Multi-nucleotide Polymorphisms Research Articles

Single-tube Ptprc SNP genotyping of JAXBoy (CD45.1) and C57BL/6J (CD45.2) mice by endpoint PCR and gel electrophoresis

Allelic variation at the Ptprc gene, which encodes the pan-leukocyte marker CD45/Ly5, is commonly exploited to track hematopoietic reconstitution by flow cytometry in mixed bone marrow chimera transplant experiments. Historically, this was accomplished using bone marrow from C57BL/6 (Ptprcb/CD45.2/Ly5.2) and congenic B6.SJL-PtprcaPepcb/Boy (Ptprca/CD45.1/Ly5.1) mice. Recently, the Jackson Laboratory directly CRISPR-engineered the Ptprca allele in C57BL/6J mice. This new isogenic strain, termed JAXBoy, differs from wild-type C57BL/6J mice by two nucleotides, compared to the biologically significant 37 megabase (Mb) SJL interval retained in B6.SJL-PtprcaPepcb/Boy/J mice. Currently, Ptprc/CD45 variants are identified by flow cytometry or allele-specific real-time PCR, both of which require specialized workflows and equipment compared to standard genotyping of endpoint PCR products by gel electrophoresis. Here, we employed allele-specific oligonucleotides in conjunction with differential incorporation of a long non-specific oligo 5′-tail to allow for simultaneous identification of the Ptprca and Ptprcb alleles using endpoint PCR and gel electrophoresis. This method allows for integration of Ptprc genotyping into standard genotyping workflows, which use a single set of thermocycling and gel electrophoresis conditions. Importantly, the strategy of primer placement and tail addition described here can be adapted to discriminate similar single- or multi-nucleotide polymorphisms at other genomic loci.

Native American genetic ancestry and pigmentation allele contributions to skin color in a Caribbean population.

Our interest in the genetic basis of skin color variation between populations led us to seek a Native American population with genetically African admixture but low frequency of European light skin alleles. Analysis of 458 genomes from individuals residing in the Kalinago Territory of the Commonwealth of Dominica showed approximately 55% Native American, 32% African, and 12% European genetic ancestry, the highest Native American genetic ancestry among Caribbean populations to date. Skin pigmentation ranged from 20 to 80 melanin units, averaging 46. Three albino individuals were determined to be homozygous for a causative multi-nucleotide polymorphism OCA2NW273KV contained within a haplotype of African origin; its allele frequency was 0.03 and single allele effect size was -8 melanin units. Derived allele frequencies of SLC24A5A111T and SLC45A2L374F were 0.14 and 0.06, with single allele effect sizes of -6 and -4, respectively. Native American genetic ancestry by itself reduced pigmentation by more than 20 melanin units (range 24-29). The responsible hypopigmenting genetic variants remain to be identified, since none of the published polymorphisms predicted in prior literature to affect skin color in Native Americans caused detectable hypopigmentation in the Kalinago.

VCF2CAPS-A high-throughput CAPS marker design from VCF files and its test-use on a genotyping-by-sequencing (GBS) dataset.

Next-generation sequencing (NGS) is a powerful tool for massive detection of DNA sequence variants such as single nucleotide polymorphisms (SNPs), multi-nucleotide polymorphisms (MNPs) and insertions/deletions (indels). For routine screening of numerous samples, these variants are often converted into cleaved amplified polymorphic sequence (CAPS) markers which are based on the presence versus absence of restriction sites within PCR products. Current computational tools for SNP to CAPS conversion are limited and usually infeasible to use for large datasets as those generated with NGS. Moreover, there is no available tool for massive conversion of MNPs and indels into CAPS markers. Here, we present VCF2CAPS–a new software for identification of restriction endonucleases that recognize SNP/MNP/indel-containing sequences from NGS experiments. Additionally, the program contains filtration utilities not available in other SNP to CAPS converters–selection of markers with a single polymorphic cut site within a user-specified sequence length, and selection of markers that differentiate up to three user-defined groups of individuals from the analyzed population. Performance of VCF2CAPS was tested on a thoroughly analyzed dataset from a genotyping-by-sequencing (GBS) experiment. A selection of CAPS markers picked by the program was subjected to experimental verification. CAPS markers, also referred to as PCR-RFLPs, belong to basic tools exploited in plant, animal and human genetics. Our new software–VCF2CAPS–fills the gap in the current inventory of genetic software by high-throughput CAPS marker design from next-generation sequencing (NGS) data. The program should be of interest to geneticists involved in molecular diagnostics. In this paper we show a successful exemplary application of VCF2CAPS and we believe that its usefulness is guaranteed by the growing availability of NGS services.

