Don't let valuable microbiome data go to waste: combined usage of merging and direct-joining of sequencing reads for low-quality paired-end amplicon data.

The pernicious nature of low-quality sequencing data warrants improvement in the bioinformatics workflow for profiling microbial diversity. The conventional merging approach, which drops a copious amount of sequencing reads when processing low-quality amplicon data, requires alternative methods. In this study, a computational workflow, a combination of merging and direct-joining where the paired-end reads lacking overlaps are concatenated and pooled with the merged sequences, is proposed to handle the low-quality amplicon data. The proposed computational strategy was compared with two workflows; the merging approach where the paired-end reads are merged, and the direct-joining approach where the reads are concatenated. The results showed that the merging approach generates a significantly low number of amplicon sequences, limits the microbiome inference, and obscures some microbial associations. In comparison to other workflows, the combination of merging and direct-joining strategy reduces the loss of amplicon data, improves the taxonomy classification, and importantly, abates the misleading results associated with the merging approach when analysing the low-quality amplicon data. The mock community analysis also supports the findings. In summary, the researchers are suggested to follow the merging and direct-joining workflow to avoid problems associated with low-quality data while profiling the microbial community structure.

Review of bioaerosols from different sources and their health impacts.

The emission of bioaerosols in the ambient atmosphere from different sources is a cause of concern for human health and the environment. Bioaerosols are a combination of biotic matter like microbes and pollens. The present review emphasizes the understanding of various sources of bioaerosols (industries, municipal solid waste, and medical facilities), their components, and their impact on human health. The study of bioaerosols is of great importance as large numbers of people are estimated to be exposed on the global scale. Bioaerosols exposure in different work environments results in health issues such as infectious diseases, allergies, toxic effects, and respiratory problems. Hence, extensive research is urged to establish an effective assessment of bioaerosols exposure in the workplace, risks involved, distribution, and validation. The present review is intended to explore the relationship between bioaerosols exposure to the atmosphere and its impacts on human health. Some of the preliminary findings, based on our analysis of bioaerosols arising from municipal solid waste at a landfill site and a waste transfer station in Hyderabad, India, are also discussed herein.

Influence of 16S rRNA reference databases in amplicon-based environmental microbiome research.

PURPOSE: The reference databases play a pivotal role in amplicon microbiome research, however these databases differ in the sequence content and taxonomic information available. Studies on mock community and human health microbiome have revealed the problems associated with the choice of reference database on the outcome. Nonetheless, the influence of reference databases in environmental microbiome studies is not explicitly illustrated. METHODS: This study analyzed the amplicon (V1V3, V3V4, V4V5 and V6V8) data of 128 soil samples and evaluated the impact of 16S rRNA databases, Genome Taxonomy Database (GTDB), Ribosomal Database Project (RDP), SILVA and Consensus Taxonomy (ConTax), on microbiome inference. RESULTS: The analyses showed that the distribution of observed amplicon sequence variants was significantly different (P-value < 2.647e-12) across four datasets, generated using different databases for each amplicon region. In addition, the beta diversity was also found to be altered by different databases. Further investigation revealed that the microbiome composition inferred by various databases differ significantly (P-value = 0.001), irrespective of amplicon regions. This study, found that the core-microbiome structure in environmental studies is influenced by the type of reference database used. CONCLUSION: In summary, this present study illustrates that the choice of reference database could influence the outcome of environmental microbiome research.

A comprehensive evaluation of single-end sequencing data analyses for environmental microbiome research.

