Direct haplotyping of bi-allelic SNPs using ARMS and RFLP analysis techniques

Haplotype analysis of single nucleotide polymorphisms (SNPs) is an important and rapidly growing approach for association studies. In recent years, statistical procedures to haplotype determination from genotypic information have employed in population studies. These procedures, even though some advantages for estimation of haplotype frequencies in large population samples, have limitations in the accuracy of the analysis. In this study, we have designed a reliable method for direct haplotyping of polymorphic sites using the amplification refractory mutation system (ARMS) and restriction fragment length polymorphism (RFLP) analysis techniques. We applied the method to determination of haplotypes composed of three SNPs within the paraoxonase1 gene promoter and found the approach can be used in many studies in population and in a variety of clinical settings.     

Global dinucleotide signatures and analysis of genomic heterogeneity

Early biochemical experiments measuring nearest neighbor frequencies established that the set of dinucleotide relative abundance values (dinucleotide biases) is a remarkably stable property of the DNA of an organism. Analyses of currently available genomic sequence data have extended these earlier results, showing that the dinucleotide biases evaluated for successive 50 kb segments of a genome are significantly more similar to each other than to those of sequences from more distant organisms. From this perspective, the set of dinucleotide biases constitutes a 'genomic signature' that can discriminate sequences from different organisms. The dinucleotide biases appear to reflect species-specific properties of DNA stacking energies, modification, replication, and repair mechanisms. The genomic signature is useful for detecting pathogenicity islands in bacterial genomes.     

Direct haplotyping of kilobase-size DNA using carbon nanotube probes

We have implemented a method for multiplexed detection of polymorphic sites and direct determination of haplotypes in 10-kilobase-size DNA fragments using single-walled carbon nanotube (SWNT) atomic force microscopy (AFM) probes. Labeled oligonucleotides are hybridized specifically to complementary target sequences in template DNA, and the positions of the tagged sequences are detected by direct SWNT tip imaging. We demonstrated this concept by detecting streptavidin and IRD800 labels at two different sequences in M13mp18. Our approach also permits haplotype determination from simple visual inspection of AFM images of individual DNA molecules, which we have done on UGT1A7, a gene under study as a cancer risk factor. The haplotypes of individuals heterozygous at two critical loci, which together influence cancer risk, can be easily and directly distinguished from AFM images. The application of this technique to haplotyping in population-based genetic disease studies and other genomic screening problems is discussed.     

Single-cell genome-wide concurrent haplotyping and copy-number profiling through genotyping-by-sequencing

Single-cell whole-genome haplotyping allows simultaneous detection of haplotypes associated with monogenic diseases, chromosome copy-numbering and subsequently, has revealed mosaicism in embryos and embryonic stem cells. Methods, such as karyomapping and haplarithmisis, were deployed as a generic and genome-wide approach for preimplantation genetic testing (PGT) and are replacing traditional PGT methods. While current methods primarily rely on single-nucleotide polymorphism (SNP) array, we envision sequencing-based methods to become more accessible and cost-efficient. Here, we developed a novel sequencing-based methodology to haplotype and copy-number profile single cells. Following DNA amplification, genomic size and complexity is reduced through restriction enzyme digestion and DNA is genotyped through sequencing. This single-cell genotyping-by-sequencing (scGBS) is the input for haplarithmisis, an algorithm we previously developed for SNP array-based single-cell haplotyping. We established technical parameters and developed an analysis pipeline enabling accurate concurrent haplotyping and copy-number profiling of single cells. We demonstrate its value in human blastomere and trophectoderm samples as application for PGT for monogenic disorders. Furthermore, we demonstrate the method to work in other species through analyzing blastomeres of bovine embryos. Our scGBS method opens up the path for single-cell haplotyping of any species with diploid genomes and could make its way into the clinic as a PGT application.     

