Viral Reads Research Articles

Traces of SARS-CoV-2 RNA in Peripheral Blood Cells of Patients with COVID-19.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the third virus that caused coronavirus-related outbreaks over the past 20 years. The outbreak was first reported in December 2019 in Wuhan, China, but rapidly progressed into a pandemic of an unprecedented scale since the 1918 flu pandemic. Besides respiratory complications in patients with COVID-19, clinical characterization of severe infection cases showed several other comorbidities, including multiple organ failure, and septic shock. To better understand the systemic pathogenesis of COVID-19, we interrogated the virus's presence in the peripheral blood cells, which might provide a form of trafficking or hiding to the virus. By analyzing >2 billion sequence reads of high-throughput transcriptome sequence data from 180 samples of patients with active SARS-CoV-2 infection or healthy controls collected from 6 studies, we found evidence of traces of SARS-CoV-2 RNA in peripheral blood mononuclear cells in two samples from two independent studies. In contrast, the viral RNA was abundant in bronchoalveolar lavage specimens from the same patients. We also devised a “viral spike-to-actin” RNA normalization as a metric to compare across various samples and minimize errors caused by intersample variability in total human RNA abundance. Our observation suggests immune presentation and discounts the possibility of extensive viral infection of lymphocytes or monocytes.

METAnnotatorX2: a Comprehensive Tool for Deep and Shallow Metagenomic Data Set Analyses.

ABSTRACTThe use of bioinformatic tools for read-based taxonomic and functional analyses of metagenomic data sets, including their assembly and management, is rather fragmentary due to the absence of an accepted gold standard. Moreover, most currently available software tools need input of millions of reads and rely on approximations in data analysis in order to reduce computing times. These issues result in suboptimal results in terms of accuracy, sensitivity, and specificity when used either for the reconstruction of taxonomic or functional profiles through read analysis or analysis of genomes reconstructed by metagenomic assembly. Moreover, the recent introduction of novel DNA sequencing technologies that generate long reads, such as Nanopore and PacBio, represent a valuable data resource that still suffers from a lack of dedicated tools to perform integrated hybrid analysis alongside short read data. In order to overcome these limitations, here we describe a comprehensive bioinformatic platform, METAnnotatorX2, aimed at providing an optimized user-friendly resource which maximizes output quality, while also allowing user-specific adaptation of the pipeline and straightforward integrated analysis of both short and long read data. To further improve performance quality and accuracy of taxonomic assignment of reads and contigs, custom preprocessed and taxonomically revised genomic databases for viruses, prokaryotes, and various eukaryotes were developed. The performance of METAnnotatorX2 was tested by analysis of artificial data sets encompassing viral, archaeal, bacterial, and eukaryotic (fungal) sequence reads that simulate different biological matrices. Moreover, real biological samples were employed to validate in silico results.IMPORTANCE We developed a novel tool, i.e., METAnnotatorX2, that includes a number of new advanced features for analysis of deep and shallow metagenomic data sets and is accompanied by (regularly updated) customized databases for archaea, bacteria, fungi, protists, and viruses. Both software and databases were developed so as to maximize sensitivity and specificity while including support for shallow metagenomic data sets. Through extensive tests performed on Illumina and Nanopore artificial data sets, we demonstrated the high performance of the software to not only extract taxonomic and functional information from sequence reads but also to assemble and process genomes from metagenomic data. The robustness of these functionalities was validated using “real-life” data sets obtained from Illumina and Nanopore sequencing of biological samples. Furthermore, the performance of METAnnotatorX2 was compared to other available software tools for analysis of shotgun metagenomics data.

Viral footprints across Gulfs of Kathiawar Peninsula and Arabian Sea: Unraveled from pelagic sediment metagenomic data

Marine biosphere is one of the largest, diverse and dynamic system hosting numerous of microorganisms. Viruses being the most abundant under explored lifeforms in ocean, represent a reservoir of great genetic diversity. We report the metagenomic insights on the viral communities in the deep sediments of the two Gulfs of Gujarat i.e. Gulf of Khambhat and Gulf of Kutch, with one sample from Arabian Sea, treated as open sea control. The viral reads were filtered from the whole dataset, assembled and studied for viral diversity, which was visualized by Pavian. The sequences were checked for the viral abundance, diversity and functionality. The resulting viral taxonomic classification contained 6 orders, 8 families and 47 genera. The results revealed that the phages infecting Cyanobacterium, Bacillus and Vibrio dominated the sediments. Further, it was observed that majority of viral sequences belonged to double-stranded DNA phages. The present study attempts to provide a primary insight of the viral signals and potential genetic content in the Gulfs of Kathiawar.

