Biology subjects

Kingan, S. B.

Publications and source records attributed to Kingan, S. B..

9 recordsLinked to original sources

bioRxiv2024

CCG interruptions are unstable and hypermethylated in DM1 patients

Myotonic dystrophy type 1 (DM1) exhibits highly heterogeneous clinical manifestations caused by an unstable CTG repeat expansion reaching up to 4,000 CTG. The dynamics of CTG repeats and clinical variability depends on the CTG repeat number, CNG repeat interruptions, DNA methylation, somatic mosaicism, but also gene modifiers. Around 10% of the DM1 population carries triplet repeat interruptions (CCG, CGG, CTC, CAG), which differ in number and nature between DM1 families. CCG interruptions have been associated with stabilization of the CTG repeat expansions and a milder phenotype in the DM1 population. However, the dynamics and precise role of interruptions in CTG repeat instability remain relatively underexplored due to the complexities of analyzing them. In this study, we showed that the number of CCG interruptions within the expansion varies within tissues and between generations using single-molecule real-time long-read sequencing. Although the interrupted expanded alleles showed global stabilization, the CCG interruptions themselves displayed variability across generations and within somatic tissues. Importantly, our findings reveal, for the first time, CCG hypermethylation within the CTG expansion, which is linked to downstream hypermethylation of the repeat. These results support the hypothesis that methylation of CCG interruptions within the expanded DMPK alleles may contribute to the stabilization of trinucleotide repeats.

molecular biology↗

bioRxiv2024

Development and extensive sequencing of a broadly-consented Genome in a Bottle matched tumor-normal pair for somatic benchmarks

The Genome in a Bottle Consortium (GIAB), hosted by the National Institute of Standards and Technology (NIST), is developing new matched tumor-normal samples, the first to be explicitly consented for public dissemination of genomic data and cell lines. Here, we describe a comprehensive genomic dataset from the first individual, HG008, including DNA from an adherent, epithelial-like pancreatic ductal adenocarcinoma (PDAC) tumor cell line and matched normal cells from duodenal and pancreatic tissues. Data for the tumor-normal matched samples comes from seventeen distinct state-of-the-art whole genome measurement technologies, including high depth short and long-read bulk whole genome sequencing (WGS), single cell WGS, and Hi-C, and karyotyping. In future publications, these data will be used by the GIAB Consortium to develop matched tumor-normal benchmarks for somatic variant detection. We expect these data to facilitate innovation for whole genome measurement technologies, de novo assembly of tumor and normal genomes, and bioinformatic tools to identify small and structural somatic mutations. This first-of-its-kind broadly consented open-access resource will facilitate further understanding of sequencing methods used for cancer biology.

genomics↗

bioRxiv2022

Design and performance of a long-read sequencing panel for pharmacogenomics

Pharmacogenomics (PGx)-guided drug treatment is one of the cornerstones of personalized medicine. However, the genes involved in drug response are highly complex and known to carry many (rare) variants. Current technologies (short-read sequencing and SNP panels) are limited in their ability to resolve these genes and characterize all variants. Moreover, these technologies cannot always phase variants to their allele of origin. Recent advance in long-read sequencing technologies have shown promise in resolving these problems. Here we present a long-read sequencing panel-based approach for PGx using PacBio HiFi sequencing. A capture based approach was developed using a custom panel of clinically-relevant pharmacogenes including up- and downstream regions. A total of 27 samples were sequenced and panel accuracy was determined using benchmarking variant calls for 3 Genome in a Bottle samples and GeT-RM star(*)-allele calls for 21 samples.. The coverage was uniform for all samples with an average of 94% of bases covered at >30x. When compared to benchmarking results, accuracy was high with an average F1 score of 0.89 for INDELs and 0.98 for SNPs. Phasing was good with an average of 68% the target region phased (compared to ~20% for short-reads) and an average phased haploblock size of 6.6kbp. Using Aldy 4, we compared our variant calls to GeT-RM data for 8 genes (CYP2B6, CYP2C19, CYP2C9, CYP2D6, CYP3A4, CYP3A5, SLCO1B1, TPMT), and observed highly accurate star(*)-allele calling with 98.2% concordance (165/168 calls), with only one discordance in CYP2C9 leading to a different predicted phenotype. We have shown that our long-read panel-based approach results in high accuracy and target phasing for SNVs as well as for clinical star(*)-alleles.

