High-throughput DNA Sequencing Methods Research Articles

Microbes matter: integrating microbial sequence information for biodiversity

The large efforts to document and map aboveground biodiversity have helped to elucidate ecological and evolutionary mechanisms and processes, predict responses to global change, and identify potential management options in response to those changes. Yet these concepts have mostly been applied to aboveground plant and animal communities, while microbial diversity remains difficult to incorporate. The ability to integrate microbial sequence data into an accessible global infrastructure has previously been limited by a few key factors: First, most of microbial diversity remains undescribed and unknown; there is just an enormous amount of biodiversity. Second, there is a lack of congruence between the many disparate microbial datasets (e.g. taxonomy, phylogeny, and methodological biases), which limits the ability to monitor and quantify global patterns of the terrestrial microbiome. Finally, there is a lack of coordination and networking between scientists studying microbes. In this presentation I will discuss two case studies that highlight how we can begin to link microbial data to the already well-established macro-knowledge and other environmental databases (like global carbon maps) Study 1 – a megameta analysis: The emergence of high-throughput DNA sequencing methods provides unprecedented opportunities to further unravel microbial ecology and its worldwide role from human health to ecosystem functioning. However, in spite of the abundance of sequencing studies, combining data from multiple individual studies to address macroecological questions of bacterial diversity remains methodically challenging and plagued with biases. While previous meta-analysis efforts have focused on diversity measures or abundances of major taxa, in a recent study(1) we show that disparate amplicon sequence data can be combined at the taxonomy-based level to assess bacterial community structure. Using a machine learning approach, we found that rarer taxa are more important for structuring soil communities than abundant taxa. We concluded that combining data from independent studies can be used to explore novel patterns in bacterial communities, identify potential ‘indicator’ taxa with an important role in structuring communities, and propose new hypotheses on the factors that shape microbial biogeography previously overlooked. Study 2 – a global soil biodiversity database: Greater access to microbial data is an important next step for biodiversity research and conservation, and for understanding the ecology and evolution of microbial communities. In collaboration with the Global Soil Biodiversity Initiative and the German Biodiversity Synthesis Centre (sDIV) we outlined steps that must be taken to ensure microbial sequence data can be included in global measures and maps of biodiversity(2). Here I will discuss how the plant associated microbiome is an optimal starting point to synthesize microbial sequence data on an open and global platform. The plant-microbiome is an optimal model system that goes across scales and time, can act as a bridge between microorganisms and macroorganisms, and as an opportunity to more thoroughly explore the synthesis of global microbial sequence data (for a global soil biodiversity database). Beyond expanding primary research, the patterns discovered in a synthesis of plant-microbiome can be used to explore and guide ecosystem restoration and sustainability. Overall, a better understanding of microbial biodiversity will help to predict consequences of (human-induced) global changes and facilitate conservation and adaptation responses. (1) Ramirez, K.S., C.G. Knight et al. and F.T. de Vries (2017). Detecting macroecological patterns in bacterial communities across independent studies of global soils. Nature Microbiology. (2) Ramirez, K.S., M. Döring, N. Eisenhauer, C. Gardi, J. Ladau, J.W. Leff, G. Lentendu, Z. Lindo, M.C. Rillig, D. Russell, S. Scheu, M.G. St. John, F.T. de Vries, T. Wubet, W.H. van der Putten, D.H. Wall, (2015). Towards a global platform for linking soil biodiversity data. Frontiers in Ecology and Evolutionary Biology 3(91). doi: 10.3389/fevo.2015.00091

16S rRNA gene metabarcoding and TEM reveals different ecological strategies within the genus Neogloboquadrina (planktonic foraminifer).