Fast sequence-based microsatellite genotyping development workflow.

Application of high-throughput sequencing technologies to microsatellite genotyping (SSRseq) has been shown to remove many of the limitations of electrophoresis-based methods and to refine inference of population genetic diversity and structure. We present here a streamlined SSRseq development workflow that includes microsatellite development, multiplexed marker amplification and sequencing, and automated bioinformatics data analysis. We illustrate its application to five groups of species across phyla (fungi, plant, insect and fish) with different levels of genomic resource availability. We found that relying on previously developed microsatellite assay is not optimal and leads to a resulting low number of reliable locus being genotyped. In contrast, de novo ad hoc primer designs gives highly multiplexed microsatellite assays that can be sequenced to produce high quality genotypes for 20–40 loci. We highlight critical upfront development factors to consider for effective SSRseq setup in a wide range of situations. Sequence analysis accounting for all linked polymorphisms along the sequence quickly generates a powerful multi-allelic haplotype-based genotypic dataset, calling to new theoretical and analytical frameworks to extract more information from multi-nucleotide polymorphism marker systems.

An improved encoding of genetic variation in a Burrows-Wheeler transform.

In resequencing experiments, a high-throughput sequencer produces DNA-fragments (called reads) and each read is then mapped to the locus in a reference genome at which it fits best. Currently dominant read mappers are based on the Burrows-Wheeler transform (BWT). A read can be mapped correctly if it is similar enough to a substring of the reference genome. However, since the reference genome does not represent all known variations, read mapping tends to be biased towards the reference and mapping errors may thus occur. To cope with this problem, Huang et al. encoded single nucleotide polymorphisms (SNPs) in a BWT by the International Union of Pure and Applied Chemistry (IUPAC) nucleotide code. In a different approach, Maciuca et al. provided a 'natural encoding' of SNPs and other genetic variations in a BWT. However, their encoding resulted in a significantly increased alphabet size (the modified alphabet can have millions of new symbols, which usually implies a loss of efficiency). Moreover, the two approaches do not handle all known kinds of variation. In this article, we propose a method that is able to encode many kinds of genetic variation (SNPs, multi-nucleotide polymorphisms, insertions or deletions, duplications, transpositions, inversions and copy-number variation) in a BWT. It takes the best of both worlds: SNPs are encoded by the IUPAC nucleotide code as in Huang et al. (2013, Short read alignment with populations of genomes. Bioinformatics, 29, i361-i370) and the encoding of the other kinds of genetic variation relies on the idea introduced in Maciuca et al. (2016, A natural encoding of genetic variation in a Burrows-Wheeler transform to enable mapping and genome inference. In: Proceedings of the 16th International Workshop on Algorithms in Bioinformatics, Volume 9838 of Lecture Notes in Computer Science, pp. 222-233. Springer). In contrast to Maciuca et al., however, we use only one additional symbol. This symbol marks variant sites in a chromosome and delimits multiple variants, which are added at the end of the 'marked chromosome'. We show how the backward search algorithm, which is used in BWT-based read mappers, can be modified in such a way that it can cope with the genetic variation encoded in the BWT. We implemented our method and compared it with BWBBLE and gramtools. https://www.uni-ulm.de/in/theo/research/seqana/.

Copy number variation and variant discovery in Bullmastiff dogs.