Expanding the taxonomic range in the fecal metagenome

BackgroundExcept for bacteria, the taxonomic diversity of the human fecal metagenome has not been widely studied, despite the potential importance of viruses and eukaryotes. Widely used bioinformatic tools contain limited numbers of non-bacterial species in their databases compared to available genomic sequences and their methodologies do not favour classification of rare sequences which may represent only a small fraction of their parent genome. In seeking to optimise identification of non-bacterial species, we evaluated five widely-used metagenome classifier programs (BURST, Kraken2, Centrifuge, MetaPhlAn2 and CCMetagen) for their ability to correctly assign and count simulations of bacterial, viral and eukaryotic DNA sequence reads, including the effect of taxonomic order of analysis of bacteria, viruses and eukaryotes and the effect of sequencing depth.ResultsWe found that the precision of metagenome classifiers varied significantly between programs and between taxonomic groups. When classifying viruses and eukaryotes, ordering the analysis such that bacteria were classified first significantly improved classification precision. Increasing sequencing depth decreased classification precision and did not improve recall of rare species.ConclusionsChoice of metagenome classifier program can have a marked effect on results with respect to precision of species assignment in different taxonomic groups. The order of taxonomic classification can markedly improve precision. Increasing sequencing depth can decrease classification precision and yields diminishing returns in probability of species detection.

Studying the virome in psychiatric disease

An overlooked aspect of current microbiome studies is the role of viruses in human health. Compared to bacterial studies, laboratory and analytical methods to study the entirety of viral communities in clinical samples are rudimentary and need further refinement. In order to address this need, we developed Virobiome-Seq, a sequence capture method and an accompanying bioinformatics analysis pipeline, that identifies viral reads in human samples. Virobiome-Seq is able to enrich for and detect multiple types of viruses in human samples, including novel subtypes that diverge at the sequence level. In addition, Virobiome-Seq is able to detect RNA transcripts from DNA viruses and may provide a sensitive method for detecting viral activity in vivo. Since Virobiome-Seq also yields the viral sequence, it makes it possible to investigate associations between viral genotype and psychiatric illness. In this proof of concept study, we detected HIV1, Torque Teno, Pegi, Herpes and Papilloma virus sequences in Peripheral Blood Mononuclear Cells, plasma and stool samples collected from individuals with psychiatric disorders. We also detected the presence of numerous novel circular RNA viruses but were unable to determine whether these viruses originate from the sample or represent contaminants. Despite this challenge, we demonstrate that our knowledge of viral diversity is incomplete and opportunities for novel virus discovery exist. Virobiome-Seq will enable a more sophisticated analysis of the virome and has the potential of uncovering complex interactions between viral activity and psychiatric disease.

An enrichment method for capturing mumps virus whole genome sequences directly from clinical specimens

Recently there has been a significant increase in the number of mumps outbreaks occurring in highly vaccinated populations. These outbreaks are often due to a mumps genotype G virus, where sequencing of the SH gene does not reveal enough genetic diversity to sufficient to resolve outbreaks. This has elevated the need to be able to sequence complete mumps viruses from clinical samples without laborious methods. Here we describe a probe enrichment method that allows for whole genome sequencing of the mumps virus directly from clinical specimens. Using 136 clinical samples, we show this method allows for a significant increase in the percentage of viral sequencing reads, resulting in the capture of mumps genomes. This method will be an asset in investigating future mumps outbreaks.

Using single cell spatial and transcriptional analysis to understand early host response to RNA viral infections.

