Integration of polygenic and gut metagenomic risk prediction for common diseases.

Multiomics has shown promise in noninvasive risk profiling and early detection of various common diseases. In the present study, in a prospective population-based cohort with ~18 years of e-health record follow-up, we investigated the incremental and combined value of genomic and gut metagenomic risk assessment compared with conventional risk factors for predicting incident coronary artery disease (CAD), type 2 diabetes (T2D), Alzheimer disease and prostate cancer. We found that polygenic risk scores (PRSs) improved prediction over conventional risk factors for all diseases. Gut microbiome scores improved predictive capacity over baseline age for CAD, T2D and prostate cancer. Integrated risk models of PRSs, gut microbiome scores and conventional risk factors achieved the highest predictive performance for all diseases studied compared with models based on conventional risk factors alone. The present study demonstrates that integrated PRSs and gut metagenomic risk models improve the predictive value over conventional risk factors for common chronic diseases.

The gut microbiome is a significant risk factor for future chronic lung disease.

BACKGROUND: The gut-lung axis is generally recognized, but there are few large studies of the gut microbiome and incident respiratory disease in adults. OBJECTIVE: We sought to investigate the association and predictive capacity of the gut microbiome for incident asthma and chronic obstructive pulmonary disease (COPD). METHODS: Shallow metagenomic sequencing was performed for stool samples from a prospective, population-based cohort (FINRISK02; N = 7115 adults) with linked national administrative health register-derived classifications for incident asthma and COPD up to 15 years after baseline. Generalized linear models and Cox regressions were used to assess associations of microbial taxa and diversity with disease occurrence. Predictive models were constructed using machine learning with extreme gradient boosting. Models considered taxa abundances individually and in combination with other risk factors, including sex, age, body mass index, and smoking status. RESULTS: A total of 695 and 392 statistically significant associations were found between baseline taxonomic groups and incident asthma and COPD, respectively. Gradient boosting decision trees of baseline gut microbiome abundance predicted incident asthma and COPD in the validation data sets with mean area under the curves of 0.608 and 0.780, respectively. Cox analysis showed that the baseline gut microbiome achieved higher predictive performance than individual conventional risk factors, with C-indices of 0.623 for asthma and 0.817 for COPD. The integration of the gut microbiome and conventional risk factors further improved prediction capacities. CONCLUSIONS: The gut microbiome is a significant risk factor for incident asthma and incident COPD and is largely independent of conventional risk factors.

Early prediction of incident liver disease using conventional risk factors and gut-microbiome-augmented gradient boosting.

The gut microbiome has shown promise as a predictive biomarker for various diseases. However, the potential of gut microbiota for prospective risk prediction of liver disease has not been assessed. Here, we utilized shallow shotgun metagenomic sequencing of a large population-based cohort (N > 7,000) with ∼15 years of follow-up in combination with machine learning to investigate the predictive capacity of gut microbial predictors individually and in conjunction with conventional risk factors for incident liver disease. Separately, conventional and microbial factors showed comparable predictive capacity. However, microbiome augmentation of conventional risk factors using machine learning significantly improved the performance. Similarly, disease-free survival analysis showed significantly improved stratification using microbiome-augmented models. Investigation of predictive microbial signatures revealed previously unknown taxa for liver disease, as well as those previously associated with hepatic function and disease. This study supports the potential clinical validity of gut metagenomic sequencing to complement conventional risk factors for prediction of liver diseases.

Integrative analysis of the plasma proteome and polygenic risk of cardiometabolic diseases.

Cardiometabolic diseases are frequently polygenic in architecture, comprising a large number of risk alleles with small effects spread across the genome1-3. Polygenic scores (PGS) aggregate these into a metric representing an individual's genetic predisposition to disease. PGS have shown promise for early risk prediction4-7 and there is an open question as to whether PGS can also be used to understand disease biology8. Here, we demonstrate that cardiometabolic disease PGS can be used to elucidate the proteins underlying disease pathogenesis. In 3,087 healthy individuals, we found that PGS for coronary artery disease, type 2 diabetes, chronic kidney disease and ischaemic stroke are associated with the levels of 49 plasma proteins. Associations were polygenic in architecture, largely independent of cis and trans protein quantitative trait loci and present for proteins without quantitative trait loci. Over a follow-up of 7.7 years, 28 of these proteins associated with future myocardial infarction or type 2 diabetes events, 16 of which were mediators between polygenic risk and incident disease. Twelve of these were druggable targets with therapeutic potential. Our results demonstrate the potential for PGS to uncover causal disease biology and targets with therapeutic potential, including those that may be missed by approaches utilizing information at a single locus.

