Project description:Secreted parasite factors from Babesia canis during acute infection were investigated using a LC-MS/MS assay which led to the discovery of potential pathogenicity factors. Combined data from genomics, transcriptomics and proteomics provide a first predicted and partially validated set of potential virulence factors exported during fatal infections.
Project description:Bacteria belonging to phylum Gemmatimonadetes are found in a wide variety of environments and are particularly abundant in soils. To date, only two Gemmatimonadetes strains have been characterized. Here we report the complete genome sequence and methylation pattern of Gemmatirosa kalamazoonensis KBS708 (ATCC BAA-2150; NCCB 100411), the first characterized Gemmatimondetes strain isolated from soil. Examination of the methylome of Gemmatirosa kalamazoonenis KBS708 using kinetic data from single-molecule, real-time (SMRT) sequencing on the PacBio RS
Project description:To understand Babesia gene regulation during tick and mammalian host infection, we performed high throughput RNA-sequencing using samples collected from calves and Rhipicephalus microplus ticks infected with Babesia bigemina. We evaluated gene expression differences between B. bigemina kinetes and blood-stage parasites
Project description:Dendrocalamus latiflorus Munro (D. latiflorus) is a woody clumping bamboo with rapid shoot growth. Both genetic transformation and CRISPR-Cas9 gene editing techniques are available for D. latiflorus, enabling reverse genetic approaches. Thus, D. latiflorus has the potential to be a model bamboo species. However, the genome sequence of D. latiflorus has remained unreported due to its polyploidy and large genome size. Here, we sequenced the D. latiflorus genome and assembled it into three allele-aware subgenomes (AABBCC), representing the largest genome of a major bamboo species. We assembled 70 allelic chromosomes (2,737 Mb) for hexaploid D. latiflorus using both single-molecule sequencing from the Pacific Biosciences (PacBio) Sequel platform and chromosome conformation capture sequencing (Hi-C). Repetitive sequences comprised 52.65% of the D. latiflorus genome. We annotated 135,231 protein-coding genes in the genome based on transcriptomes from eight different tissues. Transcriptome sequencing using RNA-Seq and PacBio single-molecule real-time (SMRT) long-read isoform sequencing (Iso-Seq) revealed highly differential alternative splicing (AS) between non-abortive and abortive shoots, suggesting that AS regulates the abortion rate of bamboo shoots. This high-quality hexaploid genome and comprehensive strand-specific transcriptome datasets for this Poaceae family member will pave the way for bamboo research using D. latiflorus as a model species.
Project description:Canine babesiosis, a tick-borne disease, is characterized by protozoan parasites invading red blood cells. It is rapidly expanding in many European countries. Examining extracellular vesicles (EVs) and their protein cargo has the potential to offer crucial insights into the response to Babesia canis infection, presenting opportunities for advancements in veterinary diagnostic and therapeutic strategies. In the present study, we have a) isolated small EVs (< 200 nm) from the serum of 15 healthy dogs and 15 dogs naturally infected with B. canis using size-exclusion chromatography (fraction 2 and 3 per each sample), (2) characterized isolated EVs by nanoparticle tracking analysis, transmission electron microscopy and Western blot (3) analysed the protein cargo of isolated EVs by mass spectrometry. We hypothesized that there will be a difference in EV characteristics (size, concentration, EV marker proteins) and profiles of luminal proteins between the two experimental groups. Our aim was to characterize proteins that can offer valuable insights into B. canis infection in dogs, thereby unravelling the complex mechanisms of B. canis infection.
Project description:Concurrent readout of sequence and base modifications from long unamplified DNA templates by PacBio single-molecule sequencing requires large amounts of input material. Here we adapt Tn5 transposition to introduce hairpin oligonucleotides and fragment (tagment) limiting quantities of DNA for generating PacBio-compatible circular molecules. We developed two methods that implement tagmentation and use 90–99% less input than current protocols: (1) single-molecule real-time sequencing by tagmentation (SMRT-Tag), which allows detection of genetic variation and CpG methylation; and (2) single-molecule adenine-methylated oligonucleosome sequencing assay by tagmentation (SAMOSA-Tag), which uses exogenous adenine methylation to add a third channel for probing chromatin accessibility. SMRT-Tag of 40 ng or more human DNA (approximately 7,000 cell equivalents) yielded data comparable to gold standard whole-genome and bisulfite sequencing. SAMOSA-Tag of 30,000–50,000 nuclei resolved single-fiber chromatin structure, CTCF binding and DNA methylation in patient-derived prostate cancer xenografts and uncovered metastasis-associated global epigenome disorganization. Tagmentation thus promises to enable sensitive, scalable and multimodal single-molecule genomics for diverse basic and clinical applications.