Illumina sequencing platforms have been widely used for amplicon-based environmental microbiome research. Analyses of amplicon data of environmental samples, generated from Illumina MiSeq platform illustrate the reverse (R2) reads in the PE datasets to have low quality towards the 3' end of the reads which affect the sequencing depth of samples and ultimately impact the sample size which may possibly lead to an altered outcome. This study evaluates the usefulness of single-end (SE) sequencing data in microbiome research when the Illumina MiSeq PE dataset shows significantly high number of low-quality reverse reads. In this study, the amplicon data (V1V3, V3V4, V4V5 and V6V8) from 128 environmental (soil) samples, downloaded from SRA, demonstrate the efficiency of single-end (SE) sequencing data analyses in microbiome research. The SE datasets were found to infer the core microbiome structure as comparable to the PE dataset. Conspicuously, the forward (R1) datasets inferred a higher number of taxa as compared to PE datasets for most of the amplicon regions, except V3V4. Thus, analyses of SE sequencing data, especially R1 reads, in environmental microbiome studies could ameliorate the problems arising on sample size of the study due to low quality reverse reads in the dataset. However, care must be taken while interpreting the microbiome structure as few taxa observed in the PE datasets were absent in the SE datasets. In conclusion, this study demonstrates the availability of choices in analyzing the amplicon data without having the need to remove samples with low quality reverse reads.

Effect of Amplicon Sequencing Depth in Environmental Microbiome Research.

Amplicon sequencing approach is commonly employed in microbiome studies and sequencing depth is considered as a major factor influencing the outcome of data analyses. As of now, the effect of amplicon sequencing depth in environmental microbiome analyses is not explicitly illustrated. In this study, microbiome data of nine aquatic samples from Sundarbans mangrove region, obtained from SRA, were analyzed to explain the influence of sequencing depth variation in environmental microbiome data analyses. Briefly, four groups based on number of reads (NOR) were created comprising of, total NOR, 75 k, 50 k and 25 k, followed by data analyses. The results showed that the observed ASVs among four groups were significantly different (P value 1.094e-06). The Bray-Curtis dissimilarity analysis showed differences in microbiome composition and also, each group exhibited slightly different core-microbiome structure. Importantly, the variation in sequencing depth was found to affect the predictions of environmental drivers associated with microbiome composition. Thus, this study emphasizes that the microbiome data are compositional and the NOR in the data could affect the microbial composition. In summary, this study demonstrates the consequences of sequencing depth variation on microbiome data analyses and suggests the researchers to take proper cautions to avoid misleading results due to sequencing depth variation.

Integrative genomic analysis identifies ancestry-related expression quantitative trait loci on DNA polymerase ß and supports the association of genetic ancestry with survival disparities in head and neck squamous cell carcinoma.

BACKGROUND: African Americans with head and neck squamous cell carcinoma (HNSCC) have a lower survival rate than whites. This study investigated the functional importance of ancestry-informative single-nucleotide polymorphisms (SNPs) in HNSCC and also examined the effect of functionally important genetic elements on racial disparities in HNSCC survival. METHODS: Ancestry-informative SNPs, RNA sequencing, methylation, and copy number variation data for 316 oral cavity and laryngeal cancer patients were analyzed across 178 DNA repair genes. The results of expression quantitative trait locus (eQTL) analyses were also replicated with a Gene Expression Omnibus (GEO) data set. The effects of eQTLs on overall survival (OS) and disease-free survival (DFS) were evaluated. RESULTS: Five ancestry-related SNPs were identified as cis-eQTLs in the DNA polymerase ß (POLB) gene (false discovery rate [FDR] < 0.01). The homozygous/heterozygous genotypes containing the African allele showed higher POLB expression than the homozygous white allele genotype (P < .001). A replication study using a GEO data set validated all 5 eQTLs and also showed a statistically significant difference in POLB expression based on genetic ancestry (P = .002). An association was observed between these eQTLs and OS (P < .037; FDR < 0.0363) as well as DFS (P = .018 to .0629; FDR < 0.079) for oral cavity and laryngeal cancer patients treated with platinum-based chemotherapy and/or radiotherapy. Genotypes containing the African allele were associated with poor OS/DFS in comparison with homozygous genotypes harboring the white allele. CONCLUSIONS: Analyses show that ancestry-related alleles could act as eQTLs in HNSCC and support the association of ancestry-related genetic factors with survival disparities in patients diagnosed with oral cavity and laryngeal cancer. Cancer 2017;123:849-60. © 2016 American Cancer Society.

Ancestral-derived effects on the mutational landscape of laryngeal cancer.