Uncovering the complexities of trophic and metabolic interactions among microorganisms is essential for the understanding of marine biogeochemical cycling and modelling climate-driven ecosystem shifts. High-throughput DNA sequencing methods provide valuable tools for examining these complex interactions, although this remains challenging, as many microorganisms are difficult to isolate, identify and culture. We use two species of planktonic foraminifera from the climatically susceptible, palaeoceanographically important genus Neogloboquadrina, as ideal test microorganisms for the application of 16S rRNA gene metabarcoding. Neogloboquadrina dutertrei and Neogloboquadrina incompta were collected from the California Current and subjected to either 16S rRNA gene metabarcoding, fluorescence microscopy, or transmission electron microscopy (TEM) to investigate their species-specific trophic interactions and potential symbiotic associations. 53–99% of 16S rRNA gene sequences recovered from two specimens of N. dutertrei were assigned to a single operational taxonomic unit (OTU) from a chloroplast of the phylum Stramenopile. TEM observations confirmed the presence of numerous intact coccoid algae within the host cell, consistent with algal symbionts. Based on sequence data and observed ultrastructure, we taxonomically assign the putative algal symbionts to Pelagophyceae and not Chrysophyceae, as previously reported in this species. In addition, our data shows that N. dutertrei feeds on protists within particulate organic matter (POM), but not on bacteria as a major food source. In total contrast, of OTUs recovered from three N. incompta specimens, 83–95% were assigned to bacterial classes Alteromonadales and Vibrionales of the order Gammaproteobacteria. TEM demonstrates that these bacteria are a food source, not putative symbionts. Contrary to the current view that non-spinose foraminifera are predominantly herbivorous, neither N. dutertrei nor N. incompta contained significant numbers of phytoplankton OTUs. We present an alternative view of their trophic interactions and discuss these results within the context of modelling global planktonic foraminiferal abundances in response to high-latitude climate change.

Structure of bacterial chromosome: An analysis of DNA-protein interactions in vivo.

According to recent reports, bacterial chromosomes exhibit a hierarchical organization. The number of proteins that bind DNA are responsible for local and global organization of the DNA ensuring proper chromosome compaction. Advanced molecular biology techniques combined with high-throughput DNA sequencing methods allow a precise analysis of bacterial chromosome structures on a local and global scale. Methods such as in vivo footprinting and ChIP-seq allow to map binding sites of analyzed proteins in certain chromosomal regions or along the whole chromosome while analysis of the spatial interactions on global scale could be performed by 3C techniques. Additional insight into complex structures created by chromosome-organizing proteins is provided by high-resolution fluorescence microscopy techniques.

Detecting macroecological patterns in bacterial communities across independent studies of global soils.

The emergence of high-throughput DNA sequencing methods provides unprecedented opportunities to further unravel bacterial biodiversity and its worldwide role from human health to ecosystem functioning. However, despite the abundance of sequencing studies, combining data from multiple individual studies to address macroecological questions of bacterial diversity remains methodically challenging and plagued with biases. Here, using a machine-learning approach that accounts for differences among studies and complex interactions among taxa, we merge 30 independent bacterial data sets comprising 1,998 soil samples from 21 countries. Whereas previous meta-analysis efforts have focused on bacterial diversity measures or abundances of major taxa, we show that disparate amplicon sequence data can be combined at the taxonomy-based level to assess bacterial community structure. We find that rarer taxa are more important for structuring soil communities than abundant taxa, and that these rarer taxa are better predictors of community structure than environmental factors, which are often confounded across studies. We conclude that combining data from independent studies can be used to explore bacterial community dynamics, identify potential 'indicator' taxa with an important role in structuring communities, and propose hypotheses on the factors that shape bacterial biogeography that have been overlooked in the past.

Unveiling the Identity of Wenwan Walnuts and Phylogenetic Relationships of Asian Juglans Species Using Restriction Site-Associated DNA-Sequencing.

