The COLO-COHORT (Colorectal Cancer Cohort) study: Protocol for a multi-centre, observational research study and development of a consent-for-contact research platform.

AIM: The COLO-COHORT study aims to produce a multi-factorial risk prediction model for colorectal neoplasia that can be used to target colonoscopy to those at greatest risk of colorectal neoplasia, ensuring that people are not investigated unnecessarily and maximizing the use of limited endoscopy resources. The study will also explore the link between neoplasia and the human gut microbiome. Additionally, the study aims to generate a cohort of colonoscopy patients who are 'research ready' through the development of a consent-for-contact (C4C) platform, to facilitate a range of colorectal cancer prevention studies to be conducted at scale and speed. METHODS AND ANALYSIS: This is a multi-centre observational study involving sites across the UK. Recruitment is over a 6-year period (2019-2025). Patients recruited to the study are those attending for colonoscopy. Patients are recruited into two groups, namely observational group A (10 000 patients) and C4C group B (10 000 patients), known as COLO-SPEED (Colorectal Cancer Screening Prevention Endoscopy and Early Diagnosis; https://colospeed.uk). Patients complete a health questionnaire, provide anthropometric measurements and submit biosamples (blood and stool-depending on the part of the study they are recruited into). Patients' colonoscopy and histology findings are also recorded. Models of factors associated with the presence of neoplasia at colonoscopy will be developed using logistic or multinomial regression. For internal validation, model discrimination and calibration will be assessed and bootstrapping and cross-validation approaches used. To enable long-term follow-up for outcomes related to colorectal cancer and polyps, patients are asked to consent to follow-up through data linkage with national databases. DISSEMINATION: In keeping with good research practice, following analysis by the study team the study investigators will make the anonymized dataset available to other researchers. The C4C platform will also be accessible to other researchers. The study findings will be submitted for publication in peer-reviewed journals and lay summaries will be disseminated to participants and the wider public.

Perturbations of the gut microbiome in anti-CCP positive individuals at risk of developing rheumatoid arthritis.

OBJECTIVE: Individuals with newly diagnosed RA have a distinct microbiome when compared with healthy controls. However, little is known as to when these microbiome perturbations begin. Using a prospective at-risk cohort of individuals positive for anti-citrullinated protein (anti-CCP) antibody with new onset musculoskeletal symptoms, but without clinical arthritis, we investigated for the presence of a gut dysbiosis before the onset of RA. METHODS: The gut microbiota of 25 anti-CCP positive individuals without clinical synovitis were sequenced targeting the V4 region of the 16S rRNA gene. Using a publicly available database, a control population of 44 individuals, approximately matched in age, gender, diet and ethnicity was selected for comparison, using the same sequencing methodology. Median interval between sample collection and progression to RA was 188 days. Taxonomic analysis was performed using QIIME and MEGAN, and statistical analysis using R software. RESULTS: There were significant differences (P =0.01) at family level in gut microbiomes of anti-CCP positive individuals vs controls. The anti-CCP positive population had an overabundance of Lachnospiraceae, Helicobacteraceae, Ruminococcaceae, Erysipelotrichaceae and Bifidobacteriaceae, among others. Five individuals progressed to RA between sample collection and analysis. Clustering of the progressor population was observed on a phylogenetic network created using a probabilistic similarity index (Goodall's index). CONCLUSIONS: Anti-CCP positive at-risk individuals without clinical synovitis appear to have a distinct gut microbiome compared with healthy controls. Phylogenetic clustering was observed in individuals who progressed to RA, suggesting that distinct taxa are associated with the development of RA many months before its onset.

Luminal Bioavailability of Orally Administered ω-3 PUFAs in the Distal Small Intestine, and Associated Changes to the Ileal Microbiome, in Humans with a Temporary Ileostomy.