Laryngeal cancer disproportionately affects more African-Americans than European-Americans. Here, we analyze the genome-wide somatic point mutations from the tumors of 13 African-Americans and 57 European-Americans from TCGA to differentiate between environmental and ancestrally-inherited factors. The mean number of mutations was different between African-Americans (151.31) and European-Americans (277.63). Other differences in the overall mutational landscape between African-American and European-American were also found. The frequency of C>A, and C>G were significantly different between the two populations (p-value<0.05). Context nucleotide signatures for some mutation types significantly differ between these two populations. Thus, the context nucleotide signatures along with other factors could be related to the observed mutational landscape differences between two races. Finally, we show that mutated genes associated with these mutational differences differ between the two populations. Thus, at the molecular level, race appears to be a factor in the progression of laryngeal cancer with ancestral genomic signatures best explaining these differences.

A 454 sequencing approach to dipteran mitochondrial genome research.

The availability of complete mitochondrial genome (mtgenome) data for Diptera, one of the largest metazoan orders, in public databases is limited. The advent of high throughput sequencing technology provides the potential to generate mtgenomes for many species affordably and quickly. However, these technologies need to be validated for dipterans as the members of this clade play important economic and research roles. Illumina and 454 sequencing platforms are widely used in genomic research involving non-model organisms. The Illumina platform has already been utilized for generating mitochondrial genomes without using conventional long range PCR for insects whereas the power of 454 sequencing for generating mitochondrial genome drafts without PCR has not yet been validated for insects. Thus, this study examines the utility of 454 sequencing approach for dipteran mtgenomic research. We generated complete or nearly complete mitochondrial genomes for Cochliomyia hominivorax, Haematobia irritans, Phormia regina and Sarcophaga crassipalpis using a 454 sequencing approach. Comparisons between newly obtained and existing assemblies for C. hominivorax and H. irritans revealed no major discrepancies and verified the utility of 454 sequencing for dipteran mitochondrial genomes. We also report the complete mitochondrial sequences for two forensically important flies, P. regina and S. crassipalpis, which could be used to provide useful information to legal personnel. Comparative analyses revealed that dipterans follow similar codon usage and nucleotide biases that could be due to mutational and selection pressures. This study illustrates the utility of 454 sequencing to obtain complete mitochondrial genomes for dipterans without the aid of conventional molecular techniques such as PCR and cloning and validates this method of mtgenome sequencing in arthropods.

Merging and concatenation of sequencing reads: a bioinformatics workflow for the comprehensive profiling of microbiome from amplicon data.

A comprehensive profiling of microbial diversity is essential to understand the ecosystem functions. Universal primer sets such as the 515Y/926R could amplify a part of 16S and 18S rRNA and infer the diversity of prokaryotes and eukaryotes. However, the analyses of mixed sequencing data pose a bioinformatics challenge; the 16S and 18S rRNA sequences need to be separated first and analysed individually/independently due to variations in the amplicon length. This study describes an alternative strategy, a merging and concatenation workflow, to analyse the mixed amplicon data without separating the 16S and 18S rRNA sequences. The workflow was tested with 24 mock community (MC) samples, and the analyses resolved the composition of prokaryotes and eukaryotes adequately. In addition, there was a strong correlation (cor = 0.950; P-value = 4.754e-10) between the observed and expected abundances in the MC samples, which suggests that the computational approach could infer the microbial proportions accurately. Further, 18 samples collected from the Sundarbans mangrove region were analysed as a case study. The analyses identified Proteobacteria, Bacteroidota, Actinobacteriota, Cyanobacteria, and Crenarchaeota as dominant bacterial phyla and eukaryotic divisions such as Metazoa, Gyrista, Cryptophyta, Chlorophyta, and Dinoflagellata were found to be dominant in the samples. Thus, the results support the applicability of the method in environmental microbiome research. The merging and concatenation workflow presented here requires considerably less computational resources and uses widely/commonly used bioinformatics packages, saving researchers analyses time (for equivalent sample numbers, compared to the conventional approach) required to infer the diversity of major microbial domains from mixed amplicon data at comparable accuracy.

Existence of rare actinobacterial forms in the Indian sector of Southern Ocean: 16 S rRNA based metabarcoding study.

