Mitochondrial metagenomics: letting the genes out of the bottle

‘Mitochondrial metagenomics’ (MMG) is a methodology for shotgun sequencing of total DNA from specimen mixtures and subsequent bioinformatic extraction of mitochondrial sequences. The approach can be applied to phylogenetic analysis of taxonomically selected taxa, as an economical alternative to mitogenome sequencing from individual species, or to environmental samples of mixed specimens, such as from mass trapping of invertebrates. The routine generation of mitochondrial genome sequences has great potential both for systematics and community phylogenetics. Mapping of reads from low-coverage shotgun sequencing of environmental samples also makes it possible to obtain data on spatial and temporal turnover in whole-community phylogenetic and species composition, even in complex ecosystems where species-level taxonomy and biodiversity patterns are poorly known. In addition, read mapping can produce information on species biomass, and potentially allows quantification of within-species genetic variation. The success of MMG relies on the formation of numerous mitochondrial genome contigs, achievable with standard genome assemblers, but various challenges for the efficiency of assembly remain, particularly in the face of variable relative species abundance and intra-specific genetic variation. Nevertheless, several studies have demonstrated the power of mitogenomes from MMG for accurate phylogenetic placement, evolutionary analysis of species traits, biodiversity discovery and the establishment of species distribution patterns; it offers a promising avenue for unifying the ecological and evolutionary understanding of species diversity

High-Throughput, Whole-Genome Sequencing

Since the completion of the Human Genome Project, research focusing on the consequence of known human genetic code has advanced by leaps and bounds. The development of personalized medicine, a field focused on enumerating the effects of individual genetic variations, termed SNPs, has become a reality for those researching the molecular basis of disease. With clinical correlates between genotype and prognosis becoming ever more common, the utility of personal genetic screening has become impossible to ignore. In this report, we present PennBio: a whole-genome sequencing company utilizing a novel single-molecule, real time sequencing-by-synthesis technology. Using unique zero-mode waveguides, which have revolutionized single-molecule detection, individual enzymes polymerizing novel phospholinked fluorescence labeled nucleotides can be observed as they sequence genomic template DNA. Modern optical techniques record these fragmented sequences, which are then analyzed by highly efficient alignment algorithms. A personal genomic code will ultimately allow consumers to be aware of their genetic predispositions as the medical community continues to discover them

MICRO$EC: Cost Effective, Whole-Genome Sequencing

While the feasibility of whole human genome sequencing was proven by the success of the Human Genome Project several years ago, the prevalence of personal genome sequencing in the medical industry is still elusive due to its unrealistic cost and time requirements. Micro$eq is a startup company with the goal of overcoming these limitations by sequencing a minimum of 12 complete human genomes per day at an error rate less than ten parts in million at a profitable market price of less than US$1000 per genome. To overcome the technology bottlenecks hindering current biotech companies from achieving these target throughput, error rate, and market price goals, Micro$eq has developed an innovative sequencing technique that uses shortread fragments with high coverage on a microfluidics platform. Short, amplified DNA fragments are generated from an input of customer saliva. 6 base pair(bp) sequence hybridization is used for sequencing each of the DNA fragments individually. The results are these hydridization reads are then assembled via de Bruijn graph theory and the graphical reconstructions of each fragment’s sequence are then assembled to a complete genome via shotgun sequencing with an expected error rate less than 1 in 100,000bp. Upon the completion of financial analysis, both a small-scale business model producing 72 genomes per day at US$999 per genome, and a largescale business model producing 52.2 genomes per year at a market price of US$299 per genome were found to be profitable, yielding Micro$eq investors return margins of ~90% and 300% for the small and large scale models, respectively. With a market price Micro$eq offers personal genome sequencing at one-tenth of its nearest potential competitor’s cost. Additionally, its ability for bulk-sequencing allows it to profitably venture into the previously untapped Pharmaceutical Industry market sector, enabling the creation of large-scale genome databases which are the next step forward in the quest for truly personalized

Recovering complete and draft population genomes from metagenome datasets.

Assembly of metagenomic sequence data into microbial genomes is of fundamental value to improving our understanding of microbial ecology and metabolism by elucidating the functional potential of hard-to-culture microorganisms. Here, we provide a synthesis of available methods to bin metagenomic contigs into species-level groups and highlight how genetic diversity, sequencing depth, and coverage influence binning success. Despite the computational cost on application to deeply sequenced complex metagenomes (e.g., soil), covarying patterns of contig coverage across multiple datasets significantly improves the binning process. We also discuss and compare current genome validation methods and reveal how these methods tackle the problem of chimeric genome bins i.e., sequences from multiple species. Finally, we explore how population genome assembly can be used to uncover biogeographic trends and to characterize the effect of in situ functional constraints on the genome-wide evolution

