HaeIII induces position-dependent chromosomal breakage in barley (Hordeum vulgare L.)

The pattern of localized chromosomal breakage induced by the restriction endonuclease HaeIII in reconstructed barley karyotypes T-1586 and T-21 was investigated. It was found that nucleolus organizing regions (NORs) of chromosomes 6 and 7 (segments 46 and 38, respectively), containing actively transcribed ribosomal (r)DNA, as well as segments 39 and 47, both containing condensed rDNA repeats, are the most pronounced aberration hot-spots in T-1586. The number of aberrations observed in these segments was three to five times higher than theoretically expected. The intrachromosomal distribution of chromatid aberrations in karyotype T-21, where the NOR-bearing segments in chromosomes 6 and 7 change their position, revealed a substantial difference in the aberration hot-spot behaviour. A position-specific increase in aberration clustering was observed, most pronounced in segments 38 and 47. On the other hand, segment 46 retained its initial sensitivity, while segment 39 in the new position lost its previous status as a mutation hot-spot. The data are indicative of the expressivity of aberration hot-spots generated after treatment with this restriction endonuclease being influenced by their distinct chromosomal location.     

Multi-laboratory proficiency testing of clinical cancer genomic profiling by next-generation sequencing

Next-generation sequencing (NGS) enables parallel analysis of multiple genomic targets. The increasing demand for NGS-based multiplexed molecular diagnostics requires standardized protocols and recommendations to ensure reproducibility and accuracy of test results for routine clinical decision making. However, the lack of clinical NGS data from multi-laboratory tests and the absence of inter-laboratory comparisons have hampered the establishment of instructive clinical NGS standards. To fill the gap, we set up Proficiency Testing (PT) for inter-laboratory comparison, in which formalin-fixed paraffin-embedded specimens from eight lung and eight colon cancers were analyzed by 15 European molecular diagnostic laboratories on three different platforms using multiple target enrichment systems. We first performed platform, test, and informatics pipeline validation and conducted sensitivity and specificity analysis by random in silico down-sampling. We then implemented a multi-level filtering strategy based on performance tests of base substitution, replicate runs, and Sanger sequencing verified variants. We finally applied the filter criteria to the NGS data from the respective PT participants and obtained high inter-laboratory agreement. We demonstrated accuracy, scalability, and robustness of NGS by means of PT, serving as a benchmark for detecting clinically actionable molecular alterations in research and diagnostic laboratories. In conclusion, this study strongly highlights the importance of establishing standards for NGS-based testing, particularly when the test results impact on clinical decisions, and systematically provides data sets from multiple different labs to infer such standards.     

Optimal conditions for the use of cDNA probes to measure the concentration of barley yellow dwarf virus in barley (Hordeum vulgare)

Experiments which optimise the conditions for the measurement of the relative concentration of BYDV in barley (Hordeum vulgare) tissues using cDNA probes are described herein. These studies have shown that both the pH of the buffer and the ratio of buffer to tissue used to homogenise plant material greatly affects the amount of cDNA probe which hybridises to leaf extracts immobilised on nitrocellulose. These studies also showed that the measurement of this virus was greatly facilitated by using a dot-blot apparatus which allows samples contact with a piece of nitrocellulose 10 mm in diameter rather than a 3 mm (approx) diameter piece of nitrocellulose as is the case with most commercial dot-blot apparatuses. Further experiments using this technique showed that there was a large difference in the rate of replication of the PAV, BYDV serotype between BYDV-resistant and BYDV-susceptible cultivars of barley. These data suggest that a BYDV-resistant cultivar can easily be distinguished from a BYDV-susceptible one if the BYDV content of leaves is measured between 7 and 14 days after inoculation.     

Complete genomic sequence of the goat prion protein gene (PRNP)

Prion protein genetics plays a central role in transmissible spongiform encephalopathies, a disease occurring in human and animals. Here we report a 27-kb genomic sequence containing the goat PRNP gene. It shows, both in structure and content, a remarkable similarity with its sheep ortholog and can serve as a basis for future (comparative) studies with reference to the regulation of PRNP gene expression and the search for genetic tools to prevent/control/eradicate (goat) TSE. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. The nucleotide sequence data reported in this paper has been submitted to the GenBank nucleotide sequence database and has been assigned the accession number EU870890.     

