October 23, 2019  |  

Adeno-associated virus genome population sequencing achieves full vector genome resolution and reveals human-vector chimeras

Recombinant adeno-associated virus (rAAV)-based gene therapy has entered a phase of clinical translation and commercialization. Despite this progress, vector integrity following production is often overlooked. Compromised vectors may negatively impact therapeutic efficacy and safety. Using single molecule, real-time (SMRT) sequencing, we can comprehensively profile packaged genomes as a single intact molecule and directly assess vector integrity without extensive preparation. We have exploited this methodology to profile all heterogeneic populations of self-complementary AAV genomes via bioinformatics pipelines and have coined this approach AAV-genome population sequencing (AAV-GPseq). The approach can reveal the relative distribution of truncated genomes versus full-length genomes in vector preparations. Preparations that seemingly show high genome homogeneity by gel electrophoresis are revealed to consist of less than 50% full-length species. With AAV-GPseq, we can also detect many reverse-packaged genomes that encompass sequences originating from plasmid backbone, as well as sequences from packaging and helper plasmids. Finally, we detect host-cell genomic sequences that are chimeric with inverted terminal repeat (ITR)-containing vector sequences. We show that vector populations can contain between 1.3% and 2.3% of this type of undesirable genome. These discoveries redefine quality control standards for viral vector preparations and highlight the degree of foreign products in rAAV-based therapeutic vectors.


September 22, 2019  |  

Novel syntrophic populations dominate an ammonia-tolerant methanogenic microbiome.

Biogas reactors operating with protein-rich substrates have high methane potential and industrial value; however, they are highly susceptible to process failure because of the accumulation of ammonia. High ammonia levels cause a decline in acetate-utilizing methanogens and instead promote the conversion of acetate via a two-step mechanism involving syntrophic acetate oxidation (SAO) to H2 and CO2, followed by hydrogenotrophic methanogenesis. Despite the key role of syntrophic acetate-oxidizing bacteria (SAOB), only a few culturable representatives have been characterized. Here we show that the microbiome of a commercial, ammonia-tolerant biogas reactor harbors a deeply branched, uncultured phylotype (unFirm_1) accounting for approximately 5% of the 16S rRNA gene inventory and sharing 88% 16S rRNA gene identity with its closest characterized relative. Reconstructed genome and quantitative metaproteomic analyses imply unFirm_1’s metabolic dominance and SAO capabilities, whereby the key enzymes required for acetate oxidation are among the most highly detected in the reactor microbiome. While culturable SAOB were identified in genomic analyses of the reactor, their limited proteomic representation suggests that unFirm_1 plays an important role in channeling acetate toward methane. Notably, unFirm_1-like populations were found in other high-ammonia biogas installations, conjecturing a broader importance for this novel clade of SAOB in anaerobic fermentations. IMPORTANCE The microbial production of methane or “biogas” is an attractive renewable energy technology that can recycle organic waste into biofuel. Biogas reactors operating with protein-rich substrates such as household municipal or agricultural wastes have significant industrial and societal value; however, they are highly unstable and frequently collapse due to the accumulation of ammonia. We report the discovery of a novel uncultured phylotype (unFirm_1) that is highly detectable in metaproteomic data generated from an ammonia-tolerant commercial reactor. Importantly, unFirm_1 is proposed to perform a key metabolic step in biogas microbiomes, whereby it syntrophically oxidizes acetate to hydrogen and carbon dioxide, which methanogens then covert to methane. Only very few culturable syntrophic acetate-oxidizing bacteria have been described, and all were detected at low in situ levels compared to unFirm_1. Broader comparisons produced the hypothesis that unFirm_1 is a key mediator toward the successful long-term stable operation of biogas production using protein-rich substrates.


September 22, 2019  |  

Evolution of selective-sequencing approaches for virus discovery and virome analysis.

