Trine B. Rounge

Reproducibility and repeatability of six high-throughput 16S rDNA sequencing protocols for microbiota profiling

Culture-independent molecular techniques and advances in next generation sequencing (NGS) technologies make large-scale epidemiological studies on microbiota feasible. A challenge using NGS is to obtain high reproducibility and repeatability, which is mostly attained through robust amplification. We aimed to assess the reproducibility of saliva microbiota by comparing triplicate samples. The microbiota was produced with simplified in-house 16S amplicon assays taking advantage of large number of barcodes. The assays included primers with Truseq (TS-tailed) or Nextera (NX-tailed) adapters and either with dual index or dual index plus a 6-nt internal index. All amplification protocols produced consistent microbial profiles for the same samples. Although, in our study, reproducibility was highest for the TS-tailed method. Five replicates of a single sample, prepared with the TS-tailed 1-step protocol without internal index sequenced on the HiSeq platform provided high alpha-diversity and low standard deviation (mean Shannon and Inverse Simpson diversity was 3.19u202f±u202f0.097 and 13.56u202f±u202f1.634 respectively). Large-scale profiling of microbiota can consistently be produced by all 16S amplicon assays. The TS-tailed-1S dual index protocol is preferred since it provides repeatable profiles on the HiSeq platform and are less labour intensive.

Genotypic diversity of anogenital human papillomavirus in women attending cervical cancer screening in Harare, Zimbabwe†

Although anogenital cancers have been on a gradual rise in developing countries in the past few decades, they have been understudied. The objective was to investigate genotypic diversity of anogenital HPV amongst women reporting for routine cervical cancer screening in Harare in Zimbabwe. A cross‐sectional study that enrolled 144 women ≥18 years from a cervical cancer‐screening clinic was performed. Each woman provided a self‐collected cervico‐vaginal swab (VS) and a clinician‐collected anal swab (CCAS). HIV testing was offered and cervical cytology was performed. Both VS and CCAS samples were HPV genotyped, using amplicon sequencing of the L1 gene region with Illumina technology. Mean age of the women was 39.9 (range 18‐83 years, SDu2009±u200911.0). HPV prevalence was 72% (104/144) in VS and 48% (69/144) in CCAS. The most common genotypes detected in both VS and CCAS were HPV18, HPV52, and HPV16. Sixty two percent of the subjects had multiple genotypic HPV infections. The odds of being HPV‐positive among HIV‐infected women were higher than in HIV‐negative women in both the vagina and the anus (CCAS ORu2009=u20094.8; CI 2.4‐9.8, Pu2009<u20090.001) and (VS ORu2009=u20092.9; CI 1.3‐6.4, Pu2009=u20090.005). High HPV prevalence and diverse genotypes were detected in both the vagina and anus. Anal oncogenic HPV infection was common. HPV 52 was one of the most common oncogenic genotypes in both the vagina and anus. HIV co‐infection played a significant role in the prevalence of HPV. These data have implications for design of primary and secondary programs for prevention of anogenital cancer in Zimbabwe.

Circulating small non-coding RNAs associated with age, sex, smoking, body mass and physical activity

Non-coding RNAs (ncRNA) are regulators of cell functions and circulating ncRNAs from the majority of RNA classes, such as miRNA, tRNA, piRNAs, lncRNA, snoRNA, snRNA and miscRNAs, are potential non-invasive biomarkers. Understanding how non-disease traits influence ncRNA expression is essential for assessing their biomarker potential. We studied associations of common traits (sex, age, smoking, body mass, physical activity, and technical factors such as sample storage and processing) with serum ncRNAs. We used RNAseq data from 526 donors from the Janus Serum Bank and traits from health examination surveys. We identified associations between all RNA classes and traits. Ageing showed the strongest association with ncRNA expression, both in terms of statistical significance and number of RNAs, regardless of RNA class. Serum processing modifications and storage times significantly altered expression levels of a number of ncRNAs. Interestingly, smoking cessation generally restored RNA expression to non-smoking levels, although for some isomiRs, mRNA fragments and tRNAs smoking-related expression levels persisted. Our results show that common traits influence circulating ncRNA expression. Therefore it is clear that ncRNA biomarker analyses should be adjusted for age and sex. In addition, for specific ncRNAs identified in our study, analyses should also be adjusted for body mass, smoking, physical activity and serum processing and storage.

