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 3.19 +/- 0.097 and 13.56 +/- 1.634 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.Peer reviewe

Gender-Specific Associations Between Saliva Microbiota and Body Size

Objective: The human intestinal microbiota likely play an important role in the development of overweight and obesity. However, the associations between saliva microbiota and body mass index (BMI) have been sparsely studied. The aim of this study was to identify the associations between saliva microbiota and body size in Finnish children. Methods: The saliva microbiota of 900 Finnish children, aged 11-14 years with measured height and weight, was characterized using 16S rRNA (V3-V4) sequencing. Results: The core saliva microbiota consisted of 14 genera that were present in more than 95% of the Finnish children. The saliva microbiota profiles were gender-specific with higher alpha-diversity in boys than girls and significant differences between the genders in community composition and abundances. Alpha-diversity differed between normal weight and overweight girls and between normal weight and obese boys. The composition was dissimilar between normal weight and obese girls, but not in boys. The relative abundance profiles differed according to body size. Decrease in commensal saliva bacteria were observed in all the body sizes when compared to normal weight children. Notably, the relative abundance of bacteria related to, Veillonella, Prevotella, Selenomonas, and Streptococcus was reduced in obese children. Conclusion: Saliva microbiota diversity and composition were significantly associated with body size and gender in Finnish children. Body size-specific saliva microbiota profiles open new avenues for studying the potential roles of microbiota in weight development and management.Peer reviewe

TaME-seq : An efficient sequencing approach for characterisation of HPV genomic variability and chromosomal integration

HPV genomic variability and chromosomal integration are important in the HPV-induced carcinogenic process. To uncover these genomic events in an HPV infection, we have developed an innovative and cost-effective sequencing approach named TaME-seq (tagmentation-assisted multiplex PCR enrichment sequencing). TaME-seq combines tagmentation and multiplex PCR enrichment for simultaneous analysis of HPV variation and chromosomal integration, and it can also be adapted to other viruses. For method validation, cell lines (n = 4), plasmids (n = 3), and HPV16, 18, 31, 33 and 45 positive clinical samples (n = 21) were analysed. Our results showed deep HPV genome-wide sequencing coverage. Chromosomal integration breakpoints and large deletions were identified in HPV positive cell lines and in one clinical sample. HPV genomic variability was observed in all samples allowing identification of low frequency variants. In contrast to other approaches, TaME-seq proved to be highly efficient in HPV target enrichment, leading to reduced sequencing costs. Comprehensive studies on HPV intra-host variability generated during a persistent infection will improve our understanding of viral carcinogenesis. Efficient identification of both HPV variability and integration sites will be important for the study of HPV evolution and adaptability and may be an important tool for use in cervical cancer diagnostics.Peer reviewe

STAT3 Mutation Is Associated with STAT3 Activation in CD30+ ALK− ALCL

Peripheral T-cell lymphomas (PTCL) are a heterogeneous, and often aggressive group of non-Hodgkin lymphomas. Recent advances in the molecular and genetic characterization of PTCLs have helped to delineate differences and similarities between the various subtypes, and the JAK/STAT pathway has been found to play an important oncogenic role. Here, we aimed to characterize the JAK/STAT pathway in PTCL subtypes and investigate whether the activation of the pathway correlates with the frequency of STAT gene mutations. Patient samples from AITL (n = 30), ALCL (n = 21) and PTCL-NOS (n = 12) cases were sequenced for STAT3, STAT5B, JAK1, JAK3, and RHOA mutations using amplicon sequencing and stained immunohistochemically for pSTAT3, pMAPK, and pAKT. We discovered STAT3 mutations in 13% of AITL, 13% of ALK+ ALCL, 38% of ALK− ALCL and 17% of PTCL-NOS cases. However, no STAT5B mutations were found and JAK mutations were only present in ALK- ALCL (15%). Concurrent mutations were found in all subgroups except ALK+ ALCL where STAT3 mutations were always seen alone. High pY-STAT3 expression was observed especially in AITL and ALCL samples. When studying JAK-STAT pathway mutations, pY-STAT3 expression was highest in PTCLs harboring either JAK1 or STAT3 mutations and CD30+ phenotype representing primarily ALK− ALCLs. Further investigation is needed to elucidate the molecular mechanisms of JAK-STAT pathway activation in PTCL