Identification of genomic variants within dogs is important for understanding genetic factors contributing to breed diversity and phenotypic traits. This study aimed to identify sources of variation in the Bullmastiff using high-density signal intensity and whole-genome sequence data. Close to 3000 copy number variants (CNVs) were identified in Bullmastiff dogs using Canine HD BeadChip data. When CNVs were collated, 82 CNV regions (CNVRs) were detected, 50% in transcribed regions encompassing 432 genes. Fifty of the CNVRs detected have not been reported in other breeds and represent potential breed-specific variants. A proportion of the CNVR variants with predicted modifying effects on gene pathways may contribute to breed traits. Approximately 5million putative variants per dog, inclusive of single nucleotide polymorphisms (SNPs), multi-nucleotide polymorphisms (MNPs) and insertion and deletions (INDELs), were identified from DNA sequence data on a small number of animals. Identification of genetic variants in the Bullmastiff highlights sources of variation in the breed and molecular markers that will assist in future trait and disease investigations in dogs.

Linked Mutations at Adjacent Nucleotides Have Shaped Human Population Differentiation and Protein Evolution

Despite the fundamental importance of single nucleotide polymorphisms (SNPs) to human evolution, there are still large gaps in our understanding of the forces that shape their distribution across the genome. SNPs have been shown to not be distributed evenly, with directly adjacent SNPs found unusually frequently. Why this is the case is unclear. We illustrate how neighboring SNPs that cannot be explained by a single mutation event (that we term here sequential dinucleotide mutations [SDMs]) are driven by distinct processes to SNPs and multinucleotide polymorphisms (MNPs). By studying variation across populations, including a novel cohort of 1,358 Scottish genomes, we show that, SDMs are over twice as common as MNPs and like SNPs display distinct mutational spectra across populations. These biases are not only different to those observed among SNPs and MNPs but are also more divergent between human population groups. We show that the changes that make up SDMs are not independent and identify a distinct mutational profile, CA → CG → TG, that is observed an order of magnitude more often than expected from background SNP rates and the numbers of other SDMs involving the gain and deamination of CpG sites. Intriguingly particular pathways through the amino acid code appear to have been favored relative to that expected from intergenic SDM rates and the occurrences of coding SNPs, and in particular those that lead to the creation of single codon amino acids. We finally present evidence that epistatic selection has potentially disfavored sequential nonsynonymous changes in the human genome.

Genome-wide discovery of DNA polymorphisms among chickpea cultivars with contrasting seed size/weight and their functional relevance

Seed size/weight is a major agronomic trait which determine crop productivity in legumes. To understand the genetic basis of seed size determination, we sought to identify DNA polymorphisms between two small (Himchana 1 and Pusa 362) and two large-seeded (JGK 3 and PG 0515) chickpea cultivars via whole genome resequencing. We identified a total of 75535 single nucleotide polymorphisms (SNPs), 6486 insertions and deletions (InDels), 1938 multi-nucleotide polymorphisms (MNPs) and 5025 complex variants between the two small and two large-seeded chickpea cultivars. Our analysis revealed 814, 244 and 72 seed-specific genes harboring DNA polymorphisms in promoter or non-synonymous and large-effect DNA polymorphisms, respectively. Gene ontology analysis revealed enrichment of cell growth and division related terms in these genes. Among them, at least 22 genes associated with quantitative trait loci, and those involved in cell growth and division and encoding transcription factors harbored promoter and/or large-effect/non-synonymous DNA polymorphisms. These also showed higher expression at late-embryogenesis and/or mid-maturation stages of seed development in the large-seeded cultivar, suggesting their role in seed size/weight determination in chickpea. Altogether, this study provided a valuable resource for large-scale genotyping applications and a few putative candidate genes that might play crucial role in governing seed size/weight in chickpea.

Comparative whole genome re-sequencing analysis in upland New Rice for Africa: insights into the breeding history and respective genome compositions

