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September 22, 2019  |  

A workflow for studying specialized metabolism in nonmodel eukaryotic organisms

Eukaryotes contain a diverse tapestry of specialized metabolites, many of which are of significant pharmaceutical and industrial importance to humans. Nevertheless, exploration of specialized metabolic pathways underlying specific chemical traits in nonmodel eukaryotic organisms has been technically challenging and historically lagged behind that of the bacterial systems. Recent advances in genomics, metabolomics, phylogenomics, and synthetic biology now enable a new workflow for interrogating unknown specialized metabolic systems in nonmodel eukaryotic hosts with greater efficiency and mechanistic depth. This chapter delineates such workflow by providing a collection of state-of-the-art approaches and tools, ranging from multiomics-guided candidate gene identification to in vitro and in vivo functional and structural characterization of specialized metabolic enzymes. As already demonstrated by several recent studies, this new workflow opens up a gateway into the largely untapped world of natural product biochemistry in eukaryotes. © 2016 Elsevier Inc. All rights reserved.

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September 22, 2019  |  

Single molecule real-time (SMRT) sequencing comes of age: applications and utilities for medical diagnostics.

Short read massive parallel sequencing has emerged as a standard diagnostic tool in the medical setting. However, short read technologies have inherent limitations such as GC bias, difficulties mapping to repetitive elements, trouble discriminating paralogous sequences, and difficulties in phasing alleles. Long read single molecule sequencers resolve these obstacles. Moreover, they offer higher consensus accuracies and can detect epigenetic modifications from native DNA. The first commercially available long read single molecule platform was the RS system based on PacBio’s single molecule real-time (SMRT) sequencing technology, which has since evolved into their RSII and Sequel systems. Here we capsulize how SMRT sequencing is revolutionizing constitutional, reproductive, cancer, microbial and viral genetic testing.© The Author(s) 2018. Published by Oxford University Press on behalf of Nucleic Acids Research.

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September 22, 2019  |  

Deciphering highly similar multigene family transcripts from Iso-Seq data with IsoCon

A significant portion of genes in vertebrate genomes belongs to multigene families, with each family containing several gene copies whose presence/absence, as well as isoform structure, can be highly variable across individuals. Existing de novo techniques for assaying the sequences of such highly-similar gene families fall short of reconstructing end-to-end transcripts with nucleotide-level precision or assigning alternatively spliced transcripts to their respective gene copies. We present IsoCon, a high-precision method using long PacBio Iso-Seq reads to tackle this challenge. We apply IsoCon to nine Y chromosome ampliconic gene families and show that it outperforms existing methods on both experimental and simulated data. IsoCon has allowed us to detect an unprecedented number of novel isoforms and has opened the door for unraveling the structure of many multigene families and gaining a deeper understanding of genome evolution and human diseases.

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September 22, 2019  |  

Long reads: their purpose and place.

In recent years long-read technologies have moved from being a niche and specialist field to a point of relative maturity likely to feature frequently in the genomic landscape. Analogous to next generation sequencing, the cost of sequencing using long-read technologies has materially dropped whilst the instrument throughput continues to increase. Together these changes present the prospect of sequencing large numbers of individuals with the aim of fully characterizing genomes at high resolution. In this article, we will endeavour to present an introduction to long-read technologies showing: what long reads are; how they are distinct from short reads; why long reads are useful and how they are being used. We will highlight the recent developments in this field, and the applications and potential of these technologies in medical research, and clinical diagnostics and therapeutics.

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September 22, 2019  |  

Conventional and single-molecule targeted sequencing method for specific variant detection in IKBKG while bypassing the IKBKGP1 pseudogene.

In addition to Sanger sequencing, next-generation sequencing of gene panels and exomes has emerged as a standard diagnostic tool in many laboratories. However, these captures can miss regions, have poor efficiency, or capture pseudogenes, which hamper proper diagnoses. One such example is the primary immunodeficiency-associated gene IKBKG. Its pseudogene IKBKGP1 makes traditional capture methods aspecific. We therefore developed a long-range PCR method to efficiently target IKBKG, as well as two associated genes (IRAK4 and MYD88), while bypassing the IKBKGP1 pseudogene. Sequencing accuracy was evaluated using both conventional short-read technology and a newer long-read, single-molecule sequencer. Different mapping and variant calling options were evaluated in their capability to bypass the pseudogene using both sequencing platforms. Based on these evaluations, we determined a robust diagnostic application for unambiguous sequencing and variant calling in IKBKG, IRAK4, and MYD88. This method allows rapid identification of selected primary immunodeficiency diseases in patients suffering from life-threatening invasive pyogenic bacterial infections. Copyright © 2018 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

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September 22, 2019  |  

Diagnostic and Therapeutic Strategies for Fluoropyrimidine Treatment of Patients Carrying Multiple DPYD Variants.

DPYD genotyping prior to fluoropyrimidine treatment is increasingly implemented in clinical care. Without phasing information (i.e., allelic location of variants), current genotype-based dosing guidelines cannot be applied to patients carrying multiple DPYD variants. The primary aim of this study is to examine diagnostic and therapeutic strategies for fluoropyrimidine treatment of patients carrying multiple DPYD variants. A case series of patients carrying multiple DPYD variants is presented. Different genotyping techniques were used to determine phasing information. Phenotyping was performed by dihydropyrimidine dehydrogenase (DPD) enzyme activity measurements. Publicly available databases were queried to explore the frequency and phasing of variants of patients carrying multiple DPYD variants. Four out of seven patients carrying multiple DPYD variants received a full dose of fluoropyrimidines and experienced severe toxicity. Phasing information could be retrieved for four patients. In three patients, variants were located on two different alleles, i.e., in trans. Recommended dose reductions based on the phased genotype differed from the phenotype-derived dose reductions in three out of four cases. Data from publicly available databases show that the frequency of patients carrying multiple DPYD variants is low (< 0.2%), but higher than the frequency of the commonly tested DPYD*13 variant (0.1%). Patients carrying multiple DPYD variants are at high risk of developing severe toxicity. Additional analyses are required to determine the correct dose of fluoropyrimidine treatment. In patients carrying multiple DPYD variants, we recommend that a DPD phenotyping assay be carried out to determine a safe starting dose.

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