Juglans species have considerable ecological and economic value worldwide. In China, Wenwan walnuts have been collected by aristocrats and noblemen for more than 2000 years. As a diversity center of Asian Juglans, five species are widely distributed in China. The most famous of these is Mahetao (J. hopeiensis), which is an uncharacterized species that is mostly cultivated. Wild J. hopeiensis individuals are very rare and are endemic to Hebei Province. Because of the minimal variations in previously used molecular markers and the heterogeneity between chloroplast and nuclear genomes, determining the phylogenetic relationships among the Juglans species has been challenging, and has hindered subsequent evolutionary inferences. In this study, we collected enough materials for both cultivated and wild Mahetao to construct well-resolved phylogenetic trees for Asian Juglans species. We used a high-throughput genome-wide restriction site-associated DNA sequencing method. Consequently, the identity of J. hopeiensis has been clearly resolved. Our results indicate that J. hopeiensis is a hybrid of J. regia and J. mandshurica. However, J. hopeiensis, J. regia and J. sigillata should be considered as a single species from section Juglans. Additionally, J. ailantifolia, J. cathayensis, and J. mandshurica likely represent one species from section Cardiocaryon according to morphological and molecular studies. These results are supported by population structure analysis and morphological comparison. We propose that J. hopeiensis trees growing in the wild should be conserved because of the economic value of their nuts. These trees may be of particular importance to impoverished communities. Furthermore, they may serve as a valuable genetic resource relevant for enhancing the production of edible walnuts. The 2b-RAD method is a viable option for future phylogenetic studies of Juglans species as well as other plant species.

Links between bacteria derived from penguin guts and deposited guano and the surrounding soil microbiota

Penguins are an important indicator of marine ecosystem health and a major contributor of nutrients to terrestrial ecosystems in Antarctica. Their stomach microbiota is influenced by both the prey consumed and their foraging environment in the sea. As penguins feed at sea and breed on land, they might be expected to transfer microbes (e.g. prey-associated and marine bacteria) as well as nutrients from their stomachs while regurgitating food or in their guano to the surrounding terrestrial environment. However, most research attention to date has focused separately on the penguin gut microbiota (via cloacal/guano samples) and the terrestrial soil microbiota, and any relationship between them has yet to be established. Here, we analysed the bacterial communities in stomach regurgitates and cloacal swabs from the same individual birds, freshly deposited guano and rookery soils of two Pygoscelis penguins that breed sympatrically on Signy Island (South Orkney Islands, maritime Antarctic) using a high-throughput DNA sequencing method. Our data do not support the hypothesis that bacteria transferred from penguin guts and/or deposited guano make a significant contribution to the communities of the surrounding terrestrial microbial ecosystem. In both penguin species, composition of bacterial communities differed between the four sample types, with Jaccard similarities ranging between 10 and 36%. Assemblages of the dominant and co-occurring bacterial communities in rookery soils were either significantly negatively correlated or not correlated with the three other sample types. Sample-specific communities were also identified in this study, contributing around 63% of the identified diversity overall.

Origins of mtDNA mutations in ageing.

MtDNA mutations are one of the hallmarks of ageing and age-related diseases. It is well established that somatic point mutations accumulate in mtDNA of multiple organs and tissues with increasing age and heteroplasmy is universal in mammals. However, the origin of these mutations remains controversial. The long-lasting hypothesis stating that mtDNA mutations emanate from oxidative damage via a self-perpetuating mechanism has been extensively challenged in recent years. Contrary to this initial ascertainment, mtDNA appears to be well protected from action of reactive oxygen species (ROS) through robust protein coating and endomitochondrial microcompartmentalization. Extensive development of scrupulous high-throughput DNA sequencing methods suggests that an imperfect replication process, rather than oxidative lesions are the main sources of mtDNA point mutations, indicating that mtDNA polymerase γ (POLG) might be responsible for the majority of mtDNA mutagenic events. Here, we summarize the recent knowledge in prevention and defence of mtDNA oxidative lesions and discuss the plausible mechanisms of mtDNA point mutation generation and fixation.

Midgut bacteria in deltamethrin-resistant, deltamethrin-susceptible, and field-caught populations of Plutella xylostella, and phenomics of the predominant midgut bacterium Enterococcus mundtii