genetics↗

bioRxiv2020

Sequence and annotation of 42 cannabis genomes reveals extensive copy number variation in cannabinoid synthesis and pathogen resistance genes

Cannabis is a diverse and polymorphic species. To better understand cannabinoid synthesis inheritance and its impact on pathogen resistance, we shotgun sequenced and assembled a Cannabis trio (sibling pair and their offspring) utilizing long read single molecule sequencing. This resulted in the most contiguous Cannabis sativa assemblies to date. These reference assemblies were further annotated with full-length male and female mRNA sequencing (Iso-Seq) to help inform isoform complexity, gene model predictions and identification of the Y chromosome. To further annotate the genetic diversity in the species, 40 male, female, and monoecious cannabis and hemp varietals were evaluated for copy number variation (CNV) and RNA expression. This identified multiple CNVs governing cannabinoid expression and 82 genes associated with resistance to Golovinomyces chicoracearum, the causal agent of powdery mildew in cannabis. Results indicated that breeding for plants with low tetrahydrocannabinolic acid (THCA) concentrations may result in deletion of pathogen resistance genes. Low THCA cultivars also have a polymorphism every 51 bases while dispensary grade high THCA cannabis exhibited a variant every 73 bases. A refined genetic map of the variation in cannabis can guide more stable and directed breeding efforts for desired chemotypes and pathogen-resistant cultivars. O_FIG O_LINKSMALLFIG WIDTH=160 HEIGHT=200 SRC="FIGDIR/small/894428v1_ufig1.gif" ALT="Figure 1"> View larger version (126K): org.highwire.dtl.DTLVardef@7a1ac3org.highwire.dtl.DTLVardef@731a4dorg.highwire.dtl.DTLVardef@c3e5acorg.highwire.dtl.DTLVardef@11babb4_HPS_FORMAT_FIGEXP M_FIG Sequence and annotation of 42 cannabis genomes reveals extensive copy number variation in cannabinoid synthesis and pathogen resistance genes C_FIG

genomics↗

bioRxiv2019

Genomic Analysis of Powdery Mildew Resistance in a Hop (Humulus lupulus L.) Bi-parental Population Segregating for R6-locus

Genetic response in hop to fungal pathogen infection has been evaluated at the chromosomal level through QTL analyses but very little information exists on the expression of genes during infection periods. Raw GBS reads and phenotypic data from a previously published QTL analysis along with a newly assembled PacBio-derived hop reference genome were used to re-evaluate resistance to races v4/v6 of powdery mildew (PM; Podosphaera humuli). QTL analyses revealed two tightly linked regions of association on a single linkage group. The three SNP markers most tightly linked to PM resistance (found on contig 000559F) were observed downstream from a putative R-gene locus for powdery mildew resistance. This 230 kb region contained a series of seven putative R-genes surrounded by seven putative peroxidase-3 genes downstream and seven putative glucan endo-1,3-beta-glucosidase upstream and an expressed F-box domain protein. RNAseq data showed all putative R-genes along with all putative glucan endo-1,3-beta-glucosidase genes were expressed under diseased conditions, while none of the peroxidase genes were expressed. The second region contained three SNPs found on contig 002916F next to two putative R-genes. RNAseq data showed complex expression of exons contained in putative isoforms of R-genes. This preliminary information will prove valuable information for development of precise markers located either within or next to genes responsible for race v4/v6 PM resistance.

genomics↗

bioRxiv2019

Haplotype-Resolved Cattle Genomes Provide Insights Into Structural Variation and Adaptation