Natural Product Biosynthesis In Uncultured Bacteria

A single gram of soil can contain thousands of unique bacterial species, only a small fraction of which is regularly cultured in the laboratory.Although the fermentation of cultured microorganisms has provided access to numerous bioactive secondary metabolites, with these same methods it is not possible to characterize the natural products encoded by the uncultured majority. The heterologous expression of biosynthetic gene clusters cloned from DNA extracted directly from environmental samples (eDNA) has begun to provide access to the chemical diversity encoded in the genomes of previously uncultured bacteria. The systematic exploration of natural product biosynthesis in uncultured bacteria, however, still faces several challenges that we sought to experimentally address. First, many natural product gene clusters cannot be detected in functional screens due to cloning and expression limitations of metagenomic library host strains. Second, the lack of robust and scalable gene cluster assembly methods precludes the functional characterization of a large number of natural product biosynthetic gene clusters from cosmid-based eDNA libraries. Third, the large-scale analysis of metagenomic natural product chemical diversity and the phylogenetic context it is found in were previously unaddressed due to the complexities of microbial communities. To address these questions, we have: 1) Demonstrated that sequence-based screens can be used to systematically discover a diverse range of natural product gene clusters by screening two of the largest recombinant eDNA libraries reported to date. This approach circumvents many of the challenges of using functional screens to discover novel biosynthetic gene clusters. (Chapter 2) 2) Shown that transformation associated recombination in S. cerevisiae can be used to functionally reassemble large natural product gene clusters that exceed conventional eDNA cloning limits. This approach overcomes a significant barrier which prevented the functional characterization of many natural product gene clusters from eDNA libraries. (Chapter 3) 3) Developed a high throughput sequencing analysis framework to characterize environmental biosynthetic capacity. These results suggest that the continued construction and screening of soil-based eDNA libraries should provide access to additional novel pools of biosynthetic enzyme diversity. (Chapter 4

A Linkage-Based Genome Assembly for the Mosquito Aedes albopictus and Identification of Chromosomal Regions Affecting Diapause

The Asian tiger mosquito, Aedes albopictus, is an invasive vector mosquito of substantial public health concern. The large genome size (similar to 1.19-1.28 Gb by cytofluorometric estimates), comprised of similar to 68% repetitive DNA sequences, has made it difficult to produce a high-quality genome assembly for this species. We constructed a high-density linkage map for Ae. albopictus based on 111,328 informative SNPs obtained by RNAseq. We then performed a linkage-map anchored reassembly of AalbF2, the genome assembly produced by Palatini et al. (2020). Our reassembled genome sequence, AalbF3, represents several improvements relative to AalbF2. First, the size of the AalbF3 assembly is 1.45 Gb, almost half the size of AalbF2. Furthermore, relative to AalbF2, AalbF3 contains a higher proportion of complete and single-copy BUSCO genes (84.3%) and a higher proportion of aligned RNAseq reads that map concordantly to a single location of the genome (46%). We demonstrate the utility of AalbF3 by using it as a reference for a bulk-segregant-based comparative genomics analysis that identifies chromosomal regions with clusters of candidate SNPs putatively associated with photoperiodic diapause, a crucial ecological adaptation underpinning the rapid range expansion and climatic adaptation of A. albopictus.Peer reviewe

Bacteroides-bakteerien eristys ja karakterisointi lasten ja äitien suolistomikrobiomista

