Menu

Auto Tag: using the PacBio

July 19, 2019  |  

ALF: a strategy for identification of unauthorized GMOs in complex mixtures by a GW-NGS method and dedicated bioinformatics analysis.

The majority of feed products in industrialised countries contains materials derived from genetically modified organisms (GMOs). In parallel, the number of reports of unauthorised GMOs (UGMOs) is gradually increasing. There is a lack of specific detection methods for UGMOs, due to the absence of detailed sequence information and reference materials. In this research, an adapted genome walking approach was developed, called ALF: Amplification of Linearly-enriched Fragments. Coupling of ALF to NGS aims for simultaneous detection and identification of all GMOs, including UGMOs, in one sample, in a single analysis. The ALF approach was assessed on a mixture made of DNA extracts from four reference materials, in an uneven distribution, mimicking a real life situation. The complete insert and genomic flanking regions were known for three of the included GMO events, while for MON15985 only partial sequence information was available. Combined with a known organisation of elements, this GMO served as a model for a UGMO. We successfully identified sequences matching with this organisation of elements serving as proof of principle for ALF as new UGMO detection strategy. Additionally, this study provides a first outline of an automated, web-based analysis pipeline for identification of UGMOs containing known GM elements.

Read more
July 19, 2019  |  

The diversity, structure, and function of heritable adaptive immunity sequences in the Aedes aegypti genome.

The Aedes aegypti mosquito transmits arboviruses, including dengue, chikungunya, and Zika virus. Understanding the mechanisms underlying mosquito immunity could provide new tools to control arbovirus spread. Insects exploit two different RNAi pathways to combat viral and transposon infection: short interfering RNAs (siRNAs) and PIWI-interacting RNAs (piRNAs) [1, 2]. Endogenous viral elements (EVEs) are sequences from non-retroviral viruses that are inserted into the mosquito genome and can act as templates for the production of piRNAs [3, 4]. EVEs therefore represent a record of past infections and a reservoir of potential immune memory [5]. The large-scale organization of EVEs has been difficult to resolve with short-read sequencing because they tend to integrate into repetitive regions of the genome. To define the diversity, organization, and function of EVEs, we took advantage of the contiguity associated with long-read sequencing to generate a high-quality assembly of the Ae. aegypti-derived Aag2 cell line genome, an important and widely used model system. We show EVEs are acquired through recombination with specific classes of long terminal repeat (LTR) retrotransposons and organize into large loci (>50 kbp) characterized by high LTR density. These EVE-containing loci have increased density of piRNAs compared to similar regions without EVEs. Furthermore, we detected EVE-derived piRNAs consistent with a targeted processing of persistently infecting virus genomes. We propose that comparisons of EVEs across mosquito populations may explain differences in vector competence, and further study of the structure and function of these elements in the genome of mosquitoes may lead to epidemiological interventions. Copyright © 2017 Elsevier Ltd. All rights reserved.

Read more
July 19, 2019  |  

Dissecting the causal mechanism of X-linked Dystonia-Parkinsonism by integrating genome and transcriptome assembly.

X-linked Dystonia-Parkinsonism (XDP) is a Mendelian neurodegenerative disease that is endemic to the Philippines and is associated with a founder haplotype. We integrated multiple genome and transcriptome assembly technologies to narrow the causal mutation to the TAF1 locus, which included a SINE-VNTR-Alu (SVA) retrotransposition into intron 32 of the gene. Transcriptome analyses identified decreased expression of the canonical cTAF1 transcript among XDP probands, and de novo assembly across multiple pluripotent stem-cell-derived neuronal lineages discovered aberrant TAF1 transcription that involved alternative splicing and intron retention (IR) in proximity to the SVA that was anti-correlated with overall TAF1 expression. CRISPR/Cas9 excision of the SVA rescued this XDP-specific transcriptional signature and normalized TAF1 expression in probands. These data suggest an SVA-mediated aberrant transcriptional mechanism associated with XDP and may provide a roadmap for layered technologies and integrated assembly-based analyses for other unsolved Mendelian disorders. Copyright © 2018 Elsevier Inc. All rights reserved.

Read more
July 19, 2019  |  

Resolving the complete genome of Kuenenia stuttgartiensis from a membrane bioreactor enrichment using Single-Molecule Real-Time sequencing.

