WisecondorFF: Improved Fetal Aneuploidy Detection from Shallow WGS through Fragment Length Analysis

In prenatal diagnostics, NIPT screening utilizing read coverage-based profiles obtained from shallow WGS data is routinely used to detect fetal CNVs. From this same data, fragment size distributions of fetal and maternal DNA fragments can be derived, which are known to be different, and often used to infer fetal fractions. We argue that the fragment size has the potential to aid in the detection of CNVs. By integrating, in parallel, fragment size and read coverage in a within-sample normalization approach, it is possible to construct a reference set encompassing both data types. This reference then allows the detection of CNVs within queried samples, utilizing both data sources. We present a new methodology, WisecondorFF, which improves sensitivity, while maintaining specificity, relative to existing approaches. WisecondorFF increases robustness of detected CNVs, and can reliably detect even at lower fetal fractions (<2%).

Comparison of Direct Sequencing with Real-time PCR High Resolution Melt and PCR Restriction Fragment Length Polymorphism Analysis to Identify Clinically Important Candida Species

Background: Candida albicans is the predominant yeast reported from human infection. Non-albicans Candida species have been recently developed as medically vital fungi. Therefore, it is essential to detect and identify the pathogens at the species level to prescribe appropriate treatment. Methods: This study assessed two complementary methods, including real-time polymerase chain reaction-high resolution melt (PCR-HRM) and polymerase chain reaction-restriction fragment length morphism (PCR-RFLP) with standard PCR and Sanger sequencing as the benchmark. Results: In total, 66 samples were tested, and two newly-advanced assays were more effective and displayed comprehensive concordance (66/66, 100%) with Sanger sequencing outcomes. Moreover, accurate and economical tests were positively advanced by real-time PCR-HRM for C. albicans and C. parapsilosis complexes. Conclusions: Given the number of studies performed on the comparison of sensitivity and specificity of phenotypic and genotypic methods to diagnose and identify invasive fungal pathogens and the findings of this study, it could be stated that the correlative PCR-HRM and PCR-RFLP methods were effectively advanced as substitutes for conventional Sanger sequencing for the reasonable identification. However, supplementary evaluations and confirming studies should be carried out with a broad range of samples to standardize this method for routine application in medical laboratories.

REDigest: a Python GUI for In-Silico Restriction Digestion Analysis of Genes or Complete Genome Sequences

Restriction fragment length polymorphism (RFLP) is a technology for the molecular characterization of DNA and widely used genome mapping, medical genetics, molecular microbiology and forensics etc. Terminal restriction fragment length polymorphism (T-RFLP), a variant of RFLP is extensively used in environmental microbiology for the microbial community profiling based on the restriction digestion profile of marker gene (16S rRNA, FTHFS etc.) amplicons. At present, there is a lack of a tool which can perform in-silico restriction digestion of a large number of sequences at a time, in an interactive way and as an output produce sequences of the restriction fragments and visualization plot. I have developed a graphical user interface based software REDigest for the in-silico restriction digestion analysis for gene or genome sequences. The REDigest software program with a graphical user interface is freely available at https://github.com/abhijeetsingh1704/REDigest.

Genotyping of Cryptosporidium species in children suffering from diarrhea in Sharkyia Governorate, Egypt

Introduction: The protozoan parasite Cryptosporidium is one of the principal reasons for childhood diarrhea around the world. This work aimed to differentiate Cryptosporidium species among children suffering from diarrhea in Sharkyia Governorate, Egypt. Methodology: A total of 97 fecal specimens were taken from children suffering from diarrhea, attending Pediatric Clinics of Zagazig University and Al-Ahrar Hospitals. Full history was taken. Stool samples were examined microscopically using modified Ziehl–Neelsen stain for detection of Cryptosporidium oocysts. To identify Cryptosporidium genotypes, positive samples were then subjected to nested Polymerase chain reaction-restriction fragment length polymorphism targeting Cryptosporidium oocyst wall protein gene. Results: The overall detection rate was 27.8% (27/97) using modified Ziehl–Neelsen stain staining method. Using nested polymerase chain reaction, the gene was amplified in 85.2% (23/27). Restriction fragment length polymorphism analysis revealed that 65.2% (15/23) were Cryptosporidium hominis, 30.4% (7/23) were Cryptosporidium parvum, and one sample was not typed (4.4%). The significant risk factors associated with Cryptosporidium infection in children were animal contact and residence in rural areas. Conclusions: Cryptosporidium is a common enteric parasite affecting children in Sharkyia Governorate, Egypt, with the predominance of C. hominis genotype in children.