We present high quality, phased genome assemblies representative of taurine and indicine cattle, subspecies that differ markedly in productivity-related traits and environmental adaptation. We report a new haplotype-aware scaffolding and polishing pipeline using contigs generated by the trio binning method to produce haplotype-resolved, chromosome-level genome assemblies of Angus (taurine) and Brahman (indicine) cattle breeds. These assemblies were used to identify structural and copy number variants that differentiate the subspecies and we found variant detection was sensitive to the specific reference genome chosen. Six gene families with immune related functions are expanded in the indicine lineage. Assembly of the genomes of both subspecies from a single individual enabled transcripts to be phased to detect allele-specific expression, and to study genome-wide selective sweeps. An indicus-specific extra copy of fatty acid desaturase is under positive selection and may contribute to indicine adaptation to heat and drought.

genomics↗

bioRxiv2019

A High-Quality Genome Assembly from a Single, Field collected Spotted Lanternfly (Lycorma delicatula) using the PacBio Sequel II System

A high-quality reference genome is an essential tool for applied and basic research on arthropods. Long-read sequencing technologies may be used to generate more complete and contiguous genome assemblies than alternate technologies, however, long-read methods have historically had greater input DNA requirements and higher costs than next generation sequencing, which are barriers to their use on many samples. Here, we present a 2.3 Gb de novo genome assembly of a field-collected adult female Spotted Lanternfly (Lycorma delicatula) using a single PacBio SMRT Cell. The Spotted Lanternfly is an invasive species recently discovered in the northeastern United States, threatening to damage economically important crop plants in the region. The DNA from one individual was used to make one standard, size-selected library with an average DNA fragment size of ~20 kb. The library was run on one Sequel II SMRT Cell 8M, generating a total of 132 Gb of long-read sequences, of which 82 Gb were from unique library molecules, representing approximately 36-fold coverage of the genome. The assembly had high contiguity (contig N50 length = 1.5 Mb), completeness, and sequence level accuracy as estimated by conserved gene set analysis (96.8% of conserved genes both complete and without frame shift errors). Further, it was possible to segregate more than half of the diploid genome into the two separate haplotypes. The assembly also recovered two microbial symbiont genomes known to be associated with L. delicatula, each microbial genome being assembled into a single contig. We demonstrate that field-collected arthropods can be used for the rapid generation of high-quality genome assemblies, an attractive approach for projects on emerging invasive species, disease vectors, or conservation efforts of endangered species.

genomics↗

bioRxiv2018

FALCON-Phase: Integrating PacBio and Hi-C data for phased diploid genomes

Haplotype-resolved genome assemblies are important for understanding how combinations of variants impact phenotypes. These assemblies can be created in various ways, such as use of tissues that contain single-haplotype (haploid) genomes, or by co-sequencing of parental genomes, but these approaches can be impractical in many situations. We present FALCON-Phase, which integrates long-read sequencing data and ultra-long-range Hi-C chromatin interaction data of a diploid individual to create high-quality, phased diploid genome assemblies. The method was evaluated by application to three datasets, including human, cattle, and zebra finch, for which high-quality, fully haplotype resolved assemblies were available for benchmarking. Phasing algorithm accuracy was affected by heterozygosity of the individual sequenced, with higher accuracy for cattle and zebra finch (>97%) compared to human (82%). In addition, scaffolding with the same Hi-C chromatin contact data resulted in phased chromosome-scale scaffolds.

genomics↗

bioRxiv2018

Complete assembly of parental haplotypes with trio binning

Reference genome projects have historically selected inbred individuals to minimize heterozygosity and simplify assembly. We challenge this dogma and present a new approach designed specifically for heterozygous genomes. \"Trio binning\" uses short reads from two parental genomes to partition long reads from an offspring into haplotype-specific sets prior to assembly. Each haplotype is then assembled independently, resulting in a complete diploid reconstruction. On a benchmark human trio, this method achieved high accuracy and recovered complex structural variants missed by alternative approaches. To demonstrate its effectiveness on a heterozygous genome, we sequenced an F1 cross between cattle subspecies Bos taurus taurus and Bos taurus indicus, and completely assembled both parental haplotypes with NG50 haplotig sizes >20 Mbp and 99.998% accuracy, surpassing the quality of current cattle reference genomes. We propose trio binning as a new best practice for diploid genome assembly that will enable new studies of haplotype variation and inheritance.

genomics↗