Complete genomic sequence of turkey coronavirus

Turkey coronavirus (TCoV), one of the least characterized of all known coronaviruses, was isolated from an outbreak of acute enteritis in young turkeys in Ontario, Canada, and the full-length genomic sequence was determined. The full-length genome was 27,632 nucleotides plus the 3' poly(A) tail. Two open reading frames, ORFs 1a and 1b, resided in the first two thirds of the genome, and nine additional downstream ORFs were identified. A gene for hemagglutinin-esterase was absent in TCoV. The region between the membrane (M) and nucleocapsid (N) protein genes contained three potential small ORFs: ORF-X, a previously uncharacterized ORF with an associated putative TRS within the M gene (apparently shared among all group III coronaviruses), and previously described ORFs 5a and 5b. The TCoV genome is organized as follows: 5' UTR--replicase (ORFs 1a, 1b)--spike (S) protein--ORF3 (ORFs 3a, 3b)--small envelop (E or 3c) protein--membrane (M) protein--ORF5 (ORFs X, 5a, 5b)--nucleocapsid (N) protein--3' UTR--poly(A). TCoV genome structure and sequence was most similar, but distinct from, avian infectious bronchitis virus (IBV). This is the first complete genome sequence for a TCoV and confirms that TCoV belongs to group III coronaviruses.     

The cost-effectiveness of returning incidental findings from next-generation genomic sequencing

PurposeThe American College of Medical Genetics and Genomics (ACMG) recommended that clinical laboratories performing next-generation sequencing analyze and return pathogenic variants for 56 specific genes it considered medically actionable. Our objective was to evaluate the clinical and economic impact of returning these results.MethodsWe developed a decision-analytic policy model to project the quality-adjusted life-years and lifetime costs associated with returning ACMG-recommended incidental findings in three hypothetical cohorts of 10,000 patients.ResultsReturning incidental findings to cardiomyopathy patients, colorectal cancer patients, or healthy individuals would increase costs by $896,000, $2.9 million, and $3.9 million, respectively, and would increase quality-adjusted life-years by 20, 25.4, and 67 years, respectively, for incremental cost-effectiveness ratios of $44,800, $115,020, and $58,600, respectively. In probabilistic analyses, returning incidental findings cost less than $100,000/quality-adjusted life-year gained in 85, 28, and 91%, respectively, of simulations. Assuming next-generation sequencing costs $500, the incremental cost-effectiveness ratio for primary screening of healthy individuals was $133,400 (<$100,000/quality-adjusted life-year gained in 10% of simulations). Results were sensitive to the cohort age and assumptions about gene penetrance.ConclusionReturning incidental findings is likely cost-effective for certain patient populations. Screening of generally healthy individuals is likely not cost-effective based on current data, unless next-generation sequencing costs less than $500.Genet Med 17 7, 587–595.     

Buccal swab genomic DNA fragmentation predicts likelihood of successful HLA genotyping by next-generation sequencing

Many clinical human leukocyte antigen (HLA) laboratories are adopting next-generation sequencing (NGS) technology for HLA genotyping. There have been several reports of the cost-benefit and reduction in turn-around-time provided by NGS. Ninety-six percent of buccal swabs and peripheral blood samples had reportable HLA genotyping by NGS. The HLA loci most likely to fail genotyping from buccal swabs were DQB1, DPB1, and DPA1. Successful buccal swab samples had significantly less genomic DNA fragmentation compared to buccal swab samples that were unsuccessful. Increasing sequencing depth of coverage for heavily fragmented samples rescued HLA genotyping. This information provides laboratories with a quality assurance parameter that reduces the amount of repeat NGS needed to achieve high-resolution HLA genotyping. This information should further reduce laboratory and patient costs for HLA genotyping.     

Complete genome sequence of a novel endornavirus in the wheat sharp eyespot pathogen Rhizoctonia cerealis

We report here the presence of a novel double-stranded RNA (dsRNA) virus in an isolate (R0959) of the fungus Rhizoctonia cerealis, the causal agent of sharp eyespot of wheat in China. Sequence analysis showed that the dsRNA segment is 17,486 bp long and contains a single open reading frame (ORF) with the potential to encode a protein of 5,747 amino acids. The predicted protein contains conserved motifs of putative viral methyltransferase, helicase 1, and RNA-dependent RNA polymerase. Sequence similarity and phylogenetic analysis clearly place it in a distinct species within the genus Endornavirus, family Endornaviridae, and therefore we propose its name to Rhizoctonia cerealis endornavirus 1 (RcEV1). This is the first report of the full-length genomic sequence of a dsRNA mycovirus in R. cerealis.     

