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September 22, 2019  |  

Revertant mosaicism repairs skin lesions in a patient with keratitis-ichthyosis-deafness syndrome by second-site mutations in connexin 26.

Revertant mosaicism (RM) is a naturally occurring phenomenon where the pathogenic effect of a germline mutation is corrected by a second somatic event. Development of healthy-looking skin due to RM has been observed in patients with various inherited skin disorders, but not in connexin-related disease. We aimed to clarify the underlying molecular mechanisms of suspected RM in the skin of a patient with keratitis-ichthyosis-deafness (KID) syndrome. The patient was diagnosed with KID syndrome due to characteristic skin lesions, hearing deficiency and keratitis. Investigation of GJB2 encoding connexin (Cx) 26 revealed heterozygosity for the recurrent de novo germline mutation, c.148G?>?A, p.Asp50Asn. At age 20, the patient developed spots of healthy-looking skin that grew in size and number within widespread erythrokeratodermic lesions. Ultra-deep sequencing of two healthy-looking skin biopsies identified five somatic nonsynonymous mutations, independently present in cis with the p.Asp50Asn mutation. Functional studies of Cx26 in HeLa cells revealed co-expression of Cx26-Asp50Asn and wild-type Cx26 in gap junction channel plaques. However, Cx26-Asp50Asn with the second-site mutations identified in the patient displayed no formation of gap junction channel plaques. We argue that the second-site mutations independently inhibit Cx26-Asp50Asn expression in gap junction channels, reverting the dominant negative effect of the p.Asp50Asn mutation. To our knowledge, this is the first time RM has been reported to result in the development of healthy-looking skin in a patient with KID syndrome. © The Author 2017. Published by Oxford University Press.

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September 22, 2019  |  

Long-read, Single Molecule, Real-Time (SMRT) DNA Sequencing for metagenomic applications

In this chapter, we describe applications of single molecule, real-time (SMRT) DNA sequencing toward metagenomic research. The long sequence reads, combined with a lack of bias with respect to DNA sequence context or GC content, facilitate a more comprehensive analysis of the genomic constitution of microbial communities. Full-length 16S RNA gene sequencing at high (>99%) accuracy allows for species-level characterization of community members concomitant with the determination of community structure. The application of SMRT sequencing to whole-community shotgun microbial metagenomics has also been discussed.

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September 22, 2019  |  

Transcript profiling of a bitter variety of narrow-leafed lupin to discover alkaloid biosynthetic genes.

Lupins (Lupinus spp.) are nitrogen-fixing legumes that accumulate toxic alkaloids in their protein-rich beans. These anti-nutritional compounds belong to the family of quinolizidine alkaloids (QAs), which are of interest to the pharmaceutical and chemical industries. To unleash the potential of lupins as protein crops and as sources of QAs, a thorough understanding of the QA pathway is needed. However, only the first enzyme in the pathway, lysine decarboxylase (LDC), is known. Here, we report the transcriptome of a high-QA variety of narrow-leafed lupin (L. angustifolius), obtained using eight different tissues and two different sequencing technologies. In addition, we present a list of 33 genes that are closely co-expressed with LDC and that represent strong candidates for involvement in lupin alkaloid biosynthesis. One of these genes encodes a copper amine oxidase able to convert the product of LDC, cadaverine, into 1-piperideine, as shown by heterologous expression and enzyme assays. Kinetic analysis revealed a low KM value for cadaverine, supporting a role as the second enzyme in the QA pathway. Our transcriptomic data set represents a crucial step towards the discovery of enzymes, transporters, and regulators involved in lupin alkaloid biosynthesis.© The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology.

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September 22, 2019  |  

Full-length mRNA sequencing uncovers a widespread coupling between transcription initiation and mRNA processing.

The multifaceted control of gene expression requires tight coordination of regulatory mechanisms at transcriptional and post-transcriptional level. Here, we studied the interdependence of transcription initiation, splicing and polyadenylation events on single mRNA molecules by full-length mRNA sequencing.In MCF-7 breast cancer cells, we find 2700 genes with interdependent alternative transcription initiation, splicing and polyadenylation events, both in proximal and distant parts of mRNA molecules, including examples of coupling between transcription start sites and polyadenylation sites. The analysis of three human primary tissues (brain, heart and liver) reveals similar patterns of interdependency between transcription initiation and mRNA processing events. We predict thousands of novel open reading frames from full-length mRNA sequences and obtained evidence for their translation by shotgun proteomics. The mapping database rescues 358 previously unassigned peptides and improves the assignment of others. By recognizing sample-specific amino-acid changes and novel splicing patterns, full-length mRNA sequencing improves proteogenomics analysis of MCF-7 cells.Our findings demonstrate that our understanding of transcriptome complexity is far from complete and provides a basis to reveal largely unresolved mechanisms that coordinate transcription initiation and mRNA processing.