Assessing genome-wide significance for the detection of differentially methylated regions

Abstract DNA methylation plays an important role in human health and disease, and methods for the identification of differently methylated regions are of increasing interest. There is currently a lack of statistical methods which properly address multiple testing, i.e. control genome-wide significance for differentially methylated regions. We introduce a scan statistic (DMRScan), which overcomes these limitations. We benchmark DMRScan against two well established methods (bumphunter, DMRcate), using a simulation study based on real methylation data. An implementation of DMRScan is available from Bioconductor. Our method has higher power than alternative methods across different simulation scenarios, particularly for small effect sizes. DMRScan exhibits greater flexibility in statistical modeling and can be used with more complex designs than current methods. DMRScan is the first dynamic approach which properly addresses the multiple-testing challenges for the identification of differently methylated regions. DMRScan outperformed alternative methods in terms of power, while keeping the false discovery rate controlled.

Intra-host sequence variability in human papillomavirus

Human papillomaviruses (HPVs) co-evolve slowly with the human host and each HPV genotype displays epithelial tropisms. We assessed the evolution of intra HPV genotype variants within samples, and their association to anogenital site, cervical cytology and HIV status. Variability in the L1 gene of 35 HPV genotypes was characterized phylogenetically using maximum likelihood, and portrayed by phenotype. Up to a thousand unique variants were identified within individual samples. In-depth analyses of the most prevalent genotypes, HPV16, HPV18 and HPV52, revealed that the high diversity was dominated by a few abundant variants. This suggests high intra-host mutation rates. Clades of HPV16, HPV18 and HPV52 were associated to anatomical site and HIV co-infection. Particularly, we observed that one HPV16 clade was specific to vaginal cells and one HPV52 clade was specific to anal cells. One major HPV52 clade, present in several samples, was strongly associated with cervical neoplasia. Overall, our data suggest that tissue tropism and HIV immunosuppression are strong shapers of HPV evolution.

Cohort Profile: The Finnish Health in Teens (Fin-HIT) study: a population-based study

Cohort Profile: The Finnish Health in Teens (Fin-HIT) study: a population-based study Rejane Augusta de Oliveira Figueiredo,* Sabina Simola-Ström, Trine B Rounge, Heli Viljakainen, Johan G Eriksson, Eva Roos and Elisabete Weiderpass Genetic Epidemiology Group, Folkhälsan Research Center, Helsinki, Finland, Faculty of Medicine, University of Helsinki, Helsinki, Finland, Department of Research, Cancer Registry of Norway, Institute of Population-Based Cancer Research, Oslo, Norway, Department of Food and Environmental Sciences, University of Helsinki, Helsinki, Finland, Department of General Practice and Primary Healthcare, University of Helsinki and Helsinki University Hospital, Helsinki, Finland, Department of Public Health, University of Helsinki, Helsinki, Finland, Department of Medical Epidemiology and Biostatistics, Karolinska Institutet, Stockholm, Sweden and Department of Community Medicine, Faculty of Health Sciences, University of Tromsø, Arctic University of Norway, Tromsø, Norway

Abstract 524: Impact of age, sex, smoking, body mass and physical activity on circulating small non-coding RNA expression profiles

Non-coding RNAs (ncRNA) are regulators of cell functions and circulating ncRNAs from the majority of the RNA classes, such as miRNA, tRNA, piRNAs, lncRNA, snoRNA, snRNA and miscRNAs, are potential non-invasive cancer biomarkers. Understanding how non-disease traits influence ncRNA expression is essential for assessing their biomarker potential. The aim of the study was to investigate associations between sex, age, smoking, body mass, physical activity, technical factors such as sample storage and processing, and serum ncRNA expression profiles. Serum samples from 526 healthy individuals in the Janus Serum Bank Cohort were included in the study. The samples were collected in the time-period 1972-2004 with varying collection procedures, and stored at - 25o C. Information on smoking habits, body mass and physical activity was linked from health examination survey data. RNA was extracted from 400 µl serum using phenol-chloroform separation and the miRNA Neasy Serum/Plasma kit (Qiagen). Small RNAseq was performed using NEBNext Small RNA Library Prep Set for Illumina with an average sequencing depth of 18 million reads per sample. The RNAseq reads were initially trimmed for adapters using Adapter Removal (v2.1.7). We then mapped the collapsed reads (generated by FASTX v0.14) to the human genome (hg38) using Bowtie2 (10 alignments per read were allowed). We compiled a comprehensive annotation set from miRBase (v21) for miRNAs, pirBAse (v1.0) for piRNAs, GENCODE (v26) for other RNAs and tRNAs. We used SeqBuster (v3.1) to get isomiR and miRNA profiles. To count the mapped reads, HTSeq (v0.7.2) was used. Differential gene expression analyses based on the negative binomial distribution and Wald significance tests were performed for each trait using the R package DESeq2 version 1.14.1. We identified associations between all RNA classes and traits. Ageing showed the strongest association with ncRNA expression, both in terms of statistical significance and number of RNAs, regardless of RNA class. Serum processing modifications and storage times significantly altered expression levels of a number of ncRNAs. Smoking cessation generally restored RNA expression to non-smoking levels, although for some isomiRs, mRNA fragments and tRNAs smoking-related expression levels persisted. sncRNA expression levels in serum are considerably age-dependent and age should be adjusted for in studies of circulating sncRNA expression. Certain biomarkers are also influenced by body mass, smoking, physical activity, serum processing and storage conditions. Citation Format: Trine Ballestad Rounge, Sinan Ugur Umu, Andreas Keller, Eckart Meese, Giske Ursin, Steinar Tretli, Robert Lyle, Hilde Langseth. Impact of age, sex, smoking, body mass and physical activity on circulating small non-coding RNA expression profiles [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 524.