BackgroundIncreasing rice demand is one of the consequences of the steadily improving socio-economic status of the African countries. New Rice for Africa (NERICA), which are interspecific hybrids between Asian and African rice varieties, are one of successful breeding products utilizing biodiversity across the two different rice crop species. Upland NERICA varieties (NU) exhibit agronomic traits of value for the harsh eco-geography, including shorter duration, higher yield and stress tolerance, compared to local African varieties. However, the molecular basis of the traits in NU varieties is largely unknown.ResultsWhole genome re-sequencing was performed of four NU lines (3, 4, 5, and 7) and for the parental Oryza sativa WAB56–104 and Oryza glaberrima CG14. The k-mer analysis predicted large genomes for the four NU lines, most likely inherited from WAB56–104. Approximately 3.1, 0.10, and 0.40 million single nucleotide polymorphisms, multi nucleotide polymorphisms, and short insertions/deletions were mined between the parental lines, respectively. Integrated analysis with another four NU lines (1, 2, 8, and 9) showed that the ratios of the donor CG14 allelic sites in the NU lines ranged from 1.3 to 9.8%. High resolution graphical genotype indicated genome-level similarities and common genetic events during the breeding process: five xyloglucan fucosyltransferase from O. glaberrima were introgressed in common. Segregation of genic segments revealed potential causal genes for some agronomic traits including grain shattering, awnness, susceptibility to bacterial leaf bright, and salt tolerance. Analysis of unmapped sequences against the reference cultivar Nipponbare indicated existence of unique genes for pathogen and abiotic stress resistance in the NU varieties.ConclusionsThe results provide understanding of NU genomes for rice improvement for Africa reinforcing local capacity for food security and insights into molecular events in breeding of interspecific hybrid crops.

Canary: an atomic pipeline for clinical amplicon assays

BackgroundHigh throughput sequencing requires bioinformatics pipelines to process large volumes of data into meaningful variants that can be translated into a clinical report. These pipelines often suffer from a number of shortcomings: they lack robustness and have many components written in multiple languages, each with a variety of resource requirements. Pipeline components must be linked together with a workflow system to achieve the processing of FASTQ files through to a VCF file of variants. Crafting these pipelines requires considerable bioinformatics and IT skills beyond the reach of many clinical laboratories.ResultsHere we present Canary, a single program that can be run on a laptop, which takes FASTQ files from amplicon assays through to an annotated VCF file ready for clinical analysis. Canary can be installed and run with a single command using Docker containerization or run as a single JAR file on a wide range of platforms. Although it is a single utility, Canary performs all the functions present in more complex and unwieldy pipelines. All variants identified by Canary are 3′ shifted and represented in their most parsimonious form to provide a consistent nomenclature, irrespective of sequencing variation. Further, proximate in-phase variants are represented as a single HGVS ‘delins’ variant. This allows for correct nomenclature and consequences to be ascribed to complex multi-nucleotide polymorphisms (MNPs), which are otherwise difficult to represent and interpret. Variants can also be annotated with hundreds of attributes sourced from MyVariant.info to give up to date details on pathogenicity, population statistics and in-silico predictors.ConclusionsCanary has been used at the Peter MacCallum Cancer Centre in Melbourne for the last 2 years for the processing of clinical sequencing data. By encapsulating clinical features in a single, easily installed executable, Canary makes sequencing more accessible to all pathology laboratories.Canary is available for download as source or a Docker image at https://github.com/PapenfussLab/Canary under a GPL-3.0 License.

The Visual Colorimetric Detection of Multi-nucleotide Polymorphisms on a Pneumatic Droplet Manipulation Platform.

A simple and visual method to detect multi-nucleotide polymorphism (MNP) was performed on a pneumatic droplet manipulation platform on an open surface. This approach to colorimetric DNA detection was based on the hybridization-mediated growth of gold nanoparticle probes (AuNP probes). The growth size and configuration of the AuNP are dominated by the number of DNA samples hybridized with the probes. Based on the specific size- and shape-dependent optical properties of the nanoparticles, the number of mismatches in a sample DNA fragment to the probes is able to be discriminated. The tests were conducted via droplets containing reagents and DNA samples respectively, and were transported and mixed on the pneumatic platform with the controlled pneumatic suction of the flexible PDMS-based superhydrophobic membrane. Droplets can be delivered simultaneously and precisely on an open-surface on the proposed pneumatic platform that is highly biocompatible with no side effect of DNA samples inside the droplets. Combining the two proposed methods, the multi-nucleotide polymorphism can be detected at sight on the pneumatic droplet manipulation platform; no additional instrument is required. The procedure from installing the droplets on the platform to the final result takes less than 5 min, much less than with existing methods. Moreover, this combined MNP detection approach requires a sample volume of only 10 µl in each operation, which is remarkably less than that of a macro system.