BACKGROUND: Oral administration of purified omega-3 (ω-3) PUFAs is associated with changes to the fecal microbiome. However, it is not known whether this effect is associated with increased PUFA concentrations in the gut. OBJECTIVES: We investigated the luminal bioavailability of oral ω-3 PUFAs (daily dose 1 g EPA and 1g DHA free fatty acid equivalents as triglycerides in soft-gel capsules, twice daily) and changes to the gut microbiome, in the ileum. METHODS: Ileostomy fluid (IF) and blood were obtained at baseline, after first capsule dosing (median 2 h), and at a similar time after final dosing on day 28, in 11 individuals (median age 63 y) with a temporary ileostomy. Fatty acids were measured by LC-tandem MS. The ileal microbiome was characterized by 16S rRNA PCR and Illumina sequencing. RESULTS: There was a mean 6.0 ± 9.8-fold and 6.6 ± 9.6-fold increase in ileal EPA and DHA concentrations (primary outcome), respectively, at 28 d, which was associated with increased RBC ω-3 PUFA content (P ≤ 0.05). The first oral dose did not increase the ileal ω-3 PUFA concentration except in 4 individuals, who displayed high luminal EPA and DHA concentrations, which reduced to concentrations similar to the overall study population at day 28, suggesting physiological adaptation. Bacteroides, Clostridium, and Streptococcus were abundant bacterial genera in the ileum. Ileal microbiome variability over time and between individuals was large, with no consistent change associated with acute ω-3 PUFA dosing. However, high concentrations of EPA and DHA in IF on day 28 were associated with higher abundance of Bacteroides (r2 > 0.86, P < 0.05) and reduced abundance of other genera, including Actinomyces (r2 > 0.94, P < 0.05). CONCLUSIONS: Oral administration of ω-3 PUFAs leads to increased luminal ω-3 PUFA concentrations and changes to the microbiome, in the ileum of individuals with a temporary ileostomy. This study is registered on the ISRCTN registry as ISRCTN14530452.

Molecular and culture-based surveys of metabolically active hydrocarbon-degrading archaeal communities in Sundarban mangrove sediments.

Archaea remain important players in global biogeochemical cycles worldwide, including in the highly productive mangrove estuarine ecosystems. In the present study, we have explored the diversity, distribution, and function of the metabolically active fraction of the resident archaeal community of the Sundarban mangrove ecosystem, using both culture-independent and culture-dependent approaches. To evaluate the diversity and distribution pattern of the active archaeal communities, RNA based analysis of the 16S rRNA gene was performed on an Illumina platform. The active Crenarchaeal community was observed to remain constant while active Euryarchaeal community underwent considerable change across the sampling sites depending on varying anthropogenic factors. Haloarchaea were the predominant group in hydrocarbon polluted sediments, leading us to successfully isolate eleven p-hydroxybenzoic acid degrading haloarchaeal species. The isolates could also survive in benzoic acid, naphthalene, and o-phthalate. Quantitative estimation of p-hydroxybenzoic acid degradation was studied on select isolates, and their ability to reduce COD of polluted saline waters of Sundarban was also evaluated. To our knowledge, this is the first ever study combining culture-independent (Next Generation sequencing and metatranscriptome) and culture-dependent analyses for an assessment of archaeal function in the sediment of Sundarban.

A randomised trial of the effect of omega-3 polyunsaturated fatty acid supplements on the human intestinal microbiota.

OBJECTIVE: Omega-3 polyunsaturated fatty acids (PUFAs) have anticolorectal cancer (CRC) activity. The intestinal microbiota has been implicated in colorectal carcinogenesis. Dietary omega-3 PUFAs alter the mouse intestinal microbiome compatible with antineoplastic activity. Therefore, we investigated the effect of omega-3 PUFA supplements on the faecal microbiome in middle-aged, healthy volunteers (n=22). DESIGN: A randomised, open-label, cross-over trial of 8 weeks' treatment with 4 g mixed eicosapentaenoic acid/docosahexaenoic acid in two formulations (soft-gel capsules and Smartfish drinks), separated by a 12-week 'washout' period. Faecal samples were collected at five time-points for microbiome analysis by 16S ribosomal RNA PCR and Illumina MiSeq sequencing. Red blood cell (RBC) fatty acid analysis was performed by liquid chromatography tandem mass spectrometry. RESULTS: Both omega-3 PUFA formulations induced similar changes in RBC fatty acid content, except that drinks were associated with a larger, and more prolonged, decrease in omega-6 PUFA arachidonic acid than the capsule intervention (p=0.02). There were no significant changes in α or ß diversity, or phyla composition, associated with omega-3 PUFA supplementation. However, a reversible increased abundance of several genera, including Bifidobacterium, Roseburia and Lactobacillus was observed with one or both omega-3 PUFA interventions. Microbiome changes did not correlate with RBC omega-3 PUFA incorporation or development of omega-3 PUFA-induced diarrhoea. There were no treatment order effects. CONCLUSION: Omega-3 PUFA supplementation induces a reversible increase in several short-chain fatty acid-producing bacteria, independently of the method of administration. There is no simple relationship between the intestinal microbiome and systemic omega-3 PUFA exposure. TRIAL REGISTRATION NUMBER: ISRCTN18662143.