The intersect of genetics, environment, and microbiota in asthma-perspectives and challenges.

In asthma, a significant portion of the interaction between genetics and environment occurs through microbiota. The proposed mechanisms behind this interaction are complex and at times contradictory. This review covers recent developments in our understanding of this interaction: the "microbial hypothesis" and the "farm effect"; the role of endotoxin and genetic variation in pattern recognition systems; the interaction with allergen exposure; the additional involvement of host gut and airway microbiota; the role of viral respiratory infections in interaction with the 17q21 and CDHR3 genetic loci; and the importance of in utero and early-life timing of exposures. We propose a unified framework for understanding how all these phenomena interact to drive asthma pathogenesis. Finally, we point out some future challenges for continued research in this field, in particular the need for multiomic integration, as well as the potential utility of asthma endotyping.

Developmental patterns in the nasopharyngeal microbiome during infancy are associated with asthma risk.

BACKGROUND: Studies indicate that the nasal microbiome may correlate strongly with the presence or future risk of childhood asthma. OBJECTIVES: In this study, we tested whether developmental trajectories of the nasopharyngeal microbiome in early life and the composition of the microbiome during illnesses were related to risk of childhood asthma. METHODS: Children participating in the Childhood Origins of Asthma study (N = 285) provided nasopharyngeal mucus samples in the first 2 years of life, during routine healthy study visits (at 2, 4, 6, 9, 12, 18, and 24 months of age), and during episodes of respiratory illnesses, all of which were analyzed for respiratory viruses and bacteria. We identified developmental trajectories of early-life microbiome composition, as well as predominant bacteria during respiratory illnesses, and we correlated these with presence of asthma at 6, 8, 11, 13, and 18 years of age. RESULTS: Of the 4 microbiome trajectories identified, a Staphylococcus-dominant microbiome in the first 6 months of life was associated with increased risk of recurrent wheezing by age 3 years and asthma that persisted throughout childhood. In addition, this trajectory was associated with the early onset of allergic sensitization. During wheezing illnesses, detection of rhinoviruses and predominance of Moraxella were associated with asthma that persisted throughout later childhood. CONCLUSION: In infancy, the developmental composition of the microbiome during healthy periods and the predominant microbes during acute wheezing illnesses are both associated with the subsequent risk of developing persistent childhood asthma.

Neonatal genetics of gene expression reveal potential origins of autoimmune and allergic disease risk.

Chronic immune-mediated diseases of adulthood often originate in early childhood. To investigate genetic associations between neonatal immunity and disease, we map expression quantitative trait loci (eQTLs) in resting myeloid cells and CD4+ T cells from cord blood samples, as well as in response to lipopolysaccharide (LPS) or phytohemagglutinin (PHA) stimulation, respectively. Cis-eQTLs are largely specific to cell type or stimulation, and 31% and 52% of genes with cis-eQTLs have response eQTLs (reQTLs) in myeloid cells and T cells, respectively. We identified cis regulatory factors acting as mediators of trans effects. There is extensive colocalisation between condition-specific neonatal cis-eQTLs and variants associated with immune-mediated diseases, in particular CTSH had widespread colocalisation across diseases. Mendelian randomisation shows causal neonatal gene expression effects on disease risk for BTN3A2, HLA-C and others. Our study elucidates the genetics of gene expression in neonatal immune cells, and aetiological origins of autoimmune and allergic diseases.

Luminal microbiota related to Crohn's disease recurrence after surgery.

BACKGROUND: Microbial factors are likely to be involved in the recurrence of Crohn's disease (CD) after bowel resection. We investigated the luminal microbiota before and longitudinally after surgery, in relation to disease recurrence, using 16S metagenomic techniques. METHODS: In the prospective Post-Operative Crohn's Endoscopic Recurrence (POCER) study, fecal samples were obtained before surgery and 6, 12, and 18 months after surgery from 130 CD patients. Endoscopy was undertaken to detect disease recurrence, defined as Rutgeerts score ≥i2, at 6 months in two-thirds of patients and all patients at 18 months after surgery. The V2 region of the 16S rRNA gene was sequenced using Illumina MiSeq. Cluster analysis was performed at family level, assessing microbiome community differences between patients with and without recurrence. RESULTS: Six microbial cluster groups were identified. The cluster associated with maintenance of remission was enriched for the Lachnospiraceae family [adjusted OR 0.47 (0.27-0.82), P = .007]. The OTU diversity of Lachnospiraceae within this cluster was significantly greater than in all other clusters. The cluster enriched for Enterobacteriaceae was associated with an increased risk of disease recurrence [adjusted OR 6.35 (1.24-32.44), P = .026]. OTU diversity of Enterobacteriaceae within this cluster was significantly greater than in other clusters. CONCLUSIONS: Luminal bacterial communities are associated with protection from, and the occurrence of, Crohn's disease recurrence after surgery. Recurrence may relate to a higher abundance of facultatively anaerobic pathobionts from the Enterobacteriaceae family. The ecologic change of depleted Lachnospiraceae, a genus of butyrate-producing bacteria, may permit expansion of Enterobacteriaceae through luminal environmental perturbation.

