Data pre-processing for analyzing microbiome data - A mini review.

The human microbiome is an emerging research frontier due to its profound impacts on health. High-throughput microbiome sequencing enables studying microbial communities but suffers from analytical challenges. In particular, the lack of dedicated preprocessing methods to improve data quality impedes effective minimization of biases prior to downstream analysis. This review aims to address this gap by providing a comprehensive overview of preprocessing techniques relevant to microbiome research. We outline a typical workflow for microbiome data analysis. Preprocessing methods discussed include quality filtering, batch effect correction, imputation of missing values, normalization, and data transformation. We highlight strengths and limitations of each technique to serve as a practical guide for researchers and identify areas needing further methodological development. Establishing robust, standardized preprocessing will be essential for drawing valid biological conclusions from microbiome studies.

Carnobacterium maltaromaticum boosts intestinal vitamin D production to suppress colorectal cancer in female mice.

Carnobacterium maltaromaticum was found to be specifically depleted in female patients with colorectal cancer (CRC). Administration of C. maltaromaticum reduces intestinal tumor formation in two murine CRC models in a female-specific manner. Estrogen increases the attachment and colonization of C. maltaromaticum via increasing the colonic expression of SLC3A2 that binds to DD-CPase of this bacterium. Metabolomic and transcriptomic profiling unveils the increased gut abundance of vitamin D-related metabolites and the mucosal activation of vitamin D receptor (VDR) signaling in C. maltaromaticum-gavaged mice in a gut microbiome- and VDR-dependent manner. In vitro fermentation system confirms the metabolic cross-feeding of C. maltaromaticum with Faecalibacterium prausnitzii to convert C. maltaromaticum-produced 7-dehydrocholesterol into vitamin D for activating the host VDR signaling. Overall, C. maltaromaticum colonizes the gut in an estrogen-dependent manner and acts along with other microbes to augment the intestinal vitamin D production to activate the host VDR for suppressing CRC.

Altered gut microbiota of obesity subjects promotes colorectal carcinogenesis in mice.

BACKGROUND: Obesity is a risk factor for colorectal cancer (CRC). The role of gut microbiota in mediating the cancer-promoting effect of obesity is unknown. METHODS: Azoxymethane (AOM)-treated, ApcMin/+ and germ-free mice were gavaged with feces from obese individuals and control subjects respectively. The colonic tumor load and number were recorded at the endpoint in two carcinogenic models. The gut microbiota composition and colonic transcriptome were assessed by metagenomic sequencing and RNA sequencing, respectively. The anticancer effects of bacteria depleted in fecal samples of obese individuals were validated. FINDINGS: Conventional AOM-treated and ApcMin/+ mice receiving feces from obese individuals showed significantly increased colon tumor formation compared with those receiving feces from control subjects. AOM-treated mice receiving feces from obese individuals showed impaired intestinal barrier function and significant upregulation of pro-inflammatory cytokines and activation of oncogenic Wnt signaling pathway. Consistently, transferring feces from obese individuals to germ-free mice led to increased colonic cell proliferation, intestinal barrier function impairment, and induction of oncogenic and proinflammatory gene expression. Moreover, germ-free mice transplanted with feces from obese human donors had increased abundance of potential pathobiont Alistipes finegoldii, and reduced abundance of commensals Bacteroides vulgatus and Akkermansia muciniphila compared with those receiving feces from human donors with normal body mass index (BMI). Validation experiments showed that B. vulgatus and A. muciniphila demonstrated anti-proliferative effects in CRC, while A. finegoldii promoted CRC tumor growth. INTERPRETATION: Our results supported the role of obesity-associated microbiota in colorectal carcinogenesis and identified putative bacterial candidates that may mediate its mechanisms. Microbiota modulation in obese individuals may provide new approaches to prevent or treat obesity-related cancers including CRC. FUNDING: This work was funded by National Key Research and Development Program of China (2020YFA0509200/2020YFA0509203), National Natural Science Foundation of China (81922082), RGC Theme-based Research Scheme Hong Kong (T21-705/20-N), RGC Research Impact Fund Hong Kong (R4632-21F), RGC-CRF Hong Kong (C4039-19GF and C7065-18GF), RGC-GRF Hong Kong (14110819, 14111621), and NTU Start-Up Grant (021337-00001).

