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Asset Tag: Circular consensus sequencing

April 21, 2020

Single-Cell Virus Sequencing of Influenza Infections That Trigger Innate Immunity.

Influenza virus-infected cells vary widely in their expression of viral genes and only occasionally activate innate immunity. Here, we develop a new method to assess how the genetic variation in viral populations contributes to this heterogeneity. We do this by determining the transcriptome and full-length sequences of all viral genes in single cells infected with a nominally “pure” stock of influenza virus. Most cells are infected by virions with defects, some of which increase the frequency of innate-immune activation. These immunostimulatory defects are diverse and include mutations that perturb the function of the viral polymerase protein PB1, large internal deletions in viral genes, and failure to express the virus’s interferon antagonist NS1. However, immune activation remains stochastic in cells infected by virions with these defects and occasionally is triggered even by virions that express unmutated copies of all genes. Our work shows that the diverse spectrum of defects in influenza virus populations contributes to-but does not completely explain-the heterogeneity in viral gene expression and immune activation in single infected cells.IMPORTANCE Because influenza virus has a high mutation rate, many cells are infected by mutated virions. But so far, it has been impossible to fully characterize the sequence of the virion infecting any given cell, since conventional techniques such as flow cytometry and single-cell transcriptome sequencing (scRNA-seq) only detect if a protein or transcript is present, not its sequence. Here we develop a new approach that uses long-read PacBio sequencing to determine the sequences of virions infecting single cells. We show that viral genetic variation explains some but not all of the cell-to-cell variability in viral gene expression and innate immune induction. Overall, our study provides the first complete picture of how viral mutations affect the course of infection in single cells.Copyright © 2019 Russell et al.

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April 21, 2020

A Highly Unusual V1 Region of Env in an Elite Controller of HIV Infection.

HIV elite controllers represent a remarkable minority of patients who maintain normal CD4+ T-cell counts and low or undetectable viral loads for decades in the absence of antiretroviral therapy. To examine the possible contribution of virus attenuation to elite control, we obtained a primary HIV-1 isolate from an elite controller who had been infected for 19?years, the last 10 of which were in the absence of antiretroviral therapy. Full-length sequencing of this isolate revealed a highly unusual V1 domain in Envelope (Env). The V1 domain in this HIV-1 strain was 49 amino acids, placing it in the top 1% of lengths among the 6,112 Env sequences in the Los Alamos National Laboratory online database. Furthermore, it included two additional N-glycosylation sites and a pair of cysteines suggestive of an extra disulfide loop. Virus with this Env retained good infectivity and replicative capacity; however, analysis of recombinant viruses suggested that other sequences in Env were adapted to accommodate the unusual V1 domain. While the long V1 domain did not confer resistance to neutralization by monoclonal antibodies of the V1/V2-glycan-dependent class, it did confer resistance to neutralization by monoclonal antibodies of the V3-glycan-dependent class. Our findings support results in the literature that suggest a role for long V1 regions in shielding HIV-1 from recognition by V3-directed broadly neutralizing antibodies. In the case of the elite controller described here, it seems likely that selective pressures from the humoral immune system were responsible for driving the highly unusual polymorphisms present in this HIV-1 Envelope.IMPORTANCE Elite controllers have long provided an avenue for researchers to reveal mechanisms underlying control of HIV-1. While the role of host genetic factors in facilitating elite control is well known, the possibility of infection by attenuated strains of HIV-1 has been much less studied. Here we describe an unusual viral feature found in an elite controller of HIV-1 infection and demonstrate its role in conferring escape from monoclonal antibodies of the V3-glycan class. Our results suggest that extreme variation may be needed by HIV-1 to escape neutralization by some antibody specificities. Copyright © 2019 Silver et al.

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April 21, 2020

Full-Length Multi-Barcoding: DNA Barcoding from Single Ingredient to Complex Mixtures.

