Jack Chen

Analytical Sensitivity Comparison between Singleplex Real-Time PCR and a Multiplex PCR Platform for Detecting Respiratory Viruses.

Multiplex PCR methods are attractive to clinical laboratories wanting to broaden their detection of respiratory viral pathogens in clinical specimens. However, multiplexed assays must be well optimized to retain or improve upon the analytic sensitivity of their singleplex counterparts. In this experiment, the lower limit of detection (LOD) of singleplex real-time PCR assays targeting respiratory viruses is compared to an equivalent panel on a multiplex PCR platform, the GenMark eSensor RVP. LODs were measured for each singleplex real-time PCR assay and expressed as the lowest copy number detected 95–100% of the time, depending on the assay. The GenMark eSensor RVP LODs were obtained by converting the TCID50/mL concentrations reported in the package insert to copies/μL using qPCR. Analytical sensitivity between the two methods varied from 1.2–1280.8 copies/μL (0.08–3.11 log differences) for all 12 assays compared. Assays targeting influenza A/H3N2, influenza A/H1N1pdm09, influenza B, and human parainfluenza 1 and 2 were most comparable (1.2–8.4 copies/μL, <1 log difference). Largest differences in LOD were demonstrated for assays targeting adenovirus group E, respiratory syncytial virus subtype A, and a generic assay for all influenza A viruses regardless of subtype (319.4–1280.8 copies/μL, 2.50–3.11 log difference). The multiplex PCR platform, the GenMark eSensor RVP, demonstrated improved analytical sensitivity for detecting influenza A/H3 viruses, influenza B virus, human parainfluenza virus 2, and human rhinovirus (1.6–94.8 copies/μL, 0.20–1.98 logs). Broader detection of influenza A/H3 viruses was demonstrated by the GenMark eSensor RVP. The relationship between TCID50/mL concentrations and the corresponding copy number related to various ATCC cultures is also reported.

Application of next generation sequencing for the detection of human viral pathogens in clinical specimens

BACKGROUND Next generation sequencing (NGS) is a new technology that can be used for broad detection of infectious pathogens and is rapidly becoming an essential platform in clinical laboratories. It is not known how NGS will displace or enhance gold standard methodologies in infectious disease diagnosis. OBJECTIVES To investigate the feasibility and application of NGS technology in public health laboratories and compare NGS technology with conventional methods. STUDY DESIGN Illumina MiSeq system was used to detect viral pathogens alongside other conventional virology methods using typical clinical specimen matrices. Sixteen clinical specimens and two CDC proficiency panels containing seventeen specimens were analyzed. RESULTS Known pathogenic viral nucleic acid was positively identified in all clinical specimens, correlating and building upon results obtained by more conventional laboratory methods. Sequencing depths ranged from 0.008X to 319 and genome coverage ranged from 0.6% to 99.9%. To substantiate the described methods used to analyze data derived from clinical specimens, the results of a clinical proficiency panel are also presented. DISCUSSION Our results reveal true scarcity of known pathogenic viral nucleic acids in clinical specimens. NGS outperforms more conventional detection methods in this study by turnaround time as well as the improved depth of knowledge in regards to serotyping and drug resistance.

Preferential Amplification of Pathogenic Sequences

The application of next generation sequencing (NGS) technology in the diagnosis of human pathogens is hindered by the fact that pathogenic sequences, especially viral, are often scarce in human clinical specimens. This known disproportion leads to the requirement of subsequent deep sequencing and extensive bioinformatics analysis. Here we report a method we called “Preferential Amplification of Pathogenic Sequences (PATHseq)” that can be used to greatly enrich pathogenic sequences. Using a computer program, we developed 8-, 9-, and 10-mer oligonucleotides called “non-human primers” that do not match the most abundant human transcripts, but instead selectively match transcripts of human pathogens. Instead of using random primers in the construction of cDNA libraries, the PATHseq method recruits these short non-human primers, which in turn, preferentially amplifies non-human, presumably pathogenic sequences. Using this method, we were able to enrich pathogenic sequences up to 200-fold in the final sequencing library. This method does not require prior knowledge of the pathogen or assumption of the infection; therefore, it provides a fast and sequence-independent approach for detection and identification of human viruses and other pathogens. The PATHseq method, coupled with NGS technology, can be broadly used in identification of known human pathogens and discovery of new pathogens.

Draft Genome Sequence of an Atypical Strain of Streptococcus pneumoniae Isolated from a Respiratory Infection

ABSTRACT Next-generation sequencing was used to investigate an unknown clinical respiratory infection. This new strain of Streptococcus pneumoniae, ASVL_JC_0001, was isolated from a clinical specimen from a patient with bronchitis and pulmonary inflammation. The draft genome sequence, obtained with an Illumina MiSeq sequencing system, consists of 83 large contigs, a total of 2,092,532 bp long, and has a GC content of 40.3%.