Genetic analysis of biological pathway data through genomic randomization

Genome Wide Association Studies (GWAS) are a standard approach for large-scale common variation characterization and for identification of single loci predisposing to disease. However, due to issues of moderate sample sizes and particularly multiple testing correction, many variants of smaller effect size are not detected within a single allele analysis framework. Thus, small main effects and potential epistatic effects are not consistently observed in GWAS using standard analytical approaches that consider only single SNP alleles. Here, we propose unique methodology that aggregates variants of interest (for example, genes in a biological pathway) using GWAS results. Multiple testing and type I error concerns are minimized using empirical genomic randomization to estimate significance. Randomization corrects for common pathway-based analysis biases, such as SNP coverage and density, linkage disequilibrium, gene size and pathway size. Pathway Analysis by Randomization Incorporating Structure (PARIS) applies this randomization and in doing so directly accounts for linkage disequilibrium effects. PARIS is independent of association analysis method and is thus applicable to GWAS datasets of all study designs. Using the KEGG database as an example, we apply PARIS to the publicly available Autism Genetic Resource Exchange GWAS dataset, revealing pathways with a significant enrichment of positive association results.     

Direct molecular haplotyping by melting curve analysis of hybridization probes: beta 2-adrenergic receptor haplotypes as an example

Direct determination of the association of multiple genetic polymorphisms, or haplotyping, in individual samples is challenging because of chromosome diploidy. Here, we describe the ability of hybridization probes, commonly used as genotyping tools, to establish single nucleotide polymorphism (SNP) haplotypes in a single step. Three haplotypes found in the beta 2-adrenergic receptor (β2AR) gene and characterized by three different SNPs combinations are presented as examples. Each combination of SNPs has a unique stability, recorded by its melting temperature, even when intervening sequences from the template must loop out during probe hybridization. In the course of this study, two haplotypes in β2AR not described previously were discovered. This approach provides a tool for molecular haplotyping that should prove useful in clinical molecular genetics diagnostics and pharmacogenetic research where methods for direct haplotyping are needed.     

Detection of genomic islands via segmental genome heterogeneity

While the recognition of genomic islands can be a powerful mechanism for identifying genes that distinguish related bacteria, few methods have been developed to identify them specifically. Rather, identification of islands often begins with cataloging individual genes likely to have been recently introduced into the genome; regions with many putative alien genes are then examined for other features suggestive of recent acquisition of a large genomic region. When few phylogenetic relatives are available, the identification of alien genes relies on their atypical features relative to the bulk of the genes in the genome. The weakness of these ‘bottom–up’ approaches lies in the difficulty in identifying robustly those genes which are atypical, or phylogenetically restricted, due to recent foreign ancestry. Herein, we apply an alternative ‘top–down’ approach where bacterial genomes are recursively divided into progressively smaller regions, each with uniform composition. In this way, large chromosomal regions with atypical features are identified with high confidence due to the simultaneous analysis of multiple genes. This approach is based on a generalized divergence measure to quantify the compositional difference between segments in a hypothesis-testing framework. We tested the proposed genome island prediction algorithm on both artificial chimeric genomes and genuine bacterial genomes.     

Intratumor heterogeneity and clonal evolution revealed in castration-resistant prostate cancer by longitudinal genomic analysis

Intratumor heterogeneity is a key driver for local relapse and treatment failure. Thus, using multifocal prostate cancer as a model to investigate tumor inter-clonal relationships and tumor evolution could aid in our understanding of drug resistance. Previous studies discovered genomic alterations by comparing hormone-sensitive prostate cancer (HSPC) with castration-resistant prostate cancer (CRPC) in large cohorts. However, most studies did not sequentially sample tumors from the same patient. In our study, we performed whole-exome sequencing (WES) on 14 specimens from five locally relapsed patients before and after androgen-deprivation therapy. We described the landscape of genomic alterations before and after treatment and identified critical driver events that could have contributed to the evolution of CRPC. In addition to confirming known cancer genes such as TP53 and CDK12, we also identified new candidate genes that may play a role in the progression of prostate cancer, including MYO15A, CHD6 and LZTR1. At copy number alteration (CNA) level, gain of 8q24.13-8q24.3 was observed in 60% of patients and was the most commonly altered locus in both HSPC and CRPC tumors. Finally, utilizing phylogenetic reconstruction, we explored the clonal progression pattern from HSPC to CRPC in each patient. Our findings highlight the complex and heterogeneous mechanisms underlying the development of drug resistance, and underscore the potential value of monitoring tumor clonal architectures during disease progression in a clinical setting.     

