A high-performance computational workflow to accelerate GATK SNP detection across a 25-genome dataset

Background: Single-nucleotide polymorphisms (SNPs) are the most widely used form of molecular genetic variation studies. As reference genomes and resequencing data sets expand exponentially, tools must be in place to call SNPs at a similar pace. The genome analysis toolkit (GATK) is one of the most widely used SNP calling software tools publicly available, but unfortunately, high-performance computing versions of this tool have yet to become widely available and affordable. Results: Here we report an open-source high-performance computing genome variant calling workflow (HPC-GVCW) for GATK that can run on multiple computing platforms from supercomputers to desktop machines. We benchmarked HPC-GVCW on multiple crop species for performance and accuracy with comparable results with previously published reports (using GATK alone). Finally, we used HPC-GVCW in production mode to call SNPs on a “subpopulation aware” 16-genome rice reference panel with ~ 3000 resequenced rice accessions. The entire process took ~ 16 weeks and resulted in the identification of an average of 27.3 M SNPs/genome and the discovery of ~ 2.3 million novel SNPs that were not present in the flagship reference genome for rice (i.e., IRGSP RefSeq). Conclusions: This study developed an open-source pipeline (HPC-GVCW) to run GATK on HPC platforms, which significantly improved the speed at which SNPs can be called. The workflow is widely applicable as demonstrated successfully for four major crop species with genomes ranging in size from 400 Mb to 2.4 Gb. Using HPC-GVCW in production mode to call SNPs on a 25 multi-crop-reference genome data set produced over 1.1 billion SNPs that were publicly released for functional and breeding studies. For rice, many novel SNPs were identified and were found to reside within genes and open chromatin regions that are predicted to have functional consequences. Combined, our results demonstrate the usefulness of combining a high-performance SNP calling architecture solution with a subpopulation-aware reference genome panel for rapid SNP discovery and public deployment. © 2024, The Author(s).Open access journalThis item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

Pseudopolyrotaxane Formation in the Synthesis of Cyclodextrin Polymers: Effects on Drug Delivery, Mechanics, and Cell Compatibility

Numerous groups have reported the use of cyclodextrin (CD)-based polymers for drug delivery applications due to their capacity to form inclusions with small molecule drugs, delaying the rate of drug release beyond that of diffusion alone (termed “affinity-based” drug delivery). Herein we demonstrate synthesis and characterization of a new family of CD-based polymers, some as pseudopolyrotaxanes, generated under mild (aqueous, room temperature) conditions. The formation of these new affinity polymers results in broad mechanical properties. Three diglycidylether cross-linkers which vary in length from 0 to 10 ethylene glycol units were examined. Pseudopolyrotaxane formation was found only with the highest-length cross-linker, noted first by a sharp change in both material properties and then confirmed by chemical signature. Materials were thoroughly evaluated by NMR, DSC, DMA, TGA, XRD, and FTIR. Cross-linker choice was also tested for impact on drug loading and delivery capacity, using antibiotics as model drugs. Chemically similar polymers without showing affinity rapidly saturated in loading experiments, while affinity materials showing high capacity for drug loading, even beyond the solubility limit of the drugs. When using the polymers with these new cross-linkers, affinity-based drug delivery is maintained: the materials are capable of antibiotic delivery, and clearance of Staphylococcus aureus, at least an order of magnitude better than diffusion-only control polymers. In cell compatibility studies, CD-based polymers were shown to have low overt cell toxicity and even resisted cell adhesion, presumably due to their highly hydrated state

Comparative evaluation of human breast milk, bovine milk, and infant milk formula on cariogenicity in children: An in vivo study

Aim: The aim of the study was to compare the cariogenicity of human breast milk (HBM), bovine milk, and infant milk formulas. Patients and Methods: Ninety children of 1–4 years were randomly selected according to the type of milk they consume and were divided into five groups: Group I – HBM, Group II – cow milk, Group III – buffalo milk, Group IV – Lactogen 2, and Group V – Dexolac 4. Three parameters were assessed (salivary pH, plaque pH, and Streptococcus mutans count). Baseline salivary pH was measured, plaque sample was collected from children before feeding, and then, children were fed with milk. The second sample was collected after 45 min of feeding and the third sample after 3 h of second sample collection. Collected plaque samples were assessed for plaque pH and were sent to microbiological laboratory and cultured on blood agar. The intergroup comparison was done by one-way analysis of variance (ANOVA) and Tukey test. Intragroup comparison was done by one-way anova and Bonferroni test. P < 0.05 was considered statistically significant. Results: No statistically significant difference in intra- and intergroup comparisons of salivary pH was noted. However, with regard to plaque pH, there is a statistically significant difference in the second sample in Groups III, IV, and V. There was an increase in colony-forming units of S. mutans in plaque samples from baseline to the third sample in Groups IV and V. Conclusion: Lactogen 2 and Dexolac 4 showed greater cariogenic activity, buffalo milk is mildly cariogenic, whereas HBM and cow milk showed least cariogenicity

