Sequence-based Polymorphisms Research Articles

Genome mutations of the Turkish strain Leishmania infantum_TR01.

Today, almost 15years have passed since the whole genome sequence (WGS) of a Leishmania parasite was completed for the first time. However, information on the genetics of these parasites remains to be elucidated. Genome-based studies may contribute to control strategies for leishmaniasis. The increase in genetically based studies, particularly whole-genome sequencing studies on Leishmania, will contribute to the control of leishmaniasis, which is a common and neglected disease worldwide. Our previous study obtained the Leishmania infantum_TR01 (Lin_TR01) genome sequence (Guldemir et al., 2020). In the present study, we focused on the mutations detected in the genome and aimed to investigate the effects of these mutations. In our previous study, the whole-genome sequence of the L. infantum_TR01 strain was obtained (Guldemir et al., 2020). In the present study, 3153 polymorphisms were detected in bioinformatics analysis performed on the Geneious 11.0.5. (www.geneious.com) platform. Herein, the L. infantum JPCM5 strain was used as the reference genome for genome mapping. Polymorphic regions were determined using the Find Variations/SNPs program on the Geneious platform. We further analyzed these polymorphisms detected in the previous study. Additionally, a literature review was performed by searching the PubMed database for proteins with polymorphisms. In our previous study (Guldemir et al., 2020), the genomic DNA sequence was submitted to the NCBI GenBank (www.ncbi.nlm.nih.gov) database and registered under the name Leishmania infantum_TR01 (Lin_TR01) and project accession number PRJNA437593. As a result of the annotation of the genome, 3153 polymorphisms were identified. In this study, 166 protein-coding polymorphisms were found among the 3153 polymorphisms, affecting 63 different proteins. Fourteen of them were studied, and the remaining 49 proteins were not studied. The 14 proteins examined in terms of the mutations detected in this study were related to virulence (n=5), vaccine candidates (n=2), diagnosis/typing (n=4), drug resistance (n=2), drug targets (n=3) and vital function (n=1). As mentioned previously, the acquisition of the Lin_TR01 genome was described in our previous study (Guldemir et al., 2020). The present meta-analytical study is the first comparison report of whole-genome sequence-based polymorphisms between the Turkish strain Leishmania infantum_TR01 and reference Leishmania infantum JPCM5 strain and evaluated polymorphisms and proteins. In this study, we focused on the mutations detected in the genome, and the effects of these mutations were investigated and evaluated together with the current literature. In our previous study, a high-quality WGS of Leishmania infantum was successfully obtained for the first time in Turkey (1). In this study, the comparison of both genomes will contribute to providing the scientific community with a solid infrastructure for postgenomic investigations of the parasite.

Enhancing forensic efficiency of STR typing using mass spectrometry-based assay

Using the Abbott PLEX-ID PCR/mass spectrometry-based STR assay covering a total of 13 CODIS loci, we typed 310 blood samples from unrelated Russian individuals all had full STR profiles generated with the conventional ABI Identifier Plus/CE system. The MS platform provides for determination of PCR product base compositions thus enabling to determine when an allele has a SNP within the amplified region. Sequence-based polymorphisms relative to the reference alleles were seen in 10 of the 13 loci tested. Up to 15 variant alleles in particular locus were observed. At the 8 loci at least one representative heterozygous individual was found for which the locus is called homozygous by conventional typing. The results demonstrate potential advantages of the sequence-based polymorphisms assay in resolution of samples when limited number of loci can be STR-typed.

Sequence-Based Polymorphisms in the Mitochondrial D-Loop and Potential SNP Predictors for Chronic Dialysis

BackgroundThe mitochondrial (mt) displacement loop (D-loop) is known to accumulate structural alterations and mutations. The aim of this study was to investigate the prevalence of single nucleotide polymorphisms (SNPs) within the D-loop among chronic dialysis patients and healthy controls.Methodology and Principal FindingsWe enrolled 193 chronic dialysis patients and 704 healthy controls. SNPs were identified by large scale D-loop sequencing and bioinformatic analysis. Chronic dialysis patients had lower body mass index, blood thiols, and cholesterol levels than controls. A total of 77 SNPs matched with the positions in reference of the Revised Cambridge Reference Sequence (CRS) were found in the study population. Chronic dialysis patients had a significantly higher incidence of 9 SNPs compared to controls. These include SNP5 (16108Y), SNP17 (16172Y), SNP21 (16223Y), SNP34 (16274R), SNP35 (16278Y), SNP55 (16463R), SNP56 (16519Y), SNP64 (185R), and SNP65 (189R) in D-loop of CRS. Among these SNPs with genotypes, SNP55-G, SNP56-C, and SNP64-A were 4.78, 1.47, and 5.15 times more frequent in dialysis patients compared to controls (P<0.05), respectively. When adjusting the covariates of demographics and comorbidities, SNP64-A was 5.13 times more frequent in dialysis patients compared to controls (P<0.01). Furthermore, SNP64-A was found to be 35.80, 3.48, 4.69, 5,55, and 4.67 times higher in female patients and in patients without diabetes, coronary artery disease, smoking, and hypertension in an independent significance manner (P<0.05), respectively. In patients older than 50 years or with hypertension, SNP34-A and SNP17-C were found to be 7.97 and 3.71 times more frequent (P<0.05) compared to patients younger than 50 years or those without hypertension, respectively.Conclusions and SignificanceThe results of large-scale sequencing suggest that specific SNPs in the mtDNA D-loop are significantly associated with chronic dialysis. These SNPs can be considered as potential predictors for chronic dialysis.