Recent advances in sequencing technologies have transformed the field of virus discovery and virome analysis. Once mostly confined to the traditional Sanger sequencing based individual virus discovery, is now entirely replaced by high throughput sequencing (HTS) based virus metagenomics that can be used to characterize the nature and composition of entire viromes. To better harness the potential of HTS for the study of viromes, sample preparation methodologies use different approaches to exclude amplification of non-viral components that can overshadow low-titer viruses. These virus-sequence enrichment approaches mostly focus on the sample preparation methods, like enzymatic digestion of non-viral nucleic acids and size exclusion of non-viral constituents by column filtration, ultrafiltration or density gradient centrifugation. However, recently a new approach of virus-sequence enrichment called virome-capture sequencing, focused on the amplification or HTS library preparation stage, was developed to increase the ability of virome characterization. This new approach has the potential to further transform the field of virus discovery and virome analysis, but its technical complexity and sequence-dependence warrants further improvements. In this review we discuss the different methods, their applications and evolution, for selective sequencing based virome analysis and also propose refinements needed to harness the full potential of HTS for virome analysis. Copyright © 2017 Elsevier B.V. All rights reserved.


September 22, 2019  |  

ABC transporter mis-splicing associated with resistance to Bt toxin Cry2Ab in laboratory- and field-selected pink bollworm.

Evolution of pest resistance threatens the benefits of genetically engineered crops that produce Bacillus thuringiensis (Bt) insecticidal proteins. Strategies intended to delay pest resistance are most effective when implemented proactively. Accordingly, researchers have selected for and analyzed resistance to Bt toxins in many laboratory strains of pests before resistance evolves in the field, but the utility of this approach depends on the largely untested assumption that laboratory- and field-selected resistance to Bt toxins are similar. Here we compared the genetic basis of resistance to Bt toxin Cry2Ab, which is widely deployed in transgenic crops, between laboratory- and field-selected populations of the pink bollworm (Pectinophora gossypiella), a global pest of cotton. We discovered that resistance to Cry2Ab is associated with mutations disrupting the same ATP-binding cassette transporter gene (PgABCA2) in a laboratory-selected strain from Arizona, USA, and in field-selected populations from India. The most common mutation, loss of exon 6 caused by alternative splicing, occurred in resistant larvae from both locations. Together with previous data, the results imply that mutations in the same gene confer Bt resistance in laboratory- and field-selected strains and suggest that focusing on ABCA2 genes may help to accelerate progress in monitoring and managing resistance to Cry2Ab.


September 22, 2019  |  

Analysis of gut microbiota – An ever changing landscape.

In the last two decades, the field of metagenomics has greatly expanded due to improvement in sequencing technologies allowing for a more comprehensive characterization of microbial communities. The use of these technologies has led to an unprecedented understanding of human, animal, and environmental microbiomes and have shown that the gut microbiota are comparable to an organ that is intrinsically linked with a variety of diseases. Characterization of microbial communities using next-generation sequencing-by-synthesis approaches have revealed important shifts in microbiota associated with debilitating diseases such as Clostridium difficile infection. But due to limitations in sequence read length, primer biases, and the quality of databases, genus- and species-level classification have been difficult. Third-generation technologies, such as Pacific Biosciences’ single molecule, real-time (SMRT) approach, allow for unbiased, more specific identification of species that are likely clinically relevant. Comparison of Illumina next-generation characterization and SMRT sequencing of samples from patients treated for C. difficile infection revealed similarities in community composition at the phylum and family levels, but SMRT sequencing further allowed for species-level characterization – permitting a better understanding of the microbial ecology of this disease. Thus, as sequencing technologies continue to advance, new species-level insights can be gained in the study of complex and clinically-relevant microbial communities.


September 22, 2019  |  

Gene activity in primary T cells infected with HIV89.6: intron retention and induction of genomic repeats.

HIV infection has been reported to alter cellular gene activity, but published studies have commonly assayed transformed cell lines and lab-adapted HIV strains, yielding inconsistent results. Here we carried out a deep RNA-Seq analysis of primary human T cells infected with the low passage HIV isolate HIV89.6.Seventeen percent of cellular genes showed altered activity 48 h after infection. In a meta-analysis including four other studies, our data differed from studies of HIV infection in cell lines but showed more parallels with infections of primary cells. We found a global trend toward retention of introns after infection, suggestive of a novel cellular response to infection. HIV89.6 infection was also associated with activation of several human endogenous retroviruses (HERVs) and retrotransposons, of interest as possible novel antigens that could serve as vaccine targets. The most highly activated group of HERVs was a subset of the ERV-9. Analysis showed that activation was associated with a particular variant of ERV-9 long terminal repeats that contains an indel near the U3-R border. These data also allowed quantification of >70 splice forms of the HIV89.6 RNA and specified the main types of chimeric HIV89.6-host RNAs. Comparison to over 100,000 integration site sequences from the same infected cell populations allowed quantification of authentic versus artifactual chimeric reads, showing that 5′ read-in, splicing out of HIV89.6 from the D4 donor and 3′ read-through were the most common HIV89.6-host cell chimeric RNA forms.Analysis of RNA abundance after infection of primary T cells with the low passage HIV89.6 isolate disclosed multiple novel features of HIV-host interactions, notably intron retention and induction of transcription of retrotransposons and endogenous retroviruses.


