Genomic Next-generation Sequencing Research Articles

Identification and validation of microsatellite markers in strawberry tree(Arbutusunedo L.)

Strawberry tree (Arbutus unedo L.), an evergreen shrub/small tree of the family Ericaceae, is a main constituent of the Mediterranean basin flora; although it is also found in southwestern France, Macaronesia, and Ireland. The small fruits are edible but mostly used for preparation of preserves and jams, and for liquors such as the Portuguese traditional "aguardente de medronho". Traditionally cultivated by small farmers, often in consociation with Quercus sp., strawberry tree is presently emerging as a new important fruit crop cultivated in large orchards by modern export-oriented enterprises. This change of paradigm requires a growing role of plant breeding, upstream of the production process. Genomic tools for this species are mostly limited to the chloroplast genome sequence and to genomic data described in this work. In order to identify strawberry tree microsatellite (SSR) loci we performed partial genome next-generation sequencing using the Ion Torrent technology. The sequenced ~24.6M nucleotides resulted in the identification of 1185 microsatellite markers mostly constituted by dinucleotide motifs. The relative amount of microsatellite dinucleotide motifs (AG/ CT - 71.7%, AC/GT - 20.5%, AT/AT - 2.9%, and CG/CG - 0.3%) is similar to the one observed in other Ericaceae species. Among a tested sample of 40 SSR primer pairs, 20 amplified well-defined PCR products, 12 (30%) were validated as polymorphic. Used in our collaborative project for molecular identification of selected and improved clones, the identified SSR loci constitute a strong tool for a large panoply of applied and fundamental studies of this emerging fruit crop.

A5 Near full-length HIV-1 genome sequencing in newly diagnosed individuals in Sweden

The Swedish HIV-1 epidemic is characterized by a high diversity in HIV subtypes and recombinants as a result of migration. To study the time from infection through viral diversification, transmission patterns, and drug resistance in minor quasispecies, a robust protocol for pan-genotypic near full-length HIV-1 genome (HIV-NFLG) next-generation sequencing (NGS) is key. Our group has established two protocols for HIV-NFLG on the Illumina platform that we aim to compare and, if necessary, modify to find a method with optimized coverage, depth, and subtype inclusivity. Zanini et al. (https://doi.org/10.7554/eLife.11282.001) have developed a method with one-step RT-PCR with six overlapping primer sets, followed by NGS and quality filtering and assembly with in-house methods. Aralaguppe et al. (https://doi.org/10.1016/j.jviromet.2016.07.010) have designed amplification in two fragments, followed by multiplexed NGS and quality control and assembly with Iterative Virus Assembler and VICUNA. Both methods have high coverage per nucleotide and low error rates in amplification and sequencing and can reliably identify SNPs at 1 per cent of the viral population with linkage within the quasispecies. Subtype inclusivity remains a challenge even though both methods show success in amplifying and sequencing subtypes B, C, and the common recombinants 01_AE and 02_AG. Therefore, we aim to evaluate and optimize our NFLG NGS methods on a panel of patient samples that more completely reflects HIV-1 diversity in Sweden. Patient samples from fifty treatment-naïve viremic individuals representing the genotypic HIV-1 panorama in Sweden, including CRFs, are being amplified and sequenced by both protocols. Coverage of the genome, error rate, and possible depth of quasispecies analysis is being evaluated. We will compare number of reads, coverage across the HIV genome, and representation of minor single nucleotide variants as well as subtype inclusivity and impact of plasma RNA levels. To do this we will use an in-house bioinformatic pipeline. The NFLG sequences will also be analyzed with phylogenetic tools for determination of subtypes including CRFs and URFs.

Sperm chromosomal abnormalities and their contribution to human embryo aneuploidy.

In this work we reviewed 18years of experience using fluorescence in situ hybridization (FISH) for sperm aneuploidy testing. We evaluated parameters associated with increased numerical sperm chromosome abnormalities and determined the male contribution to embryo aneploidies in terms of reproductive outcome by increased sperm aneuploidy. This retrospective study analyzed data from 2008 sperm samples of infertile males undergoing FISH analysis because of clinical history of repetitive implantation failure, recurrent miscarriage, impaired sperm parameters, or mixed causes. Sperm concentration was the only sperm parameter associated with FISH results-we observed a gradual increase of abnormal sperm FISH results in males with decreasing sperm concentration. However, a great proportion of normozoospermic males also showed increased sperm aneuploidies, suggesting that sperm parameters alone do not enable identification of a substantial proportion of infertile males at risk of sperm aneuploidies. Regarding reproductive outcomes, couples with normal sperm FISH results for the male had similar outcomes regardless of conventional in vitro fertilization (IVF), intracytoplasmic sperm injection (ICSI), or preimplantation genetic testing for aneuploidies (PGT-A). However, couples with abnormal sperm FISH results for the male showed better clinical outcomes after PGT-A, suggesting a potential contribution of sperm to embryo aneuploidy. Moreover, PGT-A cycles showed better clinical outcomes when 24 chromosomes were analyzed by array comparative genome hybridization (aCGH) or next-generation sequencing (NGS) instead of only nine chromosomes analyzed by FISH. In conclusion, sperm FISH analysis offers clinical prognostic value to evaluate reproductive possibilities in infertile couples. Therefore, couples with abnormal sperm FISH results should be offered genetic counseling and presented with clinical options such as PGT-A.