Complete nucleotide sequence and genomic organization of hepatopancreatic parvovirus (HPV) of Penaeus monodon

We have determined the genome of hepatopancreatic parvovirus (HPV), a minus, single-stranded DNA virus isolated from infected Penaeus monodon in Thailand. Its genome consisted of 6321 nucleotides, representing three large open reading frames (ORFs) and two non-coding termini. The left (ORF1), mid (ORF2), and right (ORF3) ORFs on the complementary (plus) strand may code for 428, 579, and 818 amino acids, equivalent to 50, 68, and 92 kDa, respectively. The 5' and 3' ends of viral genome contained hairpin-like structure length of approximately 222 and 215 bp, respectively. No inverted terminal repeat (ITR) was detected. The ORF2 contained conserved replication initiator motif, NTP-binding and helicase domain similar to NS-1 of other parvoviruses. Therefore, it most likely encoded the major nonstructural protein (NS-1). The ORF1 encoded putative nonstructural protein-2 (NS-2) with unknown function. The ORF3 of the HPV genome encoded a capsid protein (VP) of approximately 92 kDa. This may be later cleaved after arginine residue to produce a 57-kDa structural protein. A phylogenetic tree based on conserved amino acid sequences (119 aa) revealed that it is closely related to Brevidensoviruses, which are shrimp parvovirus (IHHNV) and mosquito densoviruses (AaeDNV and AalDNV). However, the overall genomic organization and genome size of HPV were different from these parvoviruses, for instance, the non-overlapping of NS1 and NS2, the larger VP gene, and the bigger genome size. This suggested that this HPV virus is a new type in Parvoviridae family. We therefore propose to rename this virus P. monodon densovirus (PmDNV).     

Complete genomic DNA sequence of rock bream iridovirus

Iridovirus is a causative agent of epizootics among cultured rock bream (Oplegnathus fasciatus) in Korea. Here, we report the complete genomic sequence of rock bream iridovirus (RBIV). The genome of RBIV was 112080 bp long and contained at least 118 putative open reading frames (ORFs), and its genome organization was similar to that of infectious spleen and kidney necrosis virus (ISKNV). Of the RBIV's 118 ORFs, 85 ORFs showed 60-99% amino acid identity to those of ISKNV. Phylogenetic analysis of major capsid protein (MCP), DNA repair protein RAD2, and DNA polymerase type-B family indicated that RBIV is closely related to red sea bream iridovirus (RSIV), Grouper sleepy disease iridovirus (GSDIV), Dwarf gourami iridovirus (DGIV), and ISKNV. The genome sequence provides useful information concerning the evolution and divergence of iridoviruses in cultured fish.     

Complete genomic sequence and an infectious BAC clone of feline herpesvirus-1 (FHV-1)

Infection with feline herpesvirus-1 (FHV-1) is a major cause of upper respiratory and ocular diseases in Felidae. We report the first complete genomic sequence of FHV-1, as well as the construction and characterization of a bacterial artificial chromosome (BAC) clone of FHV-1, which contains the entire FHV-1 genome and has the BAC vector inserted at the left end of U_L. Complete genomic sequences were derived from both the FHV-1 BAC clone and purified virion DNA. The FHV-1 genome is 135,797 bp in size with an overall G + C content of 45%. A total of 78 open reading frames were predicted, encoding 74 distinct proteins. The gene arrangement is collinear with that of most sequenced varicelloviruses. The virus regenerated from the BAC was very similar to the parental C-27 strain in vitro in terms of plaque morphology and growth characteristics and highly virulent in cats in a preliminary in vivo study.     