STAT3 Mutation Is Associated with STAT3 Activation in CD30+ ALK− ALCL

Peripheral T-cell lymphomas (PTCL) are a heterogeneous, and often aggressive group of non-Hodgkin lymphomas. Recent advances in the molecular and genetic characterization of PTCLs have helped to delineate differences and similarities between the various subtypes, and the JAK/STAT pathway has been found to play an important oncogenic role. Here, we aimed to characterize the JAK/STAT pathway in PTCL subtypes and investigate whether the activation of the pathway correlates with the frequency of STAT gene mutations. Patient samples from AITL (n = 30), ALCL (n = 21) and PTCL-NOS (n = 12) cases were sequenced for STAT3, STAT5B, JAK1, JAK3, and RHOA mutations using amplicon sequencing and stained immunohistochemically for pSTAT3, pMAPK, and pAKT. We discovered STAT3 mutations in 13% of AITL, 13% of ALK+ ALCL, 38% of ALK− ALCL and 17% of PTCL-NOS cases. However, no STAT5B mutations were found and JAK mutations were only present in ALK- ALCL (15%). Concurrent mutations were found in all subgroups except ALK+ ALCL where STAT3 mutations were always seen alone. High pY-STAT3 expression was observed especially in AITL and ALCL samples. When studying JAK-STAT pathway mutations, pY-STAT3 expression was highest in PTCLs harboring either JAK1 or STAT3 mutations and CD30+ phenotype representing primarily ALK− ALCLs. Further investigation is needed to elucidate the molecular mechanisms of JAK-STAT pathway activation in PTCL

Novel TMEM173 Mutation and the Role of Disease Modifying Alleles

Upon binding to pathogen or self-derived cytosolic nucleic acids cyclic GMP-AMP synthase (cGAS) triggers the production of cGAMP that further activates transmembrane protein STING. Upon activation STING translocates from ER via Golgi to vesicles. Monogenic STING gain-of-function mutations cause early-onset type I interferonopathy, with disease presentation ranging from fatal vasculopathy to mild chilblain lupus. Molecular mechanisms underlying the variable phenotype-genotype correlation are presently unclear. Here, we report a novel gain-of-function G207E STING mutation causing a distinct phenotype with alopecia, photosensitivity, thyroid dysfunction, and features of STING-associated vasculopathy with onset in infancy (SAVI), such as livedo reticularis, skin vasculitis, nasal septum perforation, facial erythema, and bacterial infections. Polymorphism in TMEM173 and IFIH1 showed variable penetrance in the affected family, implying contribution to varying phenotype spectrum. The G207E mutation constitutively activates inflammation-related pathways in vitro, and causes aberrant interferon signature and inflammasome activation in patient PBMCs. Treatment with Janus kinase 1 and 2 (JAK1/2) inhibitor baricitinib was beneficiary for a vasculitic ulcer, induced hair regrowth and improved overall well-being in one patient. Protein-protein interactions propose impaired cellular trafficking of G207E mutant. These findings reveal the molecular landscape of STING and propose common polymorphisms in TMEM173 and IFIH1 as likely modifiers of the phenotype.Peer reviewe

Gender-Specific Associations Between Saliva Microbiota and Body Size

ObjectiveThe human intestinal microbiota likely play an important role in the development of overweight and obesity. However, the associations between saliva microbiota and body mass index (BMI) have been sparsely studied. The aim of this study was to identify the associations between saliva microbiota and body size in Finnish children.MethodsThe saliva microbiota of 900 Finnish children, aged 11–14 years with measured height and weight, was characterized using 16S rRNA (V3–V4) sequencing.ResultsThe core saliva microbiota consisted of 14 genera that were present in more than 95% of the Finnish children. The saliva microbiota profiles were gender-specific with higher alpha-diversity in boys than girls and significant differences between the genders in community composition and abundances. Alpha-diversity differed between normal weight and overweight girls and between normal weight and obese boys. The composition was dissimilar between normal weight and obese girls, but not in boys. The relative abundance profiles differed according to body size. Decrease in commensal saliva bacteria were observed in all the body sizes when compared to normal weight children. Notably, the relative abundance of bacteria related to, Veillonella, Prevotella, Selenomonas, and Streptococcus was reduced in obese children.ConclusionSaliva microbiota diversity and composition were significantly associated with body size and gender in Finnish children. Body size–specific saliva microbiota profiles open new avenues for studying the potential roles of microbiota in weight development and management