Multivariate Genome-wide Association Analysis of a Cytokine Network Reveals Variants with Widespread Immune, Haematological, and Cardiometabolic Pleiotropy.

Cytokines are essential regulatory components of the immune system, and their aberrant levels have been linked to many disease states. Despite increasing evidence that cytokines operate in concert, many of the physiological interactions between cytokines, and the shared genetic architecture that underlies them, remain unknown. Here, we aimed to identify and characterize genetic variants with pleiotropic effects on cytokines. Using three population-based cohorts (n = 9,263), we performed multivariate genome-wide association studies (GWAS) for a correlation network of 11 circulating cytokines, then combined our results in meta-analysis. We identified a total of eight loci significantly associated with the cytokine network, of which two (PDGFRB and ABO) had not been detected previously. In addition, conditional analyses revealed a further four secondary signals at three known cytokine loci. Integration, through the use of Bayesian colocalization analysis, of publicly available GWAS summary statistics with the cytokine network associations revealed shared causal variants between the eight cytokine loci and other traits; in particular, cytokine network variants at the ABO, SERPINE2, and ZFPM2 loci showed pleiotropic effects on the production of immune-related proteins, on metabolic traits such as lipoprotein and lipid levels, on blood-cell-related traits such as platelet count, and on disease traits such as coronary artery disease and type 2 diabetes.

Trajectories of childhood immune development and respiratory health relevant to asthma and allergy.

Events in early life contribute to subsequent risk of asthma; however, the causes and trajectories of childhood wheeze are heterogeneous and do not always result in asthma. Similarly, not all atopic individuals develop wheeze, and vice versa. The reasons for these differences are unclear. Using unsupervised model-based cluster analysis, we identified latent clusters within a prospective birth cohort with deep immunological and respiratory phenotyping. We characterised each cluster in terms of immunological profile and disease risk, and replicated our results in external cohorts from the UK and USA. We discovered three distinct trajectories, one of which is a high-risk 'atopic' cluster with increased propensity for allergic diseases throughout childhood. Atopy contributes varyingly to later wheeze depending on cluster membership. Our findings demonstrate the utility of unsupervised analysis in elucidating heterogeneity in asthma pathogenesis and provide a foundation for improving management and prevention of childhood asthma.

Airway Microbiota Dynamics Uncover a Critical Window for Interplay of Pathogenic Bacteria and Allergy in Childhood Respiratory Disease.

Repeated cycles of infection-associated lower airway inflammation drive the pathogenesis of persistent wheezing disease in children. In this study, the occurrence of acute respiratory tract illnesses (ARIs) and the nasopharyngeal microbiome (NPM) were characterized in 244 infants through their first five years of life. Through this analysis, we demonstrate that >80% of infectious events involve viral pathogens, but are accompanied by a shift in the NPM toward dominance by a small range of pathogenic bacterial genera. Unexpectedly, this change frequently precedes the detection of viral pathogens and acute symptoms. Colonization of illness-associated bacteria coupled with early allergic sensitization is associated with persistent wheeze in school-aged children, which is the hallmark of the asthma phenotype. In contrast, these bacterial genera are associated with "transient wheeze" that resolves after age 3 years in non-sensitized children. Thus, to complement early allergic sensitization, monitoring NPM composition may enable early detection and intervention in high-risk children.

The lower airway microbiota in early cystic fibrosis lung disease: a longitudinal analysis.