Optimisation of 16S rRNA gut microbiota profiling of extremely low birth weight infants.

BACKGROUND: Infants born prematurely, particularly extremely low birth weight infants (ELBW) have altered gut microbial communities. Factors such as maternal health, gut immaturity, delivery mode, and antibiotic treatments are associated with microbiota disturbances, and are linked to an increased risk of certain diseases such as necrotising enterocolitis. Therefore, there is a requirement to optimally characterise microbial profiles in this at-risk cohort, via standardisation of methods, particularly for studying the influence of microbiota therapies (e.g. probiotic supplementation) on community profiles and health outcomes. Profiling of faecal samples using the 16S rRNA gene is a cost-efficient method for large-scale clinical studies to gain insights into the gut microbiota and additionally allows characterisation of cohorts were sample quantities are compromised (e.g. ELBW infants). However, DNA extraction method, and the 16S rRNA region targeted can significantly change bacterial community profiles obtained, and so confound comparisons between studies. Thus, we sought to optimise a 16S rRNA profiling protocol to allow standardisation for studying ELBW infant faecal samples, with or without probiotic supplementation. METHODS: Using ELBW faecal samples, we compared three different DNA extraction methods, and subsequently PCR amplified and sequenced three hypervariable regions of the 16S rRNA gene (V1 + V2 + V3), (V4 + V5) and (V6 + V7 + V8), and compared two bioinformatics approaches to analyse results (OTU and paired end). Paired shotgun metagenomics was used as a 'gold-standard'. RESULTS: Results indicated a longer bead-beating step was required for optimal bacterial DNA extraction and that sequencing regions (V1 + V2 + V3) and (V6 + V7 + V8) provided the most representative taxonomic profiles, which was confirmed via shotgun analysis. Samples sequenced using the (V4 + V5) region were found to be underrepresented in specific taxa including Bifidobacterium, and had altered diversity profiles. Both bioinformatics 16S rRNA pipelines used in this study (OTU and paired end) presented similar taxonomic profiles at genus level. CONCLUSIONS: We determined that DNA extraction from ELBW faecal samples, particularly those infants receiving probiotic supplementation, should include a prolonged beat-beating step. Furthermore, use of the 16S rRNA (V1 + V2 + V3) and (V6 + V7 + V8) regions provides reliable representation of ELBW microbiota profiles, while inclusion of the (V4 + V5) region may not be appropriate for studies where Bifidobacterium constitutes a resident microbiota member.

MEGAN Community Edition - Interactive Exploration and Analysis of Large-Scale Microbiome Sequencing Data.

There is increasing interest in employing shotgun sequencing, rather than amplicon sequencing, to analyze microbiome samples. Typical projects may involve hundreds of samples and billions of sequencing reads. The comparison of such samples against a protein reference database generates billions of alignments and the analysis of such data is computationally challenging. To address this, we have substantially rewritten and extended our widely-used microbiome analysis tool MEGAN so as to facilitate the interactive analysis of the taxonomic and functional content of very large microbiome datasets. Other new features include a functional classifier called InterPro2GO, gene-centric read assembly, principal coordinate analysis of taxonomy and function, and support for metadata. The new program is called MEGAN Community Edition (CE) and is open source. By integrating MEGAN CE with our high-throughput DNA-to-protein alignment tool DIAMOND and by providing a new program MeganServer that allows access to metagenome analysis files hosted on a server, we provide a straightforward, yet powerful and complete pipeline for the analysis of metagenome shotgun sequences. We illustrate how to perform a full-scale computational analysis of a metagenomic sequencing project, involving 12 samples and 800 million reads, in less than three days on a single server. All source code is available here: https://github.com/danielhuson/megan-ce.

Hickman to central venous catheter: A case of difficult venous access in a child suffering from acute lymphoblastic leukemia.

Chemotherapy in children suffering from cancer usually requires placement of an indwelling central venous catheter (CVC). A child may need to undergo repeated procedures because of infection and occlusion of previous access devices. We present a case of CVC insertion in a child suffering from acute lymphoblastic leukemia where an innovative technique was employed.

Diversity and Distribution of Archaea in the Mangrove Sediment of Sundarbans.