Abstract The innate immune system serves as the first line of defense against pathogens. One innate defense mechanism is the RIG-I like receptor pathway (RLR) which signals through the mitochondrial anti-viral signaling protein (MAVS) which plays a role in initiating the interferon (IFN) response to RNA viruses such as vesicular stomatitis virus (VSV). To investigate the mechanisms the MAVS-dependent pathway has on early viral control following VSV infection in vivo, we used a combination of two approaches: single cell RNA sequencing and multiplex imaging. These two techniques allow us to assess the individual cellular response to VSV in whole lymph node tissue. After intradermal inoculation, skin draining lymph nodes were assessed using single cell sequencing. Macrophages and plasmacytoid dendritic cells (pDCs) showed significantly elevated IFN stimulated gene (ISG) detection in C57Bl/6J (B6) mice compared to the MAVS−/− animals at 8 hours post infection (hpi), yet there was no detectable viral reads. This early initiation of ISG expression played a role in detection of viral RNA at 24 hpi which saw significantly elevated viral RNA in the MAVS−/− animals compared to the B6 animals. Interestingly, the virus replication in both conditions was contained to the macrophage populations suggesting that without the MAVS pathway active, the virus was still contained. Combining this information along with the observed spatial organization through multiplex tissue imaging, we aim to develop a model illustrating the role of the MAVS-dependent pathway in controlling early virus replication. Animal experiments were done in accordance with the guidelines of the NIAID/NIH Institutional ACUC. This research was supported by the IRP of NIH/NIAID.

K-mer-Based Metagenomics Tools Provide a Fast and Sensitive Approach for the Detection of Viral Contaminants in Biopharmaceutical and Vaccine Manufacturing Applications Using Next-Generation Sequencing.

ABSTRACTAdventitious agent detection during the production of vaccines and biotechnology-based medicines is of critical importance to ensure the final product is free from any possible viral contamination. Increasing the speed and accuracy of viral detection is beneficial as a means to accelerate development timelines and to ensure patient safety. Here, several rapid viral metagenomics approaches were tested on simulated next-generation sequencing (NGS) data sets and existing data sets from virus spike-in studies done in CHO-K1 and HeLa cell lines. It was observed that these rapid methods had comparable sensitivity to full-read alignment methods used for NGS viral detection for these data sets, but their specificity could be improved. A method that first filters host reads using KrakenUniq and then selects the virus classification tool based on the number of remaining reads is suggested as the preferred approach among those tested to detect nonlatent and nonendogenous viruses. Such an approach shows reasonable sensitivity and specificity for the data sets examined and requires less time and memory as full-read alignment methods.IMPORTANCE Next-generation sequencing (NGS) has been proposed as a complementary method to detect adventitious viruses in the production of biotherapeutics and vaccines to current in vivo and in vitro methods. Before NGS can be established in industry as a main viral detection technology, further investigation into the various aspects of bioinformatics analyses required to identify and classify viral NGS reads is needed. In this study, the ability of rapid metagenomics tools to detect viruses in biopharmaceutical relevant samples is tested and compared to recommend an efficient approach. The results showed that KrakenUniq can quickly and accurately filter host sequences and classify viral reads and had comparable sensitivity and specificity to slower full read alignment approaches, such as BLASTn, for the data sets examined.

A Versatile Processing Workflow to Enable Pathogen Detection in Clinical Samples from Organs Using VIDISCA.

In recent years, refined molecular methods coupled with powerful high throughput sequencing technologies have increased the potential of virus discovery in clinical samples. However, host genetic material remains a complicating factor that interferes with discovery of novel viruses in solid tissue samples as the relative abundance of the virus material is low. Physical enrichment processing methods, although usually complicated, labor-intensive, and costly, have proven to be successful for improving sensitivity of virus detection in complex samples. In order to further increase detectability, we studied the application of fast and simple high-throughput virus enrichment methods on tissue homogenates. Probe sonication in high EDTA concentrations, organic extraction with Vertrel™ XF, or a combination of both, were applied prior to chromatography-like enrichment using Capto™ Core 700 resin, after which effects on virus detection sensitivity by the VIDISCA method were determined. Sonication in the presence of high concentrations of EDTA showed the best performance with an increased proportion of viral reads, up to 9.4 times, yet minimal effect on the host background signal. When this sonication procedure in high EDTA concentrations was followed by organic extraction with Vertrel™ XF and two rounds of core bead chromatography enrichment, an increase up to 10.5 times in the proportion of viral reads in the processed samples was achieved, with reduction of host background sequencing. We present a simple and semi-high-throughput method that can be used to enrich homogenized tissue samples for viral reads.

The gut virome of healthy children during the first year of life is diverse and dynamic.

