Comparison of the human gastric microbiota in hypochlorhydric states arising as a result of Helicobacter pylori-induced atrophic gastritis, autoimmune atrophic gastritis and proton pump inhibitor use.

Several conditions associated with reduced gastric acid secretion confer an altered risk of developing a gastric malignancy. Helicobacter pylori-induced atrophic gastritis predisposes to gastric adenocarcinoma, autoimmune atrophic gastritis is a precursor of type I gastric neuroendocrine tumours, whereas proton pump inhibitor (PPI) use does not affect stomach cancer risk. We hypothesised that each of these conditions was associated with specific alterations in the gastric microbiota and that this influenced subsequent tumour risk. 95 patients (in groups representing normal stomach, PPI treated, H. pylori gastritis, H. pylori-induced atrophic gastritis and autoimmune atrophic gastritis) were selected from a cohort of 1400. RNA extracted from gastric corpus biopsies was analysed using 16S rRNA sequencing (MiSeq). Samples from normal stomachs and patients treated with PPIs demonstrated similarly high microbial diversity. Patients with autoimmune atrophic gastritis also exhibited relatively high microbial diversity, but with samples dominated by Streptococcus. H. pylori colonisation was associated with decreased microbial diversity and reduced complexity of co-occurrence networks. H. pylori-induced atrophic gastritis resulted in lower bacterial abundances and diversity, whereas autoimmune atrophic gastritis resulted in greater bacterial abundance and equally high diversity compared to normal stomachs. Pathway analysis suggested that glucose-6-phospahte1-dehydrogenase and D-lactate dehydrogenase were over represented in H. pylori-induced atrophic gastritis versus autoimmune atrophic gastritis, and that both these groups showed increases in fumarate reductase. Autoimmune and H. pylori-induced atrophic gastritis were associated with different gastric microbial profiles. PPI treated patients showed relatively few alterations in the gastric microbiota compared to healthy subjects.

Analysis of the bread wheat genome using whole-genome shotgun sequencing.

Bread wheat (Triticum aestivum) is a globally important crop, accounting for 20 per cent of the calories consumed by humans. Major efforts are underway worldwide to increase wheat production by extending genetic diversity and analysing key traits, and genomic resources can accelerate progress. But so far the very large size and polyploid complexity of the bread wheat genome have been substantial barriers to genome analysis. Here we report the sequencing of its large, 17-gigabase-pair, hexaploid genome using 454 pyrosequencing, and comparison of this with the sequences of diploid ancestral and progenitor genomes. We identified between 94,000 and 96,000 genes, and assigned two-thirds to the three component genomes (A, B and D) of hexaploid wheat. High-resolution synteny maps identified many small disruptions to conserved gene order. We show that the hexaploid genome is highly dynamic, with significant loss of gene family members on polyploidization and domestication, and an abundance of gene fragments. Several classes of genes involved in energy harvesting, metabolism and growth are among expanded gene families that could be associated with crop productivity. Our analyses, coupled with the identification of extensive genetic variation, provide a resource for accelerating gene discovery and improving this major crop.

Insight into biases and sequencing errors for amplicon sequencing with the Illumina MiSeq platform.

With read lengths of currently up to 2 × 300 bp, high throughput and low sequencing costs Illumina's MiSeq is becoming one of the most utilized sequencing platforms worldwide. The platform is manageable and affordable even for smaller labs. This enables quick turnaround on a broad range of applications such as targeted gene sequencing, metagenomics, small genome sequencing and clinical molecular diagnostics. However, Illumina error profiles are still poorly understood and programs are therefore not designed for the idiosyncrasies of Illumina data. A better knowledge of the error patterns is essential for sequence analysis and vital if we are to draw valid conclusions. Studying true genetic variation in a population sample is fundamental for understanding diseases, evolution and origin. We conducted a large study on the error patterns for the MiSeq based on 16S rRNA amplicon sequencing data. We tested state-of-the-art library preparation methods for amplicon sequencing and showed that the library preparation method and the choice of primers are the most significant sources of bias and cause distinct error patterns. Furthermore we tested the efficiency of various error correction strategies and identified quality trimming (Sickle) combined with error correction (BayesHammer) followed by read overlapping (PANDAseq) as the most successful approach, reducing substitution error rates on average by 93%.

Illumina error profiles: resolving fine-scale variation in metagenomic sequencing data.

