Strain-level sample characterisation using long reads and MAPQ scores

A simple but effective method for strain-level characterisation of microbial samples using long read data is presented. The method, which relies on having a non-redundant database of reference genomes, differentiates between strains within species and determines their relative abundance. It provides markedly better strain differentiation than that reported for the latest long read tools. Good estimates of relative abundances of highly similar strains present at less than 1% are achievable with as little as 1Gb of reads. Host contamination can be removed without great loss of sample characterisation performance. The method is simple and highly flexible, allowing it to be used for various different purposes, and as an extension of other characterisation tools. A code body implementing the underlying method is freely available.

A New PCR-Based Species Genotyping Differentiation Approach in Entamoeaba

The most commonly used approach for Entamoeba species differentiation up to date is the tRNA-linked STR regions of the parasite’s genome. In the present study, a new reliable, fast and easy molecular tool for species differentiation was developed. DNA was isolated from fecal samples collected from infected subjects with either Entamoeba histolytica (EH) or Entamoeba disper (ED) in Saudi Arabia. Two types of primer sets were compared in which the first targeted tRNA-linked STR regions, while the second was designed after multiple contig alignment of the two genomes using NUCmer program in aligned areas with high similarity (~90%) and difference between of ~90 bp. The selection criteria secures that designed primers should pair with both EH and ED contig sequences at homologous regions of 200-500 bp of both species except for the presence of indels that result in the recovery of amplicons of two species with different sizes. Banding patterns in the tRNA-linked STR region resulted in the occurrence of several common amplicons. We speculate that primers mismatch with regions other than the specified STR arrays of Entamoeba histolytica or Entamoeba disper with organisms other than Entamoeba existed in the fecal sample. However, the STR-based approach looked very useful in studying strain differentiation and parasite diversity. The results for the new approach complemented those of the STR-based approach, except that the latter failed to detect coinfected subjects. The new approach proved to be useful at the species level, while the tRNA-linked STR approach can still be a good choice for strain differentiation.

Diagnostics of the Presence of Viruses in the Potato and Soil

The paper presents a combined biological assay on indicator plants, a serological assay using ELISA and a molecular assay for detection of viruses that infect the potato. It also elaborates on the bioassay for PVY strain differentiation and for detection of TRV in tubers and soil samples. At IHAR-PIB Młochów Research Center, ELISA tests can be conducted for detection of PVY, PVA, PVM, PVS, PLRV, PVX, TRV and PMTV. Multiplex RT-PCR, real-time quantitative RT-PCR and sequencing methods for detection of PVY, TRV and PMTV are optimized based on a published protocol or developed in our laboratory. The primers for detection of PVY, TRV, PVM, PVS, PLRV, PVX, PMTV, PVA, AMV, CMV, PAMV, TBRV, and BMYV according to the published methods are listed. The maintenance and usage of 19 species of indicator plants are discussed.

A critical re-evaluation of multilocus sequence typing (MLST) efforts inWolbachia

Wolbachia(Alphaproteobacteria, Rickettsiales) is the most common, and arguably one of the most important inherited symbionts. Molecular differentiation ofWolbachiastrains is routinely performed with a set of five multilocus sequence typing (MLST) markers. However, since its inception in 2006, the performance of MLST inWolbachiastrain typing has not been assessed objectively. Here, we evaluate the properties ofWolbachiaMLST markers and compare it to 252 other single copy loci present in the genome of mostWolbachiastrains. Specifically, we investigated how well MLST performs at strain differentiation, at reflecting genetic diversity of strains, and as phylogenetic marker. We find that MLST loci are outperformed by other loci at all tasks they are currently employed for, and thus that they do not reflect the properties of aWolbachiastrain very well. We argue that whole genome typing approaches should be used forWolbachiatyping in the future. Alternatively, if few-loci-approaches are necessary, we provide a characterization of 252 single copy loci for a number a criteria, which may assist in designing specific typing systems or phylogenetic studies.