Gut bacteria play a significant role in host insect. This study evaluated detail difference of midgut bacteria in deltamethrin-resistant, deltamethrin-susceptible and field-caught populations of diamondback moth, and studied phenomics of the predominant midgut bacterium Enterococcus mundtii. Cultivable bacteria revealed that E. mundtii and Carnobacterium maltaromaticum dominated the bacterial populations from deltamethrin-resistant and deltamethrin-susceptible larval midguts, whereas E. mundtii was predominant in field-caught population. Illumina sequencing analysis indicated that 97% of the midgut bacteria were from the phyla Firmicutes, Proteobacteria and Cyanobacteria. Both resistant and susceptible populations had more Enterococcus and Carnobacterium. Enterococcus, Carnobacterium, Bacillus, and Pseudomonas were predominant in the field-caught population. A phenomics analysis revealed that E. mundtii was able to metabolize 25.26% of the tested carbon sources, 100% of the nitrogen sources, 100% of the phosphorus sources and 97.14% of the sulfur sources, had a wide range of osmolytes and pH conditions, and showed active deaminase activity but no decarboxylase activity. This is the first report regarding different populations of DBM midgut bacteria analyzed using both high-throughput DNA sequencing and cultivation methods, and also first report concerning the phenomics of E. mundtii. The phenomics of E. mundtii provide a basis for the future study of gut bacteria functions.

Food metagenomics: Next generation sequencing identifies species mixtures and mislabeling within highly processed cod products

Molecular identification of processed food products can be challenging due to the presence of unknown animal products or a blend of two or more species. However, with the development of high-throughput DNA sequencing methods (i.e. Next-Generation Sequencing) and DNA molecular markers libraries (e.g. DNA barcodes), it is possible to identify species using a powerful approach called metagenomics. Processed cod products, such as cakes and restaurant dishes, are very appreciated around the world and are an expensive seafood product in Brazil. Cod products are very prone to mislabeling since only four species can be legally labeled as “bacalhau” (cod) under Brazilian legislation: Gadus macrocephalus, Gadus morhua, Gadus ogac, and Boreogadus saida. The Ministry of Agriculture, Livestock and Food Supply is responsible for the inspection and certification of such products by providing an inspection stamp number (namely S.I.F.). We analyzed twenty-two processed cod products (i.e. cod pieces, frozen cakes, vacuum packaged cooked meals, a restaurant dish, and fast food cod cakes) labeled as “Bacalhau”, purchased from supermarkets, local stores, fast food outlets, and one restaurant in the city of Belo Horizonte, Brazil, with and without a S.I.F. stamp number. A mixture of two or more species were found within 31% of all products. Here, we report a mislabeling rate of 41% (N = 9) within highly processed cod products, but misidentification was less frequently found within products possessing a S.I.F stamp (4.5%). This is the first report of a metagenomic approach testing governmental certification programs and mislabeling of highly processed seafood products.

Characterization of odor-associated fungal community in automobile HVAC systems using a high-throughput DNA sequencing method

Characterization of odor-associated fungal community in automobile HVAC systems using a high-throughput DNA sequencing method

Fidelity of the DNA Ligase-Catalyzed Scaffolding of Peptide Fragments on Nucleic Acid Polymers.

We describe the development and analysis of the T4 DNA ligase-catalyzed DNA templated polymerization of pentanucleotides modified with peptide fragments toward the generation of ssDNA-scaffolded peptides. A high-throughput duplex DNA sequencing method was developed to facilitate the determination of fidelity for various codon sets and library sizes used during the polymerization process. With this process, we identified several codon sets that enable the efficient and sequence-specific incorporation of peptide fragments along a ssDNA template at fidelities up to 99% and with low sequence bias. These findings mark a significant advance in generating evolvable biomimetic polymers and should find ready application to the in vitro selection of molecular recognition.

Quantitative Baseline Circulating Tumor DNA Levels Correlate with GM-CSF Response to Idiotype Vaccine in Untreated Mantle Cell Lymphoma