Varhaiselämän suolistomikrobistolla on suuri merkitys immuunijärjestelmän kehittymisen sekä lapsen pitkäaikaisen terveyden kannalta. Yksittäisten bakteerilajien roolista suolistossa tiedetään suhteellisen vähän, koska tutkimukset ovat keskittyneet määrittelemään vastasyntyneen suolistomikrobiston monimuotoisuutta sekä yksilöllistä ja ajallista vaihtelua. Bacteroides-bakteerisuvun rooli on erityisen mielenkiintoinen, koska sen osuus on merkittävästi alentunut keisarileikkauksella syntyneillä vauvoilla, eikä suoliston varhaisten Bacteroides-kolonisoijen geneettisiä tai fenotyyppisiä ominaisuuksia tunneta hyvin, vaikka niiden epäillään olevan yhteydessä keisarinleikkauksella syntyneiden lasten korkeampaan sairastavuuteen. Tämän tutkielman tavoitteena on Bacteroides-bakteerikantojen eristäminen ja karakterisointi lasten suolistomikrobiston kehitykseen keskittyvän Health and Early Life Microbiota (HELMi) -kohorttitutkimukseen osallistuvien vauvojen ja äitien ulostenäytteistä viljely- sekä metagenomiikkapohjaisten menetelmien avulla. Lasten 9. ikäviikon näytteistä eristettiin gramnegatiivisia bakteereita, jotka tunnistettiin Sanger-sekvensoinnin avulla. Yhteensä seitsemän joko lasten ulostenäytteistä tai äitien myöhäisraskaudenaikaisista ulostenäytteistä aiemmin eristetyistä isolaateista tunnistettiin Bacteroides-lajeiksi. Isolaattien kykyä aktivoida synnynnäinen immuniteetti tutkittiin in vitro HEK-Blue™ hTLR2-hTLR6 soluilla, jotka altistettiin sekä eläville bakteereille että bakteerien suodatetulle kasvualustalle. Tutkielmassa hyödynnettiin lisäksi 88 lapsen suolistomikrobiston metagenomeja ensimmäisen elinvuoden ajalta. Metagenomipohjaisten bakteerigenomien kokoamiseksi luotiin metagenomidatan suureen kokoon skaalautuva analyysiohjelmisto. Kootut bakteerigenomit pyrittiin tunnistamaan ja lopuksi määriteltiin Bacteroides-lajien koottujen genomien toiminnallisia ominaisuuksia. Suurin osa seitsemästä HELMi-tutkimuksen äitien ja lasten ulosteista eristetyistä Bacteroides-lajeista aktivoi TLR2/6-reseptorin in vitro-olosuhteissa. Isolaattien kyky aktivoida reseptori vaihteli solujen pintamolekyylien ja kasvualustaan eritettyjen molekyylien perusteella. Lisäksi kokosimme metagenomeista yli 2500 bakteerigenomia, joista 18 tunnistettiin kuuluvan Bacteroides-sukuun, joille tehtiin edelleen myös toiminnallinen analyysi. Ennustettujen avoimien lukukehysten perusteella suurin osa koottujen Bacteroides-genomien tunnistetuista proteiineista osallistui solujen perustoimintoihin, mutta suurin osa solujen aineenvaihduntaan liittyvistä ennustetuista proteiineista osallistui hiilihydraatti-, aminohappoa- ja glykaaniaineenvaihduntaan korostaen Bacteroides-suvun roolia suolistossa tärkeänä ja monipuolisena hiilihydraattien kuluttajana. Tulokset osoittavat, että Bacteroides-lajin jäsenillä on tärkeä immunologinen ja metabolinen rooli vauvan suolistomikrobistossa. Lisää tutkimusta tarvitaan Bacteroides-bakteerilajin TLR2/6-reseptorin aktivoinnin aiheuttavan molekyylin karakterisoimiseksi sekä aktivoinnin aikaansaamien immunomodulaaristen reaktioiden tunnistamiseksi.The early life gut microbiota plays a major role in establishing neonatal immunity and child’s long-term health. However, relatively little is still known about the role of individual bacteria as most studies so far have focused on characterizing the diversity and the individual and temporal variations of the infant gut microbiome. The genus Bacteroides is of particular interest since its abundance is remarkably decreased in infants born via C-section, and relatively little is known about the genomic and phenotypic characteristics of early Bacteroides colonizers despite their anticipated role in the increased morbidity following C-section birth. This thesis aims to contribute to the isolation and characterization of Bacteroides strains from infant and mother stool samples from the Health and Early Life Microbiota (HELMi) cohort study using culture-based and metagenomic approaches. Gram-negative bacteria were isolated from stool samples of 9-week-old infants and identified by Sanger sequencing. In total, seven isolates identified as unique species of Bacteroides, isolated from infant samples or previously from mother samples in late pregnancy, were then characterized for their potential to activate innate immunity in vitro by using HEK-Blue™ hTLR2-hTLR6 reporter cells either as live cells or filtered culture media. Whole genome shotgun sequenced stool metagenomes obtained from 88 infants during the first year of life were leveraged as well. A computational pipeline able to scale to the large size of the dataset was developed to obtain metagenome assembled genomes (MAGs) from the metagenomes. MAGs obtained from Bacteroides species were further taxonomically and functionally annotated. Among the seven Bacteroides spp. isolated from HELMi mother and infant samples, the majority were able to activate the TLR2/6 receptor in vitro. The isolates varied in their potential to activate the receptor via their cell surface molecules and substances they excreted to the culture media. In addition, over 2500 MAGs could be retrieved from the infant metagenomes, of which 18 belonged to Bacteroides spp. Based on predicted open reading frames, majority of the identified proteins of these MAGs were involved in housekeeping functions. Most of predicted proteins involved in cellular metabolism were, however, related to carbohydrate metabolism, amino acid metabolism, and glycan metabolism, stressing the role of Bacteroides spp. in the gut as important and versatile carbohydrate consumers. The results indicate that the Bacteroides spp. colonizing infant gut have an immunologically and metabolically active role. Further work is needed to characterize the molecules responsible for the TLR2/6 activation as well as the nature of the downstream immune responses elicited by the isolated Bacteroides spp