Meta-analysis of mucosal microbiota reveals universal microbial signatures and dysbiosis in gastric carcinogenesis.

The consistency of the associations between gastric mucosal microbiome and gastric cancer across studies remained unexamined. We aimed to identify universal microbial signatures in gastric carcinogenesis through a meta-analysis of gastric microbiome from multiple studies. Compositional and ecological profiles of gastric microbes across stages of gastric carcinogenesis were significantly altered. Meta-analysis revealed that opportunistic pathobionts Fusobacterium, Parvimonas, Veillonella, Prevotella and Peptostreptococcus were enriched in GC, while commensals Bifidobacterium, Bacillus and Blautia were depleted in comparison to SG. The co-occurring correlation strengths of GC-enriched bacteria were increased along disease progression while those of GC-depleted bacteria were decreased. Eight bacterial taxa, including Veillonella, Dialister, Granulicatella, Herbaspirillum, Comamonas, Chryseobacterium, Shewanella and Helicobacter, were newly identified by this study as universal biomarkers for robustly discriminating GC from SG, with an area under the curve (AUC) of 0.85. Moreover, H. pylori-positive samples exhibited reduced microbial diversity, altered microbiota community and weaker interactions among gastric microbes. Our meta-analysis demonstrated comprehensive and generalizable gastric mucosa microbial features associated with histological stages of gastric carcinogenesis, including GC associated bacteria, diagnostic biomarkers, bacterial network alteration and H. pylori influence.

Forward and reverse mutations in stages of cancer development.

BACKGROUND: Massive occurrences of interstitial loss of heterozygosity (LOH) likely resulting from gene conversions were found by us in different cancers as a type of single-nucleotide variations (SNVs), comparable in abundance to the commonly investigated gain of heterozygosity (GOH) type of SNVs, raising the question of the relationships between these two opposing types of cancer mutations. METHODS: In the present study, SNVs in 12 tetra sample and 17 trio sample sets from four cancer types along with copy number variations (CNVs) were analyzed by AluScan sequencing, comparing tumor with white blood cells as well as tissues vicinal to the tumor. Four published "nontumor"-tumor metastasis trios and 246 pan-cancer pairs analyzed by whole-genome sequencing (WGS) and 67 trios by whole-exome sequencing (WES) were also examined. RESULTS: Widespread GOHs enriched with CG-to-TG changes and associated with nearby CNVs and LOHs enriched with TG-to-CG changes were observed. Occurrences of GOH were 1.9-fold higher than LOH in "nontumor" tissues more than 2 cm away from the tumors, and a majority of these GOHs and LOHs were reversed in "paratumor" tissues within 2 cm of the tumors, forming forward-reverse mutation cycles where the revertant LOHs displayed strong lineage effects that pointed to a sequential instead of parallel development from "nontumor" to "paratumor" and onto tumor cells, which was also supported by the relative frequencies of 26 distinct classes of CNVs between these three types of cell populations. CONCLUSIONS: These findings suggest that developing cancer cells undergo sequential changes that enable the "nontumor" cells to acquire a wide range of forward mutations including ones that are essential for oncogenicity, followed by revertant mutations in the "paratumor" cells to avoid growth retardation by excessive mutation load. Such utilization of forward-reverse mutation cycles as an adaptive mechanism was also observed in cultured HeLa cells upon successive replatings. An understanding of forward-reverse mutation cycles in cancer development could provide a genomic basis for improved early diagnosis, staging, and treatment of cancers.