RATIONALE: In infants and young children with cystic fibrosis, lower airway infection and inflammation are associated with adverse respiratory outcomes. However, the role of lower airway microbiota in the pathogenesis of early cystic fibrosis lung disease remains uncertain. OBJECTIVES: To assess the development of the lower airway microbiota over time in infants and young children with cystic fibrosis, and to explore its association with airway inflammation and pulmonary function at age 6 years. METHODS: Serial, semi-annual bronchoscopies and bronchoalveolar lavage (BAL) procedures were performed in infants newly diagnosed with cystic fibrosis following newborn screening. Quantitative microbiological cultures and inflammatory marker (interleukin 8 and neutrophil elastase) measurements were undertaken contemporaneously. 16S ribosomal RNA gene sequencing was conducted on stored BAL samples. Spirometry results recorded at 6 years of age were extracted from medical records. MEASUREMENTS AND MAIN RESULTS: Ninety-five BAL samples provided 16S ribosomal RNA gene data. These were collected from 48 subjects aged 1.2-78.3 months, including longitudinal samples from 27 subjects and 13 before age 6 months. The lower airway microbiota varied, but diversity decreased with advancing age. Detection of recognised cystic fibrosis bacterial pathogens was associated with reduced microbial diversity and greater lower airway inflammation. There was no association between the lower airway microbiota and pulmonary function at age 6 years. CONCLUSIONS: In infants with cystic fibrosis, the lower airway microbiota is dynamic. Dominance of the microbiota by recognised cystic fibrosis bacterial pathogens is associated with increased lower airway inflammation, however early microbial diversity is not associated with pulmonary function at 6 years of age.

Microbial Factors Associated with Postoperative Crohn's Disease Recurrence.

BACKGROUND AND AIMS: The intestinal microbiota is a key antigenic driver in Crohn's disease [CD]. We aimed to identify changes in the gut microbiome associated with, and predictive of, disease recurrence and remission. METHODS: A total of 141 mucosal biopsy samples from 34 CD patients were obtained at surgical resection and at colonoscopy 6 and/or 18 months postoperatively; 28 control samples were obtained: 12 from healthy patients [healthy controls] and 16 from hemicolectomy patients [surgical controls]. Bacterial 16S ribosomal profiling was performed using the Illumina MiSeq platform. RESULTS: CD was associated with reduced alpha diversity when compared with healthy controls but not surgical controls [p < 0.001 and p = 0.666, respectively]. Beta diversity [composition] differed significantly between CD and both healthy [p < 0.001] and surgical [p = 0.022] controls, but did not differ significantly between those with and without endoscopic recurrence. There were significant taxonomic differences between recurrence and remission. Patients experiencing recurrence demonstrated elevated Proteus genera [p = 0.008] and reduced Faecalibacterium [p< 0.001]. Active smoking was associated with elevated levels of Proteus [p = 0.013] postoperatively. Low abundance of Faecalibacterium [< 0.1%] and detectable Proteus in the postoperative ileal mucosa was associated with a higher risk of recurrence (odds ratio [OR] 14 [1.7-110], p = 0.013 and 13 [1.1-150], p = 0.039, respectively) when corrected for smoking. A model of recurrence comprising the presence of Proteus, abundance of Faecalibacterium, and smoking status showed moderate accuracy (area under the curve [AUC] 0.740, 95% confidence interval [CI] [0.69-0.79]). CONCLUSIONS: CD is associated with a microbial signature distinct from health. Microbial factors and smoking independently influence postoperative CD recurrence. The genus Proteus may play a role in the development of CD.

Vitamin D over the first decade and susceptibility to childhood allergy and asthma.

BACKGROUND: Vitamin D (25(OH)D) deficiency has been implicated as a possible risk factor for asthma development, but studies at selected time points measuring 25(OH)D levels during childhood have yielded conflicting findings. Prospective studies tracking 25(OH)D levels during the initiation phase of asthma in early childhood have not been reported. OBJECTIVE: We sought to elucidate relationships between 25(OH)D levels from birth to age 10 years and susceptibility to allergic sensitization, respiratory tract infections, and asthma. METHODS: Asthma-, allergy-, and respiratory tract infection-associated phenotypes (including pathogen identification) were characterized in a high-risk birth cohort. Plasma 25(OH)D concentrations were quantified at birth and at clinical follow-ups at the ages of 0.5, 1, 2, 3, 4, 5, and 10 years, and relationships with clinical outcomes were examined. RESULTS: Cross-sectional analyses demonstrated inverse associations between 25(OH)D concentrations and the risk for concurrent sensitization at age 0.5, 2, and 3 years, and mixed-effects regression demonstrated inverse longitudinal associations of 25(OH)D levels with both sensitization and eczema. Multivariate regression modeling suggested that the number of 25(OH)D-deficient follow-ups was positively associated with risk for asthma/wheeze, eczema, and sensitization at 10 years; adjustment for sensitization (particularly by 2 years) in the asthma/wheeze models reduced 25(OH)D associations with these latter outcomes. 25(OH)D levels were also inversely associated with early nasopharyngeal colonization with Streptococcus species and age of first febrile lower respiratory illness, both of which are known asthma risk factors. CONCLUSION: 25(OH)D deficiency in early childhood is associated with increased risk for persistent asthma, potentially through modulating susceptibility to early allergic sensitization, upper respiratory tract colonization with bacterial pathogens, or both. These relationships are only evident if 25(OH)D status is monitored prospectively and longitudinally.

