Enhanced Detection and Genotyping of Disease-Associated Tandem Repeats Using HMMSTR and Targeted Long-Read Sequencing.

Tandem repeat sequences comprise approximately 8% of the human genome and are linked to more than 50 neurodegenerative disorders. Accurate characterization of disease-associated repeat loci remains resource intensive and often lacks high resolution genotype calls. We introduce a multiplexed, targeted nanopore sequencing panel and HMMSTR, a sequence-based tandem repeat copy number caller. HMMSTR outperforms current signal- and sequence-based callers relative to two assemblies and we show it performs with high accuracy in heterozygous regions and at low read coverage. The flexible panel allows us to capture disease associated regions at an average coverage of >150x. Using these tools, we successfully characterize known or suspected repeat expansions in patient derived samples. In these samples we also identify unexpected expanded alleles at tandem repeat loci not previously associated with the underlying diagnosis. This genotyping approach for tandem repeat expansions is scalable, simple, flexible, and accurate, offering significant potential for diagnostic applications and investigation of expansion co-occurrence in neurodegenerative disorders.

Multiplexed long-read plasmid validation and analysis using OnRamp.

Recombinant plasmid vectors are versatile tools that have facilitated discoveries in molecular biology, genetics, proteomics, and many other fields. As the enzymatic and bacterial processes used to create recombinant DNA can introduce errors, sequence validation is an essential step in plasmid assembly. Sanger sequencing is the current standard for plasmid validation; however, this method is limited by an inability to sequence through complex secondary structure and lacks scalability when applied to full-plasmid sequencing of multiple plasmids owing to read-length limits. Although high-throughput sequencing does provide full-plasmid sequencing at scale, it is impractical and costly when used outside of library-scale validation. Here, we present Oxford nanopore-based rapid analysis of multiplexed plasmids (OnRamp), an alternative method for routine plasmid validation that combines the advantages of high-throughput sequencing's full-plasmid coverage and scalability with Sanger's affordability and accessibility by leveraging nanopore's long-read sequencing technology. We include customized wet-laboratory protocols for plasmid preparation along with a pipeline designed for analysis of read data obtained using these protocols. This analysis pipeline is deployed on the OnRamp web app, which generates alignments between actual and predicted plasmid sequences, quality scores, and read-level views. OnRamp is designed to be broadly accessible regardless of programming experience to facilitate more widespread adoption of long-read sequencing for routine plasmid validation. Here we describe the OnRamp protocols and pipeline and show our ability to obtain full sequences from pooled plasmids while detecting sequence variation even in regions of high secondary structure at less than half the cost of equivalent Sanger sequencing.

The Inducible lac Operator-Repressor System Is Functional in Zebrafish Cells.

BACKGROUND: Zebrafish are a foundational model organism for studying the spatio-temporal activity of genes and their regulatory sequences. A variety of approaches are currently available for editing genes and modifying gene expression in zebrafish, including RNAi, Cre/lox, and CRISPR-Cas9. However, the lac operator-repressor system, an E. coli lac operon component which has been adapted for use in many other species and is a valuable, flexible tool for inducible modulation of gene expression studies, has not been previously tested in zebrafish. RESULTS: Here we demonstrate that the lac operator-repressor system robustly decreases expression of firefly luciferase in cultured zebrafish fibroblast cells. Our work establishes the lac operator-repressor system as a promising tool for the manipulation of gene expression in whole zebrafish. CONCLUSION: Our results lay the groundwork for the development of lac-based reporter assays in zebrafish, and adds to the tools available for investigating dynamic gene expression in embryogenesis. We believe this work will catalyze the development of new reporter assay systems to investigate uncharacterized regulatory elements and their cell-type specific activities.

Cas9 targeted enrichment of mobile elements using nanopore sequencing.

Mobile element insertions (MEIs) are repetitive genomic sequences that contribute to genetic variation and can lead to genetic disorders. Targeted and whole-genome approaches using short-read sequencing have been developed to identify reference and non-reference MEIs; however, the read length hampers detection of these elements in complex genomic regions. Here, we pair Cas9-targeted nanopore sequencing with computational methodologies to capture active MEIs in human genomes. We demonstrate parallel enrichment for distinct classes of MEIs, averaging 44% of reads on-targeted signals and exhibiting a 13.4-54x enrichment over whole-genome approaches. We show an individual flow cell can recover most MEIs (97% L1Hs, 93% AluYb, 51% AluYa, 99% SVA_F, and 65% SVA_E). We identify seventeen non-reference MEIs in GM12878 overlooked by modern, long-read analysis pipelines, primarily in repetitive genomic regions. This work introduces the utility of nanopore sequencing for MEI enrichment and lays the foundation for rapid discovery of elusive, repetitive genetic elements.