Analysis of Mitochondrial Genome from Labrador (Canis lupus familiaris) with Mammary Gland Tumour Reveals Novel Mutations and Polymorphisms

Abstract The aim of the study was to find associations between the process of neoplastic transformation and mtDNA mutations/polymorphisms, i.e. factors with potential prognostic significance, and to determine their impact on the biochemical properties, as well as structural, and functional properties of proteins. Blood and neoplastic tissue samples were collected from a 9-year-old Labrador dog with a diagnosed malignant mammary tumour. Next-generation genome sequencing (NGS) of the entire mitochondrial genome was performed using Illumina technology, and bioinformatics analyses were carried out. This is the first report demonstrating the application of NGS in the analysis of the canine mtDNA genome in neoplastic disease. The proposed strategy is innovative and promising. For the first time in the literature, the sequence of 29 genes was analysed to determine their association with the prevalence of tumour. In total, 32 polymorphic loci and 15 mutations were identified. For the first time, as many as 24 polymorphisms and all the mutations have been described to be associated with the neoplastic process in dogs. Most polymorphisms/mutations were found in the D-loop (31% of the polymorphisms and 93% of the mutations) and the COX1 gene sequence (16% of the polymorphisms). Blood or cancer heteroplasmy was noted in 93% of the mutations. Four of the 18 polymorphisms detected in the protein-coding genes were non-synonymous polymorphisms that have not been described in the literature so far (m.T7593C in COX2, m.G8807A in COX3, m.A9911G in ND4L, and m.T13299A in ND5) but resulted in changes in amino acids in proteins. These mutations and polymorphisms can affect mitochondrial functions and may be a result of cell adaptation to the changes in the environment occurring during carcinogenesis. The replacement of “wild type” mtDNA by a mutated molecule may be an important phenomenon accompanying carcinogenesis.

Abstract 727: Comprehensive genomic profiling of >1000 plasma and tumor tissue samples from metastatic castration-resistant prostate cancer (mCRPC) patients gives insight into targeted treatment strategies

Abstract Background: The phase 2 TRITON2 (NCT02952534) study is evaluating the poly(ADP-ribose) polymerase inhibitor rucaparib in mCRPC patients with a deleterious germline or somatic mutation in BRCA1, BRCA2, ATM, or 1 of 12 other DNA damage repair (DDR) genes who have progressed on prior androgen receptor-directed therapy and 1 taxane-based therapy. Here we present results from central genomic screening of plasma and tissue samples for TRITON2. Methods: Plasma samples were profiled for genomic alterations (GAs) in 70 genes and tumor tissue FFPE samples for GAs in 395 genes by Foundation Medicine, Inc., using next-generation sequencing (NGS) assays. Deleterious alterations included frameshift, nonsense, and deleterious missense mutations, protein-truncating rearrangements, and (for tissue samples) homozygous loss. Tissue samples included both archival and more recent specimens, whereas plasma samples were collected at the time of disease progression on prior therapy. Results: As of July 2, 2018, 359 plasma samples from patients with mCRPC were screened for deleterious GAs in a DDR gene. Of the plasma samples, 96% (343/359) were successfully sequenced for a comprehensive genomic profile. The most commonly altered genes were AR (49%) and TP53 (48%). Deleterious GAs were detected in BRCA1 (2%), BRCA2 (10%), ATM (15%), CDK12 (6%), or other DDR genes (6%). GAs in the MAPK pathway were observed in 5% of plasma samples. Additionally, 738 tissue samples from primary prostate cancer tumors (78%) or metastases (22%) were profiled. Only 66% of tissue samples (487/738) were sequenced successfully, highlighting the challenges associated with NGS of predominantly archival prostate tissues (median sample age, 2.5 years [range, 4 days to 21 years]). The most commonly altered genes were TP53 (38%), PTEN (33%), ERG-TEMPRSS2 fusions (28%), AR (15%), and MYC (9%). Deleterious GAs were observed in BRCA1(2%), BRCA2 (9%), ATM (7%), CDK12 (7%), or other DDR genes (5%), as well as in the PI3K/AKT (41%), Wnt (11%), MAPK (5%), and mismatch repair (3%) pathways. Further, GAs in the DDR, PI3K/AKT, and Wnt pathways were observed at a higher rate in metastatic (42%, 40%, and 15%, respectively) than in primary prostate tissues (32%, 34%, and 12%, respectively). Updated and expanded genomic analyses and plasma-tissue concordance analyses will be presented. Conclusions: Genomic NGS profiling of plasma and FFPE tumor tissue samples successfully identified patients with GAs in a DDR gene for the evaluation of rucaparib in mCRPC. The plasma assay is convenient for patients and has a low NGS failure rate, whereas the tumor tissue assay has a higher failure rate but can detect more alteration types. Taken together, these data highlight the strengths and limitations of tissue and plasma testing to identify GAs for targeted therapies in mCRPC. Citation Format: Foad Green, Jeremy D. Shapiro, Ray McDermott, Josep Maria Piulats, Alison Reid, Peter Ostler, Jingsong Zhang, David Campbell, Dominique Spaeth, Ivor Percent, Arif Hussain, Andrew D. Simmons, Tony Golsorkhi, Simon P. Watkins, Andrea Loehr, Simon Chowdhury, Wassim Abida. Comprehensive genomic profiling of >1000 plasma and tumor tissue samples from metastatic castration-resistant prostate cancer (mCRPC) patients gives insight into targeted treatment strategies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 727.