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September 22, 2019  |  

Full-length isoform sequencing reveals novel transcripts and substantial transcriptional overlaps in a herpesvirus.

Whole transcriptome studies have become essential for understanding the complexity of genetic regulation. However, the conventionally applied short-read sequencing platforms cannot be used to reliably distinguish between many transcript isoforms. The Pacific Biosciences (PacBio) RS II platform is capable of reading long nucleic acid stretches in a single sequencing run. The pseudorabies virus (PRV) is an excellent system to study herpesvirus gene expression and potential interactions between the transcriptional units. In this work, non-amplified and amplified isoform sequencing protocols were used to characterize the poly(A+) fraction of the lytic transcriptome of PRV, with the aim of a complete transcriptional annotation of the viral genes. The analyses revealed a previously unrecognized complexity of the PRV transcriptome including the discovery of novel protein-coding and non-coding genes, novel mono- and polycistronic transcription units, as well as extensive transcriptional overlaps between neighboring and distal genes. This study identified non-coding transcripts overlapping all three replication origins of the PRV, which might play a role in the control of DNA synthesis. We additionally established the relative expression levels of gene products. Our investigations revealed that the whole PRV genome is utilized for transcription, including both DNA strands in all coding and intergenic regions. The genome-wide occurrence of transcript overlaps suggests a crosstalk between genes through a network formed by interacting transcriptional machineries with a potential function in the control of gene expression.

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September 22, 2019  |  

Next generation sequencing technology: Advances and applications.

Impressive progress has been made in the field of Next Generation Sequencing (NGS). Through advancements in the fields of molecular biology and technical engineering, parallelization of the sequencing reaction has profoundly increased the total number of produced sequence reads per run. Current sequencing platforms allow for a previously unprecedented view into complex mixtures of RNA and DNA samples. NGS is currently evolving into a molecular microscope finding its way into virtually every fields of biomedical research. In this chapter we review the technical background of the different commercially available NGS platforms with respect to template generation and the sequencing reaction and take a small step towards what the upcoming NGS technologies will bring. We close with an overview of different implementations of NGS into biomedical research. This article is part of a Special Issue entitled: From Genome to Function. Copyright © 2014 Elsevier B.V. All rights reserved.

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September 22, 2019  |  

Multi-platform analysis reveals a complex transcriptome architecture of a circovirus.

In this study, we used Pacific Biosciences RS II long-read and Illumina HiScanSQ short-read sequencing technologies for the characterization of porcine circovirus type 1 (PCV-1) transcripts. Our aim was to identify novel RNA molecules and transcript isoforms, as well as to determine the exact 5′- and 3′-end sequences of previously described transcripts with single base-pair accuracy. We discovered a novel 3′-UTR length isoform of the Cap transcript, and a non-spliced Cap transcript variant. Additionally, our analysis has revealed a 3′-UTR isoform of Rep and two 5′-UTR isoforms of Rep’ transcripts, and a novel splice variant of the longer Rep’ transcript. We also explored two novel long transcripts, one with a previously identified splice site, and a formerly undetected mRNA of ORF3. Altogether, our methods have identified nine novel RNA molecules, doubling the size of PCV-1 transcriptome that had been known before. Additionally, our investigations revealed an intricate pattern of transcript overlapping, which might produce transcriptional interference between the transcriptional machineries of adjacent genes, and thereby may potentially play a role in the regulation of gene expression in circoviruses. Copyright © 2017 Elsevier B.V. All rights reserved.

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September 22, 2019  |  

Discovery of the fourth mobile sulfonamide resistance gene.

Over the past 75 years, human pathogens have acquired antibiotic resistance genes (ARGs), often from environmental bacteria. Integrons play a major role in the acquisition of antibiotic resistance genes. We therefore hypothesized that focused exploration of integron gene cassettes from microbial communities could be an efficient way to find novel mobile resistance genes. DNA from polluted Indian river sediments were amplified using three sets of primers targeting class 1 integrons and sequenced by long- and short-read technologies to maintain both accuracy and context.Up to 89% of identified open reading frames encode known resistance genes, or variations thereof (>?1000). We identified putative novel ARGs to aminoglycosides, beta-lactams, trimethoprim, rifampicin, and chloramphenicol, including several novel OXA variants, providing reduced susceptibility to carbapenems. One dihydropteroate synthase gene, with less than 34% amino acid identity to the three known mobile sulfonamide resistance genes (sul1-3), provided complete resistance when expressed in Escherichia coli. The mobilized gene, here named sul4, is the first mobile sulfonamide resistance gene discovered since 2003. Analyses of adjacent DNA suggest that sul4 has been decontextualized from a set of chromosomal genes involved in folate synthesis in its original host, likely within the phylum Chloroflexi. The presence of an insertion sequence common region element could provide mobility to the entire integron. Screening of 6489 metagenomic datasets revealed that sul4 is already widespread in seven countries across Asia and Europe.Our findings show that exploring integrons from environmental communities with a history of antibiotic exposure can provide an efficient way to find novel, mobile resistance genes. The mobilization of a fourth sulfonamide resistance gene is likely to provide expanded opportunities for sulfonamide resistance to spread, with potential impacts on both human and animal health.