Mangroves are among the most diverse and productive coastal ecosystems in the tropical and subtropical regions. Environmental conditions particular to this biome make mangroves hotspots for microbial diversity, and the resident microbial communities play essential roles in maintenance of the ecosystem. Recently, there has been increasing interest to understand the composition and contribution of microorganisms in mangroves. In the present study, we have analyzed the diversity and distribution of archaea in the tropical mangrove sediments of Sundarbans using 16S rRNA gene amplicon sequencing. The extraction of DNA from sediment samples and the direct application of 16S rRNA gene amplicon sequencing resulted in approximately 142 Mb of data from three distinct mangrove areas (Godkhali, Bonnie camp, and Dhulibhashani). The taxonomic analysis revealed the dominance of phyla Euryarchaeota and Thaumarchaeota (Marine Group I) within our dataset. The distribution of different archaeal taxa and respective statistical analysis (SIMPER, NMDS) revealed a clear community shift along the sampling stations. The sampling stations (Godkhali and Bonnie camp) with history of higher hydrocarbon/oil pollution showed different archaeal community pattern (dominated by haloarchaea) compared to station (Dhulibhashani) with nearly pristine environment (dominated by methanogens). It is indicated that sediment archaeal community patterns were influenced by environmental conditions.

Ecogenomics reveals metals and land-use pressures on microbial communities in the waterways of a megacity.

Networks of engineered waterways are critical in meeting the growing water demands in megacities. To capture and treat rainwater in an energy-efficient manner, approaches can be developed for such networks that use ecological services from microbial communities. Traditionally, engineered waterways were regarded as homogeneous systems with little responsiveness of ecological communities and ensuing processes. This study provides ecogenomics-derived key information to explain the complexity of urban aquatic ecosystems in well-managed watersheds with densely interspersed land-use patterns. Overall, sedimentary microbial communities had higher richness and evenness compared to the suspended communities in water phase. On the basis of PERMANOVA analysis, variation in structure and functions of microbial communities over space within same land-use type was not significant. In contrast, this difference was significant between different land-use types, which had similar chemical profiles. Of the 36 environmental parameters from spatial analysis, only three metals, namely potassium, copper and aluminum significantly explained between 7% and 11% of the variation in taxa and functions, based on distance-based linear models (DistLM). The ecogenomics approach adopted here allows the identification of key drivers of microbial communities and their functions at watershed-scale. These findings can be used to enhance microbial services, which are critical to develop ecologically friendly waterways in rapidly urbanizing environments.

Integrative analysis of environmental sequences using MEGAN4.

A major challenge in the analysis of environmental sequences is data integration. The question is how to analyze different types of data in a unified approach, addressing both the taxonomic and functional aspects. To facilitate such analyses, we have substantially extended MEGAN, a widely used taxonomic analysis program. The new program, MEGAN4, provides an integrated approach to the taxonomic and functional analysis of metagenomic, metatranscriptomic, metaproteomic, and rRNA data. While taxonomic analysis is performed based on the NCBI taxonomy, functional analysis is performed using the SEED classification of subsystems and functional roles or the KEGG classification of pathways and enzymes. A number of examples illustrate how such analyses can be performed, and show that one can also import and compare classification results obtained using others' tools. MEGAN4 is freely available for academic purposes, and installers for all three major operating systems can be downloaded from www-ab.informatik.uni-tuebingen.de/software/megan.

Analysis of the intestinal microbiota using SOLiD 16S rRNA gene sequencing and SOLiD shotgun sequencing.

BACKGROUND: Metagenomics seeks to understand microbial communities and assemblages by DNA sequencing. Technological advances in next generation sequencing technologies are fuelling a rapid growth in the number and scope of projects aiming to analyze complex microbial environments such as marine, soil or the gut. Recent improvements in longer read lengths and paired-sequencing allow better resolution in profiling microbial communities. While both 454 sequencing and Illumina sequencing have been used in numerous metagenomic studies, SOLiD sequencing is not commonly used in this area, as it is believed to be more suitable in the context of reference-guided projects. RESULTS: To investigate the performance of SOLiD sequencing in a metagenomic context, we compared taxonomic profiles of SOLiD mate-pair sequencing reads with Sanger paired reads and 454 single reads. All sequences were obtained from the bacterial 16S rRNA gene, which was amplified from microbial DNA extracted from a human fecal sample. Additionally, from the same fecal sample, complete genomic microbial DNA was extracted and shotgun sequenced using SOLiD sequencing to study the composition of the intestinal microbiota and the existing microbial metabolism. We found that the microbiota composition of 16S rRNA gene sequences obtained using Sanger, 454 and SOLiD sequencing provide results comparable to the result based on shotgun sequencing. Moreover, with SOLiD sequences we obtained more resolution down to the species level. In addition, the shotgun data allowed us to determine a functional profile using the databases SEED and KEGG. CONCLUSIONS: This study shows that SOLiD mate-pair sequencing is a viable and cost-efficient option for analyzing a complex microbiome. To the best of our knowledge, this is the first time that SOLiD sequencing has been used in a human sample.