Differential requirements of singleplex and multiplex recombineering of large DNA constructs

Recombineering is an in vivo genetic engineering technique involving homologous recombination mediated by phage recombination proteins. The use of recombineering methodology is not limited by size and sequence constraints and therefore has enabled the streamlined construction of bacterial strains and multi-component plasmids. Recombineering applications commonly utilize singleplex strategies and the parameters are extensively tested. However, singleplex recombineering is not suitable for the modification of several loci in genome recoding and strain engineering exercises, which requires a multiplex recombineering design. Defining the main parameters affecting multiplex efficiency especially the insertion of multiple large genes is necessary to enable efficient large-scale modification of the genome. Here, we have tested different recombineering operational parameters of the lambda phage Red recombination system and compared singleplex and multiplex recombineering of large gene sized DNA cassettes. We have found that optimal multiplex recombination required long homology lengths in excess of 120 bp. However, efficient multiplexing was possible with only 60 bp of homology. Multiplex recombination was more limited by lower amounts of DNA than singleplex recombineering and was greatly enhanced by use of phosphorothioate protection of DNA. Exploring the mechanism of multiplexing revealed that efficient recombination required co-selection of an antibiotic marker and the presence of all three Red proteins. Building on these results, we substantially increased multiplex efficiency using an ExoVII deletion strain. Our findings elucidate key differences between singleplex and multiplex recombineering and provide important clues for further improving multiplex recombination efficiency

Lambda red mediated gap repair utilizes a novel replicative intermediate in Escherichia coli

The lambda phage Red recombination system can mediate efficient homologous recombination in Escherichia coli, which is the basis of the DNA engineering technique termed recombineering. Red mediated insertion of DNA requires DNA replication, involves a single-stranded DNA intermediate and is more efficient on the lagging strand of the replication fork. Lagging strand recombination has also been postulated to explain the Red mediated repair of gapped plasmids by an Okazaki fragment gap filling model. Here, we demonstrate that gap repair involves a different strand independent mechanism. Gap repair assays examining the strand asymmetry of recombination did not show a lagging strand bias. Directly testing an ssDNA plasmid showed lagging strand recombination is possible but dsDNA plasmids did not employ this mechanism. Insertional recombination combined with gap repair also did not demonstrate preferential lagging strand bias, supporting a different gap repair mechanism. The predominant recombination route involved concerted insertion and subcloning though other routes also operated at lower frequencies. Simultaneous insertion of DNA resulted in modification of both strands and was unaffected by mutations to DNA polymerase I, responsible for Okazaki fragment maturation. The lower efficiency of an alternate Red mediated ends-in recombination pathway and the apparent lack of a Holliday junction intermediate suggested that gap repair does not involve a different Red recombination pathway. Our results may be explained by a novel replicative intermediate in gap repair that does not involve a replication fork. We exploited these observations by developing a new recombineering application based on concerted insertion and gap repair, termed SPI (subcloning plus insertion). SPI selected against empty vector background and selected for correct gap repair recombinants. We used SPI to simultaneously insert up to four different gene cassettes in a single recombineering reaction. Consequently, our findings have important implications for the understanding of E. coli replication and Red recombination

Multiplex recombination is limited by availability of DNA template.

<p>(A) Insertion assay. A Gentamicin lagging strand protected cassette was inserted at a site of the <i>P2rx1</i> gene. (B) Multiplex insertion. Two different Gentamicin and Zeocin lagging strand protected cassettes were inserted at two different sites of the <i>P2rx1</i> gene. Recombination assays were performed with different amounts of each DNA cassette in the electroporation. Values represent averages; error bars indicate standard error of mean (<i>n</i> = 4). (C) Multiplex insertion PTO assay. Three different Zeocin, Gentamicin and Blasticidin resistance cassettes were PTO protected or unmodified and inserted at three different sites of the <i>P2rx1</i> gene (<i>n</i> = 3). (D) SPI PTO assay. SPI was performed using a p15A <i>dhfrII</i> subcloning plasmid and the Zeocin and Gentamicin cassettes used in (A) (<i>n</i> = 3).</p

Different types of recombineering processes.

<p>(A) Insertional recombination. A selection marker (sm) containing homology to a target site is inserted during the process of DNA replication (dashed line). Gap repair cloning. A gapped plasmid with terminal homology regions to a target site is used to subclone a sequence of interest. (C) Subcloning plus insertion (SPI). Insertion of a cassette occurs simultaneously during subcloning and generates a targeted subcloned plasmid. The template DNA remains unmodified.</p