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September 22, 2019  |  

MEGAN-LR: new algorithms allow accurate binning and easy interactive exploration of metagenomic long reads and contigs.

There are numerous computational tools for taxonomic or functional analysis of microbiome samples, optimized to run on hundreds of millions of short, high quality sequencing reads. Programs such as MEGAN allow the user to interactively navigate these large datasets. Long read sequencing technologies continue to improve and produce increasing numbers of longer reads (of varying lengths in the range of 10k-1M bps, say), but of low quality. There is an increasing interest in using long reads in microbiome sequencing, and there is a need to adapt short read tools to long read datasets.We describe a new LCA-based algorithm for taxonomic binning, and an interval-tree based algorithm for functional binning, that are explicitly designed for long reads and assembled contigs. We provide a new interactive tool for investigating the alignment of long reads against reference sequences. For taxonomic and functional binning, we propose to use LAST to compare long reads against the NCBI-nr protein reference database so as to obtain frame-shift aware alignments, and then to process the results using our new methods.All presented methods are implemented in the open source edition of MEGAN, and we refer to this new extension as MEGAN-LR (MEGAN long read). We evaluate the LAST+MEGAN-LR approach in a simulation study, and on a number of mock community datasets consisting of Nanopore reads, PacBio reads and assembled PacBio reads. We also illustrate the practical application on a Nanopore dataset that we sequenced from an anammox bio-rector community.This article was reviewed by Nicola Segata together with Moreno Zolfo, Pete James Lockhart and Serghei Mangul.This work extends the applicability of the widely-used metagenomic analysis software MEGAN to long reads. Our study suggests that the presented LAST+MEGAN-LR pipeline is sufficiently fast and accurate.

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September 22, 2019  |  

Revealing the transcriptomic complexity of switchgrass by PacBio long-read sequencing.

Switchgrass (Panicum virgatum L.) is an important bioenergy crop widely used for lignocellulosic research. While extensive transcriptomic analyses have been conducted on this species using short read-based sequencing techniques, very little has been reliably derived regarding alternatively spliced (AS) transcripts.We present an analysis of transcriptomes of six switchgrass tissue types pooled together, sequenced using Pacific Biosciences (PacBio) single-molecular long-read technology. Our analysis identified 105,419 unique transcripts covering 43,570 known genes and 8795 previously unknown genes. 45,168 are novel transcripts of known genes. A total of 60,096 AS transcripts are identified, 45,628 being novel. We have also predicted 1549 transcripts of genes involved in cell wall construction and remodeling, 639 being novel transcripts of known cell wall genes. Most of the predicted transcripts are validated against Illumina-based short reads. Specifically, 96% of the splice junction sites in all the unique transcripts are validated by at least five Illumina reads. Comparisons between genes derived from our identified transcripts and the current genome annotation revealed that among the gene set predicted by both analyses, 16,640 have different exon-intron structures.Overall, substantial amount of new information is derived from the PacBio RNA data regarding both the transcriptome and the genome of switchgrass.

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September 22, 2019  |  

Full-length transcriptome sequences and the identification of putative genes for flavonoid biosynthesis in safflower.

The flower of the safflower (Carthamus tinctorius L.) has been widely used in traditional Chinese medicine for the ability to improve cerebral blood flow. Flavonoids are the primary bioactive components in safflower, and their biosynthesis has attracted widespread interest. Previous studies mostly used second-generation sequencing platforms to survey the putative flavonoid biosynthesis genes. For a better understanding of transcription data and the putative genes involved in flavonoid biosynthesis in safflower, we carry our study.High-quality RNA was extracted from six types of safflower tissue. The RNAs of different tissues were mixed equally and used for multiple size-fractionated libraries (1-2, 2-3 and 3-6 k) library construction. Five cells were carried (2 cells for 1-2 and for 2-3 k libraries and 1 cell for 3-6 k libraries). 10.43Gb clean data and 38,302 de-redundant sequences were captured. 44 unique isoforms were annotated as encoding enzymes involved in flavonoid biosynthesis. The full length flavonoid genes were characterized and their evolutional relationship and expressional pattern were analyzed. They can be divided into eight families, with a large differences in the tissue expression. The temporal expressions under MeJA treatment were also measured, 9 genes are significantly up-regulated and 2 genes are significantly down-regulated. The genes involved in flavonoid synthesis in safflower were predicted in our study. Besides, the SSR and lncRNA are also analyzed in our study.Full-length transcriptome sequences were used in our study. The genes involved in flavonoid synthesis in safflower were predicted in our study. Combined the determination of flavonoids, CtC4H2, CtCHS3, CtCHI3, CtF3H3, CtF3H1 are mainly participated in MeJA promoting the synthesis of flavonoids. Our results also provide a valuable resource for further study on safflower.