In this work, we determined the diversity and dynamics of the gut virome of infants during the first year of life. Fecal samples were collected monthly, from birth to one year of age, from three healthy children living in a semi-rural village in Mexico. Most of the viral reads were classified into six families of bacteriophages including five dsDNA virus families of the order Caudovirales, with Siphoviridae and Podoviridae being the most abundant. Eukaryotic viruses were detected as early as two weeks after birth and remained present all along the first year of life. Thirty-four different eukaryotic virus families were found, where eight of these families accounted for 98% of all eukaryotic viral reads: Anelloviridae, Astroviridae, Caliciviridae, Genomoviridae, Parvoviridae, Picornaviridae, Reoviridae and the plant-infecting viruses of the Virgaviridae family. Some viruses in these families are known human pathogens, and it is surprising that they were found during the first year of life in infants without gastrointestinal symptoms. The eukaryotic virus species richness found in this work was higher than that observed in previous studies; on average between 7 and 24 virus species were identified per sample. The richness and abundance of the eukaryotic virome significantly increased during the second semester of life, probably because of an increased environmental exposure of infants with age. Our findings suggest an early and permanent contact of infants with a diverse array of bacteriophages and eukaryotic viruses, whose composition changes over time. The bacteriophages and eukaryotic viruses found in these children could represent a metastable virome, whose potential influence on the development of the infant’s immune system or on the health of the infants later in life, remains to be investigated.

Presence of complete murine viral genome sequences in patient-derived xenografts

Patient-derived xenografts are crucial for drug development but their use is challenged by issues such as murine viral infection. We evaluate the scope of viral infection and its impact on patient-derived xenografts by taking an unbiased data-driven approach to analyze unmapped RNA-Seq reads from 184 experiments. We find and experimentally validate the extensive presence of murine viral sequence reads covering entire viral genomes in patient-derived xenografts. The existence of viral sequences inside tumor cells is further confirmed by single cell sequencing data. Extensive chimeric reads containing both viral and human sequences are also observed. Furthermore, we find significantly changed expression levels of many cancer-, immune-, and drug metabolism-related genes in samples with high virus load. Our analyses indicate a need to carefully evaluate the impact of viral infection on patient-derived xenografts for drug development. They also point to a need for attention to quality control of patient-derived xenograft experiments.

Deep viral blood metagenomics reveals extensive anellovirus diversity in healthy humans

Human blood metagenomics has revealed the presence of different types of viruses in apparently healthy subjects. By far, anelloviruses constitute the viral family that is more frequently found in human blood, although amplification biases and contaminations pose a major challenge in this field. To investigate this further, we subjected pooled plasma samples from 120 healthy donors in Spain to high-speed centrifugation, RNA and DNA extraction, random amplification, and massive parallel sequencing. Our results confirm the extensive presence of anelloviruses in such samples, which represented nearly 97% of the total viral sequence reads obtained. We assembled 114 different viral genomes belonging to this family, revealing remarkable diversity. Phylogenetic analysis of ORF1 suggested 28 potentially novel anellovirus species, 24 of which were validated by Sanger sequencing to discard artifacts. These findings underscore the importance of implementing more efficient purification procedures that enrich the viral fraction as an essential step in virome studies and question the suggested pathological role of anelloviruses.

HHV-8 positive clinically advanced castrate-resistant prostate cancer (mCRPC): A potentially distinct molecular subset.

