forc) using PacBio SMRT sequencing. All but one of the core chromosomes were assembled into single contigs, and a chromosome that shows all the hallmarks of a pathogenicity chromosome comprised two contigs. A central part of this chromosome contains all identified candidate effector genes, including homologs of SIX6, SIX9, SIX11 and SIX 13. We show that SIX6 contributes to virulence of Forc. Through horizontal chromosome transfer (HCT) to a non-pathogenic strain, we also show that the accessory chromosome containing the SIX gene homologs is indeed a pathogenicity chromosome for cucurbit infection. Conversely, complete loss of virulence was observed in Forc016 strains that lost this chromosome. We conclude that also a non-wilt-inducing Fo pathogen relies on effector proteins for successful infection and that the Forc pathogenicity chromosome contains all the information necessary for causing root rot of cucurbits. Three out of nine HCT strains investigated have undergone large-scale chromosome alterations, reflecting the remarkable plasticity of Fo genomes.

Read more
July 19, 2019

Polylox barcoding reveals haematopoietic stem cell fates realized in vivo.

Developmental deconvolution of complex organs and tissues at the level of individual cells remains challenging. Non-invasive genetic fate mapping has been widely used, but the low number of distinct fluorescent marker proteins limits its resolution. Much higher numbers of cell markers have been generated using viral integration sites, viral barcodes, and strategies based on transposons and CRISPR-Cas9 genome editing; however, temporal and tissue-specific induction of barcodes in situ has not been achieved. Here we report the development of an artificial DNA recombination locus (termed Polylox) that enables broadly applicable endogenous barcoding based on the Cre-loxP recombination system. Polylox recombination in situ reaches a practical diversity of several hundred thousand barcodes, allowing tagging of single cells. We have used this experimental system, combined with fate mapping, to assess haematopoietic stem cell (HSC) fates in vivo. Classical models of haematopoietic lineage specification assume a tree with few major branches. More recently, driven in part by the development of more efficient single-cell assays and improved transplantation efficiencies, different models have been proposed, in which unilineage priming may occur in mice and humans at the level of HSCs. We have introduced barcodes into HSC progenitors in embryonic mice, and found that the adult HSC compartment is a mosaic of embryo-derived HSC clones, some of which are unexpectedly large. Most HSC clones gave rise to multilineage or oligolineage fates, arguing against unilineage priming, and suggesting coherent usage of the potential of cells in a clone. The spreading of barcodes, both after induction in embryos and in adult mice, revealed a basic split between common myeloid-erythroid development and common lymphocyte development, supporting the long-held but contested view of a tree-like haematopoietic structure.

Read more
July 19, 2019

Increased risk of low birth weight in women with placental malaria associated with P. falciparum VAR2CSA clade.

Pregnancy associated malaria (PAM) causes adverse pregnancy and birth outcomes owing to Plasmodium falciparum accumulation in the placenta. Placental accumulation is mediated by P. falciparum protein VAR2CSA, a leading PAM-specific vaccine target. The extent of its antigen diversity and impact on clinical outcomes remain poorly understood. Through amplicon deep-sequencing placental malaria samples from women in Malawi and Benin, we assessed sequence diversity of VAR2CSA’s ID1-DBL2x region, containing putative vaccine targets and estimated associations of specific clades with adverse birth outcomes. Overall, var2csa diversity was high and haplotypes subdivided into five clades, the largest two defined by homology to parasites strains, 3D7 or FCR3. Across both cohorts, compared to women infected with only FCR3-like variants, women infected with only 3D7-like variants delivered infants with lower birthweight (difference: -267.99?g; 95% Confidence Interval [CI]: -466.43?g,-69.55?g) and higher odds of low birthweight (<2500?g) (Odds Ratio [OR] 5.41; 95% CI:0.99,29.52) and small-for-gestational-age (OR: 3.65; 95% CI: 1.01,13.38). In two distinct malaria-endemic African settings, parasites harboring 3D7-like variants of VAR2CSA were associated with worse birth outcomes, supporting differential effects of infection with specific parasite strains. The immense diversity coupled with differential clinical effects of this diversity suggest that an effective VAR2CSA-based vaccine may require multivalent activity.

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.