DNA barcoding has been used for decades, although it has mostly been applied to somesingle-species. Traditional Chinese medicine (TCM), which is mainly used in the form ofcombination-one type of the multi-species, identification is crucial for clinical usage.Next-generation Sequencing (NGS) has been used to address this authentication issue for the pastfew years, but conventional NGS technology is hampered in application due to its short sequencingreads and systematic errors. Here, a novel method, Full-length multi-barcoding (FLMB) vialong-read sequencing, is employed for the identification of biological compositions in herbalcompound formulas in adequate and well controlled studies. By directly sequencing the full-lengthamplicons of ITS2 and psbA-trnH through single-molecule real-time (SMRT) technology, thebiological composition of a classical prescription Sheng-Mai-San (SMS) was analyzed. At the sametime, clone-dependent Sanger sequencing was carried out as a parallel control. Further, anotherformula-Sanwei-Jili-San (SJS)-was analyzed with genes of ITS2 and CO1. All the ingredients inthe samples of SMS and SJS were successfully authenticated at the species level, and 11 exogenousspecies were also checked, some of which were considered as common contaminations in theseproducts. Methodology analysis demonstrated that this method was sensitive, accurate andreliable. FLMB, a superior but feasible approach for the identification of biological complexmixture, was established and elucidated, which shows perfect interpretation for DNA barcodingthat could lead its application in multi-species mixtures.

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April 21, 2020

Genome sequence of the corn leaf aphid (Rhopalosiphum maidis Fitch).

The corn leaf aphid (Rhopalosiphum maidis Fitch) is the most economically damaging aphid pest on maize (Zea mays), one of the world’s most important grain crops. In addition to causing direct damage by removing photoassimilates, R. maidis transmits several destructive maize viruses, including maize yellow dwarf virus, barley yellow dwarf virus, sugarcane mosaic virus, and cucumber mosaic virus.The genome of a parthenogenetically reproducing R. maidis clone was assembled with a combination of Pacific Biosciences (207-fold coverage) and Illumina (83-fold coverage) sequencing. The 689 assembled contigs, which have an N50 size of 9.0 megabases (Mb) and a low level of heterozygosity, were clustered using Phase Genomics Hi-C interaction maps. Consistent with the commonly observed 2n = 8 karyotype of R. maidis, most of the contigs (473 spanning 321 Mb) were successfully oriented into 4 scaffolds. The genome assembly captured the full length of 95.8% of the core eukaryotic genes, indicating that it is highly complete. Repetitive sequences accounted for 21.2% of the assembly, and a total of 17,629 protein-coding genes were predicted with integrated evidence from ab initio and homology-based gene predictions and transcriptome sequences generated with both Pacific Biosciences and Illumina. An analysis of likely horizontally transferred genes identified 2 from bacteria, 7 from fungi, 2 from protozoa, and 9 from algae. Repeat elements, transposons, and genes encoding likely detoxification enzymes (cytochrome P450s, glutathione S-transferases, carboxylesterases, uridine diphosphate-glucosyltransferases, and ABC transporters) were identified in the genome sequence. Other than Buchnera aphidicola (642,929 base pairs, 602 genes), no endosymbiont bacteria were found in R. maidis.A high-quality R. maidis genome was assembled at the chromosome level. This genome sequence will enable further research related to ecological interactions, virus transmission, pesticide resistance, and other aspects of R. maidis biology. It also serves as a valuable resource for comparative investigation of other aphid species. © The Author(s) 2019. Published by Oxford University Press.

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April 21, 2020

A personalized platform identifies trametinib plus zoledronate for a patient with KRAS-mutant metastatic colorectal cancer.

Colorectal cancer remains a leading source of cancer mortality worldwide. Initial response is often followed by emergent resistance that is poorly responsive to targeted therapies, reflecting currently undruggable cancer drivers such as KRAS and overall genomic complexity. Here, we report a novel approach to developing a personalized therapy for a patient with treatment-resistant metastatic KRAS-mutant colorectal cancer. An extensive genomic analysis of the tumor’s genomic landscape identified nine key drivers. A transgenic model that altered orthologs of these nine genes in the Drosophila hindgut was developed; a robotics-based screen using this platform identified trametinib plus zoledronate as a candidate treatment combination. Treating the patient led to a significant response: Target and nontarget lesions displayed a strong partial response and remained stable for 11 months. By addressing a disease’s genomic complexity, this personalized approach may provide an alternative treatment option for recalcitrant disease such as KRAS-mutant colorectal cancer.