Genome-centric metatranscriptomes and ecological roles of the active microbial populations during cellulosic biomass anaerobic digestion.

BACKGROUND: Although anaerobic digestion for biogas production is used worldwide in treatment processes to recover energy from carbon-rich waste such as cellulosic biomass, the activities and interactions among the microbial populations that perform anaerobic digestion deserve further investigations, especially at the population genome level. To understand the cellulosic biomass-degrading potentials in two full-scale digesters, this study examined five methanogenic enrichment cultures derived from the digesters that anaerobically digested cellulose or xylan for more than 2 years under 35 or 55 °C conditions. RESULTS: Metagenomics and metatranscriptomics were used to capture the active microbial populations in each enrichment culture and reconstruct their meta-metabolic network and ecological roles. 107 population genomes were reconstructed from the five enrichment cultures using a differential coverage binning approach, of which only a subset was highly transcribed in the metatranscriptomes. Phylogenetic and functional convergence of communities by enrichment condition and phase of fermentation was observed for the highly transcribed populations in the metatranscriptomes. In the 35 °C cultures grown on cellulose, Clostridium cellulolyticum-related and Ruminococcus-related bacteria were identified as major hydrolyzers and primary fermenters in the early growth phase, while Clostridium leptum-related bacteria were major secondary fermenters and potential fatty acid scavengers in the late growth phase. While the meta-metabolism and trophic roles of the cultures were similar, the bacterial populations performing each function were distinct between the enrichment conditions. CONCLUSIONS: Overall, a population genome-centric view of the meta-metabolism and functional roles of key active players in anaerobic digestion of cellulosic biomass was obtained. This study represents a major step forward towards understanding the microbial functions and interactions at population genome level during the microbial conversion of lignocellulosic biomass to methane. The knowledge of this study can facilitate development of potential biomarkers and rational design of the microbiome in anaerobic digesters.

Physiological and Metagenomic Characterizations of the Synergistic Relationships between Ammonia- and Nitrite-Oxidizing Bacteria in Freshwater Nitrification.

Nitrification plays a crucial role in global nitrogen cycling and treatment processes. However, the relationships between the nitrifier guilds of ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) are still poorly understood, especially in freshwater habitats. This study examined the physiological interactions between the AOB and NOB present in a freshwater aquarium biofilter by culturing them, either together or separately, in a synthetic medium. Metagenomic and 16S rRNA gene sequencing revealed the presence and the draft genomes of Nitrosomonas-like AOB as well as Nitrobacter-like NOB in the cultures, including the first draft genome of Nitrobacter vulgaris. The nitrifiers exhibited different growth rates with different ammonium (NH4+) or nitrite concentrations (50-1,500 µM) and the growth rates were elevated under a high bicarbonate (HCO3-) concentration. The half-saturation constant (Ks for NH4+), the maximum growth rate (µmax), and the lag duration indicated a strong dependence on the synergistic relationships between the two guilds. Overall, the ecophysiological and metagenomic results in this study provided insights into the phylogeny of the key nitrifying players in a freshwater biofilter and showed that interactions between the two nitrifying guilds in a microbial community enhanced nitrification.

Physiological and molecular characterizations of the interactions in two cellulose-to-methane cocultures.