The Visual Colorimetric Detection of Multi-nucleotide Polymorphisms on a Pneumatic Droplet Manipulation Platform

The Visual Colorimetric Detection of Multi-nucleotide Polymorphisms on a Pneumatic Droplet Manipulation Platform

Abstract B21: Targeted next-generation sequencing of hotspots in cancer genes in normal-tumor breast cancer patients of African ancestry reveals novel and known mutations

Abstract Introduction: Genomic profiling of primary tumors and their concomitant normal tissues has provided critical information about the mechanisms associated with carcinogenesis. Furthermore, while comprehensive analysis of breast tumors have yielded actionable findings, breast cancer (BCa) remains one of the most common cancers worldwide and BCa incidences and mortality rates vary by population reflecting health disparities resulting from socioeconomic and biologic factors. Given that African American (AA) BCa cases often display distinctly worse clinicopathological features, we hypothesize that interrogating tumors from AAs could significantly aid in understanding the drivers of BCa carcinogenesis specifically in this population. These findings would aid in the identification of significantly mutated genes leading to the development of interventions for improved drug targeting of tumors in these populations with African Ancestry that display an increase propensity for harboring these previously unknown somatic variants. Methods: For this study, DNA from 16 normal and tumor tissue pairs from the “African American Familial Breast Cancer Study” (AAFBC) were utilized. Using targeted next generation sequencing (NGS) technologies, libraries for the Ion AmpliSeq™Cancer Hotspot Panel v2 (CHPv2) panel were created using 20ng of DNA and each sample was assigned a unique barcode. The CHPv2 is a commercially available primer pool for targeted NGS that amplifies 207 regions within 50 oncogenes for a total of 31kb. The resulting libraries were quantified and pooled with unequal volumes to allow varying depth of coverage between the matched tumor and normal pairs. The samples were sequenced on the Ion Personal Genome Machine™ (PGM™) System with a 316v2 chip. Sequence data from the PGM was processed using the Ion Torrent Suite v4 software package. Single nucleotide variants (SNVs), multi-nucleotide polymorphisms (MNPs), insertions or deletions were identified using Torrent Variant Caller Plugin v.4.6.0.7. Results: Sequencing resulted in 3,241,580 total usable reads with an average length of 126bp. The average coverage was 667x for tumor tissues and 165x for matched normal tissues. There were 88 unique variants representing 528 total variants with an average of 16.5 variants per sample. Of these, there were 52 germline variants that were expressed by both the normal and tumor samples. Five of these variants displayed a loss of one allele resulting in one homozygous (FGFR3) and 4 heterozygous mutations (PDGFRA, STK11 and ERBB4). The remaining 36 variants were somatic mutations of which 20 were novel and 16 have been described previously. The coding consequences were 56% missense mutations, 28% synonymous mutations, 8% frameshifts and 6% in-frame deletions. The most common coding mutations found in at least 3 patients were in TP53, SMARCB1, CDKN2A, NOTCH1 and PIK3CA. Citation Format: Luisel Ricks-Santi, John McDonald, Cha'Tonya Brown, Latiffany Prince, Muneer Abbas, Georgia Dunston. Targeted next-generation sequencing of hotspots in cancer genes in normal-tumor breast cancer patients of African ancestry reveals novel and known mutations. [abstract]. In: Proceedings of the Eighth AACR Conference on The Science of Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; Nov 13-16, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2016;25(3 Suppl):Abstract nr B21.

Introgression browser: high-throughput whole-genome SNP visualization.

