Project description:Low-pass sequencing (sequencing a genome to an average depth less than 1× coverage) combined with genotype imputation has been proposed as an alternative to genotyping arrays for trait mapping and calculation of polygenic scores. To empirically assess the relative performance of these technologies for different applications, we performed low-pass sequencing (targeting coverage levels of 0.5× and 1×) and array genotyping (using the Illumina Global Screening Array (GSA)) on 120 DNA samples derived from African and European-ancestry individuals that are part of the 1000 Genomes Project. We then imputed both the sequencing data and the genotyping array data to the 1000 Genomes Phase 3 haplotype reference panel using a leave- one-out design. We evaluated overall imputation accuracy from these different assays as well as overall power for GWAS from imputed data, and computed polygenic risk scores for coronary artery disease and breast cancer using previously derived weights. We conclude that low-pass sequencing plus imputation, in addition to providing a substantial increase in statistical power for genome wide association studies, provides increased accuracy for polygenic risk prediction at effective coverages of ∼ 0.5× and higher compared to the Illumina GSA.
Project description:Bottom-up proteomics database search algorithms used for peptide identification cannot comprehensively identify posttranslational modifications (PTMs) in a single-pass because of high false discovery rates (FDRs). A new approach to database searching enables Global PTM (G-PTM) identification by exclusively looking for curated PTMs, thereby avoiding the FDR penalty experienced during conventional variable modification searches. We identified nearly 2500 unique, high-confidence modified peptides comprising 31 different PTM types in single-pass database searches.
Project description:We present a strategy termed PASS-DIA (Precursor ion And Small Slice-DIA) in which MS/MS spectra are acquired with small isolation window (slices) and MS/MS spectra are interpreted with accurately measured precursor ion masses. This method enables direct application of conventional spectrum-centric analysis pipelines for peptide identification and precursor ion-based quantitation. The performance of PASS-DIA was superior to both DDA and conventional DIA experiment with regard to identification of peptides. Application of PASS-DIA for analysis of samples with post-translationally modified peptides such as phosphorylation and N-glycosylation again revealed its superior performance. Finally, use of PASS-DIA to characterize a rare proteome of human fallopian tube organoid samples revealed biologically relevant and low abundance proteins. Overall, PASS-DIA is a novel DIA approach for use as a discovery tool which outperforms both conventional DDA and DIA experiments to provide additional protein information. We believe that PASS-DIA method could become an important strategy for discovery type studies when deeper proteome characterization is required.
Project description:Bottom-up proteomics database search algorithms used for peptide identification cannot comprehensively identify posttranslational modifications (PTMs) in a single-pass because of high false discovery rates (FDRs). A new approach to database searching enables Global PTM (G-PTM) identification by exclusively looking for curated PTMs, thereby avoiding the FDR penalty experienced during conventional variable modification searches. We identified nearly 2500 unique, high-confidence modified peptides comprising 31 different PTM types in single-pass database searches. Male C57BL/6J (B6) and CAST/EiJ (CAST) mice were purchased from The Jackson Laboratories (Bar Harbor, Maine) and housed in an environmentally controlled vivarium at the University of Wisconsin Biochemistry Department. Mice were provided standard rodent chow (Purina no. 5008) and water ad libitum, and maintained on a 12-hour light/dark cycle (6 AM – 6 PM). At 10 weeks of age, mice were sacrificed by CO2 asphyxiation. All animal procedures were preapproved by the University of Wisconsin Animal Care and Use Committee.
Project description:Transgenic plants carrying an estradiol-inducible ROS1-YFP construct (XVE:ROS1-YFP) were subjected to long-read sequencing (Oxford Nanopore Technologies) to assess the global impacts of ROS1 activity on the methylome of Arabidopsis thaliana (ecotype Col-0).
Project description:Low-pass sequencing increases the power of GWAS and decreases measurement error of polygenic risk scores compared to genotyping arrays
Project description:We sequenced DNA from the leaves of ten Col x Ler F1 hybrid plants (WT and recq4) using Nanopore long-read sequencing and identified crossover sites with COmapper. These data were used as a negative control for COmapper, as no crossover sites were expected to be detected. For nanopore sequencing of gDNA from leaves, leaves from 10 5-week-old plants were ground in liquid nitrogen using a mortar and pestle. The ground tissue was resuspended in four volumes of CTAB buffer (1% [w/v] CTAB, 50 mM Tris-HCl pH 8.0, 0.7 M NaCl, 10 mM EDTA) and incubated at 65°C for 30 min. Following chloroform extraction, isopropanol precipitation and removal of RNAs as above, the gDNA pellet was resuspended in 150 μl TE (10 mM Tris-HCl pH 8.0, 0.1 mM EDTA) buffer and gDNA was quantified using a Qubit dsDNA Broad Range assay kit (Thermo Fisher, Q32853). Nine micrograms of gDNA from pollen or seedlings was used to construct a nanopore long-read sequencing library using a Ligation Sequencing Kit V14 (Nanopore, SQK-LSK114). The libraries were sequenced using a PromethION platform (BGI, Hong Kong).
Project description:We sequenced DNA from a bulk of Col x Ler F2 hybrid plants (WT and recq4) using Nanopore long-read sequencing and identified crossover sites with COmapper. For nanopore sequencing of gDNA from 1,000 pooled seedlings, 10-day-old seedlings were ground in liquid nitrogen using a mortar and pestle. The ground tissue was resuspended in four volumes of CTAB buffer (1% [w/v] CTAB, 50 mM Tris-HCl pH 8.0, 0.7 M NaCl, 10 mM EDTA) and incubated at 65°C for 30 min. Following chloroform extraction, isopropanol precipitation and removal of RNAs as above, the gDNA pellet was resuspended in 150 μl TE (10 mM Tris-HCl pH 8.0, 0.1 mM EDTA) buffer and gDNA was quantified using a Qubit dsDNA Broad Range assay kit (Thermo Fisher, Q32853). Nine micrograms of gDNA from pollen or seedlings was used to construct a nanopore long-read sequencing library using a Ligation Sequencing Kit V14 (Nanopore, SQK-LSK114). The libraries were sequenced using a PromethION platform (BGI, Hong Kong).