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April 21, 2020

A new full-length circular DNA sequencing method for viral-sized genomes reveals that RNAi transgenic plants provoke a shift in geminivirus populations in the field.

We present a new method, CIDER-Seq (Circular DNA Enrichment sequencing) for the unbiased enrichment and long-read sequencing of viral-sized circular DNA molecules. We used CIDER-Seq to produce single-read full-length virus genomes for the first time. CIDER-Seq combines PCR-free virus enrichment with Single Molecule Real Time sequencing and a new sequence de-concatenation algorithm. We apply our technique to produce >1200 full-length, highly accurate geminivirus genomes from RNAi-transgenic and control plants in a field trial in Kenya. Using CIDER-Seq we can demonstrate for the first time that the expression of antiviral double-stranded RNA (dsRNA) in transgenic plants causes a consistent shift in virus populations towards species sharing low homology to the transgene derived dsRNA. Our method and its application in an economically important crop plant opens new possibilities in periodic virus sequence surveillance and accurate profiling of diverse circular DNA elements.

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April 21, 2020

Full-length 16S rRNA gene classification of Atlantic salmon bacteria and effects of using different 16S variable regions on community structure analysis.

Understanding fish-microbial relationships may be of great value for fish producers as fish growth, development and welfare are influenced by the microbial community associated with the rearing systems and fish surfaces. Accurate methods to generate and analyze these microbial communities would be an important tool to help improve understanding of microbial effects in the industry. In this study, we performed taxonomic classification and determination of operational taxonomic units on Atlantic salmon microbiota by taking advantage of full-length 16S rRNA gene sequences. Skin mucus was dominated by the genera Flavobacterium and Psychrobacter. Intestinal samples were dominated by the genera Carnobacterium, Aeromonas, Mycoplasma and by sequences assigned to the order Clostridiales. Applying Sanger sequencing on the full-length bacterial 16S rRNA gene from the pool of 46 isolates obtained in this study showed a clear assignment of the PacBio full-length bacterial 16S rRNA gene sequences down to the genus level. One of the bottlenecks in comparing microbial profiles is that different studies use different 16S rRNA gene regions. Comparisons of sequence assignments between full-length and in silico derived variable 16S rRNA gene regions showed different microbial profiles with variable effects between phylogenetic groups and taxonomic ranks. © 2019 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

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April 21, 2020

Long-read amplicon denoising.

Long-read next-generation amplicon sequencing shows promise for studying complete genes or genomes from complex and diverse populations. Current long-read sequencing technologies have challenging error profiles, hindering data processing and incorporation into downstream analyses. Here we consider the problem of how to reconstruct, free of sequencing error, the true sequence variants and their associated frequencies from PacBio reads. Called ‘amplicon denoising’, this problem has been extensively studied for short-read sequencing technologies, but current solutions do not always successfully generalize to long reads with high indel error rates. We introduce two methods: one that runs nearly instantly and is very accurate for medium length reads and high template coverage, and another, slower method that is more robust when reads are very long or coverage is lower. On two Mock Virus Community datasets with ground truth, each sequenced on a different PacBio instrument, and on a number of simulated datasets, we compare our two approaches to each other and to existing algorithms. We outperform all tested methods in accuracy, with competitive run times even for our slower method, successfully discriminating templates that differ by a just single nucleotide. Julia implementations of Fast Amplicon Denoising (FAD) and Robust Amplicon Denoising (RAD), and a webserver interface, are freely available. © The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.

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April 21, 2020

High-throughput amplicon sequencing of the full-length 16S rRNA gene with single-nucleotide resolution.