Breeding by introgressive hybridization is a pivotal strategy to broaden the genetic basis of crops. Usually, the desired traits are monitored in consecutive crossing generations by marker-assisted selection, but their analyses fail in chromosome regions where crossover recombinants are rare or not viable. Here, we present the Introgression Browser (iBrowser), a bioinformatics tool aimed at visualizing introgressions at nucleotide or SNP (Single Nucleotide Polymorphisms) accuracy. The software selects homozygous SNPs from Variant Call Format (VCF) information and filters out heterozygous SNPs, multi-nucleotide polymorphisms (MNPs) and insertion-deletions (InDels). For data analysis iBrowser makes use of sliding windows, but if needed it can generate any desired fragmentation pattern through General Feature Format (GFF) information. In an example of tomato (Solanum lycopersicum) accessions we visualize SNP patterns and elucidate both position and boundaries of the introgressions. We also show that our tool is capable of identifying alien DNA in a panel of the closely related S. pimpinellifolium by examining phylogenetic relationships of the introgressed segments in tomato. In a third example, we demonstrate the power of the iBrowser in a panel of 597 Arabidopsis accessions, detecting the boundaries of a SNP-free region around a polymorphic 1.17 Mbp inverted segment on the short arm of chromosome 4. The architecture and functionality of iBrowser makes the software appropriate for a broad set of analyses including SNP mining, genome structure analysis, and pedigree analysis. Its functionality, together with the capability to process large data sets and efficient visualization of sequence variation, makes iBrowser a valuable breeding tool.

PCR-CTPP design for enzyme-free SNP genotyping using memetic algorithm.

In recent years, many single nucleotide polymorphisms (SNPs) have been successfully genotyped by polymerase chain reaction with confronting two-pair primers (PCR-CTPP). However, computation experiments of feasible CTPP primers are still challenging. The melting temperatures between four primers must be within a very narrow range, and many primer constraints need to be conformed to. PCR-CTPP is a simple, time- and cost-effective SNP genotyping method applied in molecular biology and biomedical fields. In this study, an MA (memetic algorithm)-based method is proposed to enable the design of feasible CTPP primer sets. Overall, 288 SNPs which exclude the deletion/insertion polymorphisms (DIPs) and multi-nucleotide polymorphisms (MNPs) in the SLC6A4 gene were tested in silico. The results were compared with a GA (genetic algorithm)-based method and indicate that the proposed method provides more feasible CTPP primers than the GA-based method. The MA-based CTPP primer design method provides critical melting temperatures and all kinds of evaluation of the common primer constraints. It could conceivably assist biologists and other researchers in obtaining feasible CTTP primer sets. The MA-CTPP algorithm is implemented in JAVA and a user-friendly input interface is freely available at http://bio.kuas.edu.tw/ma-ctpp/.

A biocompatible open-surface droplet manipulation platform for detection of multi-nucleotide polymorphism.

We present a novel and simple method to manipulate droplets applicable to an open-surface microfluidic platform. The platform comprised a control module for pneumatic droplets and a superhydrophobic polydimethylsiloxane (PDMS) membrane. With pneumatic suction to cause deflection of the flexible PDMS-based superhydrophobic membrane, the sample and reagent droplets on the membrane become transported and mixed. A facile one-step laser micromachining technique serves to fabricate a superhydrophobic surface; a contact angle of 150° and a hysteresis angle of 4° were achieved without chemical modification. Relative to previous open-surface microfluidic systems, this platform is capable of simultaneous and precise delivery of droplets in two-dimensional (2D) manipulation. Droplets were manipulated with suction, which avoided interference from an external driving energy (e.g. heat, light, electricity) to affect the bio-sample inside the droplets. Two common bio-samples, namely protein and DNA, verified the performance of the platform. Based on the experimental results, operations on protein can be implemented without adsorption on the surface of the platform. Another striking result is the visual screening for multi-nucleotide polymorphism with hybridization-mediated growth of gold-nanoparticle (AuNP) probes. The detection results are observable with the naked eye, without the aid of advanced instruments. The entire procedure only takes 5 min from the addition of the sample and reagent to obtaining the results, which is much quicker than the traditional method. The total sample volume consumed in each operation is only 10 μL, which is significantly less than what is required in a large system. According to this approach, the proposed platform is suitable for biological and chemical applications.