Project description:Concurrent readout of sequence and base modifications from long unamplified DNA templates by PacBio single-molecule sequencing requires large amounts of input material. Here we adapt Tn5 transposition to introduce hairpin oligonucleotides and fragment (tagment) limiting quantities of DNA for generating PacBio-compatible circular molecules. We developed two methods that implement tagmentation and use 90–99% less input than current protocols: (1) single-molecule real-time sequencing by tagmentation (SMRT-Tag), which allows detection of genetic variation and CpG methylation; and (2) single-molecule adenine-methylated oligonucleosome sequencing assay by tagmentation (SAMOSA-Tag), which uses exogenous adenine methylation to add a third channel for probing chromatin accessibility. SMRT-Tag of 40 ng or more human DNA (approximately 7,000 cell equivalents) yielded data comparable to gold standard whole-genome and bisulfite sequencing. SAMOSA-Tag of 30,000–50,000 nuclei resolved single-fiber chromatin structure, CTCF binding and DNA methylation in patient-derived prostate cancer xenografts and uncovered metastasis-associated global epigenome disorganization. Tagmentation thus promises to enable sensitive, scalable and multimodal single-molecule genomics for diverse basic and clinical applications.
Project description:Concurrent readout of sequence and base modifications from long unamplified DNA templates by PacBio single-molecule sequencing requires large amounts of input material. Here we adapt Tn5 transposition to introduce hairpin oligonucleotides and fragment (tagment) limiting quantities of DNA for generating PacBio-compatible circular molecules. We developed two methods that implement tagmentation and use 90–99% less input than current protocols: (1) single-molecule real-time sequencing by tagmentation (SMRT-Tag), which allows detection of genetic variation and CpG methylation; and (2) single-molecule adenine-methylated oligonucleosome sequencing assay by tagmentation (SAMOSA-Tag), which uses exogenous adenine methylation to add a third channel for probing chromatin accessibility. SMRT-Tag of 40 ng or more human DNA (approximately 7,000 cell equivalents) yielded data comparable to gold standard whole-genome and bisulfite sequencing. SAMOSA-Tag of 30,000–50,000 nuclei resolved single-fiber chromatin structure, CTCF binding and DNA methylation in patient-derived prostate cancer xenografts and uncovered metastasis-associated global epigenome disorganization. Tagmentation thus promises to enable sensitive, scalable and multimodal single-molecule genomics for diverse basic and clinical applications.
Project description:Concurrent readout of sequence and base modifications from long unamplified DNA templates by PacBio single-molecule sequencing requires large amounts of input material. Here we adapt Tn5 transposition to introduce hairpin oligonucleotides and fragment (tagment) limiting quantities of DNA for generating PacBio-compatible circular molecules. We developed two methods that implement tagmentation and use 90–99% less input than current protocols: (1) single-molecule real-time sequencing by tagmentation (SMRT-Tag), which allows detection of genetic variation and CpG methylation; and (2) single-molecule adenine-methylated oligonucleosome sequencing assay by tagmentation (SAMOSA-Tag), which uses exogenous adenine methylation to add a third channel for probing chromatin accessibility. SMRT-Tag of 40 ng or more human DNA (approximately 7,000 cell equivalents) yielded data comparable to gold standard whole-genome and bisulfite sequencing. SAMOSA-Tag of 30,000–50,000 nuclei resolved single-fiber chromatin structure, CTCF binding and DNA methylation in patient-derived prostate cancer xenografts and uncovered metastasis-associated global epigenome disorganization. Tagmentation thus promises to enable sensitive, scalable and multimodal single-molecule genomics for diverse basic and clinical applications.
Project description:Six bacterial genomes, Geobacter metallireducens GS-15, Chromohalobacter salexigens, Vibrio breoganii 1C-10, Bacillus cereus ATCC 10987, Campylobacter jejuni subsp. jejuni 81-176 and Campylobacter jejuni NCTC 11168, all of which had previously been sequenced using other platforms were re-sequenced using single-molecule, real-time (SMRT) sequencing specifically to analyze their methylomes. In every case a number of new N6-methyladenine (m6A) and N4-methylcytosine (m4C) methylation patterns were discovered and the DNA methyltransferases (MTases) responsible for those methylation patterns were assigned. In 15 cases it was possible to match MTase genes with MTase recognition sequences without further sub-cloning. Two Type I restriction systems required sub-cloning to differentiate their recognition sequences, while four MTases genes that were not expressed in the native organism were sub-cloned to test for viability and recognition sequences. No attempt was made to detect 5-methylcytosine (m5C) recognition motifs from the SMRT sequencing data because this modification produces weaker signals using current methods. However, all predicted m6A and m4C MTases were detected unambiguously. This study shows that the addition of SMRT sequencing to traditional sequencing approaches gives a wealth of useful functional information about a genome showing not only which MTase genes are active, but also revealing their recognition sequences. Examination of the methylomes of six different strains of bacteria using kinetic data from single-molecule, real-time (SMRT) sequencing on the PacBio RS.