BACKGROUND: The interspecies interactions in a biomethanation community play a vital role in substrate degradation and methane (CH4) formation. However, the physiological and molecular mechanisms of interaction among the microbial members of this community remain poorly understood due to the lack of an experimentally tractable model system. In this study, we successfully established two coculture models combining the cellulose-degrading bacterium Clostridium cellulovorans 743B with Methanosarcina barkeri Fusaro or Methanosarcina mazei Gö1 for the direct conversion of cellulose to CH4. RESULTS: Physiological characterizations of these models revealed that the methanogens in both cocultures were able to efficiently utilize the products produced by C. cellulovorans during cellulose degradation. In particular, the simultaneous utilization of hydrogen, formate, and acetate for methanogenesis was observed in the C. cellulovorans-M. barkeri cocultures, whereas monocultures of M. barkeri were unable to grow with formate alone. Enhanced cellulose degradation was observed in both cocultures, and the CH4 yield of the C. cellulovorans-M. barkeri cocultures (0.87 ± 0.02 mol CH4/mol glucose equivalent) was among the highest compared to other coculture studies. A metabolic shift in the fermentation pattern of C. cellulovorans was observed in both cocultures. The expression levels of genes in key pathways that are important to the regulation and metabolism of the interactions in cocultures were examined by reverse transcription-quantitative PCR, and the expression profiles largely matched the physiological observations. CONCLUSIONS: The physiological and molecular characteristics of the interactions of two CH4-producing cocultures are reported. Coculturing C. cellulovorans with M. barkeri or M. mazei not only enabled direct conversion of cellulose to CH4, but also stabilized pH for C. cellulovorans, resulting in a metabolic shift and enhanced cellulose degradation. This study deepens our understanding of interspecies interactions for CH4 production from cellulose, providing useful insights for assembling consortia as inocula for industrial biomethanation processes.

Metagenomic Reconstruction of Key Anaerobic Digestion Pathways in Municipal Sludge and Industrial Wastewater Biogas-Producing Systems.

Anaerobic digestion (AD) is a microbial process widely used to treat organic wastes. While the microbes involved in digestion of municipal sludge are increasingly well characterized, the taxonomic and functional compositions of AD digesters treating industrial wastewater have been understudied. This study examined metagenomes from a biogas-producing digester treating municipal sludge in Shek Wu Hui (SWH), Hong Kong and an industrial wastewater digester in Guangzhou (GZ), China, and compared their taxonomic composition and reconstructed biochemical pathways. Genes encoding carbohydrate metabolism and protein metabolism functions were overrepresented in GZ, while genes encoding functions related to fatty acids, lipids and isoprenoids were overrepresented in SWH, reflecting the plants' feedstocks. Mapping of genera to functions in each community indicated that both digesters had a high level of functional redundancy, and a more even distribution of genera in GZ suggested that it was more functionally stable. While fermentation in both samples was dominated by Clostridia, SWH had an overrepresentation of Proteobacteria, including syntrophic acetogens, reflecting its more complex substrate. Considering the growing importance of biogas as an alternative fuel source, a detailed mechanistic understanding of AD is important and this report will be a basis for further study of industrial wastewater AD.

Coevolution Theory of the Genetic Code at Age Forty: Pathway to Translation and Synthetic Life.

The origins of the components of genetic coding are examined in the present study. Genetic information arose from replicator induction by metabolite in accordance with the metabolic expansion law. Messenger RNA and transfer RNA stemmed from a template for binding the aminoacyl-RNA synthetase ribozymes employed to synthesize peptide prosthetic groups on RNAs in the Peptidated RNA World. Coevolution of the genetic code with amino acid biosynthesis generated tRNA paralogs that identify a last universal common ancestor (LUCA) of extant life close to Methanopyrus, which in turn points to archaeal tRNA introns as the most primitive introns and the anticodon usage of Methanopyrus as an ancient mode of wobble. The prediction of the coevolution theory of the genetic code that the code should be a mutable code has led to the isolation of optional and mandatory synthetic life forms with altered protein alphabets.

Feature co-localization landscape of the human genome.