Targeted PCR amplification and high-throughput sequencing (amplicon sequencing) of 16S rRNA gene fragments is widely used to profile microbial communities. New long-read sequencing technologies can sequence the entire 16S rRNA gene, but higher error rates have limited their attractiveness when accuracy is important. Here we present a high-throughput amplicon sequencing methodology based on PacBio circular consensus sequencing and the DADA2 sample inference method that measures the full-length 16S rRNA gene with single-nucleotide resolution and a near-zero error rate. In two artificial communities of known composition, our method recovered the full complement of full-length 16S sequence variants from expected community members without residual errors. The measured abundances of intra-genomic sequence variants were in the integral ratios expected from the genuine allelic variants within a genome. The full-length 16S gene sequences recovered by our approach allowed Escherichia coli strains to be correctly classified to the O157:H7 and K12 sub-species clades. In human fecal samples, our method showed strong technical replication and was able to recover the full complement of 16S rRNA alleles in several E. coli strains. There are likely many applications beyond microbial profiling for which high-throughput amplicon sequencing of complete genes with single-nucleotide resolution will be of use. © The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.

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April 21, 2020

Targeted Long-Read RNA Sequencing Demonstrates Transcriptional Diversity Driven by Splice-Site Variation in MYBPC3.

To date, clinical sequencing has focused on genomic DNA using targeted panels and exome sequencing. Sequencing of a large hypertrophic cardiomyopathy (HCM) cohort revealed that positive identification of a disease-associated variant was returned in only 32% of patients, with an additional 15% receiving inconclusive results. When genome sequencing fails to reveal causative variants, the transcriptome may provide additional diagnostic clarity. A recent study examining patients with genetically undiagnosed muscle disorders found that RNA sequencing, when used as a complement to exome and whole genome sequencing, had an overall diagnosis rate of 35%.

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April 21, 2020

Application of long read sequencing to determine expressed antigen diversity in Trypanosoma brucei infections.

Antigenic variation is employed by many pathogens to evade the host immune response, and Trypanosoma brucei has evolved a complex system to achieve this phenotype, involving sequential use of variant surface glycoprotein (VSG) genes encoded from a large repertoire of ~2,000 genes. T. brucei express multiple, sometimes closely related, VSGs in a population at any one time, and the ability to resolve and analyse this diversity has been limited. We applied long read sequencing (PacBio) to VSG amplicons generated from blood extracted from batches of mice sacrificed at time points (days 3, 6, 10 and 12) post-infection with T. brucei TREU927. The data showed that long read sequencing is reliable for resolving variant differences between VSGs, and demonstrated that there is significant expressed diversity (449 VSGs detected across 20 mice) and across the timeframe of study there was a clear semi-reproducible pattern of expressed diversity (median of 27 VSGs per sample at day 3 post infection (p.i.), 82 VSGs at day 6 p.i., 187 VSGs at day 10 p.i. and 132 VSGs by day 12 p.i.). There was also consistent detection of one VSG dominating expression across replicates at days 3 and 6, and emergence of a second dominant VSG across replicates by day 12. The innovative application of ecological diversity analysis to VSG reads enabled characterisation of hierarchical VSG expression in the dataset, and resulted in a novel method for analysing such patterns of variation. Additionally, the long read approach allowed detection of mosaic VSG expression from very few reads-the earliest in infection that such events have been detected. Therefore, our results indicate that long read analysis is a reliable tool for resolving diverse gene expression profiles, and provides novel insights into the complexity and nature of VSG expression in trypanosomes, revealing significantly higher diversity than previously shown and the ability to identify mosaic gene formation early during the infection process.

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April 21, 2020

FLAM-seq: full-length mRNA sequencing reveals principles of poly(A) tail length control.