Anaerobic ammonium-oxidizing (anammox) bacteria are a group of strictly anaerobic chemolithoautotrophic microorganisms. They are capable of oxidizing ammonium to nitrogen gas using nitrite as a terminal electron acceptor, thereby facilitating the release of fixed nitrogen into the atmosphere. The anammox process is thought to exert a profound impact on the global nitrogen cycle and has been harnessed as an environment-friendly method for nitrogen removal from wastewater. In this study, we present the first closed genome sequence of an anammox bacterium, Kuenenia stuttgartiensis MBR1. It was obtained through Single-Molecule Real-Time (SMRT) sequencing of an enrichment culture constituting a mixture of at least two highly similar Kuenenia strains. The genome of the novel MBR1 strain is different from the previously reported Kuenenia KUST reference genome as it contains numerous structural variations and unique genomic regions. We find new proteins, such as a type 3b (sulf)hydrogenase and an additional copy of the hydrazine synthase gene cluster. Moreover, multiple copies of ammonium transporters and proteins regulating nitrogen uptake were identified, suggesting functional differences in metabolism. This assembly, including the genome-wide methylation profile, provides a new foundation for comparative and functional studies aiming to elucidate the biochemical and metabolic processes of these organisms.

Read more
July 19, 2019  |  

Coupling of single molecule, long read sequencing with IMGT/HighV-QUEST analysis expedites identification of SIV gp140-specific antibodies from scFv phage display libraries.

The simian immunodeficiency virus (SIV)/macaque model of human immunodeficiency virus (HIV)/acquired immunodeficiency syndrome pathogenesis is critical for furthering our understanding of the role of antibody responses in the prevention of HIV infection, and will only increase in importance as macaque immunoglobulin (IG) gene databases are expanded. We have previously reported the construction of a phage display library from a SIV-infected rhesus macaque (Macaca mulatta) using oligonucleotide primers based on human IG gene sequences. Our previous screening relied on Sanger sequencing, which was inefficient and generated only a few dozen sequences. Here, we re-analyzed this library using single molecule, real-time (SMRT) sequencing on the Pacific Biosciences (PacBio) platform to generate thousands of highly accurate circular consensus sequencing (CCS) reads corresponding to full length single chain fragment variable. CCS data were then analyzed through the international ImMunoGeneTics information system®(IMGT®)/HighV-QUEST (www.imgt.org) to identify variable genes and perform statistical analyses. Overall the library was very diverse, with 2,569 different IMGT clonotypes called for the 5,238 IGHV sequences assigned to an IMGT clonotype. Within the library, SIV-specific antibodies represented a relatively limited number of clones, with only 135 different IMGT clonotypes called from 4,594 IGHV-assigned sequences. Our data did confirm that the IGHV4 and IGHV3 gene usage was the most abundant within the rhesus antibodies screened, and that these genes were even more enriched among SIV gp140-specific antibodies. Although a broad range of VH CDR3 amino acid (AA) lengths was observed in the unpanned library, the vast majority of SIV gp140-specific antibodies demonstrated a more uniform VH CDR3 length (20 AA). This uniformity was far less apparent when VH CDR3 were classified according to their clonotype (range: 9-25 AA), which we believe is more relevant for specific antibody identification. Only 174 IGKV and 588 IGLV clonotypes were identified within the VL sequences associated with SIV gp140-specific VH. Together, these data strongly suggest that the combination of SMRT sequencing with the IMGT/HighV-QUEST querying tool will facilitate and expedite our understanding of polyclonal antibody responses during SIV infection and may serve to rapidly expand the known scope of macaque V genes utilized during these responses.

Read more
July 19, 2019  |  

Biomonitoring for traditional herbal medicinal products using DNA metabarcoding and single molecule, real-time sequencing.

Global concerns have been paid to the potential hazard of traditional herbal medicinal products (THMPs). Substandard and counterfeit THMPs, including traditional Chinese patent medicine, health foods, dietary supplements, etc. are potential threats to public health. Recent marketplace studies using DNA barcoding have determined that the current quality control methods are not sufficient for ensuring the presence of authentic herbal ingredients and detection of contaminants/adulterants. An efficient biomonitoring method for THMPs is of great needed. Herein, metabarcoding and single-molecule, real-time (SMRT) sequencing were used to detect the multiple ingredients in Jiuwei Qianghuo Wan (JWQHW), a classical herbal prescription widely used in China for the last 800 years. Reference experimental mixtures and commercial JWQHW products from the marketplace were used to confirm the method. Successful SMRT sequencing results recovered 5416 and 4342 circular-consensus sequencing (CCS) reads belonging to the ITS2 and psbA-trnH regions. The results suggest that with the combination of metabarcoding and SMRT sequencing, it is repeatable, reliable, and sensitive enough to detect species in the THMPs, and the error in SMRT sequencing did not affect the ability to identify multiple prescribed species and several adulterants/contaminants. It has the potential for becoming a valuable tool for the biomonitoring of multi-ingredient THMPs.