Although feature co-localizations could serve as useful guide-posts to genome architecture, a comprehensive and quantitative feature co-localization map of the human genome has been lacking. Herein we show that, in contrast to the conventional bipartite division of genomic sequences into genic and inter-genic regions, pairwise co-localizations of forty-two genomic features in the twenty-two autosomes based on 50-kb to 2,000-kb sequence windows indicate a tripartite zonal architecture comprising Genic zones enriched with gene-related features and Alu-elements; Proximal zones enriched with MIR- and L2-elements, transcription-factor-binding-sites (TFBSs), and conserved-indels (CIDs); and Distal zones enriched with L1-elements. Co-localizations between single-nucleotide-polymorphisms (SNPs) and copy-number-variations (CNVs) reveal a fraction of sequence windows displaying steeply enhanced levels of SNPs, CNVs and recombination rates that point to active adaptive evolution in such pathways as immune response, sensory perceptions, and cognition. The strongest positive co-localization observed between TFBSs and CIDs suggests a regulatory role of CIDs in cooperation with TFBSs. The positive co-localizations of cancer somatic CNVs (CNVT) with all Proximal zone and most Genic zone features, in contrast to the distinctly more restricted co-localizations exhibited by germline CNVs (CNVG), reveal disparate distributions of CNVTs and CNVGs indicative of dissimilarity in their underlying mechanisms.

GABRB2 Haplotype Association with Heroin Dependence in Chinese Population.

Substance dependence is a frequently observed comorbid disorder in schizophrenia, but little is known about genetic factors possibly shared between the two psychotic disorders. GABRB2, a schizophrenia candidate gene coding for GABAA receptor ß2 subunit, is examined for possible association with heroin dependence in Han Chinese population. Four single nucleotide polymorphisms (SNPs) in GABRB2, namely rs6556547 (S1), rs1816071 (S3), rs18016072 (S5), and rs187269 (S29), previously associated with schizophrenia, were examined for their association with heroin dependence. Two additional SNPs, rs10051667 (S31) and rs967771 (S32), previously associated with alcohol dependence and bipolar disorder respectively, were also analyzed. The six SNPs were genotyped by direct sequencing of PCR amplicons of target regions for 564 heroin dependent individuals and 498 controls of Han Chinese origin. Interestingly, it was found that recombination between the haplotypes of all-derived-allele (H1; OR = 1.00) and all-ancestral-allele (H2; OR = 0.74) at S5-S29 junction generated two recombinants H3 (OR = 8.51) and H4 (OR = 5.58), both conferring high susceptibility to heroin dependence. Additional recombination between H2 and H3 haplotypes at S1-S3 junction resulted in a risk-conferring haplotype H5 (OR = 1.94x109). In contrast, recombination between H1 and H2 haplotypes at S3-S5 junction rescued the risk-conferring effect of recombination at S5-S29 junction, giving rise to the protective haplotype H6 (OR = 0.68). Risk-conferring effects of S1-S3 and S5-S29 crossovers and protective effects of S3-S5 crossover were seen in both pure heroin dependent and multiple substance dependence subgroups. In conclusion, significant association was found with haplotypes of the S1-S29 segment in GABRB2 for heroin dependence in Han Chinese population. Local recombination was an important determining factor for switching haplotypes between risk-conferring and protective statuses. The present study provide evidence for the schizophrenia candidate gene GABRB2 to play a role in heroin dependence, but replication of these findings is required.

Massive interstitial copy-neutral loss-of-heterozygosity as evidence for cancer being a disease of the DNA-damage response.