BACKGROUND: Illumina's sequencing platforms are currently the most utilised sequencing systems worldwide. The technology has rapidly evolved over recent years and provides high throughput at low costs with increasing read-lengths and true paired-end reads. However, data from any sequencing technology contains noise and our understanding of the peculiarities and sequencing errors encountered in Illumina data has lagged behind this rapid development. RESULTS: We conducted a systematic investigation of errors and biases in Illumina data based on the largest collection of in vitro metagenomic data sets to date. We evaluated the Genome Analyzer II, HiSeq and MiSeq and tested state-of-the-art low input library preparation methods. Analysing in vitro metagenomic sequencing data allowed us to determine biases directly associated with the actual sequencing process. The position- and nucleotide-specific analysis revealed a substantial bias related to motifs (3mers preceding errors) ending in "GG". On average the top three motifs were linked to 16 % of all substitution errors. Furthermore, a preferential incorporation of ddGTPs was recorded. We hypothesise that all of these biases are related to the engineered polymerase and ddNTPs which are intrinsic to any sequencing-by-synthesis method. We show that quality-score-based error removal strategies can on average remove 69 % of the substitution errors - however, the motif-bias remains. CONCLUSION: Single-nucleotide polymorphism changes in bacterial genomes can cause significant changes in phenotype, including antibiotic resistance and virulence, detecting them within metagenomes is therefore vital. Current error removal techniques are not designed to target the peculiarities encountered in Illumina sequencing data and other sequencing-by-synthesis methods, causing biases to persist and potentially affect any conclusions drawn from the data. In order to develop effective diagnostic and therapeutic approaches we need to be able to identify systematic sequencing errors and distinguish these errors from true genetic variation.

A comprehensive benchmarking study of protocols and sequencing platforms for 16S rRNA community profiling.

BACKGROUND: In the last 5 years, the rapid pace of innovations and improvements in sequencing technologies has completely changed the landscape of metagenomic and metagenetic experiments. Therefore, it is critical to benchmark the various methodologies for interrogating the composition of microbial communities, so that we can assess their strengths and limitations. The most common phylogenetic marker for microbial community diversity studies is the 16S ribosomal RNA gene and in the last 10 years the field has moved from sequencing a small number of amplicons and samples to more complex studies where thousands of samples and multiple different gene regions are interrogated. RESULTS: We assembled 2 synthetic communities with an even (EM) and uneven (UM) distribution of archaeal and bacterial strains and species, as metagenomic control material, to assess performance of different experimental strategies. The 2 synthetic communities were used in this study, to highlight the limitations and the advantages of the leading sequencing platforms: MiSeq (Illumina), The Pacific Biosciences RSII, 454 GS-FLX/+ (Roche), and IonTorrent (Life Technologies). We describe an extensive survey based on synthetic communities using 3 experimental designs (fusion primers, universal tailed tag, ligated adaptors) across the 9 hypervariable 16S rDNA regions. We demonstrate that library preparation methodology can affect data interpretation due to different error and chimera rates generated during the procedure. The observed community composition was always biased, to a degree that depended on the platform, sequenced region and primer choice. However, crucially, our analysis suggests that 16S rRNA sequencing is still quantitative, in that relative changes in abundance of taxa between samples can be recovered, despite these biases. CONCLUSION: We have assessed a range of experimental conditions across several next generation sequencing platforms using the most up-to-date configurations. We propose that the choice of sequencing platform and experimental design needs to be taken into consideration in the early stage of a project by running a small trial consisting of several hypervariable regions to quantify the discriminatory power of each region. We also suggest that the use of a synthetic community as a positive control would be beneficial to identify the potential biases and procedural drawbacks that may lead to data misinterpretation. The results of this study will serve as a guideline for making decisions on which experimental condition and sequencing platform to consider to achieve the best microbial profiling.

GeneMill: A 21st century platform for innovation.

GeneMill officially launched on 4th February 2016 and is an open access academic facility located at The University of Liverpool that has been established for the high-throughput construction and testing of synthetic DNA constructs. GeneMill provides end-to-end design, construction and phenotypic characterization of small to large gene constructs or genetic circuits/pathways for academic and industrial applications. Thus, GeneMill is equipping the scientific community with easy access to the validated tools required to explore the possibilities of Synthetic Biology.

Targeted re-sequencing of the allohexaploid wheat exome.