Brain-derived neurotrophic factor genetic polymorphism (rs6265) is protective against chemotherapy-associated cognitive impairment in patients with early-stage breast cancer.

BACKGROUND: Brain-derived neurotrophic factor (BDNF), a neurotrophin that regulates neuronal function and development, is implicated in several neurodegenerative conditions. Preliminary data suggest that a reduction of BDNF concentrations may lead to postchemotherapy cognitive impairment. We hypothesized that a single nucleotide polymorphism (rs6265) of the BDNF gene may predispose patients to cognitive impairment. This study aimed to evaluate the effect of BDNF gene polymorphism on chemotherapy-associated cognitive impairment. METHODS: Overall, 145 patients receiving chemotherapy for early-stage breast cancer (mean age: 50.8 ± 8.8 y; 82.1% Chinese) were recruited. Patients' cognitive functions were assessed longitudinally using the validated Functional Assessment of Cancer Therapy-Cognitive Function (v.3) and an objective computerized tool, Headminder. Genotyping was performed using Sanger sequencing. Logistic regression was used to evaluate the association between BDNF Val66Met polymorphism and cognition after adjusting for ethnicity and clinically important covariates. RESULTS: Of the 145 patients, 54 (37%) reported cognitive impairment postchemotherapy. The Met/Met genotype was associated with statistically significant lower odds of developing cognitive impairment (odds ratio [OR] = 0.26; 95% CI: 0.08-0.92; P = .036). The Met carriers were less likely to experience impairment in the domains of verbal fluency (OR = 0.34; 95% CI: 0.12-0.90; P = .031) and multitasking ability (OR = 0.37; 95% CI: 0.15-0.91; P = .030) compared with the Val/Val homozygote. No associations were observed between Headminder and the BDNF Val66Met polymorphism. CONCLUSIONS: This is the first study to provide evidence that carriers of the BDNF Met allele are protected against chemotherapy-associated cognitive impairment. Further studies are required to validate the findings.

The infant nasopharyngeal microbiome impacts severity of lower respiratory infection and risk of asthma development.

The nasopharynx (NP) is a reservoir for microbes associated with acute respiratory infections (ARIs). Lung inflammation resulting from ARIs during infancy is linked to asthma development. We examined the NP microbiome during the critical first year of life in a prospective cohort of 234 children, capturing both the viral and bacterial communities and documenting all incidents of ARIs. Most infants were initially colonized with Staphylococcus or Corynebacterium before stable colonization with Alloiococcus or Moraxella. Transient incursions of Streptococcus, Moraxella, or Haemophilus marked virus-associated ARIs. Our data identify the NP microbiome as a determinant for infection spread to the lower airways, severity of accompanying inflammatory symptoms, and risk for future asthma development. Early asymptomatic colonization with Streptococcus was a strong asthma predictor, and antibiotic usage disrupted asymptomatic colonization patterns. In the absence of effective anti-viral therapies, targeting pathogenic bacteria within the NP microbiome could represent a prophylactic approach to asthma.

Recent advances in characterizing the gastrointestinal microbiome in Crohn's disease: a systematic review.