Background: Mantle cell lymphoma (MCL) is an incurable lymphoma that responds to various forms of immunotherapy. Previously, we reported that specific anti-idiotype tumor responses as measured by GM-CSF levels after vaccine were associated with overall survival (OS) and time to next treatment (TTNT) following DA-EPOCH-R in untreated MCL at 11 years of follow-up (Dunleavy ASCO 2012). Levels of GM-CSF prior to idiotype vaccine also were correlated with post-vaccine GM-CSF levels suggesting that pre-existing anti-tumor immune response predict idiotype vaccine responsiveness. High-throughput DNA sequencing methods can detect and quantify circulating tumor-specific DNA (ctDNA) in peripheral blood of MCL patients prior to therapy and can be monitored for clearance after therapy. We hypothesized that pre-treatment ctDNA levels may be a surrogate marker for ongoing host anti-tumor immunity. Here, we analyzed the relationship of pre- and post-treatment levels of ctDNA to predict GM-CSF response and clinical outcomes in untreated MCL.Methods: DA-EPOCH-R was administered x 6, followed by 5 cycles of Id-vaccine beginning at least 12 weeks later in untreated MCL. Id protein was produced by hybridoma technology, conjugated to keyhole limpet hemocyanin (KLH), and administered together with GM-CSF x 5 over 6 months. Pre- and post-vaccine samples were tested in parallel to assess humoral and cellular immune responses. Pre-treatment formalin-fixed paraffin embedded tissue specimens were de-identified and sent for identification of tumor-specific clonotypes. Tumor DNA was amplified using locus-specific primer sets for the immunoglobulin heavy-chain locus (IGH) complete (IGH-VDJH), IGH incomplete (IGH-DJH), and immunoglobulin k locus (IGK). Amplified products were sequenced and tumor-specific clonotypes were quantitated in peripheral blood mononuclear cells before treatment as ctDNA and after treatment as MRD.Results: Characteristics of all 26 pts: median age 57 (r 22-73), male sex 73%, PS 1 (0-2), MIPI (low-65%; interm-16%; high-19%), and blastoid 15%. Responses to DA-EPOCH-R: CR-92%, PR-8%. Immune analyses were performed in 24 pts; vaccine not produced in 1 pt and 1 pt progressed before immune analyses. Baseline tumor was available for clonotype calibration in 20 patients. With a potential follow-up of 14 years, the median OS of the entire cohort is 9.0 years and 10/26 (38%) patients are still alive. Pre-treatment levels of ctDNA were inversely correlated with normalized antitumor GM-CSF response (r=0.49, p=0.035) but not with white blood cell count (r=0.18, p=0.46), LDH (r=0.21, p=0.39) or MIPI scores (r=22, p=0.37). Patients with low pre-treatment ctDNA levels had longer OS than patients with high levels of ctDNA, but this did not reach statistical significance (11.01 y vs. 5.71 y, p=0.67). Eight of 20 of patients (40%) were MRD positive after DA-EPOCH-R despite achieving CR/CRu. Patients who achieved MRD-zero had a trend towards improved OS (NR vs. 8.2 years, p=0.15).Conclusions: Lower pre-treatment ctDNA levels correlate with normalized GM-CSF response to idiotype vaccine and may be a surrogate marker of ongoing host anti-tumor immunity. In this small study, ctDNA levels did not correlate with traditional markers of tumor proliferation. There is a trend towards improvement in OS in patients who achieve MRD-zero. Future studies are underway to explore the biologic significance of pre- and post-treatment ctDNA levels in MCL.This work was supported by the Intramural Research Program of NCI at NIH and Adaptive Biotechnologies, Inc. DisclosuresCrossley:Adaptive: Employment, Equity Ownership. Kong:Adaptive: Employment, Equity Ownership. Jacob:Adaptive: Employment, Equity Ownership. Kwak:Celltrion, Inc.: Consultancy.

Pompe disease: A case report

Pompe disease is inherited in an autosomal recessive manner, and is usually observed in the children of asymptomatic carriers. Pompe disease, known as Glycogen Storage Disorder type II, is caused by pathogenic mutations in the gene encoding lysosomal acid alpha-glucosidase ( GAA ). There are three types of Pompe disease: classical infantile form, non-classical infantile form and late-onset Pompe disease. Age of onset and severity of the disease determine the type of Pompe disease. We aimed to identify a mutation in GAA gene in parents who were first cousins and their baby girl was passed away due to the Pompe disease. The baby girl had reduced acid alpha-glucosidase activity, but genetic analysis had not been performed. Mutation analysis of parents was performed using high-throughput DNA sequencing method. Heterozygous mutation of c.896 T>C in exon 5 was found in parents, and prenatal diagnosis was performed for their next pregnancy. In conclusion, c.896 T>C substitution in GAA gene may lead to the severe type of Pompe disease. Using a relatively fast and reliable molecular genetic analysis method to confirm the early diagnosis of the Pompe disease is important for the management of the disease. Key words: Pompe disease, GSD2, GAA deficiency, high-throughput DNA sequencing

High diversity of skin-associated bacterial communities of marine fishes is promoted by their high variability among body parts, individuals and species.