Direct observation of positive supercoils introduced by reverse gyrase through atomic force microscopy

Reverse gyrase is a hyperthermophilic enzyme that can introduce positive supercoiling in substrate DNA. It is showed in our studies that positive DNA supercoils were induced in both pBR322 vector and an artificially synthesized mini-plasmid DNA by reverse gyrase. The left-handed structures adopted by positively supercoiled DNA molecules could be identified from their right-handed topoisomers through atomic force microscopic examination. Additional structural comparisons revealed that positively supercoiled DNA molecule AFM images exhibited increased contour lengths. Moreover, enzymatic assays showed that the positively supercoiled DNA could not be cleaved by T7 endonuclease. Together, this suggests that the overwound structure of positive supercoils could prevent genomic duplex DNA from randomly forming single-stranded DNA regions and intra-stranded secondary structures.     

Insights on genomic diversity of Vibrio spp. through Pan-genome analysis

Purpose

The aquaculture sector is a major contributor to the economic and nutritional security for a number of countries. India’s total seafood exports for the year 2017–2018 accounted for US$ Million 7082. One of the major setbacks in this sector is the frequent outbreaks of diseases often due to bacterial pathogens. Vibriosis is one of the major diseases caused by bacteria of Vibrio spp., causing significant economic loss to the aquaculture sector. The objective of this study was to understand the genetic composition of Vibrio spp.

Methods

Thirty-five complete genomes were downloaded from GenBank comprising seven vibrio species, namely, Vibrio alginolyticus, V. anguillarum, V. campbellii, V. harveyi, V. furnissii, V. parahaemolyticus, and V. vulnificus. Pan-genome analysis was carried out with coding sequences (CDS) generated from all the Vibrio genomes. In addition, genomes were mined for genes coding for toxin-antitoxin systems, antibiotic resistance, genomic islands, and virulence factors.

Results

Results revealed an open pan-genome comprising of 2004 core, 8249 accessory, and 6780 unique genes. Downstream analysis of genomes and the identified unique genes resulted in 312 antibiotic resistance genes, 430 genes coding for toxin and antitoxin systems along with 4802, and 4825 putative virulent genes from genomic island regions and unique gene sets, respectively.

Conclusion

Pan-genome and other downstream analytical procedures followed in this study have the potential to predict strain-specific genes and their association with habitat and pathogenicity.

     

Genotyping and haplotyping of polymorphisms directly from genomic DNA via coupled amplification and sequencing (CAS).

Coupled amplification and sequencing (CAS) allows a segment of DNA to be sequenced directly from genomic DNA. An initial PCR amplification stage selects and amplifies the target. During a subsequent stage both strands of the target segment are sequenced simultaneously and amplified further. We show that CAS can readily identify variant base pairs. Genotyping of a population for known sequence variation can be achieved simply and directly from genomic DNA of each organism by performing CAS only for the variant bases. The procedure supercedes development and optimization of alternative typing assays based on oligonucleotide hybridization or ligation. In addition, we show that competitive oligonucleotide priming with allelic primers can be readily performed in concert with the second stage of CAS. The combination of techniques allows sequencing of a single chromosome from a heterozygous genomic sample and direct haplotyping of the polymorphism at the priming site with any others encompassed within the amplified segment.     

