Improved detection of artifactual viral minority variants in high-throughput sequencing data

textabstractHigh-throughput sequencing (HTS) of viral samples provides important information on the presence of viral minority variants. However, detection and accurate quantification is limited by the capacity to distinguish biological from artificial variation. In this study, errors related to the Illumina HiSeq2000 library generation and HTS process were investigated by determining minority variant frequencies in an influenza A/WSN/1933(H1N1) virus reverse-genetics plasmid pool. Errors related to amplification and sequencing were determined using the same plasmid pool, by generation of infectious virus using reverse genetics followed by in duplo reverse-transcriptase PCR (RT-PCR) amplification and HTS in the same sequence run. Results showed that after "best practice" quality control (QC), within the plasmid pool, one minority variant with a frequency >0.5% was identified, while 84 and 139 were identified in the RT-PCR amplified samples, indicating RT-PCR amplification artificially increased variation. Detailed analysis showed that artifactual minority variants could be identified by two major technical characteristics: their predominant presence in a single read orientation and uneven distribution of mismatches over the length of the reads. We demonstrate that by addition of two QC steps 95% of the artifactual minority variants could be identified. When our analysis approach was applied to three clinical samples 68% of the initially identified minority variants were identified as artifacts. Our study clearly demonstrated that, without additional QC steps, overestimation of viral minority variants is very likely to occur, mainly as a consequence of the required RT-PCR amplification step. The improved ability to detect and correct for artifactual minority variants, increases data resolution and could aid both past and future studies incorporating HTS. The source code has been made available through Sourceforge (https://sourceforge.net/projects/mva-ngs)

Middle East respiratory syndrome coronavirus (MERS-CoV) infections in two returning travellers in the Netherlands, May 2014

Two patients, returning to the Netherlands from pilgrimage in Medina and Mecca, Kingdom of Saudi Arabia, were diagnosed with Middle East respiratory syndrome coronavirus (MERS-CoV) infection in May 2014. The source and mode of transmission have not yet been determined. Hospital-acquired infection and community-acquired infection are both possible

Middle East respiratory syndrome coronavirus (MERS-CoV) infections in two returning travellers in the Netherlands, May 2014

Two patients, returning to the Netherlands from pilgrimage in Medina and Mecca, Kingdom of Saudi Arabia, were diagnosed with Middle East respiratory syndrome coronavirus (MERS-CoV) infection in May 2014. The source and mode of transmission have not yet been determined. Hospital-acquired infection and community-acquired infection are both possible

Mutations in LRRC50 Predispose Zebrafish and Humans to Seminomas

Seminoma is a subclass of human testicular germ cell tumors (TGCT), the most frequently observed cancer in young men with a rising incidence. Here we describe the identification of a novel gene predisposing specifically to seminoma formation in a vertebrate model organism. Zebrafish carrying a heterozygous nonsense mutation in Leucine-Rich Repeat Containing protein 50 (lrrc50 also called dnaaf1), associated previously with ciliary function, are found to be highly susceptible to the formation of seminomas. Genotyping of these zebrafish tumors shows loss of heterozygosity (LOH) of the wild-type lrrc50 allele in 44.4% of tumor samples, correlating with tumor progression. In humans we identified heterozygous germline LRRC50 mutations in two different pedigrees with a family history of seminomas, resulting in a nonsense Arg488* change and a missense Thr590Met change, which show reduced expression of the wild-type allele in seminomas. Zebrafish in vivo complementation studies indicate the Thr590Met to be a loss-of-function mutation. Moreover, we show that a pathogenic Gln307Glu change is significantly enriched in individuals with seminoma tumors (13% of our cohort). Together, our study introduces an animal model for seminoma and suggests LRRC50 to be a novel tumor suppressor implicated in human seminoma pathogenesis

In vivo Raman spectroscopy for bladder cancer detection using a superficial Raman probe compared to a nonsuperficial Raman probe

Raman spectroscopy is promising as a noninvasive tool for cancer diagnosis. A superficial Raman probe might improve the classification of bladder cancer, because information is gained solely from the diseased tissue and irrelevant information from deeper layers is omitted. We compared Raman measurements of a superficial to a nonsuperficial probe, in bladder cancer diagnosis. Two-hundred sixteen Raman measurements and biopsies were taken in vivo from at least one suspicious and one unsuspicious bladder location in 104 patients. A Raman classification model was constructed based on histopathology, using a principal-component fed linear-discriminant-analysis and leave-one-person-out cross-validation. The diagnostic ability measured in area under the receiver operating characteristics curve was 0.95 and 0.80, the sensitivity was 90% and 85% and the specificity was 87% and 88% for the superficial and the nonsuperficial probe, respectively. We found inflammation to be a confounder and additionally we found a gradual transition from benign to low-grade to high-grade urothelial carcinoma. Raman spectroscopy provides additional information to histopathology and the diagnostic value using a superficial probe. </p

Intratumoural and peripheral blood lymphocyte subsets in patients with metastatic renal cell carcinoma undergoing interleukin-2 based immunotherapy: association to objective response and survival

The aim of the present study was to analyse lymphocyte subsets in consecutive peripheral blood samples and consecutive tumour tissue core needle biopsies performed before and during interleukin-2 based immunotherapy, and to correlate the findings with objective response and survival. Twenty-six patients with metastatic renal cell carcinoma were treated with low dose s.c. interleukin-2, interferon-α and histamine. A total of 250 blood samples and 62 core needle biopsies from 23 and 19 of these patients, respectively, were analysed. After 2 weeks of treatment, a significant positive correlation between absolute number of peripheral blood lymphocytes (P=0.028), CD3 (P=0.017), CD57 (P=0.041) and objective response was demonstrated. There was no correlation between any peripheral blood leukocyte subsets and survival. Cytotoxicity of peripheral blood mononuclear cells was not correlated to objective response or survival. Within the tumour tissue at baseline, a significant positive correlation between CD4 (P=0.027), CD8 (P=0.028), CD57 (P=0.007) and objective response was demonstrated. After one month of immunotherapy, a significant positive correlation between intratumoral CD3 (P=0.026), CD8 (P=0.015), CD57 (P=0.009) and objective response was demonstrated. A significant positive correlation between intratumoral baseline CD4 (P=0.047), baseline CD57 (P=0.035), CD3 at one month (P=0.049) and survival was demonstrated. These data provide novel in vivo evidence of the possible contribution of lymphocyte subsets in the tumour reduction in responding patients during interleukin-2 based immunotherapy. Confirmation of the results requires further studies including a larger number of patients

Recommendations for the introduction of metagenomic high-throughput sequencing in clinical virology, part I:Wet lab procedure

Metagenomic high-throughput sequencing (mHTS) is a hypothesis-free, universal pathogen detection technique for determination of the DNA/RNA sequences in a variety of sample types and infectious syndromes. mHTS is still in its early stages of translating into clinical application. To support the development, implementation and standardization of mHTS procedures for virus diagnostics, the European Society for Clinical Virology (ESCV) Network on Next-Generation Sequencing (ENNGS) has been established. The aim of ENNGS is to bring together professionals involved in mHTS for viral diagnostics to share methodologies and experiences, and to develop application recommendations. This manuscript aims to provide practical recommendations for the wet lab procedures necessary for implementation of mHTS for virus diagnostics and to give recommendations for development and validation of laboratory methods, including mHTS quality assurance, control and quality assessment protocols