The significance of the Southern Ocean (SO) as a sink of atmospheric CO2 and other greenhouse gases is well established. Earlier studies have highlighted the role of microbes in various SO ecosystem processes. However, the diversity and role of actinobacteria in the Indian sector of SO (ISO) water and sediments are unknown. This study aimed to analyze the diversity of actinobacteria in water and sediment samples of SO based on amplicon microbiome analyses. The taxonomic analysis identified a total number of 27 phyla of which Proteobacteria (40.2%), Actinobacteria (13.6%), and Firmicutes (8.7%) were found to be dominant. The comparative study of water and sediment samples revealed the dominance of different actinobacteria in water and sediments. While the order Streptomycetales was dominant in the water samples, Micrococcales was found to be dominant in the sediment samples. The genus level analysis found the presence of eight and seventeen genera in the sediment and water samples, respectively. The genus Streptomyces, Saccharopolyspora, Nocardioides, Sva0996 marine group, and Mycobacterium were seen both in sediment and water samples. Marmoricola, Ilumatobacter, and Glaciihabitans were observed only in sediment samples whereas Rhodococcus, Corynebacterium, Micrococcus, Turicella, Pseudonocardia, Bifidobacterium, Nesterenkonia, Collinsella, Knoellia, Cadidatus, Actinomarina, Libanicoccus and Cutibacterium were noticed exclusively in water samples. Our study also emphasizes the need for further detailed study to understand the links between actinobacterial diversity and their ecological functions in the ISO. The available metabarcoding data paves the way for future research in cultivable forms of novel and rare Actinobacteria for their bioprospecting applications.

Extensive set of African ancestry-informative markers (AIMs) to study ancestry and population health.

Introduction: Human populations are often highly structured due to differences in genetic ancestry among groups, posing difficulties in associating genes with diseases. Ancestry-informative markers (AIMs) aid in the detection of population stratification and provide an alternative approach to map population-specific alleles to disease. Here, we identify and characterize a novel set of African AIMs that separate populations of African ancestry from other global populations including those of European ancestry. Methods: Using data from the 1000 Genomes Project, highly informative SNP markers from five African subpopulations were selected based on estimates of informativeness (In) and compared against the European population to generate a final set of 46,737 African ancestry-informative markers (AIMs). The AIMs identified were validated using an independent set and functionally annotated using tools like SIFT, PolyPhen. They were also investigated for representation of commonly used SNP arrays. Results: This set of African AIMs effectively separates populations of African ancestry from other global populations and further identifies substructure between populations of African ancestry. When a subset of these AIMs was studied in an independent dataset, they differentiated people who self-identify as African American or Black from those who identify their ancestry as primarily European. Most of the AIMs were found to be in their intergenic and intronic regions with only 0.6% in the coding regions of the genome. Most of the commonly used SNP array investigated contained less than 10% of the AIMs. Discussion: While several functional annotations of both coding and non-coding African AIMs are supported by the literature and linked these high-frequency African alleles to diseases in African populations, more effort is needed to map genes to diseases in these genetically diverse subpopulations. The relative dearth of these African AIMs on current genotyping platforms (the array with the highest fraction, llumina's Omni 5, harbors less than a quarter of AIMs), further demonstrates a greater need to better represent historically understudied populations.

Salivary Microbiome Diversity in Kuwaiti Adolescents with Varied Body Mass Index-A Pilot Study.

The potential role of the salivary microbiome in human diseases has increasingly been explored. The salivary microbiome has been characterized in several global populations, except the Arabian Gulf region. Hence, in this pilot study, we profiled the salivary microbiome of Kuwaiti adolescents with varied body mass indexes (BMI). The analyses of core microbiome composition showed Firmicutes, Bacteroidota, Proteobacteria, Patescibacteria, Fusobacteriota, Actinobacteriota, and Campylobacterota as the common phylum found in the Kuwaiti adolescent population. We also illustrated a diverse microbial community among the sampled individuals grouped according to their BMI. Notably, the overweight group was found with a higher number of distinct taxa than other groups. As such, the core microbiome composition was found to be significantly different (p-value < 0.001) across different BMI groups. Overall, this pilot investigation outlined the microbial diversity and suggested that changes in salivary microbiome composition in people with obese or overweight BMI might reflect their susceptibility to oral diseases.