Bread wheat, Triticum aestivum, is an allohexaploid composed of the three distinct ancestral genomes, A, B and D. The polyploid nature of the wheat genome together with its large size has limited our ability to generate the significant amount of sequence data required for whole genome studies. Even with the advent of next-generation sequencing technology, it is still relatively expensive to generate whole genome sequences for more than a few wheat genomes at any one time. To overcome this problem, we have developed a targeted-capture re-sequencing protocol based upon NimbleGen array technology to capture and characterize 56.5 Mb of genomic DNA with sequence similarity to over 100 000 transcripts from eight different UK allohexaploid wheat varieties. Using this procedure in conjunction with a carefully designed bioinformatic procedure, we have identified more than 500 000 putative single-nucleotide polymorphisms (SNPs). While 80% of these were variants between the homoeologous genomes, A, B and D, a significant number (20%) were putative varietal SNPs between the eight varieties studied. A small number of these latter polymorphisms were experimentally validated using KASPar technology and 94% proved to be genuine. The procedures described here to sequence a large proportion of the wheat genome, and the various SNPs identified should be of considerable use to the wider wheat community.

Transcript-specific, single-nucleotide polymorphism discovery and linkage analysis in hexaploid bread wheat (Triticum aestivum L.).

Food security is a global concern and substantial yield increases in cereal crops are required to feed the growing world population. Wheat is one of the three most important crops for human and livestock feed. However, the complexity of the genome coupled with a decline in genetic diversity within modern elite cultivars has hindered the application of marker-assisted selection (MAS) in breeding programmes. A crucial step in the successful application of MAS in breeding programmes is the development of cheap and easy to use molecular markers, such as single-nucleotide polymorphisms. To mine selected elite wheat germplasm for intervarietal single-nucleotide polymorphisms, we have used expressed sequence tags derived from public sequencing programmes and next-generation sequencing of normalized wheat complementary DNA libraries, in combination with a novel sequence alignment and assembly approach. Here, we describe the development and validation of a panel of 1114 single-nucleotide polymorphisms in hexaploid bread wheat using competitive allele-specific polymerase chain reaction genotyping technology. We report the genotyping results of these markers on 23 wheat varieties, selected to represent a broad cross-section of wheat germplasm including a number of elite UK varieties. Finally, we show that, using relatively simple technology, it is possible to rapidly generate a linkage map containing several hundred single-nucleotide polymorphism markers in the doubled haploid mapping population of Avalon × Cadenza.

Novel hydroxycinnamoyl-coenzyme A quinate transferase genes from artichoke are involved in the synthesis of chlorogenic acid.

Artichoke (Cynara cardunculus subsp. scolymus) extracts have high antioxidant capacity, due primarily to flavonoids and phenolic acids, particularly chlorogenic acid (5-caffeoylquinic acid [CGA]), dicaffeoylquinic acids, and caffeic acid, which are abundant in flower bracts and bioavailable to humans in the diet. The synthesis of CGA can occur following different routes in plant species, and hydroxycinnamoyl-coenzyme A transferases are important enzymes in these pathways. Here, we report on the isolation and characterization of two novel genes both encoding hydroxycinnamoyl-coenzyme A quinate transferases (HQT) from artichoke. The recombinant proteins (HQT1 and HQT2) were assayed after expression in Escherichia coli, and both showed higher affinity for quinate over shikimate. Their preferences for acyl donors, caffeoyl-coenzyme A or p-coumaroyl-coenzyme A, were examined. Modeling and docking analyses were used to propose possible pockets and residues involved in determining substrate specificities in the HQT enzyme family. Quantitative real-time polymerase chain reaction analysis of gene expression indicated that HQT1 might be more directly associated with CGA content. Transient and stable expression of HQT1 in Nicotiana resulted in a higher production of CGA and cynarin (1,3-dicaffeoylquinic acid). These findings suggest that several isoforms of HQT contribute to the synthesis of CGA in artichoke according to physiological needs and possibly following various metabolic routes.

SMRT Gate: A method for validation of synthetic constructs on Pacific Biosciences sequencing platforms.

Current DNA assembly methods are prone to sequence errors, requiring rigorous quality control (QC) to identify incorrect assemblies or synthesized constructs. Such errors can lead to misinterpretation of phenotypes. Because of this intrinsic problem, routine QC analysis is generally performed on three or more clones using a combination of restriction endonuclease assays, colony PCR, and Sanger sequencing. However, as new automation methods emerge that enable high-throughput assembly, QC using these techniques has become a major bottleneck. Here, we describe a quick and affordable methodology for the QC of synthetic constructs. Our method involves a one-pot digestion-ligation DNA assembly reaction, based on the Golden Gate assembly methodology, that is coupled with Pacific Biosciences' Single Molecule, Real-Time (PacBio SMRT) sequencing technology.