Direct detection of methylation in genomic DNA

The identification of methylated sites on bacterial genomic DNA would be a useful tool to study the major roles of DNA methylation in prokaryotes: distinction of self and nonself DNA, direction of post-replicative mismatch repair, control of DNA replication and cell cycle, and regulation of gene expression. Three types of methylated nucleobases are known: N⁶-methyladenine, 5-methylcytosine and N⁴-methylcytosine. The aim of this study was to develop a method to detect all three types of DNA methylation in complete genomic DNA. It was previously shown that N⁶-methyladenine and 5-methylcytosine in plasmid and viral DNA can be detected by intersequence trace comparison of methylated and unmethylated DNA. We extended this method to include N⁴-methylcytosine detection in both in vitro and in vivo methylated DNA. Furthermore, application of intersequence trace comparison was extended to bacterial genomic DNA. Finally, we present evidence that intrasequence comparison suffices to detect methylated sites in genomic DNA. In conclusion, we present a method to detect all three natural types of DNA methylation in bacterial genomic DNA. This provides the possibility to define the complete methylome of any prokaryote.     

Structural heterogeneity and genomic distribution of Drosophila melanogaster LTR-retrotransposons

Structural heterogeneity of five long terminal repeat (LTR) retrotransposon families (297, mdg 1, 412, copia, and 1731) was investigated in Drosophila melanogaster. The genomic distribution of canonical and rearranged elements was studied by comparing hybridization patterns of Southern blots on salivary glands from adult females and males with in situ hybridization on polytene chromosomes. The proportion and genomic distribution of noncanonical copies is distinctive to each family and presents constant features in the four different D. melanogaster strains studied. Most elements of families 297 and mdg 1 were noncanonical and presented large interstock and intrastock polymorphism. Noncanonical elements of these two families were mostly located in euchromatin, although not restricted to it. The elements of families 412 and copia were better conserved. The proportion of noncanonical elements was lower. The 1731 family is mainly composed of noncanonical, beta-heterochromatic elements that are highly conserved among stocks. The relation of structural polymorphism to phylogeny, transpositional activity and the role of natural selection in the maintenance of transposable elements are discussed.     

Probing the S100 protein family through genomic and functional analysis

The EF-hand superfamily of calcium binding proteins includes the S100, calcium binding protein, and troponin subfamilies. This study represents a genome, structure, and expression analysis of the S100 protein family, in mouse, human, and rat. We confirm the high level of conservation between mammalian sequences but show that four members, including S100A12, are present only in the human genome. We describe three new members of the S100 family in the three species and their locations within the S100 genomic clusters and propose a revised nomenclature and phylogenetic relationship between members of the EF-hand superfamily. Two of the three new genes were induced in bone-marrow-derived macrophages activated with bacterial lipopolysaccharide, suggesting a role in inflammation. Normal human and murine tissue distribution profiles indicate that some members of the family are expressed in a specific manner, whereas others are more ubiquitous. Structure-function analysis of the chemotactic properties of murine S100A8 and human S100A12, particularly within the active hinge domain, suggests that the human protein is the functional homolog of the murine protein. Strong similarities between the promoter regions of human S100A12 and murine S100A8 support this possibility. This study provides insights into the possible processes of evolution of the EF-hand protein superfamily. Evolution of the S100 proteins appears to have occurred in a modular fashion, also seen in other protein families such as the C2H2-type zinc-finger family.     

Direct genomic fluorescent on-line sequencing and analysis using in vitro amplification of DNA.

In vitro amplification of genomic DNA and total RNA, as well as recombinant DNA, using one fluorescently labelled and one unlabelled primer during amplification, together with on-line analysis of the products on the EMBL fluorescent DNA sequencer, is described. Further is reported direct sequencing of fluorescently labelled amplified probes by solid-phase chemical degradation, without subcloning and purification steps involved. At present up to 350 bases in 4 hours are determined with this technique. The fluorescent dye and its bond to the oligonucleotide are stable during the amplification cycles, and do not interfere with the enzymatic polymerization. High sensitivity of the detection device, down to 10(-18) moles, corresponding to less than 10(6) molecules makes possible analyses of the non-radioactive amplified probes after only 10 amplification cycles, starting with about 5 x 10(4) copies of recombinant DNA.