Vector modifications to eliminate transposase expression following piggyBac-mediated transgenesis

Transgene insertion plays an important role in gene therapy and in biological studies. Transposon-based systems that integrate transgenes by transposase-catalyzed “cut-and-paste” mechanism have emerged as an attractive system for transgenesis. Hyperactive piggyBac transposon is particularly promising due to its ability to integrate large transgenes with high efficiency. However, prolonged expression of transposase can become a potential source of genotoxic effects due to uncontrolled transposition of the integrated transgene from one chromosomal locus to another. In this study we propose a vector design to decrease post-transposition expression of transposase and to eliminate the cells that have residual transposase expression. We design a single plasmid construct that combines the transposase and the transpositioning transgene element to share a single polyA sequence for termination. Consequently, the separation of the transposase element from the polyA sequence after transposition leads to its deactivation. We also co-express Herpes Simplex Virus thymidine kinase (HSV-tk) with the transposase. Therefore, cells having residual transposase expression can be eliminated by the administration of ganciclovir. We demonstrate the utility of this combination transposon system by integrating and expressing a model therapeutic gene, human coagulation Factor IX, in HEK293T cells.

Investigation of a Canine Parvovirus Outbreak using Next Generation Sequencing

Canine parvovirus (CPV) outbreaks can have a devastating effect in communities with dense dog populations. The interior region of Alaska experienced a CPV outbreak in the winter of 2016 leading to the further investigation of the virus due to reports of increased morbidity and mortality occurring at dog mushing kennels in the area. Twelve rectal-swab specimens from dogs displaying clinical signs consistent with parvoviral-associated disease were processed using next-generation sequencing (NGS) methodologies by targeting RNA transcripts, and therefore detecting only replicating virus. All twelve specimens demonstrated the presence of the CPV transcriptome, with read depths ranging from 2.2X – 12,381X, genome coverage ranging from 44.8–96.5%, and representation of CPV sequencing reads to those of the metagenome background ranging from 0.0015–6.7%. Using the data generated by NGS, the presence of newly evolved, yet known, strains of both CPV-2a and CPV-2b were identified and grouped geographically. Deep-sequencing data provided additional diagnostic information in terms of investigating novel CPV in this outbreak. NGS data in addition to limited serological data provided strong diagnostic evidence that this outbreak most likely arose from unvaccinated or under-vaccinated canines, not from a novel CPV strain incapable of being neutralized by current vaccination efforts.

Identification of the interaction of VP1 with GM130 which may implicate in the pathogenesis of CVB3-induced acute pancreatitis

Coxsackievirus B3 (CVB3) is a causative agent of viral myocarditis, pancreatitis, and meningitis in humans. Although the susceptibility of CVB3-induced acute pancreatitis is age-dependent, the underlying mechanisms remain unclear. Here we identified the host factor Golgi matrix protein 130 (GM130) as a novel target of CVB3 during CVB3-induced acute pancreatitis. The viral protein VP1 interacted with GM130, disrupted GM130-GRASP65 complexes, and caused GM130 degradation, which may lead to disruption of the Golgi ribbon and development of acute pancreatitis in mice. Interestingly, the expression level of GM130 in mouse pancreas was age-dependent, which was nicely correlated with the age-associated susceptibility of CVB3-induced acute pancreatitis. Furthermore, interference RNA-mediated knockdown of GM130 significantly reduced CVB3 replication in HeLa cells. Taken together, the study identified GM130 as a novel target of CVB3, which may implicate in the pathogenesis of CVB3-induced acute pancreatitis.

Draft Genome Sequence of a Taxonomically Unique Neisseria Strain Isolated from a Greater White-Fronted Goose (Anser albifrons) Egg on the North Slope of Alaska

ABSTRACT We report here the draft genome sequence of a unique Neisseria strain that was isolated from a greater white-fronted goose (Anser albifrons) egg. The sequencing was performed with an Illumina MiSeq system, and the sequence consists of 275 contigs. The total genome is 2,397,978 bp long and has a G+C content of 46.4%.

Correction for Hansen et al., Draft Genome Sequence of a Taxonomically Unique Neisseria Strain Isolated from a Greater White-Fronted Goose (Anser albifrons) Egg on the North Slope of Alaska.

Volume 3, no. 4, [e00772-15][1], 2015. Page 1: The byline should read as given above, and a present address footnote for Sang Chul Choi should be included. [1]: /lookup/doi/10.1128/genomeA.00772-15