Predicting the effects of SNPs on transcription factor binding affinity.

MOTIVATION: Genome-wide association studies have revealed that 88% of disease-associated single-nucleotide polymorphisms (SNPs) reside in noncoding regions. However, noncoding SNPs remain understudied, partly because they are challenging to prioritize for experimental validation. To address this deficiency, we developed the SNP effect matrix pipeline (SEMpl). RESULTS: SEMpl estimates transcription factor-binding affinity by observing differences in chromatin immunoprecipitation followed by deep sequencing signal intensity for SNPs within functional transcription factor-binding sites (TFBSs) genome-wide. By cataloging the effects of every possible mutation within the TFBS motif, SEMpl can predict the consequences of SNPs to transcription factor binding. This knowledge can be used to identify potential disease-causing regulatory loci. AVAILABILITY AND IMPLEMENTATION: SEMpl is available from https://github.com/Boyle-Lab/SEM_CPP. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Effect of CRC::etr1-1 transgene expression on ethylene production, sex expression, fruit set and fruit ripening in transgenic melon (Cucumis melo L.).

Ethylene is a key factor regulating sex expression in cucurbits. Commercial melons (Cucumis melo L.) are typically andromonoecious, producing male and bisexual flowers. Our prior greenhouse studies of transgenic melon plants expressing the dominant negative ethylene perception mutant gene, etr1-1, under control of the carpel- and nectary-primordia targeted CRAB'S CLAW (CRC) promoter showed increased number and earlier appearance of carpel-bearing flowers. To further investigate this phenomenon which could be potentially useful for earlier fruit production, we observed CRC::etr1-1 plants in the field for sex expression, fruit set, fruit development, and ripening. CRC::etr1-1 melon plants showed increased number of carpel-bearing open flowers on the main stem and earlier onset by 7-10 nodes. Additional phenotypes observed in the greenhouse and field were conversion of approximately 50% of bisexual buds to female, and elongated ovaries and fruits. Earlier and greater fruit set occurred on the transgenic plants. However, CRC::etr1-1 plants had greater abscission of young fruit, and smaller fruit, so that final yield (kg/plot) was equivalent to wild type. Earlier fruit set in line M5 was accompanied by earlier appearance of ripe fruit. Fruit from line M15 frequently did not exhibit external ripening processes of rind color change and abscission, but when cut open, the majority showed a ripe or overripe interior accompanied by elevated internal ethylene. The non-ripening external phenotype in M15 fruit corresponded with elevated etr1-1 transgene expression in the exocarp. These results provide insight into the role of ethylene perception in carpel-bearing flower production, fruit set, and ripening.

Floral primordia-targeted ACS (1-aminocyclopropane-1-carboxylate synthase) expression in transgenic Cucumis melo implicates fine tuning of ethylene production mediating unisexual flower development.

MAIN CONCLUSION: Floral primordia-targeted expression of the ethylene biosynthetic gene, ACS , in melon suggests that differential timing and ethylene response thresholds combine to promote carpels, inhibit stamens, and prevent asexual bud formation. Typical angiosperm flowers produce both male and female reproductive organs. However, numerous species have evolved unisexuality. Melons (Cucumis melo L.) can produce varying combinations of male, female or bisexual flowers. Regardless of final sex, floral development begins with sequential initiation of all four floral whorls; unisexuality results from carpel or stamen primordia arrest regulated by the G and A loci, respectively. Ethylene, which promotes femaleness, is a key factor regulating sex expression. We sought to further understand the location, timing, level, and relationship to sex gene expression required for ethylene to promote carpel development or inhibit stamen development. Andromonoecious melons (GGaa) were transformed with the ethylene biosynthetic enzyme gene, ACS (1-aminocyclopropane-1-carboxylate synthase), targeted for expression in stamen and petal, or carpel and nectary, primordia using Arabidopsis APETALA3 (AP3) or CRABSCLAW (CRC) promoters, respectively. CRC::ACS plants did not exhibit altered sex phenotype. AP3::ACS melons showed increased femaleness manifested by gain of a bisexual-only phase not seen in wild type, decreased male buds and flowers, and loss of the initial male-only phase. In extreme cases, plants became phenotypically hermaphrodite, rather than andromonoecious. A reduced portion of buds progressed beyond initial whorl formation. Both the ACS transgene and exogenous ethylene reduced the expression of the native carpel-suppressing gene, G, while elevating expression of the stamen-suppressing gene, A. These results show ethylene-mediated regulation of key sex expression genes and suggest a mechanism by which temporally regulated ethylene production and differential ethylene response thresholds can promote carpels, inhibit stamens, and prevent the formation of asexual buds.