September 22, 2019  |  

Extensive alternative splicing of KIR transcripts.

The killer-cell Ig-like receptors (KIR) form a multigene entity involved in modulating immune responses through interactions with MHC class I molecules. The complexity of the KIR cluster is reflected by, for instance, abundant levels of allelic polymorphism, gene copy number variation, and stochastic expression profiles. The current transcriptome study involving human and macaque families demonstrates that KIR family members are also subjected to differential levels of alternative splicing, and this seems to be gene dependent. Alternative splicing may result in the partial or complete skipping of exons, or the partial inclusion of introns, as documented at the transcription level. This post-transcriptional process can generate multiple isoforms from a single KIR gene, which diversifies the characteristics of the encoded proteins. For example, alternative splicing could modify ligand interactions, cellular localization, signaling properties, and the number of extracellular domains of the receptor. In humans, we observed abundant splicing for KIR2DL4, and to a lesser extent in the lineage III KIR genes. All experimentally documented splice events are substantiated by in silico splicing strength predictions. To a similar extent, alternative splicing is observed in rhesus macaques, a species that shares a close evolutionary relationship with humans. Splicing profiles of Mamu-KIR1D and Mamu-KIR2DL04 displayed a great diversity, whereas Mamu-KIR3DL20 (lineage V) is consistently spliced to generate a homolog of human KIR2DL5 (lineage I). The latter case represents an example of convergent evolution. Although just a single KIR splice event is shared between humans and macaques, the splicing mechanisms are similar, and the predicted consequences are comparable. In conclusion, alternative splicing adds an additional layer of complexity to the KIR gene system in primates, and results in a wide structural and functional variety of KIR receptors and its isoforms, which may play a role in health and disease.


September 22, 2019  |  

Metataxonomic and metagenomic approaches vs. culture-based techniques for clinical pathology.

Diagnoses that are both timely and accurate are critically important for patients with life-threatening or drug resistant infections. Technological improvements in High-Throughput Sequencing (HTS) have led to its use in pathogen detection and its application in clinical diagnoses of infectious diseases. The present study compares two HTS methods, 16S rRNA marker gene sequencing (metataxonomics) and whole metagenomic shotgun sequencing (metagenomics), in their respective abilities to match the same diagnosis as traditional culture methods (culture inference) for patients with ventilator associated pneumonia (VAP). The metagenomic analysis was able to produce the same diagnosis as culture methods at the species-level for five of the six samples, while the metataxonomic analysis was only able to produce results with the same species-level identification as culture for two of the six samples. These results indicate that metagenomic analyses have the accuracy needed for a clinical diagnostic tool, but full integration in diagnostic protocols is contingent on technological improvements to decrease turnaround time and lower costs.


September 22, 2019  |  

Genomic and metabolic diversity of Marine Group I Thaumarchaeota in the mesopelagic of two subtropical gyres.

Marine Group I (MGI) Thaumarchaeota are one of the most abundant and cosmopolitan chemoautotrophs within the global dark ocean. To date, no representatives of this archaeal group retrieved from the dark ocean have been successfully cultured. We used single cell genomics to investigate the genomic and metabolic diversity of thaumarchaea within the mesopelagic of the subtropical North Pacific and South Atlantic Ocean. Phylogenetic and metagenomic recruitment analysis revealed that MGI single amplified genomes (SAGs) are genetically and biogeographically distinct from existing thaumarchaea cultures obtained from surface waters. Confirming prior studies, we found genes encoding proteins for aerobic ammonia oxidation and the hydrolysis of urea, which may be used for energy production, as well as genes involved in 3-hydroxypropionate/4-hydroxybutyrate and oxidative tricarboxylic acid pathways. A large proportion of protein sequences identified in MGI SAGs were absent in the marine cultures Cenarchaeum symbiosum and Nitrosopumilus maritimus, thus expanding the predicted protein space for this archaeal group. Identifiable genes located on genomic islands with low metagenome recruitment capacity were enriched in cellular defense functions, likely in response to viral infections or grazing. We show that MGI Thaumarchaeota in the dark ocean may have more flexibility in potential energy sources and adaptations to biotic interactions than the existing, surface-ocean cultures.