Read more
July 19, 2019  |  

The complete and fully assembled genome sequence of Aeromonas salmonicida subsp. pectinolytica and its comparative analysis with other Aeromonas species: investigation of the mobilome in environmental and pathogenic strains.

Due to the predominant usage of short-read sequencing to date, most bacterial genome sequences reported in the last years remain at the draft level. This precludes certain types of analyses, such as the in-depth analysis of genome plasticity.Here we report the finalized genome sequence of the environmental strain Aeromonas salmonicida subsp. pectinolytica 34mel, for which only a draft genome with 253 contigs is currently available. Successful completion of the transposon-rich genome critically depended on the PacBio long read sequencing technology. Using finalized genome sequences of A. salmonicida subsp. pectinolytica and other Aeromonads, we report the detailed analysis of the transposon composition of these bacterial species. Mobilome evolution is exemplified by a complex transposon, which has shifted from pathogenicity-related to environmental-related gene content in A. salmonicida subsp. pectinolytica 34mel.Obtaining the complete, circular genome of A. salmonicida subsp. pectinolytica allowed us to perform an in-depth analysis of its mobilome. We demonstrate the mobilome-dependent evolution of this strain’s genetic profile from pathogenic to environmental.

Read more
July 19, 2019  |  

Expanding an expanded genome: long-read sequencing of Trypanosoma cruzi.

Although the genome of Trypanosoma cruzi, the causative agent of Chagas disease, was first made available in 2005, with additional strains reported later, the intrinsic genome complexity of this parasite (the abundance of repetitive sequences and genes organized in tandem) has traditionally hindered high-quality genome assembly and annotation. This also limits diverse types of analyses that require high degrees of precision. Long reads generated by third-generation sequencing technologies are particularly suitable to address the challenges associated with T. cruzi’s genome since they permit direct determination of the full sequence of large clusters of repetitive sequences without collapsing them. This, in turn, not only allows accurate estimation of gene copy numbers but also circumvents assembly fragmentation. Here, we present the analysis of the genome sequences of two T. cruzi clones: the hybrid TCC (TcVI) and the non-hybrid Dm28c (TcI), determined by PacBio Single Molecular Real-Time (SMRT) technology. The improved assemblies herein obtained permitted us to accurately estimate gene copy numbers, abundance and distribution of repetitive sequences (including satellites and retroelements). We found that the genome of T. cruzi is composed of a ‘core compartment’ and a ‘disruptive compartment’ which exhibit opposite GC content and gene composition. Novel tandem and dispersed repetitive sequences were identified, including some located inside coding sequences. Additionally, homologous chromosomes were separately assembled, allowing us to retrieve haplotypes as separate contigs instead of a unique mosaic sequence. Finally, manual annotation of surface multigene families, mucins and trans-sialidases allows now a better overview of these complex groups of genes.

Read more
July 19, 2019  |  

Ultradeep single-molecule real-time sequencing of HIV envelope reveals complete compartmentalization of highly macrophage-tropic R5 proviral variants in brain and CXCR4-using variants in immune and peripheral tissues.