Sequence based polymorphic (SBP) marker technology for targeted genomic regions: its application in generating a molecular map of the Arabidopsis thaliana genome

BackgroundMolecular markers facilitate both genotype identification, essential for modern animal and plant breeding, and the isolation of genes based on their map positions. Advancements in sequencing technology have made possible the identification of single nucleotide polymorphisms (SNPs) for any genomic regions. Here a sequence based polymorphic (SBP) marker technology for generating molecular markers for targeted genomic regions in Arabidopsis is described.ResultsA ~3X genome coverage sequence of the Arabidopsis thaliana ecotype, Niederzenz (Nd-0) was obtained by applying Illumina's sequencing by synthesis (Solexa) technology. Comparison of the Nd-0 genome sequence with the assembled Columbia-0 (Col-0) genome sequence identified putative single nucleotide polymorphisms (SNPs) throughout the entire genome. Multiple 75 base pair Nd-0 sequence reads containing SNPs and originating from individual genomic DNA molecules were the basis for developing co-dominant SBP markers. SNPs containing Col-0 sequences, supported by transcript sequences or sequences from multiple BAC clones, were compared to the respective Nd-0 sequences to identify possible restriction endonuclease enzyme site variations. Small amplicons, PCR amplified from both ecotypes, were digested with suitable restriction enzymes and resolved on a gel to reveal the sequence based polymorphisms. By applying this technology, 21 SBP markers for the marker poor regions of the Arabidopsis map representing polymorphisms between Col-0 and Nd-0 ecotypes were generated.ConclusionsThe SBP marker technology described here allowed the development of molecular markers for targeted genomic regions of Arabidopsis. It should facilitate isolation of co-dominant molecular markers for targeted genomic regions of any animal or plant species, whose genomic sequences have been assembled. This technology will particularly facilitate the development of high density molecular marker maps, essential for cloning genes based on their genetic map positions and identifying tightly linked molecular markers for selecting desirable genotypes in animal and plant breeding experiments.

Assignment of Reference 5`-end 16S rDNA Sequences and Species-Specific Sequence Polymorphisms Improves Species Identification of Nocardia

16S rDNA sequence analysis is the most accurate method for definitive species identification of nocardiae. However, conflicting results can be found due to sequence errors in gene databases. This study tested the feasibility of species identification of Nocardia by partial (5’-end 606-bp) 16S rDNA sequencing, based on sequence comparison with “reference” sequences of well-annotated strains. This new approach was evaluated using 96 American Type Culture Collection (n=6), and clinical (n=90) Nocardia isolates. Nucleotide sequence-based polymorphisms within species were indicative of “sequence types” for that species. Sequences were compared with those in the GenBank, Bioinformatics Bacteria Identification and Ribosomal Database Project databases. Compared with the reference sequence set, all 96 isolates were correctly identified using the criterion of ≥99% sequence similarity. Seventy-eight (81.3%) were speciated by database comparison; alignment with reference sequences resolved the identity of 14 (15%) isolates whose sequences yielded 100% similarity to sequences in GenBank under >1 species designation. Of 90 clinical isolates, the commonest species was Nocardia nova (33.3%) followed by Nocardia cyriacigeorgica (26.7%). Recently-described or uncommon species included Nocardia veterana (4.4%), Nocarida bejingensis (2.2%) and, Nocardia abscessus and Nocardia arthriditis (each n=1). Nocardia asteroides sensu stricto was rare (n=1). There were nine sequence types of N. nova, three of Nocardia brasiliensis with two each of N. cyriacigeorgica and Nocardia farcinica. Thirteen novel sequences were identified. Alignment of sequences with reference sequences facilitated species identification of Nocardia and allowed delineation of sequence types within species, suggesting that such a barcoding approach can be clinically useful for identification of bacteria.