Isolation, characterization and identification of antibiofouling metabolite from mangrove derived Streptomyces sampsonii PM33.

In this study, we report the production, bioassay guided isolation and identification of antibiofouling metabolite from mangrove derived actinobacterium, Streptomyces sampsonii (PM33). The actinobacterial strain PM33 yields maximum amount of antifouling compounds through agar surface fermentation. In optimization, carbohydrates such as glucose, fructose and xylose, are suitable for maximum production of the active compound. In addition, other compounds such as malt extract, glutamine, and sodium chloride concentrations (2.5, 5 and 7.5%) and parameters such as pH 7.0 and temperature range 30 °C to 40 °C enhanced the production of antifouling metabolite. The antifouling metabolite was extracted in ethyl acetate. TLC and bioautography was used to separate and detect the antifouling metabolite present in the crude extract. The physico chemical features revealed that the antifouling metabolite PM33 - B as taxifolin (C15H12O7). The purified taxifolin was found to be active against biofouling bacteria, algal spore germination and mollusc foot adherence, respectively. Toxicity nature of taxifolin was also determined by adopting zebrafish embryos. The taxifolin isolated from mangrove-derived Streptomyces sampsonii PM33 is a promising candidate for the development of eco-friendly antifouling preparation.

Multiple lineages of ancient CR1 retroposons shaped the early genome evolution of amniotes.

Chicken repeat 1 (CR1) retroposons are long interspersed elements (LINEs) that are ubiquitous within amniote genomes and constitute the most abundant family of transposed elements in birds, crocodilians, turtles, and snakes. They are also present in mammalian genomes, where they reside as numerous relics of ancient retroposition events. Yet, despite their relevance for understanding amniote genome evolution, the diversity and evolution of CR1 elements has never been studied on an amniote-wide level. We reconstruct the temporal and quantitative activity of CR1 subfamilies via presence/absence analyses across crocodilian phylogeny and comparative analyses of 12 crocodilian genomes, revealing relative genomic stasis of retroposition during genome evolution of extant Crocodylia. Our large-scale phylogenetic analysis of amniote CR1 subfamilies suggests the presence of at least seven ancient CR1 lineages in the amniote ancestor; and amniote-wide analyses of CR1 successions and quantities reveal differential retention (presence of ancient relics or recent activity) of these CR1 lineages across amniote genome evolution. Interestingly, birds and lepidosaurs retained the fewest ancient CR1 lineages among amniotes and also exhibit smaller genome sizes. Our study is the first to analyze CR1 evolution in a genome-wide and amniote-wide context and the data strongly suggest that the ancestral amniote genome contained myriad CR1 elements from multiple ancient lineages, and remnants of these are still detectable in the relatively stable genomes of crocodilians and turtles. Early mammalian genome evolution was thus characterized by a drastic shift from CR1 prevalence to dominance and hyperactivity of L2 LINEs in monotremes and L1 LINEs in therians.

Three crocodilian genomes reveal ancestral patterns of evolution among archosaurs.

To provide context for the diversification of archosaurs--the group that includes crocodilians, dinosaurs, and birds--we generated draft genomes of three crocodilians: Alligator mississippiensis (the American alligator), Crocodylus porosus (the saltwater crocodile), and Gavialis gangeticus (the Indian gharial). We observed an exceptionally slow rate of genome evolution within crocodilians at all levels, including nucleotide substitutions, indels, transposable element content and movement, gene family evolution, and chromosomal synteny. When placed within the context of related taxa including birds and turtles, this suggests that the common ancestor of all of these taxa also exhibited slow genome evolution and that the comparatively rapid evolution is derived in birds. The data also provided the opportunity to analyze heterozygosity in crocodilians, which indicates a likely reduction in population size for all three taxa through the Pleistocene. Finally, these data combined with newly published bird genomes allowed us to reconstruct the partial genome of the common ancestor of archosaurs, thereby providing a tool to investigate the genetic starting material of crocodilians, birds, and dinosaurs.