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September 22, 2019  |  

Transcriptome-wide survey of pseudorabies virus using next- and third-generation sequencing platforms.

Pseudorabies virus (PRV) is an alphaherpesvirus of swine. PRV has a large double-stranded DNA genome and, as the latest investigations have revealed, a very complex transcriptome. Here, we present a large RNA-Seq dataset, derived from both short- and long-read sequencing. The dataset contains 1.3 million 100?bp paired-end reads that were obtained from the Illumina random-primed libraries, as well as 10 million 50?bp single-end reads generated by the Illumina polyA-seq. The Pacific Biosciences RSII non-amplified method yielded 57,021 reads of inserts (ROIs) aligned to the viral genome, the amplified method resulted in 158,396 PRV-specific ROIs, while we obtained 12,555 ROIs using the Sequel platform. The Oxford Nanopore’s MinION device generated 44,006 reads using their regular cDNA-sequencing method, whereas 29,832 and 120,394 reads were produced by using the direct RNA-sequencing and the Cap-selection protocols, respectively. The raw reads were aligned to the PRV reference genome (KJ717942.1). Our provided dataset can be used to compare different sequencing approaches, library preparation methods, as well as for validation and testing bioinformatic pipelines.

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September 22, 2019  |  

Single-molecule real-time transcript sequencing facilitates common wheat genome annotation and grain transcriptome research.

The large and complex hexaploid genome has greatly hindered genomics studies of common wheat (Triticum aestivum, AABBDD). Here, we investigated transcripts in common wheat developing caryopses using the emerging single-molecule real-time (SMRT) sequencing technology PacBio RSII, and assessed the resultant data for improving common wheat genome annotation and grain transcriptome research.We obtained 197,709 full-length non-chimeric (FLNC) reads, 74.6 % of which were estimated to carry complete open reading frame. A total of 91,881 high-quality FLNC reads were identified and mapped to 16,188 chromosomal loci, corresponding to 13,162 known genes and 3026 new genes not annotated previously. Although some FLNC reads could not be unambiguously mapped to the current draft genome sequence, many of them are likely useful for studying highly similar homoeologous or paralogous loci or for improving chromosomal contig assembly in further research. The 91,881 high-quality FLNC reads represented 22,768 unique transcripts, 9591 of which were newly discovered. We found 180 transcripts each spanning two or three previously annotated adjacent loci, suggesting that they should be merged to form correct gene models. Finally, our data facilitated the identification of 6030 genes differentially regulated during caryopsis development, and full-length transcripts for 72 transcribed gluten gene members that are important for the end-use quality control of common wheat.Our work demonstrated the value of PacBio transcript sequencing for improving common wheat genome annotation through uncovering the loci and full-length transcripts not discovered previously. The resource obtained may aid further structural genomics and grain transcriptome studies of common wheat.

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September 22, 2019  |  

Multiplex amplicon sequencing for microbe identification in community-based culture collections.

Microbiome analysis using metagenomic sequencing has revealed a vast microbial diversity associated with plants. Identifying the molecular functions associated with microbiome-plant interaction is a significant challenge concerning the development of microbiome-derived technologies applied to agriculture. An alternative to accelerate the discovery of the microbiome benefits to plants is to construct microbial culture collections concomitant with accessing microbial community structure and abundance. However, traditional methods of isolation, cultivation, and identification of microbes are time-consuming and expensive. Here we describe a method for identification of microbes in culture collections constructed by picking colonies from primary platings that may contain single or multiple microorganisms, which we named community-based culture collections (CBC). A multiplexing 16S rRNA gene amplicon sequencing based on two-step PCR amplifications with tagged primers for plates, rows, and columns allowed the identification of the microbial composition regardless if the well contains single or multiple microorganisms. The multiplexing system enables pooling amplicons into a single tube. The sequencing performed on the PacBio platform led to recovery near-full-length 16S rRNA gene sequences allowing accurate identification of microorganism composition in each plate well. Cross-referencing with plant microbiome structure and abundance allowed the estimation of diversity and abundance representation of microorganism in the CBC.

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