BACKGROUND: The intestinal microbiota is involved in the pathogenesis of inflammatory bowel disease. A reduction in the diversity of the intestinal microbiota as well as specific taxonomic and functional shifts have been reported in Crohn's disease and may play a central role in the inflammatory process. The aim was to systematically review recent developments in the structural and functional changes observed in the gastrointestinal microbiome in patients with Crohn's Disease. RESULTS: Seventy-two abstracts were included in this review. The effects of host genetics, disease phenotype, and inflammatory bowel disease treatment on the gastrointestinal microbiome in Crohn's disease were reviewed, and taxonomic shifts in patients with early and established disease were described. The relative abundance of Bacteroidetes is increased and Firmicutes decreased in Crohn's disease compared with healthy controls. Enterobacteriaceae, specifically Eschericia coli, is enriched in Crohn's disease. Faecalibacterium prausnitzii is found at lower abundance in Crohn's disease and in those with postoperative recurrence. Observed functional changes include major shifts in oxidative stress pathways, a decrease in butanoate and propanoate metabolism gene expression, lower levels of butyrate, and other short-chain fatty acids, decreased carbohydrate metabolism, and decreased amino acid biosynthesis. CONCLUSIONS: Changes in microbial composition and function have been described, although a causative role remains to be established. Larger, prospective, and longitudinal studies are required with deep interrogation of the microbiome if causality is to be determined, and refined microbial manipulation is to emerge as a focused therapy.

Elucidation of pathways driving asthma pathogenesis: development of a systems-level analytic strategy.

Asthma is a genetically complex, chronic lung disease defined clinically as episodic airflow limitation and breathlessness that is at least partially reversible, either spontaneously or in response to therapy. Whereas asthma was rare in the late 1800s and early 1900s, the marked increase in its incidence and prevalence since the 1960s points to substantial gene × environment interactions occurring over a period of years, but these interactions are very poorly understood (1-6). It is widely believed that the majority of asthma begins during childhood and manifests first as intermittent wheeze. However, wheeze is also very common in infancy and only a subset of wheezy children progress to persistent asthma for reasons that are largely obscure. Here, we review the current literature regarding causal pathways leading to early asthma development and chronicity. Given the complex interactions of many risk factors over time eventually leading to apparently multiple asthma phenotypes, we suggest that deeply phenotyped cohort studies combined with sophisticated network models will be required to derive the next generation of biological and clinical insights in asthma pathogenesis.

Deep whole-genome sequencing of 100 southeast Asian Malays.

Whole-genome sequencing across multiple samples in a population provides an unprecedented opportunity for comprehensively characterizing the polymorphic variants in the population. Although the 1000 Genomes Project (1KGP) has offered brief insights into the value of population-level sequencing, the low coverage has compromised the ability to confidently detect rare and low-frequency variants. In addition, the composition of populations in the 1KGP is not complete, despite the fact that the study design has been extended to more than 2,500 samples from more than 20 population groups. The Malays are one of the Austronesian groups predominantly present in Southeast Asia and Oceania, and the Singapore Sequencing Malay Project (SSMP) aims to perform deep whole-genome sequencing of 100 healthy Malays. By sequencing at a minimum of 30× coverage, we have illustrated the higher sensitivity at detecting low-frequency and rare variants and the ability to investigate the presence of hotspots of functional mutations. Compared to the low-pass sequencing in the 1KGP, the deeper coverage allows more functional variants to be identified for each person. A comparison of the fidelity of genotype imputation of Malays indicated that a population-specific reference panel, such as the SSMP, outperforms a cosmopolitan panel with larger number of individuals for common SNPs. For lower-frequency (<5%) markers, a larger number of individuals might have to be whole-genome sequenced so that the accuracy currently afforded by the 1KGP can be achieved. The SSMP data are expected to be the benchmark for evaluating the value of deep population-level sequencing versus low-pass sequencing, especially in populations that are poorly represented in population-genetics studies.

Statistical challenges associated with detecting copy number variations with next-generation sequencing.

MOTIVATION: Analysing next-generation sequencing (NGS) data for copy number variations (CNVs) detection is a relatively new and challenging field, with no accepted standard protocols or quality control measures so far. There are by now several algorithms developed for each of the four broad methods for CNV detection using NGS, namely the depth of coverage (DOC), read-pair, split-read and assembly-based methods. However, because of the complexity of the genome and the short read lengths from NGS technology, there are still many challenges associated with the analysis of NGS data for CNVs, no matter which method or algorithm is used. RESULTS: In this review, we describe and discuss areas of potential biases in CNV detection for each of the four methods. In particular, we focus on issues pertaining to (i) mappability, (ii) GC-content bias, (iii) quality control measures of reads and (iv) difficulty in identifying duplications. To gain insights to some of the issues discussed, we also download real data from the 1000 Genomes Project and analyse its DOC data. We show examples of how reads in repeated regions can affect CNV detection, demonstrate current GC-correction algorithms, investigate sensitivity of DOC algorithm before and after quality control of reads and discuss reasons for which duplications are harder to detect than deletions.