Although messenger RNAs are key molecules for understanding life, until now, no method has existed to determine the full-length sequence of endogenous mRNAs including their poly(A) tails. Moreover, although non-A nucleotides can be incorporated in poly(A) tails, there also exists no method to accurately sequence them. Here, we present full-length poly(A) and mRNA sequencing (FLAM-seq), a rapid and simple method for high-quality sequencing of entire mRNAs. We report a complementary DNA library preparation method coupled to single-molecule sequencing to perform FLAM-seq. Using human cell lines, brain organoids and Caenorhabditis elegans we show that FLAM-seq delivers high-quality full-length mRNA sequences for thousands of different genes per sample. We find that 3′ untranslated region length is correlated with poly(A) tail length, that alternative polyadenylation sites and alternative promoters for the same gene are linked to different tail lengths, and that tails contain a substantial number of cytosines.

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April 21, 2020

Genome-wide de novo L1 Retrotransposition Connects Endonuclease Activity with Replication.

L1 retrotransposon-derived sequences comprise approximately 17% of the human genome. Darwinian selective pressures alter L1 genomic distributions during evolution, confounding the ability to determine initial L1 integration preferences. Here, we generated high-confidence datasets of greater than 88,000 engineered L1 insertions in human cell lines that act as proxies for cells that accommodate retrotransposition in vivo. Comparing these insertions to a null model, in which L1 endonuclease activity is the sole determinant dictating L1 integration preferences, demonstrated that L1 insertions are not significantly enriched in genes, transcribed regions, or open chromatin. By comparison, we provide compelling evidence that the L1 endonuclease disproportionately cleaves predominant lagging strand DNA replication templates, while lagging strand 3′-hydroxyl groups may prime endonuclease-independent L1 retrotransposition in a Fanconi anemia cell line. Thus, acquisition of an endonuclease domain, in conjunction with the ability to integrate into replicating DNA, allowed L1 to become an autonomous, interspersed retrotransposon.Copyright © 2019 Elsevier Inc. All rights reserved.

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April 21, 2020

Double PIK3CA mutations in cis increase oncogenicity and sensitivity to PI3Ka inhibitors.

Activating mutations in PIK3CA are frequent in human breast cancer, and phosphoinositide 3-kinase alpha (PI3Ka) inhibitors have been approved for therapy. To characterize determinants of sensitivity to these agents, we analyzed PIK3CA-mutant cancer genomes and observed the presence of multiple PIK3CA mutations in 12 to 15% of breast cancers and other tumor types, most of which (95%) are double mutations. Double PIK3CA mutations are in cis on the same allele and result in increased PI3K activity, enhanced downstream signaling, increased cell proliferation, and tumor growth. The biochemical mechanisms of dual mutations include increased disruption of p110a binding to the inhibitory subunit p85a, which relieves its catalytic inhibition, and increased p110a membrane lipid binding. Double PIK3CA mutations predict increased sensitivity to PI3Ka inhibitors compared with single-hotspot mutations.Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

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April 21, 2020

Extreme resistance to Potato virus Y in potato carrying the Rysto gene is mediated by a TIR-NLR immune receptor.

Potato virus Y (PVY) is a major potato (Solanum tuberosum L.) pathogen that causes severe annual crop losses worth billions of dollars worldwide. PVY is transmitted by aphids, and successful control of virus transmission requires the extensive use of environmentally damaging insecticides to reduce vector populations. Rysto , from the wild relative S. stoloniferum, confers extreme resistance (ER) to PVY and related viruses and is a valuable trait that is widely employed in potato resistance breeding programmes. Rysto was previously mapped to a region of potato chromosome XII, but the specific gene has not been identified to date. In this study, we isolated Rysto using resistance gene enrichment sequencing (RenSeq) and PacBio SMRT (Pacific Biosciences single-molecule real-time sequencing). Rysto was found to encode a nucleotide-binding leucine-rich repeat (NLR) protein with an N-terminal TIR domain and was sufficient for PVY perception and ER in transgenic potato plants. Rysto -dependent extreme resistance was temperature-independent and requires EDS1 and NRG1 proteins. Rysto may prove valuable for creating PVY-resistant cultivars of potato and other Solanaceae crops. © 2019 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

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