Population-Based Resequencing Reveals That the Flowering Time Adaptation of Cultivated Barley Originated East of the Fertile Crescent

Gene resequencing and association analysis present new opportunities to study the evolution of adaptive traits in crop plants. Here we apply these tools to an extensive set of barley accessions to identify a component of the molecular basis of the flowering time adaptation, a trait critical to plant survival. Using an association-based study to relate variation in flowering time to sequence-based polymorphisms in the Ppd-H1 gene, we identify a causative polymorphism (SNP48) that accounts for the observed variation in barley flowering time. This polymorphism also shows latitude-dependent geographical distribution, consistent with the expected clinal variation in phenotype with the nonresponsive form predominating in the north. Networks, genealogies, and phylogenetic trees drawn for the Ppd-H1 haplotypes reveal population structure both in wild barley and in domesticated barley landraces. The spatial distribution of these population groups indicates that phylogeographical analysis of European landraces can provide information relevant to the Neolithic spread of barley cultivation and also has implications for the origins of domesticated barley, including those with the nonresponsive ppd-H1 phenotype. Haplotypes containing the nonresponsive version of SNP48 are present in wild barley accessions, indicating that the nonresponsive phenotype of European landraces originated in wild barley. The wild accessions whose nonresponsive haplotypes are most closely similar to those of landraces are found in Iran, within a region suggested as an area for domestication of barley east of the Fertile Crescent but which has previously been thought to have contributed relatively little to the diversity of European cultivars.

Sequence divergence of rice microsatellites in Oryza and other plant species.

To determine the basis of genetic variation at microsatellite loci, eleven primer pairs, developed to amplify microsatellite markers in rice, were evaluated for their ability to amplify a PCR product and for both electromorphic and sequence-based polymorphism of the resulting products in 12 plant samples, including representatives from six different species within the genus Oryza and one genotype each from Zea (maize), Triticum (wheat) and Arabidopsis. PCR amplification was reliable in the four O. sativa samples as well as in the closely related Oryza relatives with AA genomes, while only 73% (8/11) of primers amplified in the BB/CC and CC genomes of Oryza, and 27% (3/11) amplified in the other genera. Three out of seven DNA fragments that were amplified from all genera were determined to be orthologous to their rice counterparts. A total of 115 amplicons were detected using polyacrylamide gel electrophoresis and these clustered into 74 distinct electromorphs. Sequencing of 108 amplicons revealed size homoplasy, exposing 13 new sequence-based variants. Allelic diversity within a species was predominantly due to changes in the number of repeats in the microsatellite region, but the frequency of insertions/deletions (indels) and base substitutions increased as the genetic distance between samples increased. This study suggests that electromorph size polymorphism is an adequate measure of genetic difference in studies involving closely-related individuals, but that when phylogenetic or evolutionary inferences are being made over longer time scales, evaluation of SSR variation at the sequence level is essential.

The Application of Genetic Diagnostics to Plant Genome Analysis and Plant Breeding

DNA-based diagnostics are now well-established as a means to assay diversity at the locus, chromosome, and whole-genome levels. As technology has advanced, DNA sequence-based assays have become easier to use, more efficient at screening for nucleotide sequence-based polymorphisms, and available to a wider cross-section of the research community. A review of the use of molecular markers in several different areas of genetics and plant breeding will be presented, as well as a discussion about their advantages and limitations. Recent advances in several areas of technology development and laboratory automation will also be presented, including a summary of direct comparison of different DNA marker systems against a common set of soybean cultivars.

High Prevalence of Cleft Lip among the Barí Indians of Western Venezuela

Cleft lip with or without cleft palate (CL +/- P) is of high prevalence in an isolated, inbred indigenous population of the Venezuelan rain forest: 12 contemporary probands are known in a population of 1200. CL +/- P is familial in this population. One kindred contains four affected siblings and a total of eight probands. The mode of transmission of CL +/- P is under investigation through the collection and biological verification of genealogies and through screening for selected sequence-based polymorphisms. Polymerase chain reaction (PCR) assays were successfully applied to recover DNA from blood dried onto filter paper in the field. Eleven samples from probands and their first-degree relatives have been analyzed for DNA sequence-based polymorphisms for TGF-A and HOX7. No significant linkages for either locus were found. Evaluation of the distribution of probands in four genealogies indicates that CL +/- P could be transmitted as an autosomal recessive trait in this population. We believe this is the first report of this mode of transmission for nonsyndromic CL +/- P.

High prevalence of cleft lip among the Bari Indians of western Venezuela.

Cleft lip with or without cleft palate (CL +/- P) is of high prevalence in an isolated, inbred indigenous population of the Venezuelan rain forest: 12 contemporary probands are known in a population of 1200. CL +/- P is familial in this population. One kindred contains four affected siblings and a total of eight probands. The mode of transmission of CL +/- P is under investigation through the collection and biological verification of genealogies and through screening for selected sequence-based polymorphisms. Polymerase chain reaction (PCR) assays were successfully applied to recover DNA from blood dried onto filter paper in the field. Eleven samples from probands and their first-degree relatives have been analyzed for DNA sequence-based polymorphisms for TGF-A and HOX7. No significant linkages for either locus were found. Evaluation of the distribution of probands in four genealogies indicates that CL +/- P could be transmitted as an autosomal recessive trait in this population. We believe this is the first report of this mode of transmission for nonsyndromic CL +/- P.