From Genomics to Metagenomics in the Era of Recent Sequencing Technologies.

Metagenomics, also known as environmental genomics, is the study of the genomic content of a sample of organisms obtained from a common habitat. Metagenomics and other "omics" disciplines have captured the attention of researchers for several decades. The effect of microbes in our body is a relevant concern for health studies. Through sampling the sequences of microbial genomes within a certain environment, metagenomics allows study of the functional metabolic capacity of a community as well as its structure based upon distribution and richness of species. Exponentially increasing number of microbiome literatures illustrate the importance of sequencing techniques which have allowed the expansion of microbial research into areas, including the human gut, antibiotics, enzymes, and more. This chapter illustrates how metagenomics field has evolved with the progress of sequencing technologies.Further, from this chapter, researchers will be able to learn about all current options for sequencing techniques and comparison of their cost and read statistics, which will be helpful for planning their own studies.

Metagenomics Data Visualization Using R.

Communicating key finds is a crucial part of the research process. Data visualization is the field of graphically representing data to help communicate key findings. Building on previous chapters around data manipulating using the R programming language this, chapter will explore how to use R to plot data and generate high-quality graphics. It will cover plotting using the base R plotting functionality and introduce the famous ggplot2 package [2] that is widely used for data visualization in R. After this general introduction to data visualization tools, the chapter will explore more specific data visualization techniques for metagenomics data and their use cases using these basic packages.

Noninvasive Fecal Cytokine and Microbiota Profiles Predict Commencement of Necrotizing Enterocolitis in a Proof-of-Concept Study.

Background and Aims: Necrotizing enterocolitis (NEC) is a life-threatening disease and the most common gastrointestinal emergency in premature infants. Accurate early diagnosis is challenging. Modified Bell's staging is routinely used to guide diagnosis, but early diagnostic signs are nonspecific, potentially leading to unobserved disease progression, which is problematic given the often rapid deterioration observed. We investigated fecal cytokine levels, coupled with gut microbiota profiles, as a noninvasive method to discover specific NEC-associated signatures that can be applied as potential diagnostic markers. Methods: Premature babies born below 32 weeks of gestation were admitted to the 2-site neonatal intensive care unit (NICU) of Imperial College hospitals (St. Mary's or Queen Charlotte's & Chelsea) between January 2011 and December 2012. During the NICU stay, expert neonatologists grouped individuals by modified Bell's staging (healthy, NEC1, NEC2/3) and fecal samples from diapers were collected consecutively. Microbiota profiles were assessed by 16S rRNA gene amplicon sequencing and cytokine concentrations were measured by V-Plex multiplex assays. Results: Early evaluation of microbiota profiles revealed only minor differences. However, at later time points, significant changes in microbiota composition were observed for Bacillota (adj. P = .0396), with Enterococcus being the least abundant in Bell stage 2/3 NEC. Evaluation of fecal cytokine levels revealed significantly higher concentrations of IL-1α (P = .045), IL-5 (P = .0074), and IL-10 (P = .032) in Bell stage 1 NEC compared to healthy individuals. Conclusion: Differences in certain fecal cytokine profiles in patients with NEC indicate their potential use as diagnostic biomarkers to facilitate earlier diagnosis. Additionally, associations between microbial and cytokine profiles contribute to improving knowledge about NEC pathogenesis.

Functional analysis of metagenomes and metatranscriptomes using SEED and KEGG.

BACKGROUND: Metagenomics is the study of microbial organisms using sequencing applied directly to environmental samples. Technological advances in next-generation sequencing methods are fueling a rapid increase in the number and scope of metagenome projects. While metagenomics provides information on the gene content, metatranscriptomics aims at understanding gene expression patterns in microbial communities. The initial computational analysis of a metagenome or metatranscriptome addresses three questions: (1) Who is out there? (2) What are they doing? and (3) How do different datasets compare? There is a need for new computational tools to answer these questions. In 2007, the program MEGAN (MEtaGenome ANalyzer) was released, as a standalone interactive tool for analyzing the taxonomic content of a single metagenome dataset. The program has subsequently been extended to support comparative analyses of multiple datasets. RESULTS: The focus of this paper is to report on new features of MEGAN that allow the functional analysis of multiple metagenomes (and metatranscriptomes) based on the SEED hierarchy and KEGG pathways. We have compared our results with the MG-RAST service for different datasets. CONCLUSIONS: The MEGAN program now allows the interactive analysis and comparison of the taxonomical and functional content of multiple datasets. As a stand-alone tool, MEGAN provides an alternative to web portals for scientists that have concerns about uploading their unpublished data to a website.