Animal-associated microbiotas form complex communities, which are suspected to play crucial functions for their host fitness. However, the biodiversity of these communities, including their differences between host species and individuals, has been scarcely studied, especially in case of skin-associated communities. In addition, the intraindividual variability (i.e. between body parts) has never been assessed to date. The objective of this study was to characterize skin bacterial communities of two teleostean fish species, namely the European seabass (Dicentrarchus labrax) and gilthead seabream (Sparus aurata), using a high-throughput DNA sequencing method. In order to focus on intrinsic factors of host-associated bacterial community variability, individuals of the two species were raised in controlled conditions. Bacterial diversity was assessed using a set of four complementary indices, describing the taxonomic and phylogenetic facets of biodiversity and their respective composition (based on presence/absence data) and structure (based on species relative abundances) components. Variability of bacterial diversity was quantified at the interspecific, interindividual and intraindividual scales. We demonstrated that fish surfaces host highly diverse bacterial communities, whose composition was very different from that of surrounding bacterioplankton. This high total biodiversity of skin-associated communities was supported by the important variability, between host species, individuals and the different body parts (dorsal, anal, pectoral and caudal fins).

Monitoring of Circulating Tumor DNA As Minimal Residual Disease in Diffuse Large B-Cell Lymphoma

Background: Diffuse large B-cell lymphoma (DLBCL) can be cured with frontline therapy, but the prognosis of patients with relapse in the modern era is poor. Radiology scans (CT and PET) are commonly used as surveillance tools after therapy but do not improve outcomes and are associated with long-term health risks. Interim scans have also been used to identify early treatment failures, but are limited by false positives. Reliable predictive biomarkers that predict relapse may improve clinical outcomes. LymphoSIGHTª is a high-throughput DNA sequencing method that can detect and quantify circulating tumor DNA as minimal residual disease (MRD). We have previously reported its ability to detect circulating tumor DNA in serum after frontline therapy in DLBCL (Roschewski ASH 2013). Here, we analyzed the ability of serum MRD to predict both early treatment failure and relapse from remission in a large cohort of newly diagnosed DLBCL patients treated with curative intent.Methods: Patients with DLBCL and treated at the NCI with EPOCH-based chemotherapy +/-rituximab between July 1996 and October 2013 had research serum samples collected pre-Tx, at each cycle, and at each surveillance visit. Surveillance visits included serum samples with paired CT scans at 3, 4, 6, and 12-month intervals for the first 5 years. Pre-treatment formalin-fixed paraffin embedded (FFPE) tissue specimens and/or baseline serum were de-identified and sent for identification of tumor-specific clonotypes. Tumor DNA was amplified using locus-specific primer sets for the immunoglobulin heavy-chain locus (IGH) complete (IGH-VDJH), IGH incomplete (IGH-DJH), and immunoglobulin k locus (IGK). Amplified products were sequenced, and patients in whom a high-frequency tumor clone (.5%) was not identified were excluded from the MRD analysis. Tumor-specific clonotypes were quantitated in the serum as MRD and analyzed for the ability to predict early treatment failure (interim monitoring) and relapse from remission (surveillance monitoring). Disease progression during therapy or <6 mos from completion was considered early treatment failure.Results: 198 patients with DLBCL had pre-treatment specimens screened for a pre-treatment tumor clonotype with 3 excluded due to <10ng input DNA. When FFPE was available, 94 of 112 samples (86%) identified a clone while only 32 of 86 samples (37%) identified a clone without FFPE. The median age was 44 years (14-85) and an IPI risk groups included 56 low, 39 low-INT, 20 high-INT and 11 high. All patients who were MRD positive after therapy progressed. 101 patients achieved remission lasting > 6 mos and underwent surveillance monitoring. Eleven of these relapsed and 10/11 had a positive serum MRD at a median of 7.4 months prior to progression. The other 90 patients had sustained remissions and underwent a median of 11 surveillance CT scans (range 1-18). Dynamic monitoring of interim MRD (each cycle) identified early treatment failure (induction failure or relapse within 6 months) in 25 patients with a sensitivity of 81.8% and specificity of 87.5%. The positive predictive value (PPV) of interim MRD was 60% for predicting early failure while the negative predictive value (NPV) was 95.9%. The 5-year EFS of patients who were interim MRD positive was significantly worse than those who were negative (45.9% vs 83.0%, (p<0.0001)) (Figure).Conclusions: Non-invasive dynamic monitoring of serum MRD during therapy predicts early treatment failure, while serial MRD monitoring during surveillance predicts relapse from remission in DLBCL.This work was supported by the Intramural Research Program of NCI at NIH and Sequenta, Inc. [Display omitted] DisclosuresKong:Sequenta: Employment. Zheng:Sequenta: Employment. Willis:Sequenta: Employment, Equity Ownership. Faham:Sequenta: Employment, Equity Ownership.