September 22, 2019  |  

Single-molecule long-read 16S sequencing to characterize the lung microbiome from mechanically ventilated patients with suspected pneumonia.

In critically ill patients, the development of pneumonia results in significant morbidity and mortality and additional health care costs. The accurate and rapid identification of the microbial pathogens in patients with pulmonary infections might lead to targeted antimicrobial therapy with potentially fewer adverse effects and lower costs. Major advances in next-generation sequencing (NGS) allow culture-independent identification of pathogens. The present study used NGS of essentially full-length PCR-amplified 16S ribosomal DNA from the bronchial aspirates of intubated patients with suspected pneumonia. The results from 61 patients demonstrated that sufficient DNA was obtained from 72% of samples, 44% of which (27 samples) yielded PCR amplimers suitable for NGS. Out of the 27 sequenced samples, only 20 had bacterial culture growth, while the microbiological and NGS identification of bacteria coincided in 17 (85%) of these samples. Despite the lack of bacterial growth in 7 samples that yielded amplimers and were sequenced, the NGS identified a number of bacterial species in these samples. Overall, a significant diversity of bacterial species was identified from the same genus as the predominant cultured pathogens. The numbers of NGS-identifiable bacterial genera were consistently higher than identified by standard microbiological methods. As technical advances reduce the processing and sequencing times, NGS-based methods will ultimately be able to provide clinicians with rapid, precise, culture-independent identification of bacterial, fungal, and viral pathogens and their antimicrobial sensitivity profiles. Copyright © 2014, American Society for Microbiology. All Rights Reserved.


September 22, 2019  |  

Extensive horizontal gene transfer in cheese-associated bacteria.

Acquisition of genes through horizontal gene transfer (HGT) allows microbes to rapidly gain new capabilities and adapt to new or changing environments. Identifying widespread HGT regions within multispecies microbiomes can pinpoint the molecular mechanisms that play key roles in microbiome assembly. We sought to identify horizontally transferred genes within a model microbiome, the cheese rind. Comparing 31 newly sequenced and 134 previously sequenced bacterial isolates from cheese rinds, we identified over 200 putative horizontally transferred genomic regions containing 4733 protein coding genes. The largest of these regions are enriched for genes involved in siderophore acquisition, and are widely distributed in cheese rinds in both Europe and the US. These results suggest that HGT is prevalent in cheese rind microbiomes, and that identification of genes that are frequently transferred in a particular environment may provide insight into the selective forces shaping microbial communities.


September 22, 2019  |  

Plant 24-nt reproductive phasiRNAs from intramolecular duplex mRNAs in diverse monocots.

In grasses, two pathways that generate diverse and numerous 21-nt (premeiotic) and 24-nt (meiotic) phased siRNAs are highly enriched in anthers, the male reproductive organs. These “phasiRNAs” are analogous to mammalian piRNAs, yet their functions and evolutionary origins remain largely unknown. The 24-nt meiotic phasiRNAs have only been described in grasses, wherein their biogenesis is dependent on a specialized Dicer (DCL5). To assess how evolution gave rise to this pathway, we examined reproductive phasiRNA pathways in nongrass monocots: garden asparagus, daylily, and lily. The common ancestors of these species diverged approximately 115-117 million years ago (MYA). We found that premeiotic 21-nt and meiotic 24-nt phasiRNAs were abundant in all three species and displayed spatial localization and temporal dynamics similar to grasses. The miR2275-triggered pathway was also present, yielding 24-nt reproductive phasiRNAs, and thus originated more than 117 MYA. In asparagus, unlike in grasses, these siRNAs are largely derived from inverted repeats (IRs); analyses in lily identified thousands of precursor loci, and many were also predicted to form foldback substrates for Dicer processing. Additionally, reproductive phasiRNAs were present in female reproductive organs and thus may function in both male and female germinal development. These data describe several distinct mechanisms of production for 24-nt meiotic phasiRNAs and provide new insights into the evolution of reproductive phasiRNA pathways in monocots.© 2018 Kakrana et al.; Published by Cold Spring Harbor Laboratory Press.