Despite combined antiretroviral therapy (cART), HIV+ patients still develop neurological disorders, which may be due to persistent HIV infection and selective evolution in brain tissues. Single-molecule real-time (SMRT) sequencing technology offers an improved opportunity to study the relationship among HIV isolates in the brain and lymphoid tissues because it is capable of generating thousands of long sequence reads in a single run. Here, we used SMRT sequencing to generate ~?50,000 high-quality full-length HIV envelope sequences (>?2200 bp) from seven autopsy tissues from an HIV+/cART+ subject, including three brain and four non-brain sites. Sanger sequencing was used for comparison with SMRT data and to clone functional pseudoviruses for in vitro tropism assays. Phylogenetic analysis demonstrated that brain-derived HIV was compartmentalized from HIV outside the brain and that the variants from each of the three brain tissues grouped independently. Variants from all peripheral tissues were intermixed on the tree but independent of the brain clades. Due to the large number of sequences, a clustering analysis at three similarity thresholds (99, 99.5, and 99.9%) was also performed. All brain sequences clustered exclusive of any non-brain sequences at all thresholds; however, frontal lobe sequences clustered independently of occipital and parietal lobes. Translated sequences revealed potentially functional differences between brain and non-brain sequences in the location of putative N-linked glycosylation sites (N-sites), V1 length, V3 charge, and the number of V4 N-sites. All brain sequences were predicted to use the CCR5 co-receptor, while most non-brain sequences were predicted to use CXCR4 co-receptor. Tropism results were confirmed by in vitro infection assays. The study is the first to use a SMRT sequencing approach to study HIV compartmentalization in tissues and supports other reports of limited trafficking between brain and non-brain sequences during cART. Due to the long sequence length, we could observe changes along the entire envelope gene, likely caused by differential selective pressure in the brain that may contribute to neurological disease.

Read more
July 19, 2019  |  

HIV envelope glycoform heterogeneity and localized diversity govern the initiation and maturation of a V2 apex broadly neutralizing antibody lineage.

Understanding how broadly neutralizing antibodies (bnAbs) to HIV envelope (Env) develop during natural infection can help guide the rational design of an HIV vaccine. Here, we described a bnAb lineage targeting the Env V2 apex and the Ab-Env co-evolution that led to development of neutralization breadth. The lineage Abs bore an anionic heavy chain complementarity-determining region 3 (CDRH3) of 25 amino acids, among the shortest known for this class of Abs, and achieved breadth with only 10% nucleotide somatic hypermutation and no insertions or deletions. The data suggested a role for Env glycoform heterogeneity in the activation of the lineage germline B cell. Finally, we showed that localized diversity at key V2 epitope residues drove bnAb maturation toward breadth, mirroring the Env evolution pattern described for another donor who developed V2-apex targeting bnAbs. Overall, these findings suggest potential strategies for vaccine approaches based on germline-targeting and serial immunogen design. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Read more
July 19, 2019  |  

Antigenic variation in the lyme spirochete: Insights into recombinational switching with a suggested role for error-prone repair.

The Lyme disease spirochete, Borrelia burgdorferi, uses antigenic variation as a strategy to evade the host’s acquired immune response. New variants of surface-localized VlsE are generated efficiently by unidirectional recombination from 15 unexpressed vls cassettes into the vlsE locus. Using algorithms to analyze switching from vlsE sequencing data, we characterize a population of over 45,000 inferred recombination events generated during mouse infection. We present evidence for clustering of these recombination events within the population and along the vlsE gene, a role for the direct repeats flanking the variable region in vlsE, and the importance of sequence homology in determining the location of recombination, despite RecA’s dispensability. Finally, we report that non-templated sequence variation is strongly associated with recombinational switching and occurs predominantly at the 5′ end of conversion tracts. This likely results from an error-prone repair mechanism operational during recombinational switching that elevates the mutation rate > 5,000-fold in switched regions. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

Read more
July 19, 2019  |  

Long-read sequencing and de novo genome assembly of Ammopiptanthus nanus, a desert shrub.

Ammopiptanthus nanus is a rare broad-leaved shrub that is found in the desert and arid regions of Central Asia. This plant species exhibits extremely high tolerance to drought and freezing and has been used in abiotic tolerance research in plants. As a relic of the tertiary period, A. nanus is of great significance to plant biogeographic research in the ancient Mediterranean region. Here, we report a draft genome assembly using the Pacific Biosciences (PacBio) platform and gene annotation for A. nanus.A total of 64.72 Gb of raw PacBio sequel reads were generated from four 20-kb libraries. After filtering, 64.53 Gb of clean reads were obtained, giving 72.59× coverage depth. Assembly using Canu gave an assembly length of 823.74 Mb, with a contig N50 of 2.76 Mb. The final size of the assembled A. nanus genome was close to the 889 Mb estimated by k-mer analysis. The gene annotation completeness was evaluated using Benchmarking Universal Single-Copy Orthologs; 1,327 of the 1,440 conserved genes (92.15%) could be found in the A. nanus assembly. Genome annotation revealed that 74.08% of the A. nanus genome is composed of repetitive elements and 53.44% is composed of long terminal repeat elements. We predicted ?37,188 protein-coding genes, of which 96.53% were functionally annotated.The genomic sequences of A. nanus could be a valuable source for comparative genomic analysis in the legume family and will be useful for understanding the phylogenetic relationships of the Thermopsideae and the evolutionary response of plant species to the Qinghai Tibetan Plateau uplift.