Identification of structural variation in mouse genomes.

Structural variation is variation in structure of DNA regions affecting DNA sequence length and/or orientation. It generally includes deletions, insertions, copy-number gains, inversions, and transposable elements. Traditionally, the identification of structural variation in genomes has been challenging. However, with the recent advances in high-throughput DNA sequencing and paired-end mapping (PEM) methods, the ability to identify structural variation and their respective association to human diseases has improved considerably. In this review, we describe our current knowledge of structural variation in the mouse, one of the prime model systems for studying human diseases and mammalian biology. We further present the evolutionary implications of structural variation on transposable elements. We conclude with future directions on the study of structural variation in mouse genomes that will increase our understanding of molecular architecture and functional consequences of structural variation.

Single-cell genomics of marine plankton: Studying the single life of eukaryotic microbes

The oceans are full of innumerable numbers of single cells living in microenvironments. Understanding who they are, what they eat and what infects them can inform us about the true diversity of plankton, their biotic interactions and how they may respond to a changing environment. Analysing to significant depth the genomes and ‘gut’ (i.e. the food vacuole and other contents) of individual wild-caught cells would have been thought impossible only a few years ago. However, the rapidly expanding field of single-cell genomics, powered by modern cell-sorting procedures, high-throughput DNA sequencing and bioinformatics methods holds the promise to revolutionize understanding of the biodiversity and ecology of eukaryotic microbes and their places in the tree of life.

Visualization of genome signatures of eukaryote genomes by batch-learning self-organizing map with a special emphasis on Drosophila genomes.

A strategy of evolutionary studies that can compare vast numbers of genome sequences is becoming increasingly important with the remarkable progress of high-throughput DNA sequencing methods. We previously established a sequence alignment-free clustering method “BLSOM” for di-, tri-, and tetranucleotide compositions in genome sequences, which can characterize sequence characteristics (genome signatures) of a wide range of species. In the present study, we generated BLSOMs for tetra- and pentanucleotide compositions in approximately one million sequence fragments derived from 101 eukaryotes, for which almost complete genome sequences were available. BLSOM recognized phylotype-specific characteristics (e.g., key combinations of oligonucleotide frequencies) in the genome sequences, permitting phylotype-specific clustering of the sequences without any information regarding the species. In our detailed examination of 12 Drosophila species, the correlation between their phylogenetic classification and the classification on the BLSOMs was observed to visualize oligonucleotides diagnostic for species-specific clustering.

