Long-read RNA sequencing of human and animal filarial parasites improves gene models and discovers operons

Filarial parasitic nematodes (Filarioidea) cause substantial disease burden to humans and animals around the world. Recently there has been a coordinated global effort to generate, annotate, and curate genomic data from nematode species of medical and veterinary importance. This has resulted in two chromosome-level assemblies (Brugia malayi and Onchocerca volvulus) and 11 additional draft genomes from Filarioidea. These reference assemblies facilitate comparative genomics to explore basic helminth biology and prioritize new drug and vaccine targets. While the continual improvement of genome contiguity and completeness advances these goals, experimental functional annotation of genes is often hindered by poor gene models. Short-read RNA sequencing data and expressed sequence tags, in cooperation with ab initio prediction algorithms, are employed for gene prediction, but these can result in missing clade-specific genes, fragmented models, imperfect mapping of gene ends, and lack of isoform resolution. Long-read RNA sequencing can overcome these drawbacks and greatly improve gene model quality. Here, we present Iso-Seq data for B. malayi and Dirofilaria immitis, etiological agents of lymphatic filariasis and canine heartworm disease, respectively. These data cover approximately half of the known coding genomes and substantially improve gene models by extending untranslated regions, cataloging novel splice junctions from novel isoforms, and correcting mispredicted junctions. Furthermore, we validated computationally predicted operons, manually curated new operons, and merged fragmented gene models. We carried out analyses of poly(A) tails in both species, leading to the identification of non-canonical poly(A) signals. Finally, we prioritized and assessed known and putative anthelmintic targets, correcting or validating gene models for molecular cloning and target-based anthelmintic screening efforts. Overall, these data significantly improve the catalog of gene models for two important parasites, and they demonstrate how long-read RNA sequencing should be prioritized for ongoing improvement of parasitic nematode genome assemblies.     

An optimized FM-index library for nucleotide and amino acid search

In computational structural biology, structure comparison is fundamental for our understanding of proteins. Structure comparison is, e.g., algorithmically the starting point for computational studies of structural evolution and it guides our efforts to predict protein structures from their amino acid sequences. Most methods for structural alignment of protein structures optimize the distances between aligned and superimposed residue pairs, i.e., the distances traveled by the aligned and superimposed residues during linear interpolation. Considering such a linear interpolation, these methods do not differentiate if there is room for the interpolation, if it causes steric clashes, or more severely, if it changes the topology of the compared protein backbone curves. To distinguish such cases, we analyze the linear interpolation between two aligned and superimposed backbones. We quantify the amount of steric clashes and find all self-intersections in a linear backbone interpolation. To determine if the self-intersections alter the protein’s backbone curve significantly or not, we present a path-finding algorithm that checks if there exists a self-avoiding path in a neighborhood of the linear interpolation. A new path is constructed by altering the linear interpolation using a novel interpretation of Reidemeister moves from knot theory working on three-dimensional curves rather than on knot diagrams. Either the algorithm finds a self-avoiding path or it returns a smallest set of essential self-intersections. Each of these indicates a significant difference between the folds of the aligned protein structures. As expected, we find at least one essential self-intersection separating most unknotted structures from a knotted structure, and we find even larger motions in proteins connected by obstruction free linear interpolations. We also find examples of homologous proteins that are differently threaded, and we find many distinct folds connected by longer but simple deformations. TM-align is one of the most restrictive alignment programs. With standard parameters, it only aligns residues superimposed within 5 Ångström distance. We find 42165 topological obstructions between aligned parts in 142068 TM-alignments. Thus, this restrictive alignment procedure still allows topological dissimilarity of the aligned parts. Based on the data we conclude that our program ProteinAlignmentObstruction provides significant additional information to alignment scores based solely on distances between aligned and superimposed residue pairs.     

Markers on Bovine Chromosome 20 Associated with Carcass Quality and Composition Traits and Incidence of Contracting Infectious Bovine Keratoconjunctivitis

The objective of this study was to use single nucleotide polymorphisms (SNP) located on bovine chromosome 20 to fine map a previously identified QTL associated with the incidence of infectious bovine keratoconjunctivitis (IBK). Crossbred steers (GPE 7; n = 539) derived from sires of 7 Bos taurus breeds and having veterinary records related to IBK were used to test the association of a total of 105 SNP located under the most relevant region of the QTL. Five SNP were significantly associated with IBK (P < 0.05), as animals inheriting differing genotypes from individual SNP exhibited significantly different incidence rates of IBK. The population also had numerous other phenotypes, supporting evaluation of association of the 105 markers with carcass traits to identify potential antagonistic effects of implementing a marker-assisted selection program for IBK susceptibility. An association of 2 SNP for marbling and tenderness was identified, along with 3 SNP associated with the percentage of carcasses classified as choice. Four SNP were significantly associated with fat yield, 2 SNP with longissimus muscle area, and 2 additional SNP with dressing percentage. The association of these markers indicates that the evaluated QTL region may, in fact, harbor the causative mutations responsible for the variation observed in IBK susceptibility and carcass quality and composition traits. Thus, further evaluation of SNP in this region is necessary in order to identify mutations accounting for the largest degree of variation for IBK and carcass traits.     

Use of Phenylacetyl Disulfide (PADS) in the Synthesis of Oligodeoxyribonucleotide Phosphorothioates

Abstract

Investigations into the use of phenylacetyl disulfide (PADS) as an efficient sulfur transfer agent in the solid phase synthesis of oligodeoxyribonucleotide phosphorothioates showed that under suitable solvent conditions, this relatively inexpensive reagent rapidly and efficiently sulfurizes internucleotide phosphite linkages.     

Remote Source Document Verification in Two National Clinical Trials Networks: A Pilot Study

Objective

Barriers to executing large-scale randomized controlled trials include costs, complexity, and regulatory requirements. We hypothesized that source document verification (SDV) via remote electronic monitoring is feasible.

Methods

Five hospitals from two NIH sponsored networks provided remote electronic access to study monitors. We evaluated pre-visit remote SDV compared to traditional on-site SDV using a randomized convenience sample of all study subjects due for a monitoring visit. The number of data values verified and the time to perform remote and on-site SDV was collected.

Results

Thirty-two study subjects were randomized to either remote SDV (N=16) or traditional on-site SDV (N=16). Technical capabilities, remote access policies and regulatory requirements varied widely across sites. In the adult network, only 14 of 2965 data values (0.47%) could not be located remotely. In the traditional on-site SDV arm, 3 of 2608 data values (0.12%) required coordinator help. In the pediatric network, all 198 data values in the remote SDV arm and all 183 data values in the on-site SDV arm were located. Although not statistically significant there was a consistent trend for more time consumed per data value (minutes +/- SD): Adult 0.50 +/- 0.17 min vs. 0.39 +/- 0.10 min (two-tailed t-test p=0.11); Pediatric 0.99 +/- 1.07 min vs. 0.56 +/- 0.61 min (p=0.37) and time per case report form: Adult: 4.60 +/- 1.42 min vs. 3.60 +/- 0.96 min (p=0.10); Pediatric: 11.64 +/- 7.54 min vs. 6.07 +/- 3.18 min (p=0.10) using remote SDV.

Conclusions

Because each site had different policies, requirements, and technologies, a common approach to assimilating monitors into the access management system could not be implemented. Despite substantial technology differences, more than 99% of data values were successfully monitored remotely. This pilot study demonstrates the feasibility of remote monitoring and the need to develop consistent access policies for remote study monitoring.