Read more
July 19, 2019  |  

De novo repeat interruptions are associated with reduced somatic instability and mild or absent clinical features in myotonic dystrophy type 1.

Myotonic dystrophy type 1 (DM1) is a multisystem disorder, caused by expansion of a CTG trinucleotide repeat in the 3′-untranslated region of the DMPK gene. The repeat expansion is somatically unstable and tends to increase in length with time, contributing to disease progression. In some individuals, the repeat array is interrupted by variant repeats such as CCG and CGG, stabilising the expansion and often leading to milder symptoms. We have characterised three families, each including one person with variant repeats that had arisen de novo on paternal transmission of the repeat expansion. Two individuals were identified for screening due to an unusual result in the laboratory diagnostic test, and the third due to exceptionally mild symptoms. The presence of variant repeats in all three expanded alleles was confirmed by restriction digestion of small pool PCR products, and allele structures were determined by PacBio sequencing. Each was different, but all contained CCG repeats close to the 3′-end of the repeat expansion. All other family members had inherited pure CTG repeats. The variant repeat-containing alleles were more stable in the blood than pure alleles of similar length, which may in part account for the mild symptoms observed in all three individuals. This emphasises the importance of somatic instability as a disease mechanism in DM1. Further, since patients with variant repeats may have unusually mild symptoms, identification of these individuals has important implications for genetic counselling and for patient stratification in DM1 clinical trials.

Read more
July 19, 2019  |  

A high-quality, long-read de novo genome assembly to aid conservation of Hawaii’s last remaining crow species

Genome-level data can provide researchers with unprecedented precision to examine the causes and genetic consequences of population declines, which can inform conservation management. Here, we present a high-quality, long-read, de novo genome assembly for one of the world’s most endangered bird species, the ?Alala (Corvus hawaiiensis; Hawaiian crow). As the only remaining native crow species in Hawai?i, the ?Alala survived solely in a captive-breeding program from 2002 until 2016, at which point a long-term reintroduction program was initiated. The high-quality genome assembly was generated to lay the foundation for both comparative genomics studies and the development of population-level genomic tools that will aid conservation and recovery efforts. We illustrate how the quality of this assembly places it amongst the very best avian genomes assembled to date, comparable to intensively studied model systems. We describe the genome architecture in terms of repetitive elements and runs of homozygosity, and we show that compared with more outbred species, the ?Alala genome is substantially more homozygous. We also provide annotations for a subset of immunity genes that are likely to be important in conservation management, and we discuss how this genome is currently being used as a roadmap for downstream conservation applications.

Read more
July 19, 2019  |  

A near complete, chromosome-scale assembly of the black raspberry (Rubus occidentalis) genome.

The fragmented nature of most draft plant genomes has hindered downstream gene discovery, trait mapping for breeding, and other functional genomics applications. There is a pressing need to improve or finish draft plant genome assemblies.Here, we present a chromosome-scale assembly of the black raspberry genome using single-molecule real-time Pacific Biosciences sequencing and high-throughput chromatin conformation capture (Hi-C) genome scaffolding. The updated V3 assembly has a contig N50 of 5.1 Mb, representing an ~200-fold improvement over the previous Illumina-based version. Each of the 235 contigs was anchored and oriented into seven chromosomes, correcting several major misassemblies. Black raspberry V3 contains 47 Mb of new sequences including large pericentromeric regions and thousands of previously unannotated protein-coding genes. Among the new genes are hundreds of expanded tandem gene arrays that were collapsed in the Illumina-based assembly. Detailed comparative genomics with the high-quality V4 woodland strawberry genome (Fragaria vesca) revealed near-perfect 1:1 synteny with dramatic divergence in tandem gene array composition. Lineage-specific tandem gene arrays in black raspberry are related to agronomic traits such as disease resistance and secondary metabolite biosynthesis.The improved resolution of tandem gene arrays highlights the need to reassemble these highly complex and biologically important regions in draft plant genomes. The updated, high-quality black raspberry reference genome will be useful for comparative genomics across the horticulturally important Rosaceae family and enable the development of marker assisted breeding in Rubus.

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.