Red foxes harbor two genetically distinct, spatially separated Echinococcus multilocularis clusters in Brandenburg, Germany

Background Alveolar echinococcosis (AE) is a clinically serious zoonosis caused by the fox tapeworm Echinococcus multilocularis. We studied the diversity and the distribution of genotypes of E. multilocularis isolated from foxes in Brandenburg, Germany, and in comparison to a hunting ground in North Rhine-Westphalia. Methods Echinococcus multilocularis specimens from 101 foxes, 91 derived from Brandenburg and 10 derived from North Rhine-Westphalia, were examined. To detect potential mixed infections with different genotypes of E. multilocularis, five worms per fox were analyzed. For genotyping, three mitochondrial markers, namely cytochrome c oxidase subunit 1 (Cox1), NADH dehydrogenase subunit 1 (Nad1), and ATP synthase subunit 6 (ATP6), and the nuclear microsatellite marker EmsB were used. To identify nucleotide polymorphisms, the mitochondrial markers were sequenced and the data were compared, including with published sequences from other regions. EmsB fragment length profiles were determined and confirmed by Kohonen network analysis and grouping of Sammon’s nonlinear mapping with k-means clustering. The spatial distribution of genotypes was analyzed by SaTScan for the EmsB profiles found in Brandenburg. Results With both the mitochondrial makers and the EmsB microsatellite fragment length profile analyses, mixed infections with different E. multilocularis genotypes were detected in foxes from Brandenburg and North Rhine-Westphalia. Genotyping using the mitochondrial markers showed that the examined parasite specimens belong to the European haplotype of E. multilocularis, but a detailed spatial analysis was not possible due to the limited heterogeneity of these markers in the parasite population. Four (D, E, G, and H) out of the five EmsB profiles described in Europe so far were detected in the samples from Brandenburg and North Rhine-Westphalia. The EmsB profile G was the most common. A spatial cluster of the E. multilocularis genotype with the EmsB profile G was found in northeastern Brandenburg, and a cluster of profile D was found in southern parts of this state. Conclusions Genotyping of E. multilocularis showed that individual foxes may harbor different genotypes of the parasite. EmsB profiles allowed the identification of spatial clusters, which may help in understanding the distribution and spread of the infection in wildlife, and in relatively small endemic areas. Graphical Abstract

Restriction fragment length polymorphism analysis of genes of virulent strain isolate of Toxoplasma gondii using enzyme DdeI

Background and Aim: Toxoplasma gondii is a unicellular coccidian parasite distributed globally and is an important zoonotic pathogen. Approximately 30% of the human population worldwide is chronically infected with T. gondii. The pathogenicity of this species depends on the type originating from the clonal population. Techniques for more accurately determining the type of T. gondii have recently been developed using genetic markers. Specifically, T. gondii has been typed using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). This study aimed to identify sets of PCR-RFLP markers that have high power to discriminate genotyping of T. gondii and are easy to use and are easy to use. The objective of this study was to characterize virulent strain isolates of T. gondii by PCR-RFLP using 10 markers with DdeI. Materials and Methods: T. gondii tachyzoites (RH virulent strain) were derived from culture cells at the Indonesian Research Center for Veterinary Sciences. Genotyping was performed on T. gondii DNA extracted from cell cultured tachyzoites using 10 genetic markers of PCR-RFLP, namely, B1#1, B1#2, B1#3, SAG1#1, SAG1#2, P30, BAG1, ROP1, GRA1, and GRA7, with digestion using the restriction enzyme DdeI. Results: The 10 genes were amplified by PCR. Among them, three genetic markers, B1#3, ROP1, and GRA1, were genotyped by the PCR-RFLP using restriction enzyme DdeI. Overall, the findings showed that the specific RFLP profile of digestion of gene regions by DdeI could be used as a specific marker for the virulent biotype causative of toxoplasmosis. In addition, virulent strains of T. gondii can be easily detected by these markers. Conclusion: Three pairs of primers (B1#3, ROP1, and GRA1) with DdeI have proven useful for the diagnosis of acute toxoplasmosis (virulent strain biotype I). This proposed method is relatively simple, rapid, cheap, and can be performed in most laboratories, providing a practical approach for the routine analysis of T. gondii strains.