Analysis of 16S rRNA environmental sequences using MEGAN.

BACKGROUND: Metagenomics is a rapidly growing field of research aimed at studying assemblages of uncultured organisms using various sequencing technologies, with the hope of understanding the true diversity of microbes, their functions, cooperation and evolution. There are two main approaches to metagenomics: amplicon sequencing, which involves PCR-targeted sequencing of a specific locus, often 16S rRNA, and random shotgun sequencing. Several tools or packages have been developed for analyzing communities using 16S rRNA sequences. Similarly, a number of tools exist for analyzing randomly sequenced DNA reads. RESULTS: We describe an extension of the metagenome analysis tool MEGAN, which allows one to analyze 16S sequences. For the analysis all 16S sequences are blasted against the SILVA database. The result output is imported into MEGAN, using a synonym file that maps the SILVA accession numbers onto the NCBI taxonomy. CONCLUSIONS: Environmental samples are often studied using both targeted 16S rRNA sequencing and random shotgun sequencing. Hence tools are needed that allow one to analyze both types of data together, and one such tool is MEGAN. The ideas presented in this paper are implemented in MEGAN 4, which is available from: http://www-ab.informatik.uni-tuebingen.de/software/megan.

Trehalose-Induced Remodelling of the Human Microbiota Affects Clostridioides difficile Infection Outcome in an In Vitro Colonic Model: A Pilot Study.

Within the human intestinal tract, dietary, microbial- and host-derived compounds are used as signals by many pathogenic organisms, including Clostridioides difficile. Trehalose has been reported to enhance virulence of certain C. difficile ribotypes; however, such variants are widespread and not correlated with clinical outcomes for patients suffering from C. difficile infection (CDI). Here, we make preliminary observations on how trehalose supplementation affects the microbiota in an in vitro model and show that trehalose-induced changes can reduce the outgrowth of C. difficile, preventing simulated CDI. Three clinically reflective human gut models simulated the effects of sugar (trehalose or glucose) or saline ingestion on the microbiota. Models were instilled with sugar or saline and further exposed to C. difficile spores. The recovery of the microbiota following antibiotic treatment and CDI induction was monitored in each model. The human microbiota remodelled to utilise the bioavailable trehalose. Clindamycin induction caused simulated CDI in models supplemented with either glucose or saline; however, trehalose supplementation did not result in CDI, although limited spore germination did occur. The absence of CDI in trehalose model was associated with enhanced abundances of Finegoldia, Faecalibacterium and Oscillospira, and reduced abundances of Klebsiella and Clostridium spp., compared with the other models. Functional analysis of the microbiota in the trehalose model revealed differences in the metabolic pathways, such as amino acid metabolism, which could be attributed to prevention of CDI. Our data show that trehalose supplementation remodelled the microbiota, which prevented simulated CDI, potentially due to enhanced recovery of nutritionally competitive microbiota against C. difficile.

Short clones or long clones? A simulation study on the use of paired reads in metagenomics.

BACKGROUND: Metagenomics is the study of environmental samples using sequencing. Rapid advances in sequencing technology are fueling a vast increase in the number and scope of metagenomics projects. Most metagenome sequencing projects so far have been based on Sanger or Roche-454 sequencing, as only these technologies provide long enough reads, while Illumina sequencing has not been considered suitable for metagenomic studies due to a short read length of only 35 bp. However, now that reads of length 75 bp can be sequenced in pairs, Illumina sequencing has become a viable option for metagenome studies. RESULTS: This paper addresses the problem of taxonomical analysis of paired reads. We describe a new feature of our metagenome analysis software MEGAN that allows one to process sequencing reads in pairs and makes assignments of such reads based on the combined bit scores of their matches to reference sequences. Using this new software in a simulation study, we investigate the use of Illumina paired-sequencing in taxonomical analysis and compare the performance of single reads, short clones and long clones. In addition, we also compare against simulated Roche-454 sequencing runs. CONCLUSION: This work shows that paired reads perform better than single reads, as expected, but also, perhaps slightly less obviously, that long clones allow more specific assignments than short ones. A new version of the program MEGAN that explicitly takes paired reads into account is available from our website.