HPV Genotyping of Modified General Primer-Amplicons Is More Analytically Sensitive and Specific by Sequencing than by Hybridization.

Sensitive and specific genotyping of human papillomaviruses (HPVs) is important for population-based surveillance of carcinogenic HPV types and for monitoring vaccine effectiveness. Here we compare HPV genotyping by Next Generation Sequencing (NGS) to an established DNA hybridization method. In DNA isolated from urine, the overall analytical sensitivity of NGS was found to be 22% higher than that of hybridization. NGS was also found to be the most specific method and expanded the detection repertoire beyond the 37 types of the DNA hybridization assay. Furthermore, NGS provided an increased resolution by identifying genetic variants of individual HPV types. The same Modified General Primers (MGP)-amplicon was used in both methods. The NGS method is described in detail to facilitate implementation in the clinical microbiology laboratory and includes suggestions for new standards for detection and calling of types and variants with improved resolution.

Validity of home-measured height, weight and waist circumference among adolescents

This study assesses the validity of home-measured height, weight and waist circumference among Finnish adolescents from the Fin-HIT cohort. The adolescents were measured by fieldworkers, and were instructed to measure themselves at home with an adults assistance. Paired t-test was used for statistical analyses. Home-measured mean height, weight and waist circumference were slightly higher, but BMI lower than measured by the fieldworker. The difference in means was statistically significant for weight (0.51 kg) and waist circumference (1.6 cm), but not for height and BMI. Home-measured height, weight, waist circumference and BMI are sufficiently accurate to be used in epidemiologic studies.

Abstract LB-296: Small RNA profiling of human testicular germ cell tumor tissue samples shows abundant tRNA fragments and global loss of piRNA

Small non-coding RNAs play essential roles in tumorigenesis. However, their role in testicular germ cell tumor (TGCT), the most common malignancy in young males in most western countries, is largely unknown. In particular, a comprehensive analysis of the small RNA composition, including microRNAs (miRNAs), PIWI-interacting RNAs (piRNAs), and tRNA-derived small RNAs and their interactions, are lacking. tRNA-derived small RNAs have been shown to bind to the piRNA-associated protein HIWI2, indicating crosstalk between small RNA pathways. We have employed small RNA sequencing on 22 human TGCT samples from 5 histological subtypes, 3 carcinoma in situ, and 12 normal testis samples. The sequences from each sample group were analyzed according to sequence length, base composition, and their association with repeats, exons, tRNAs, piRNAs, and miRNAs. Most sequences 24-32 nucleotides in length, displayed piRNA characteristics. Expression analyses of the small RNA contigs demonstrated global loss of small RNAs in TGCT compared to normal testis. A large proportion of the 33-36 nt RNA sequences derives from mature tRNAs, predominantly from the 5′ end. We did not identify differences in the expression of 5′ tRNA halves between normal and cancer tissue samples; however, 3 shorter 5′ tRNA fragments showed differential expression. Although components of the same pathways might be involved in piRNA and tRNA-derived small RNAs biogenesis, expression differences in TGCT samples suggest independent regulation of key components in response to the carcinogenic process. Our results show subtype-specific microRNA expression, as well as a global loss of piRNAs in all TGCT histological subtypes when compared to normal testicular tissue. In addition, we document the presence of large amounts of tRNA-derived sequences in both TGCT and normal samples, which may provide new insight into the association between the tRNA fragments and piRNA biogenesis. These results may be valuable in the identification of new biomarkers for TGCT. Citation Format: Trine B. Rounge, Kari Furu, Rolf Skotheim, Trine B. Haugen, Espen Enerly, Tom Grotmol. Small RNA profiling of human testicular germ cell tumor tissue samples shows abundant tRNA fragments and global loss of piRNA. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-296. doi:10.1158/1538-7445.AM2015-LB-296