September 22, 2019  |  

A quantitative SMRT cell sequencing method for ribosomal amplicons.

Advances in sequencing technologies continue to provide unprecedented opportunities to characterize microbial communities. For example, the Pacific Biosciences Single Molecule Real-Time (SMRT) platform has emerged as a unique approach harnessing DNA polymerase activity to sequence template molecules, enabling long reads at low costs. With the aim to simultaneously classify and enumerate in situ microbial populations, we developed a quantitative SMRT (qSMRT) approach that involves the addition of exogenous standards to quantify ribosomal amplicons derived from environmental samples. The V7-9 regions of 18S SSU rDNA were targeted and quantified from protistan community samples collected in the Ross Sea during the Austral summer of 2011. We used three standards of different length and optimized conditions to obtain accurate quantitative retrieval across the range of expected amplicon sizes, a necessary criterion for analyzing taxonomically diverse 18S rDNA molecules from natural environments. The ability to concurrently identify and quantify microorganisms in their natural environment makes qSMRT a powerful, rapid and cost-effective approach for defining ecosystem diversity and function. Copyright © 2017 Elsevier B.V. All rights reserved.


September 22, 2019  |  

Human and rhesus macaque KIR haplotypes defined by their transcriptomes.

The killer-cell Ig-like receptors (KIRs) play a central role in the immune recognition in infection, pregnancy, and transplantation through their interactions with MHC class I molecules. KIR genes display abundant copy number variation as well as high levels of polymorphism. As a result, it is challenging to characterize this structurally dynamic region. KIR haplotypes have been analyzed in different species using conventional characterization methods, such as Sanger sequencing and Roche/454 pyrosequencing. However, these methods are time-consuming and often failed to define complete haplotypes, or do not reach allele-level resolution. In addition, most analyses were performed on genomic DNA, and thus were lacking substantial information about transcription and its corresponding modifications. In this paper, we present a single-molecule real-time sequencing approach, using Pacific Biosciences Sequel platform to characterize the KIR transcriptomes in human and rhesus macaque (Macaca mulatta) families. This high-resolution approach allowed the identification of novel Mamu-KIR alleles, the extension of reported allele sequences, and the determination of human and macaque KIR haplotypes. In addition, multiple recombinant KIR genes were discovered, all located on contracted haplotypes, which were likely the result of chromosomal rearrangements. The relatively high number of contracted haplotypes discovered might be indicative of selection on small KIR repertoires and/or novel fusion gene products. This next-generation method provides an improved high-resolution characterization of the KIR cluster in humans and macaques, which eventually may aid in a better understanding and interpretation of KIR allele-associated diseases, as well as the immune response in transplantation and reproduction. Copyright © 2018 by The American Association of Immunologists, Inc.


September 22, 2019  |  

Nearly finished genomes produced using gel microdroplet culturing reveal substantial intraspecies genomic diversity within the human microbiome.

The majority of microbial genomic diversity remains unexplored. This is largely due to our inability to culture most microorganisms in isolation, which is a prerequisite for traditional genome sequencing. Single-cell sequencing has allowed researchers to circumvent this limitation. DNA is amplified directly from a single cell using the whole-genome amplification technique of multiple displacement amplification (MDA). However, MDA from a single chromosome copy suffers from amplification bias and a large loss of specificity from even very small amounts of DNA contamination, which makes assembling a genome difficult and completely finishing a genome impossible except in extraordinary circumstances. Gel microdrop cultivation allows culturing of a diverse microbial community and provides hundreds to thousands of genetically identical cells as input for an MDA reaction. We demonstrate the utility of this approach by comparing sequencing results of gel microdroplets and single cells following MDA. Bias is reduced in the MDA reaction and genome sequencing, and assembly is greatly improved when using gel microdroplets. We acquired multiple near-complete genomes for two bacterial species from human oral and stool microbiome samples. A significant amount of genome diversity, including single nucleotide polymorphisms and genome recombination, is discovered. Gel microdroplets offer a powerful and high-throughput technology for assembling whole genomes from complex samples and for probing the pan-genome of naturally occurring populations.


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