A calibrated human Y-chromosomal phylogeny based on resequencing

We have identified variants present in high-coverage complete sequences of 36 diverse human Y chromosomes from Africa, Europe, South Asia, East Asia, and the Americas, representing eight major haplogroups. After restricting our analysis to 8.97 Mb of the unique male-specific Y sequence, we identified 6662 high-confidence variants, including single-nucleotide polymorphisms (SNPs), multi-nucleotide polymorphisms (MNPs), and indels. We constructed phylogenetic trees using these variants, or subsets of them, and recapitulated the known structure of the tree. Assuming a male mutation rate of 1 × 10−9 per base pair per year, the time depth of the tree (haplogroups A3-R) was ∼101,000–115,000 yr, and the lineages found outside Africa dated to 57,000–74,000 yr, both as expected. In addition, we dated a striking Paleolithic male lineage expansion to 41,000–52,000 yr ago and the node representing the major European Y lineage, R1b, to 4000–13,000 yr ago, supporting a Neolithic origin for these modern European Y chromosomes. In all, we provide a nearly 10-fold increase in the number of Y markers with phylogenetic information, and novel historical insights derived from placing them on a calibrated phylogenetic tree.

Defining the Role of Single Nucleotide Polymorphic (SNP) in Drug Development and Toxicityc

The sequencing of human genome has lead to define the variations among humans, which is quite astonishing. Before sequencing of human genome, variant among genes were observed as mutations. It was known that mutations in key regulatory and functional genes cause diseases. With the advent of single nucleotide polymorphism (SNP) among genes, which is believed to be present in every 1.0 kb in human genome (3X10 6 SNP in 3X10 9 bps genome), has changed the very paradigm of disease progression, drug action and toxicity. Before sequencing of the human genome, such variations were thought far less to exist. Although SNPs are found every 1.0 kb sequence but they are not uniformly distributed. In addition to be present in regulatory sequences, SNPs are found in introns and intergenic sequences. Determining the effects of these variants on biological process is a daunting task. Utilizing the modern molecular biological techniques, the role(s) of the regulatory SNPs has been defined for many genes. However, the structural SNPs, which are part of the coding sequences of genes, have been difficult to define. Models to describe the messenger RNA stability (Mfold analysis) due to such SNPs are welcome and can be used to define mRNA stability. However, such a correlation in terms of end product as protein is still confusing. Attempts have been made to decipher the gene function of individual SNP variant what is referred as “allelic expression variant” of haplotype as compared to wild type. In this context it is noteworthy that allelic variants have been found to possess varying activities. In addition to SNPs, multinucleotide polymorphism in terms of double nucleotide polymorphism (DNPs) and triple nucleotide polymorphism (TNPs) have also been observed in human genome. Frequency of DNPs and TNPs are far less as compare to SNPs. Comparison of DNPs and TNPs from Chinese and Venter data demonstrate that a total of 164 in Chinese and 127 in Venter as DNPs were found. A far less TNPs (3 with Chinese and 6 with Venter genome) were found. It is predicted that SNPs leads to nonsynonimous substitution with 50-50 occurrence while DNPs cause higher nonsynonimous substitutions. DNPs are predicted to convert stop codon into read through codon but both Chinese and Venture genome did not possess any read through DNPs [1].

Novel multi-nucleotide polymorphisms in the human genome characterized by whole genome and exome sequencing

Genomic sequence comparisons between individuals are usually restricted to the analysis of single nucleotide polymorphisms (SNPs). While the interrogation of SNPs is efficient, they are not the only form of divergence between genomes. In this report, we expand the scope of polymorphism detection by investigating the occurrence of double nucleotide polymorphisms (DNPs) and triple nucleotide polymorphisms (TNPs), in which two or three consecutive nucleotides are altered compared to the reference sequence. We have found such DNPs and TNPs throughout two complete genomes and eight exomes. Within exons, these novel polymorphisms are over-represented amongst protein-altering variants; nearly all DNPs and TNPs result in a change in amino acid sequence and, in some cases, two adjacent amino acids are changed. DNPs and TNPs represent a potentially important new source of genetic variation which may underlie human disease and they should be included in future medical genetics studies. As a confirmation of the damaging nature of xNPs, we have identified changes in the exome of a glioblastoma cell line that are important in glioblastoma pathogenesis. We have found a TNP causing a single amino acid change in LAMC2 and a TNP causing a truncation of HUWE1.

SNPs, Haplotypes, and Cancer: Applications in Molecular Epidemiology

SNPs, Haplotypes, and Cancer: Applications in Molecular Epidemiology