Study of Plant Genetic Variation through Molecular Markers: An Overview

This article refers to viewing the role of molecular markers during analyzing the genome of plants and their importance in plant biotechnology. In recent years, we observed the role of molecular techniques in programs for improving plant breeding and preserving genetic resources has been observed, and molecular and biochemical indicators which represent basic material through determining the diversity between genotypes for indicators it is never affected by external surrounding conditions as always in the phenotype features. Molecular markers of DNA have been widely applied to answer a range of questions related to taxonomy, molecular evolution, population genetics, and genetic diversity, as well as monitoring trade in plants and food products , in addition to its having a role in studying gene expression , genetic mapping, and studies of species evolution providing fast and accurate results. In this work, the advantages and limitations of the molecular techniques applied in plant sciences such as: RAPD (Random Amplification Polymorphic DNA Marker); ISSR (Inter Simple Sequence Repeat Marker); SSR (Simple Sequence Repeat Marker); AFLP (Amplified Fragment Length Polymorphic Marker); RFLP (Restriction Fragment Length Polymorphism Marker); SNP (Single Nucleotide Polymorphism) and Real Time PCR.

Reconstruction of a Symbolic Periodic Sequence from a Sequence with Noise

The problem of constructing a periodic sequence consisting of at least eight periods is considered, based on a given sequence obtained from an unknown periodic sequence, also containing at least eight periods, by introducing noise of deletion, replacement, and insertion of symbols. To construct a periodic sequence that approximates a given one, distorted by noise, it is first required to estimate the length of the repeating fragment (period). Further, the distorted original sequence is divided into successive sections of equal length; the length takes on integer values from 80 to 120 % of the period estimate. Each obtained section is compared with each of the remaining sections, a section is selected to build a periodic sequence that has the minimum edit distance (Levenshtein distance) to any of the remaining sections, minimization is carried out over all sections of a fixed length, and then along all lengths from 80 to 120 % of period estimates. For correct comparison of fragments of different lengths, we consider the ration between the edit distance and the length of the fragment. The length of a fragment that minimizes the ratio of the edit distance to another fragment of the same length to the fragment length is considered the period of the approximating periodic sequence, and the fragment itself, repeating the required number of times, forms an approximating sequence. The constructed sequence may contain an incomplete repeating fragment at the end. The quality of the approximation is estimated by the ratio of the edit distance from the original distorted sequence to the constructed periodic sequence of the same length and this length.

Comparative lactic acid bacteria (LAB) profiles during dadih fermentation with spontaneous and back-slopping methods, as identified by terminal-restriction fragment length polymorphism (T-RFLP)

The diversity of lactic acid bacteria (LAB) present during the manufacture of traditional fermented buffalo milk from West Sumatra, known as dadih, was studied via a culture-independent approach using terminal-restriction fragment length polymorphism (T-RFLP) to compare the dynamic diversity in back-slopping and spontaneous fermentation methods. Total LAB and pH were measured in freshly prepared buffalo milk and in \textit{dadih} fermented for 24 and 48 hours. The results indicated significant differences between the fermentation methods, with higher total LAB, and greater phylotype richness and relative abundance being identified in the back-slopping method. Terminal fragment lengths (TRFs) of 68 and 310 bp were common to both techniques, similar to those of Lactobacillus fermentum, Fructobacillus pseudoficulneus, Leuconostoc citreum, Leuconostoc kimchii, and Leuconostoc sp. The changes in phylotype number (species number) and relative abundances of LAB communities identified are expected to produce data needed to formulate the best fermentation process for dadih manufacturing. A 24-hour back-slopping fermentation method is recommended, as fermentation time of longer than 24 hours reduced viable LAB significantly. Our results also indicated that the T-RFLP technique is not only clearly sensitive enough and adequate for segregating LAB diversity in both fermentation methods, but that it also provides good information regarding the structure of microbial communities and their composition change during the fermentation process.

Genetic Variation and Phylogeny of Wabisuke Camellias by Amplified Fragment Length Polymorphism (AFLP) Analysis

Amplified fragment length polymorphism (AFLP) analysis was conducted on the wabisuke camellia and its relative camellia species. Genetic polymorphism was identified among the ‘Uraku’ camellia, its offspring ‘Tosa-uraku’ and Camellia japonica, whereas the two accessions of the old ‘Uraku’ showed monomorphism in all the fragments. The results suggested that the two old ‘Uraku’ trees are asexually-propagated clonal strains. The genetic distance between wabisuke cultivars and Chinese camellias and between wabisuke camellias and C. sinensis was much further than that between wabisuke cultivars and Camellia japonica. It has also been suggested that wabisuke camellias can be classified into two subgroups, I-1 and I-2, and that Subgroup I-2 originated from C. japonica, while Subgroup I-1, including ‘Uraku’ (synonym: ‘Tarokaja’), was developed by the repeated hybridization of C. japonica to interspecific hybrids with the Chinese camellias, e.g., C. pitardii var. pitardii, or by the involvement of related species not investigated in this study.