BACKGROUND: The presence of loss-of-heterozygosity (LOH) mutations in cancer cell genomes is commonly encountered. Moreover, the occurrences of LOHs in tumor suppressor genes play important roles in oncogenesis. However, because the causative mechanisms underlying LOH mutations in cancer cells yet remain to be elucidated, enquiry into the nature of these mechanisms based on a comprehensive examination of the characteristics of LOHs in multiple types of cancers has become a necessity. METHODS: We performed next-generation sequencing on inter-Alu sequences of five different types of solid tumors and acute myeloid leukemias, employing the AluScan platform which entailed amplification of such sequences using multiple PCR primers based on the consensus sequences of Alu elements; as well as the whole genome sequences of a lung-to-liver metastatic cancer and a primary liver cancer. Paired-end sequencing reads were aligned to the reference human genome to identify major and minor alleles so that the partition of LOH products between homozygous-major vs. homozygous-minor alleles could be determined at single-base resolution. Strict filtering conditions were employed to avoid false positives. Measurements of LOH occurrences in copy number variation (CNV)-neutral regions were obtained through removal of CNV-associated LOHs. RESULTS: We found: (a) average occurrence of copy-neutral LOHs amounting to 6.9% of heterologous loci in the various cancers; (b) the mainly interstitial nature of the LOHs; and (c) preference for formation of homozygous-major over homozygous-minor, and transitional over transversional, LOHs. CONCLUSIONS: The characteristics of the cancer LOHs, observed in both AluScan and whole genome sequencings, point to the formation of LOHs through repair of double-strand breaks by interhomolog recombination, or gene conversion, as the consequence of a defective DNA-damage response, leading to a unified mechanism for generating the mutations required for oncogenesis as well as the progression of cancer cells.

Application of Machine Learning to Development of Copy Number Variation-based Prediction of Cancer Risk.

In the present study, recurrent copy number variations (CNVs) from non-tumor blood cell DNAs of Caucasian non-cancer subjects and glioma, myeloma, and colorectal cancer-patients, and Korean non-cancer subjects and hepatocellular carcinoma, gastric cancer, and colorectal cancer patients, were found to reveal for each of the two ethnic cohorts highly significant differences between cancer patients and controls with respect to the number of CN-losses and size-distribution of CN-gains, suggesting the existence of recurrent constitutional CNV-features useful for prediction of predisposition to cancer. Upon identification by machine learning, such CNV-features could extensively discriminate between cancer-patient and control DNAs. When the CNV-features selected from a learning-group of Caucasian or Korean mixed DNAs consisting of both cancer-patient and control DNAs were employed to make predictions on the cancer predisposition of an unseen test group of mixed DNAs, the average prediction accuracy was 93.6% for the Caucasian cohort and 86.5% for the Korean cohort.

Social cognitive role of schizophrenia candidate gene GABRB2.

The occurrence of positive selection in schizophrenia-associated GABRB2 suggests a broader impact of the gene product on population fitness. The present study considered the possibility of cognition-related GABRB2 involvement by examining the association of GABRB2 with psychosis and altruism, respectively representing psychiatric and psychological facets of social cognition. Four single nucleotide polymorphisms (SNPs) were genotyped for quantitative trait analyses and population-based association studies. Psychosis was measured by either the Positive and Negative Syndrome Scale (PANSS) or antipsychotics dosage, and altruism was based on a self-report altruism scale. The minor alleles of SNPs rs6556547, rs1816071 and rs187269 in GABRB2 were correlated with high PANSS score for positive symptoms in a Han Chinese schizophrenic cohort, whereas those of rs1816071 and rs1816072 were associated with high antipsychotics dosage in a US Caucasian schizophrenic cohort. Moreover, strongly significant GABRB2-disease associations were found among schizophrenics with severe psychosis based on high PANSS positive score, but no significant association was observed for schizophrenics with only mild psychosis. Interestingly, in addition to association with psychosis in schizophrenics, rs187269 was also associated with altruism in healthy Han Chinese. Furthermore, parallel to correlation with severe psychosis, its minor allele was correlated with high altruism scores. These findings revealed that GABRB2 is associated with psychosis, the core symptom and an endophenotype of schizophrenia. Importantly, the association was found across the breadth of the psychiatric (psychosis) to psychological (altruism) spectrum of social cognition suggesting GABRB2 involvement in human cognition.

Epigenetic regulation on GABRB2 isoforms expression: developmental variations and disruptions in psychotic disorders.