DNA Sequencing-Based Monitoring Of Serum Predicts Clinical Relapse Before CT Imaging in Diffuse Large B-Cell Lymphoma

BackgroundDiffuse large B-cell lymphoma (DLBCL) is the most common lymphoma with >40,000 cases annually in the US. While most patients are cured by frontline immunochemotherapy, up to 20% recur and salvage therapy is poor. Relapse detection relies on CT imaging, which is hampered by radiation exposure, high costs, high false positive rates, and infrequently detects relapse prior to symptoms. Novel methods to detect early relapse may improve outcomes. The LymphoSIGHT™ platform is a high-throughput DNA sequencing method that can detect minimal residual disease (MRD) of lymphoid malignancies in peripheral blood (PB) (Faham et al. Blood 2012) with a sensitivity of one lymphoma cell per million leukocytes. We showed it can also detect circulating tumor DNA in the PB of DLBCL pts (Armand Br J Haematol 2013). We assessed the sensitivity and specificity of LymphoSIGHT™ to detect circulating tumor DNA in serum after frontline therapy in DLBCL. MethodsAll pts were newly diagnosed DLBCL undergoing frontline EPOCH-based chemotherapy. Post-therapy surveillance included clinic visits, PB samples, and contrast CT scans at 3, 4, 6, 12 and 12 mos intervals for the first 5 years, respectively. Pre-treatment lymph node biopsies and PB samples collected at initial diagnosis and during surveillance visits were sent for VDJ amplification and MRD sequencing assessment. Using universal primer sets, we amplified immunoglobulin heavy and kappa chain (IGH and IGK) variable, diversity, and joining gene segments from genomic DNA in tumor biopsy and PB samples. Amplified products were sequenced, and tumor-specific clonotypes were identified for each pt based on their high frequency within the B-cell repertoire in the lymph node. The presence of the tumor-specific clonotype was then quantitated in PB samples. ResultsTwenty-seven pts, treated between July 1996 and July 2005, had adequate DNA from pre-treatment FFPE lymph node biopsies, and 25 (92.6%) had a high-frequency clonal rearrangement (MRD) detected. The median pt age was 49 years (20-75) and an IPI risk groups included 8 low, 3 low-intermediate, 11 high-intermediate and 3 high. The pts had a median of 12 surveillance CT scans (range 1-13) and 221 serum samples analyzed for MRD. Two pts progressed early after cycles 1 and 6. The first pt had progressive disease by CT scan after cycle 1 and remained MRD positive. The second pt remained MRD positive at the end of cycle 6, at which time a CT showed CR-unconfirmed, and progressed one month later. In the remaining 23 pts who achieved CR, seven relapsed at a median of 20.3 (range 5.6-151.6) months. The 16 pts in sustained CR had a median follow-up of 99.7 months (range 3.0-136.3) and none were MRD positive post-treatment (specificity 100%; 88.9-100%). There were 0 false positives in 200 post-treatment samples tested (specificity 100%, 99-100%). Of 7 relapsing pts, 6 had serum samples within 1 year of recurrence. Of these, 5 pts had a positive MRD (range 3.2 to 11.2 months) (sensitivity 83%; 44-98%) at a median of 9.2 months (range 3.2 to 11.2 months) prior to a positive CT scan (Figure). One relapsing pt only had a serum sample obtained 18 mos before CT recurrence and it was MRD negative. ConclusionsOur data suggest that a DNA sequencing-based MRD test of serum can predict clinical relapse with high sensitivity and specificity in DLBCL prior to CT progression. MRD assay detection may significantly reduce the cost, inconvenience and toxicity of surveillance. Importantly, early detection of recurrence may significantly improve pt outcomes. Additional pts are being analyzed and updated results will be available.This work was supported by the Intramural Research Program of NCI at NIH and Sequenta, Inc.▪ Disclosures:Kong:Sequenta: Employment, Equity Ownership. Zheng:Sequenta: Employment, Equity Ownership. Willis:Sequenta: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Faham:Sequenta: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

The Gut Microbiome: A New Frontier in Autism Research

The human gut harbors a complex community of microbes that profoundly influence many aspects of growth and development, including development of the nervous system. Advances in high-throughput DNA sequencing methods have led to rapidly expanding knowledge about this gut microbiome. Here, we review fundamental emerging data on the human gut microbiome, with a focus on potential interactions between the microbiome and autism spectrum disorders (ASD) and consider research on atypical patterns of feeding and nutrition in ASD and how they might interact with the microbiome. Finally we selectively survey results from studies in rodents on the impact of the microbiome on neurobehavioral development. The evidence reviewed here suggests that a deeper understanding of the gut microbiome could open up new avenues of research on ASD, including potential novel treatment strategies.