163 Background: Herpesvirus HHV-8 has been detected in normal prostate tissue and has been linked to the acquisition of androgen-independent growth of prostate cancer cells in vitro, early development of anti-androgen therapy resistance, and more aggressive disease in a subset of patients with mCRPC. We used comprehensive genomic profiling (CGP) to test the hypothesis that HHV-8 associated mCRPC is a distinct molecular subset of mCRPC featuring altered AR signaling compared to HHV-8 negative mCRPC. Methods: Using a hybrid capture-based FDA-approved CGP assay, a series of 4,918 mCRPC were sequenced to evaluate all classes of genomic alterations (GAs). Tumor mutational burden (TMB) was determined on up to 1.1 Mb of sequenced DNA and microsatellite instability (MSI) was determined on 114 loci. PD-L1 expression was determined by IHC (Dako 22C3). HHV-8 status was determined by CGP-based identification of unique viral reads. Results: Overall, 128 (2.6%) of the mCRPC cases harbored HHV-8 sequences (Table). The patient age distribution was similar in the HHV-8+ and HHV-8− groups. GAs in AR, predominantly amplifications, and RAD21 were slightly reduced in HHV-8+ mCRPC cases (P = .01 for both). The frequencies of GAs in genes associated with mCRPC including PTEN, BRCA2, ATM, CDK12, and the TMPRSS2: ERG fusion were similar in the two groups. HHV-8+ patients were less likely to be of European ancestry (P = 0.001). Putative biomarkers associated with immunotherapy response (MSI status, TMB, and PD-L1 expression) were also similar in the two groups. Conclusions: HHV-8+ mCRPC is a rare sub-type of mCRPC with lower frequency of AR GAs compared to HHV-8− mCRPC cases, suggesting a potential role of HHV-8 in inducing androgen-independent prostate cancer growth. Additional alterations associated with potentially enhanced PARP inhibitor response seen in HHV-8+ mCRPC (e.g., RAD21) suggest that early performance of CGP may inform precision treatment strategies to be tested in clinical trials. [Table: see text]

A Metagenomic Analysis of Mosquito Virome Collected From Different Animal Farms at Yunnan-Myanmar Border of China.

Metagenomic analysis of mosquito-borne and mosquito-specific viruses is useful to understand the viral diversity and for the surveillance of pathogens of medical and veterinary importance. Yunnan province is located at the southwest of China and has rich abundance of mosquitoes. Arbovirus surveillance is not conducted regularly in this province particularly at animal farms, which have public health as well as veterinary importance. Here, we have analyzed 10 pools of mosquitoes belonging to Culex tritaeniorhyncus, Aedes aegypti, Anopheles sinensis, and Armigeres subalbatus species, collected from different animal farms located at Yunnan province of China by using metagenomic next-generation sequencing technique. The generated viral metagenomic data reveal that the viral community matched by the reads was highly diverse and varied in abundance among animal farms, which contained more than 19 viral taxonomic families, specific to vertebrates, invertebrates, fungi, plants, protozoa, and bacteria. Additionally, a large number of viral reads were related to viruses that are non-classified. The viral reads related to animal viruses included parvoviruses, anelloviruses, circoviruses, flaviviruses, rhabdoviruses, and seadornaviruses, which might be taken by mosquitoes from viremic animal hosts during blood feeding. Notably, the presence of viral reads matched with Japanese encephalitis virus, Getah virus, and porcine parvoviruses in mosquitoes collected from different geographic sites suggested a potential circulation of these viruses in their vertebrate hosts. Overall, this study provides a comprehensive knowledge of diverse viral populations present at animal farms of Yunnan province of China, which might be a potential source of diseases for humans and domestic animals.

Comparative metavirome analysis in polytransfused patients.

Due to the high transfusion volume, polytransfused patients with sickle cell disease (SCD) and beta-thalassemia are constantly exposed to parenterally transmitted infections. Currently, we have little information about the virome of such patients and how the virological composition might be influenced by the hemotherapy procedures that these patients receive. The objective of this study was to compare the viral diversity between these two groups with respect to the viral abundance and how it might be affected by the specific conditions of these groups. We sequenced by next-generation sequencing (NGS) and compared the virome of 30 patients with beta-thalassemia major, 45 with SCD, and 16 blood donors from the Blood Center of Ribeirão Preto, Brazil. Predominantly, commensal viruses including Torque teno virus (TTV) genotypes and human pegiviris-1 (HPgV-1) were identified in each group. Strikingly, while HPgV-1 reads were dominant in the SCD group, thalassemic patients showed high TTV abundance, expressed both in viral reads and genotypes. We speculated that the commensal virome of polytransfused patients might be influenced by the transfusion frequency and disease characteristics and that commensal viruses might be used as important genetic biomarkers for these hematological disturbances. Nevertheless, more specific studies are necessary to confirm a relationship between blood virome and transfusion treatment.