INTRODUCTION: To improve the understanding of psychotic abnormalities and their non-Mendelian inheritance patterns, the epigenetic regulation of the psychotic disorder-associated GABRB2, gene for the type A γ-aminobutyric acid receptor ß(2)-subunit, was investigated. METHODS: Expression of GABRB2, and the epigenetic regulatory enzymes histone deacetylases (HDACs) and DNA methyltransferases (DNMTs) in mouse and postmortem human brains was analyzed using real-time PCR. RESULTS: Results showed that expression of GABRB2 isoforms significantly increased over time in both mouse and human, especially for the long splicing isoform. In the brains of non-psychiatric controls (CON), a significant positive correlation of GABRB2 expression with age was observed in individuals with MM genotypes of the single nucleotide polymorphisms (SNPs) rs187269 and rs1816072. This was reversed to a significant negative correlation in schizophrenics (SCZ). A similar reversal was also displayed by bipolar disorder (BPD) patients. In parallel, a significant co-variation of HDAC1 with GABRB2 expression observed in CON remained significant in BPD but not in SCZ; comparably, a significant co-variation of HDAC2 with GABRB2 expression observed in CON became non-significant in both SCZ and BPD. Moreover, co-variations of DNMT1 and DNMT3B with GABRB2, not observable in CON, became significant in BPD. CONCLUSION: These findings demonstrated that GABRB2 expression was under epigenetic regulation that varied with development, genotype and disease status, and these regulatory mechanisms were observably disrupted in SCZ and BPD. This study provided insight into the complex inheritance patterns of psychiatric disorders, and pointed to the involvement of epigenetic dysregulation in the disease process of major psychotic disorders.

AluScan: a method for genome-wide scanning of sequence and structure variations in the human genome.

BACKGROUND: To complement next-generation sequencing technologies, there is a pressing need for efficient pre-sequencing capture methods with reduced costs and DNA requirement. The Alu family of short interspersed nucleotide elements is the most abundant type of transposable elements in the human genome and a recognized source of genome instability. With over one million Alu elements distributed throughout the genome, they are well positioned to facilitate genome-wide sequence amplification and capture of regions likely to harbor genetic variation hotspots of biological relevance. RESULTS: Here we report on the use of inter-Alu PCR with an enhanced range of amplicons in conjunction with next-generation sequencing to generate an Alu-anchored scan, or 'AluScan', of DNA sequences between Alu transposons, where Alu consensus sequence-based 'H-type' PCR primers that elongate outward from the head of an Alu element are combined with 'T-type' primers elongating from the poly-A containing tail to achieve huge amplicon range. To illustrate the method, glioma DNA was compared with white blood cell control DNA of the same patient by means of AluScan. The over 10 Mb sequences obtained, derived from more than 8,000 genes spread over all the chromosomes, revealed a highly reproducible capture of genomic sequences enriched in genic sequences and cancer candidate gene regions. Requiring only sub-micrograms of sample DNA, the power of AluScan as a discovery tool for genetic variations was demonstrated by the identification of 357 instances of loss of heterozygosity, 341 somatic indels, 274 somatic SNVs, and seven potential somatic SNV hotspots between control and glioma DNA. CONCLUSIONS: AluScan, implemented with just a small number of H-type and T-type inter-Alu PCR primers, provides an effective capture of a diversity of genome-wide sequences for analysis. The method, by enabling an examination of gene-enriched regions containing exons, introns, and intergenic sequences with modest capture and sequencing costs, computation workload and DNA sample requirement is particularly well suited for accelerating the discovery of somatic mutations, as well as analysis of disease-predisposing germline polymorphisms, by making possible the comparative genome-wide scanning of DNA sequences from large human cohorts.

A recombination hotspot in a schizophrenia-associated region of GABRB2.