Leaf LIMS: A Flexible Laboratory Information Management System with a Synthetic Biology Focus.

This paper presents Leaf LIMS, a flexible laboratory information management system (LIMS) designed to address the complexity of synthetic biology workflows. At the project's inception there was a lack of a LIMS designed specifically to address synthetic biology processes, with most systems focused on either next generation sequencing or biobanks and clinical sample handling. Leaf LIMS implements integrated project, item, and laboratory stock tracking, offering complete sample and construct genealogy, materials and lot tracking, and modular assay data capture. Hence, it enables highly configurable task-based workflows and supports data capture from project inception to completion. As such, in addition to it supporting synthetic biology it is ideal for many laboratory environments with multiple projects and users. The system is deployed as a web application through Docker and is provided under a permissive MIT license. It is freely available for download at https://leaflims.github.io .

Stable-isotope probing and metagenomics reveal predation by protozoa drives E. coli removal in slow sand filters.

Stable-isotope probing and metagenomics were applied to study samples taken from laboratory-scale slow sand filters 0.5, 1, 2, 3 and 4 h after challenging with (13)C-labelled Escherichia coli to determine the mechanisms and organisms responsible for coliform removal. Before spiking, the filters had been continuously operated for 7 weeks using water from the River Kelvin, Glasgow as their influent source. Direct counts and quantitative PCR assays revealed a clear predator-prey response between protozoa and E. coli. The importance of top-down trophic-interactions was confirmed by metagenomic analysis, identifying several protozoan and viral species connected to E. coli attrition, with protozoan grazing responsible for the majority of the removal. In addition to top-down mechanisms, indirect mechanisms, such as algal reactive oxygen species-induced lysis, and mutualistic interactions between algae and fungi, were also associated with coliform removal. The findings significantly further our understanding of the processes and trophic interactions underpinning E. coli removal. This study provides an example for similar studies, and the opportunity to better understand, manage and enhance E. coli removal by allowing the creation of more complex trophic interaction models.

Draft Genome Sequence of Bacillus Species from the Rhizosphere of the Desert Plant Rhazya stricta.

In order to better understand the ecology and diversity of microbes in the rhizosphere of desert plants, we undertook a survey of Bacillus species isolated from soil around Rhazya stricta plants from the area around Jeddah, in The Kingdom, Saudi Arabia. We have sequenced the genomes of 8 Bacillus isolates representing four different species.

Full genome re-sequencing reveals a novel circadian clock mutation in Arabidopsis.

Map based cloning in Arabidopsis thaliana can be a difficult and time-consuming process, specifically if the phenotype is subtle and scoring labour intensive. Here, we have re-sequenced the 120-Mb genome of a novel Arabidopsis clock mutant early bird (ebi-1) in Wassilewskija (Ws-2). We demonstrate the utility of sequencing a backcrossed line in limiting the number of SNPs considered. We identify a SNP in the gene AtNFXL-2 as the likely cause of the ebi-1 phenotype.

Molecular and functional analysis of new members of the wheat PR4 gene family.

Five new genes belonging to the pathogenesis-related (PR) 4 family have been cloned and characterised in Triticum aestivum. Two full-length genes, named wPR4e and wPR4f-b, were isolated by library screening, demonstrating the presence of a small intron only in wPR4f-b. Two other PR4 genes (wPR4f-a and wPR4f-c) were isolated by PCR, showing very high sequence identity with wPR4f-b and constituting a new sub-family. Transcription start analysis was performed by RLM-RACE, leading to the isolation of a fifth gene, named wPR4g, that is highly homologous to wPR4e; both encode putative vacuolar PR4 proteins (Wheatwin7 and Wheatwin5, respectively). wPR4e and wPR4f sub-family genes are induced by F. culmorum infection, by chemicals that lead to systemic acquired resistance and by wounding, showing different spatial and temporal induction pathways. In silico analysis of the 5' untranslated regions of wPR4e and wPR4f-b revealed the presence of several abiotic and biotic stress-responsive elements. wPR4e and wPR4f-b putative promoters were fused to the beta-glucuronidase (GUS) reporter gene, and transient and stable expression assays demonstrated that both are able to drive expression of GUS. Characterisation of these new PR4 genes and particularly of their 5' untranslated regions, as well as the determination of their expression patterns, will contribute to our understanding of the responsiveness of this gene family to various stress conditions and of its role in plant defence.