A theoretical and generalized approach for the assessment of the sample-specific limit of detection for clinical metagenomics

Metagenomics is a powerful tool to identify novel or unexpected pathogens, since it is generic and relatively unbiased. The limit of detection (LOD) is a critical parameter for the routine application of methods in the clinical diagnostic context. Although attempts for the determination of LODs for metagenomics next-generation sequencing (mNGS) have been made previously, these were only applicable for specific target species in defined samples matrices. Therefore, we developed and validated a generalized probability-based model to assess the sample-specific LOD of mNGS experiments (LODmNGS). Initial rarefaction analyses with datasets of Borna disease virus 1 human encephalitis cases revealed a stochastic behavior of virus read detection. Based on this, we transformed the Bernoulli formula to predict the minimal necessary dataset size to detect one virus read with a probability of 99%. We validated the formula with 30 datasets from diseased individuals, resulting in an accuracy of 99.1% and an average of 4.5 ± 0.4 viral reads found in the calculated minimal dataset size. We demonstrated by modeling the virus genome size, virus-, and total RNA-concentration that the main determinant of mNGS sensitivity is the virus-sample background ratio. The predicted LODmNGS for the respective pathogenic virus in the datasets were congruent with the virus-concentration determined by RT-qPCR. Theoretical assumptions were further confirmed by correlation analysis of mNGS and RT-qPCR data from the samples of the analyzed datasets. This approach should guide standardization of mNGS application, due to the generalized concept of LODmNGS.

Sequencing facility and DNA source associated patterns of virus-mappable reads in whole-genome sequencing data

Numerous viral sequences have been reported in the whole-genome sequencing (WGS) data of human blood. However, it is not clear to what degree the virus-mappable reads represent true viral sequences rather than random-mapping or noise originating from sample preparation, sequencing processes, or other sources. Identification of patterns of virus-mappable reads may generate novel indicators for evaluating the origins of these viral sequences. We characterized paired-end unmapped reads and reads aligned to viral references in human WGS datasets, then compared patterns of the virus-mappable reads among DNA sources and sequencing facilities which produced these datasets. We then examined potential origins of the source- and facility-associated viral reads. The proportions of clean unmapped reads among the seven sequencing facilities were significantly different (P < 2 × 10−16). We identified 260,339 reads that were mappable to a total of 99 viral references in 2535 samples. The majority (86.7%) of these virus-mappable reads (corresponding to 47 viral references), which can be classified into four groups based on their distinct patterns, were strongly associated with sequencing facility or DNA source (adjusted P value <0.01). Possible origins of these reads include artificial sequences in library preparation, recombinant vectors in cell culture, and phages co-contaminated with their host bacteria. The sequencing facility-associated virus-mappable reads and patterns were repeatedly observed in other datasets produced in the same facilities. We have constructed an analytic framework and profiled the unmapped reads mappable to viral references. The results provide a new understanding of sequencing facility- and DNA source-associated batch effects in deep sequencing data and may facilitate improved bioinformatics filtering of reads.

Soybean Viromes in the Republic of Korea Revealed by RT-PCR and Next-Generation Sequencing.

Soybean (Glycine max L.) is one of the most important crop plants in the Republic of Korea. Here, we conducted a soybean virome study. We harvested a total of 172 soybean leaf samples showing disease symptoms from major soybean-growing regions in the Republic of Korea. Individual samples were examined for virus infection by RT-PCR. Moreover, we generated eight libraries representing eight provinces by pooling samples and four libraries from single samples. RNA-seq followed by bioinformatics analyses revealed 10 different RNA viruses infecting soybean. The proportion of viral reads in each transcriptome ranged from 0.2 to 31.7%. Coinfection of different viruses in soybean plants was very common. There was a single dominant virus in each province, and this geographical difference might be related to the soybean seeds that transmit viruses. In this study, 32 viral genome sequences were assembled and successfully used to analyze the phylogenetic relationships and quasispecies nature of the identified RNA viruses. Moreover, RT-PCR with newly developed primers confirmed infection of the identified viruses in each library. Taken together, our soybean virome study provides a comprehensive overview of viruses infecting soybean in eight geographical regions in the Republic of Korea and four single soybean plants in detail.

