June 1, 2021  |  

Complete resequencing of extended genomic regions using fosmid target capture and single molecule real-time (SMRT) long read sequencing technology.

A longstanding goal of genomic analysis is the identification of causal genetic factors contributing to disease. While the common disease/common variant hypothesis has been tested in many genome-wide association studies, few advancements in identifying causal variation have been realized, and instead recent findings point away from common variants towards aggregate rare variants as causal. A challenge is obtaining complete phased genomic sequences over extended genomic regions from sufficient numbers of cases and controls to identify all potential variation causal of a disease. To address this, we modified methods for targeted DNA isolation using fosmid technology and single-molecule, long-sequence-read generaton that combine for complete, haplotype-resolved resequencing across extended genomic subregions. As proof of principal, we validated the approach by resequencing four 800 kbp segments that span a major histocompatibility complex (MHC) common extended haplotype (CEH) associated with disease. The data revealed the extent of conservation exposing a near identity among four DR4 CEHs over conserved regions, detailing rare variation and measuring sequence accuracy. In a second test, we sequenced the complete KIR haplotypes from 8 individuals within a specific timeframe and cost. Single molecule long-read sequencing technology generated contiguous full-­length fosmid sequences of 30 to 40 kb in a single read, allowing assembly of resolved haplotypes with very little data processing. All of the sequences produced from these projects were contiguous, phased, with accuracy above 99.99%. The results demonstrated that cost-effective scale-­up is possible to generate scores to hundreds of phased chromosomal sequences of extended lengths that can encompass genomic regions associated with disease.

April 21, 2020  |  

A First Study of the Virulence Potential of a Bacillus subtilis Isolate From Deep-Sea Hydrothermal Vent.

Bacillus subtilis is the best studied Gram-positive bacterium, primarily as a model of cell differentiation and industrial exploitation. To date, little is known about the virulence of B. subtilis. In this study, we examined the virulence potential of a B. subtilis strain (G7) isolated from the Iheya North hydrothermal field of Okinawa Trough. G7 is aerobic, motile, endospore-forming, and requires NaCl for growth. The genome of G7 is composed of one circular chromosome of 4,216,133 base pairs with an average GC content of 43.72%. G7 contains 4,416 coding genes, 27.5% of which could not be annotated, and the remaining 72.5% were annotated with known or predicted functions in 25 different COG categories. Ten sets of 23S, 5S, and 16S ribosomal RNA operons, 86 tRNA and 14 sRNA genes, 50 tandem repeats, 41 mini-satellites, one microsatellite, and 42 transposons were identified in G7. Comparing to the genome of the B. subtilis wild type strain NCIB 3610T, G7 genome contains many genomic translocations, inversions, and insertions, and twice the amount of genomic Islands (GIs), with 42.5% of GI genes encoding hypothetical proteins. G7 possesses abundant putative virulence genes associated with adhesion, invasion, dissemination, anti-phagocytosis, and intracellular survival. Experimental studies showed that G7 was able to cause mortality in fish and mice following intramuscular/intraperitoneal injection, resist the killing effect of serum complement, and replicate in mouse macrophages and fish peripheral blood leukocytes. Taken together, our study indicates that G7 is a B. subtilis isolate with unique genetic features and can be lethal to vertebrate animals once being introduced into the animals by artificial means. These results provide the first insight into the potential harmfulness of deep-sea B. subtilis.

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