BACKGROUND: Schizophrenia is a major disorder with complex genetic mechanisms. Earlier, population genetic studies revealed the occurrence of strong positive selection in the GABRB2 gene encoding the beta(2) subunit of GABA(A) receptors, within a segment of 3,551 bp harboring twenty-nine single nucleotide polymorphisms (SNPs) and containing schizophrenia-associated SNPs and haplotypes. METHODOLOGY/PRINCIPAL FINDINGS: In the present study, the possible occurrence of recombination in this 'S1-S29' segment was assessed. The occurrence of hotspot recombination was indicated by high resolution recombination rate estimation, haplotype diversity, abundance of rare haplotypes, recurrent mutations and torsos in haplotype networks, and experimental haplotyping of somatic and sperm DNA. The sub-segment distribution of relative recombination strength, measured by the ratio of haplotype diversity (H(d)) over mutation rate (theta), was indicative of a human specific Alu-Yi6 insertion serving as a central recombining sequence facilitating homologous recombination. Local anomalous DNA conformation attributable to the Alu-Yi6 element, as suggested by enhanced DNase I sensitivity and obstruction to DNA sequencing, could be a contributing factor of the increased sequence diversity. Linkage disequilibrium (LD) analysis yielded prominent low LD points that supported ongoing recombination. LD contrast revealed significant dissimilarity between control and schizophrenic cohorts. Among the large array of inferred haplotypes, H26 and H73 were identified to be protective, and H19 and H81 risk-conferring, toward the development of schizophrenia. CONCLUSIONS/SIGNIFICANCE: The co-occurrence of hotspot recombination and positive selection in the S1-S29 segment of GABRB2 has provided a plausible contribution to the molecular genetics mechanisms for schizophrenia. The present findings therefore suggest that genome regions characterized by the co-occurrence of positive selection and hotspot recombination, two interacting factors both affecting genetic diversity, merit close scrutiny with respect to the etiology of common complex disorders.

Alternative-splicing in the exon-10 region of GABA(A) receptor beta(2) subunit gene: relationships between novel isoforms and psychotic disorders.

BACKGROUND: Non-coding single nucleotide polymorphisms (SNPs) in GABRB2, the gene for beta(2)-subunit of gamma-aminobutyric acid type A (GABA(A)) receptor, have been associated with schizophrenia (SCZ) and quantitatively correlated to mRNA expression and alternative splicing. METHODS AND FINDINGS: Expression of the Exon 10 region of GABRB2 from minigene constructs revealed this region to be an "alternative splicing hotspot" that readily gave rise to differently spliced isoforms depending on intron sequences. This led to a search in human brain cDNA libraries, and the discovery of two novel isoforms, beta(2S1) and beta(2S2), bearing variations in the neighborhood of Exon-10. Quantitative real-time PCR analysis of postmortem brain samples showed increased beta(2S1) expression and decreased beta(2S2) expression in both SCZ and bipolar disorder (BPD) compared to controls. Disease-control differences were significantly correlated with SNP rs187269 in BPD males for both beta(2S1) and beta(2S2) expressions, and significantly correlated with SNPs rs2546620 and rs187269 in SCZ males for beta(2S2) expression. Moreover, site-directed mutagenesis indicated that Thr(365), a potential phosphorylation site in Exon-10, played a key role in determining the time profile of the ATP-dependent electrophysiological current run-down. CONCLUSION: This study therefore provided experimental evidence for the importance of non-coding sequences in the Exon-10 region in GABRB2 with respect to beta(2)-subunit splicing diversity and the etiologies of SCZ and BPD.

Polyphasic evidence delineating the root of life and roots of biological domains.

Twenty different lines of polyphasic evidence obtained from tRNA and protein sequences, anticodon usages, gene contents, metabolism and geochemistry have made possible the identification of a Last Universal Common Ancestor (LUCA) phylogenetically located proximal to the hyperthermophilic methanogenic archaeon Methanopyrus. Combined with analysis of high-similarity cross-domain tRNA pairs, the evidence also suggests a Thermotoga-proximal Last Bacterial Common Ancestor (LBACA) that originated from Crenarchaeota close to Aeropyrum, and a Plasmodium-proximal Last Eukaryotic Common Ancestor (LECA) derived from Ferroplasma through endosymbiosis.