Characterisation of human papillomavirus genomic variation and chromosomal integration in cervical samples

Persistent infection with a high-risk human papillomavirus (HPV) type is necessary for cervical cancer development, causing nearly 5% of all cancers worldwide. Nevertheless, only a small fraction of HPV infections progress to cancer, indicating that additional molecular factors contribute to the development of cervical cancer. The thesis aimed to characterise and explore mutations in the HPV genome and viral integrations into the human genome contributing to HPV-induced carcinogenesis. This can reveal new insight into cervical cancer development. A unique next-generation sequencing protocol, TaME-seq, was developed for analysis of HPV genomic variation and integration. The results show that the overall intra-host HPV genomic variability is higher than previously assumed, with a high number of HPV genome variants found in all samples from early infections to cancer. A noticeable part of the mutations in HPV16, which is the most carcinogenic HPV type, was associated with the APOBEC3-enzyme that is suggested to be involved in viral clearance. The findings revealed integration sites that located both in previously reported and novel genomic sites. A large number of integrations was observed in or close to human cancer-related genes, which could be an indication of a more aggressive infection. The TaME-seq method could potentially be a valuable method for assessing the risk of developing cervical cancer. An additional HPV screening test would enable more personalised follow-up, improving detection of lesions with higher risk of progression and reducing unnecessary follow-up and treatment of women with minimal risk of developing high-grade lesions or cancer

Erratum to "HPV16 whole genome minority variants in persistent infections from young Dutch women" [J. Clin. Virol. 119 (2019) 24-30].

The publisher regrets that there was a typing error in the second paragraph of the section entitled “3.3. HPV16 minor nucleotide variations”. The correct text should read: In order to explore unusual mutational patterns in any gene region, the number of synonymous and non-synonymous MNVs was mapped against the consensus sequence of each infection (Table 2). On average there were 1.67 times (STDEV ± 0.19) more non-synonymous than synonymous mutations. No genomic region could be singled out as notably different from other regions. The incorrect text had previously read: In order to explore unusual mutational patterns in any gene region, the number of synonymous and non-synonymous MNVs was mapped against the consensus sequence of each infection (Table 2). On average there were 167 times (STDEV ± 019) more non-synonymous than synonymous mutations. No genomic region could be singled out as notably different from other regions. The publisher would like to apologise for any inconvenience caused

HPV16 whole genome minority variants in persistent infections from young Dutch women.

Background: Chronic infections by one of the oncogenic human papillomaviruses (HPVs) are responsible for near 5% of the global cancer burden and HPV16 is the type most often found in cancers. HPV genomes displayunexpected levels of variation when deep-sequenced. Minor nucleotide variations (MNVs) may reveal HPVgenomic instability and HPV-related carcinogenic transformation of host cells. Objectives: The objective of this study was to investigate HPV16 genome variation at the minor variant level onpersisting HPV16 cervical infections from a population of young Dutch women. Study design:15 HPV16 infections were sequenced using a whole-HPV genome deep sequencing protocol (TaME-seq). One infection was followed over a three-year period, eight were followed over a two-year period, threewere followed over a one-year period and three infections had a single sampling point. Results and conclusions: Using a 1% variant frequency cutoff, we find on average 48 MNVs per HPV16 genomeand 1717 MNVs in total when sequencing coverage was > 100 × . Wefind the transition mutation T > C to be the most common, in contrast to other studies detecting APOBEC-related C > T mutation profiles in pre-cancerous and cancer samples. Our results suggest that the relative mutagenic footprint of HPV16 genomes may differ between the infections in this study and transforming lesions. In addition, we identify a number of MNVs that have previously been associated with higher incidence of high-grade lesions (CIN3+) in a population study. These findings may provide a starting point for future studies exploring causality between emerging HPV minorgenomic variants and cancer development