Array-based sequence capture and next-generation sequencing for the identification of primary immunodeficiencies

Primary immunodeficiencies are genetic disorders in which components of immunological pathways are either missing or dysregulated. With the advent of next-generation sequencing, testing for genes in conditions with a heterogeneous genetic background seems more promising. We designed a custom microarray with 385K probe capacity to capture exons of 395 human genes, known or predicted to be associated with primary immunodeficiency and immune regulation. Enriched target DNA was sequenced using a GS FLX Titanium 454 platform. The patients selected were likely to have an underlying immunodeficiency. In one patient with hepatosplenomegaly, recurrent infections and an elevated IgM level, sequence analysis of the patient and his two unaffected parents identified ATM (ataxia telangiectasia mutated) as the underlying defect. In a second child with a clinical SCID phenotype, we detected a mutation in the ARTEMIS gene after focusing on SCID-associated genes. 454 sequencing yielded 152,000-397,000 high-quality reads per patient. 78-99% of the targeted nucleotides were covered at least one time, 76-82% at least five times. Array-based sequence capture expands our capacities to sequence large targeted DNA regions in a less laborious and time-consuming approach. Our array was capable to find the underlying genetic defect in two patients with suspected primary immunodeficiency. Upcoming whole-exome sequencing definitely will add more valuable data, but bioinformatical analysis and validation of variants already pose major challenges.     

Transcriptome analysis of IL-10-stimulated (M2c) macrophages by next-generation sequencing

Alternatively activated “M2” macrophages are believed to function during late stages of wound healing, behaving in an anti-inflammatory manner to mediate the resolution of the pro-inflammatory response caused by “M1” macrophages. However, the differences between two main subtypes of M2 macrophages, namely interleukin-4 (IL-4)-stimulated “M2a” macrophages and IL-10-stimulated “M2c” macrophages, are not well understood. M2a macrophages are characterized by their ability to inhibit inflammation and contribute to the stabilization of angiogenesis. However, the role and temporal profile of M2c macrophages in wound healing are not known. Therefore, we performed next generation sequencing (RNA-seq) to identify biological functions and gene expression signatures of macrophages polarized in vitro with IL-10 to the M2c phenotype in comparison to M1 and M2a macrophages and an unactivated control (M0). We then explored the expression of these gene signatures in a publicly available data set of human wound healing. RNA-seq analysis showed that hundreds of genes were upregulated in M2c macrophages compared to the M0 control, with thousands of alternative splicing events. Following validation by Nanostring, 39 genes were found to be upregulated by M2c macrophages compared to the M0 control, and 17 genes were significantly upregulated relative to the M0, M1, and M2a phenotypes (using an adjusted p-value cutoff of 0.05 and fold change cutoff of 1.5). Many of the identified M2c-specific genes are associated with angiogenesis, matrix remodeling, and phagocytosis, including CD163, MMP8, TIMP1, VCAN, SERPINA1, MARCO, PLOD2, PCOCLE2 and F5. Analysis of the macrophage-conditioned media for secretion of matrix-remodeling proteins showed that M2c macrophages secreted higher levels of MMP7, MMP8, and TIMP1 compared to the other phenotypes. Interestingly, temporal gene expression analysis of a publicly available microarray data set of human wound healing showed that M2c-related genes were upregulated at early times after injury, similar to M1-related genes, while M2a-related genes appeared at later stages or were downregulated after injury. While further studies are required to confirm the timing and role of M2c macrophages in vivo, these results suggest that M2c macrophages may function at early stages of wound healing. Identification of markers of the M2c phenotype will allow more detailed investigations into the role of M2c macrophages in vivo.     

Complete nucleotide sequence and genome organization of an endornavirus from bottle gourd (Lagenaria siceraria) in California,U.S.A.

The full-length nucleotide sequence and genome organization of an Endornavirus isolated from ornamental hard shell bottle gourd plants (Lagenaria siceraria (Molina) Standl.) in California (CA), USA tentatively named L. siceraria endornavirus-California (LsEV-CA) was determined. The LsEV-CA genome was 15088 bp in length, with a G + C content of 36.55 %. The lengths of the 5′ and 3′ untranslated regions were 111 and 52 bp, respectively. The genome of LsEV-CA contained one large ORF encoding a 576 kDa polyprotein. The predicted protein contains two glycosyltransferase motifs, as well as RNA-dependent RNA polymerase and helicase domains. LsEV-CA was detected in healthy-looking field-grown gourd plants, as well as plants expressing yellows symptoms. It was also detected in non-symptomatic greenhouse-grown gourd seedlings grown from seed obtained from the same field sites. These preliminary data indicate that LsEV-CA is likely not associated with the gourd-yellows syndrome observed in the field.