Characterization of the Blood and Bone Marrow Microbiome of MDS Patients and Associations with Clinical Features

To date, several malignancies of the lymphoid lineage have been linked with microbial infection. Indeed, lymphomas have been associated with the bacteria Helicobacter pylori, and T-cell leukemia and Burkitt's lymphoma have long been known to be commonly caused by viral infections (human T-cell lymphotropic virus 1 and Epstein-Barr virus (EBV), respectively). However, metagenomics research in myeloid malignancies has primarily focused on the roles of gut flora rather than on the tumor site itself (blood and bone marrow). Traditionally, human blood and bone marrow was considered to be normally sterile, and therefore microbiome analysis of these entities would only be performed when deleterious infection was suspected. However, evidence is now accumulating for a normal blood microbiome in healthy individuals. Given the growing number of established relationships in other malignancies between the microbiome at the tumor site and clinical characteristics, we hypothesized roles for the bone marrow microbiome in MDS patients. To this end, we extracted bacterial, fungal, and viral sequence from the bone marrow and blood of 640 MDS patients taken at diagnosis. Briefly, whole-genome sequencing (minimum coverage 100x) was performed on DNA extracted from each patient sample. Reads not aligning to the human genome were aligned to NCBI's database of known microbial reference genomes, and microbial species were assessed for presence and abundance in each sample. As expected, bacterial reads dominated, comprising 99.8% of all microbial reads, while viral reads were 2.73-fold as prevalent as fungal reads. We calculated microbial diversity (Shannon diversity index) on the genus and phylum levels for each patient. As has been reported in solid tumors, higher alpha-diversity is associated with favorable overall survival (age- and sex-adjusted P = 8.6 x 10-3; Figure 1A). Interestingly, individuals with normal karyotype had significantly higher alpha-diversity at the phylum level (Wilcoxon P = 0.002; Figure 1B), while those with deletion of chromosome 5q had significantly lower alpha-diversity (P = 2.2 x 10-5; Figure 1C). Among viral infections, EBV (formally Human gammaherpesvirus 4) was the most common, with 192 patients (30%) harboring EBV DNA sequence. Human cytomegalovirus (HCMV; formally Human gammaherpesvirus 5) and Human betaherpesvirus 6 infections were also observed, in 23% and 2% of patients, respectively. EBV infection was associated with worse overall survival (age- and sex-adjusted P = 0.037; Figure 1D). There was also a substantial dose effect, with higher EBV levels corresponding to poorer outcomes (P = 1.96 x 10-6). Mining the paired-end DNA reads for human-viral chimeras, we found multiple instances of EBV integration into the patient genome, suggesting a possible mechanism for EBV infection impacting patient outcomes. Bacterial composition was also analyzed, showing strong concordance between several pairs of phyla, suggesting synergy between these bacterial communities (Figure 1E). On the other hand, the Proteobacteria and Actinobacteria phyla were strongly discordant with one another (Figure 1E). Multiple studies have shown dominance of Proteobacteria in the circulating bacteriome of healthy individuals. In contrast, the MDS patients in our study showed a continuum between complete dominance of the Proteobacteria phylum and complete dominance of the Actinobacteria phylum (Figure 1F). Intriguingly, lower levels of Actinobacteria was strongly associated with the presence of various chromosomal aberrations, while the opposite was largely true for Proteobacterial abundance. As a result, the ratio of these two phyla abundances was strongly positively associated with normal karyotype and negatively associated with del(5q) (Table 1). Overall, our study provides evidence for roles of the bone marrow microbiome in phenotypes and may even influence outcomes of patients with MDS. Further research is required to determine: a) whether microbial content is cause or consequence of patient characteristics, and b) what the mechanisms are for these microbiome-clinical associations. Disclosures Maciejewski: Alexion, BMS: Speakers Bureau; Novartis, Roche: Consultancy, Honoraria.

Viral metagenomes of Lake Soyang, the largest freshwater lake in South Korea

A high number of viral metagenomes have revealed countless genomes of putative bacteriophages that have not yet been identified due to limitations in bacteriophage cultures. However, most virome studies have been focused on marine or gut environments, thereby leaving the viral community structure of freshwater lakes unclear. Because the lakes located around the globe have independent ecosystems with unique characteristics, viral community structures are also distinctive but comparable. Here, we present data on viral metagenomes that were seasonally collected at a depth of 1 m from Lake Soyang, the largest freshwater reservoir in South Korea. Through shotgun metagenome sequencing using the Illumina MiSeq platform, 3.08 to 5.54-Gbps of reads per virome were obtained. To predict the viral genome sequences within Lake Soyang, contigs were constructed and 648 to 1,004 putative viral contigs were obtained per sample. We expect that both viral metagenome reads and viral contigs would contribute in comparing and understanding of viral communities among different freshwater lakes depending on seasonal changes.