HIV-1 DNA sequence diversity and evolution during acute subtype C infection

Little is known about the genotypic make-up of HIV-1 DNA genomes during the earliest stages of HIV-1 infection. Here, we use near-full-length, single genome next-generation sequencing to longitudinally genotype and quantify subtype C HIV-1 DNA in four women identified during acute HIV-1 infection in Durban, South Africa, through twice-weekly screening of high-risk participants. In contrast to chronically HIV-1-infected patients, we found that at the earliest phases of infection in these four participants, the majority of viral DNA genomes are intact, lack APOBEC-3G/F-associated hypermutations, have limited genome truncations, and over one year show little indication of cytotoxic T cell-driven immune selections. Viral sequence divergence during acute infection is predominantly fueled by single-base substitutions and is limited by treatment initiation during the earliest stages of disease. Our observations provide rare longitudinal insights of HIV-1 DNA sequence profiles during the first year of infection to inform future HIV cure research.

The Pros and Cons of Incorporating Transcriptomics in the Age of Precision Oncology.

The treatment of cancer continues to evolve toward personalized therapies based on individual patient and tumor characteristics. Our successes and failures in adopting a precision-oncology approach have demonstrated the utmost importance in identifying the proper predictive biomarkers of response. Until recently, most biomarkers were identified using immunohistochemistry for protein expression or single-gene analysis to identify targetable alterations. With the rapid propagation of next-generation sequencing to evaluate tumor tissue and "liquid biopsies," identification of genomic biomarkers is now standard, particularly in non-small cell lung cancer, for which there is now an extensive catalog of biomarker-directed therapies with more anticipated to come. Despite these great strides, it has also become apparent that using genomic biomarkers alone will be insufficient, as it has been consistently shown that at least one-half of patients who undergo tumor genomic profiling have no actionable alteration. This is perhaps to be expected given the remarkable breadth of nongenetic factors that contribute to tumor initiation and progression. Some have proposed that the next logical step is to use transcriptome profiling to define new biomarkers of response to targeted agents. Recently, results from the WINTHER trial were published, specifically investigating the use of transcriptomics to improve match rates over genomic next-generation sequencing alone. In this review, we discuss the complexities of precision-oncology efforts and appraise the available evidence supporting the incorporation of transcriptomic data into the precision-oncology framework in the historical context of the development of biomarkers for directing cancer therapy.

Low diversity of Angiostrongylus cantonensis complete mitochondrial DNA sequences from Australia, Hawaii, French Polynesia and the Canary Islands revealed using whole genome next-generation sequencing

BackgroundRats (Rattus spp.) invaded most of the world as stowaways including some that carried the rat lungworm, Angiostrongylus cantonensis, the cause of eosinophilic meningoencephalitis in humans and other warm-blooded animals. A high genetic diversity of A. cantonensis based on short mitochondrial DNA regions is reported from Southeast Asia. However, the identity of invasive A. cantonensis is known for only a minority of countries. The affordability of next-generation sequencing for characterisation of A. cantonensis genomes should enable new insights into rat lung worm invasion and parasite identification in experimental studies.MethodsGenomic DNA from morphologically verified A. cantonensis (two laboratory-maintained strains and two field isolates) was sequenced using low coverage whole genome sequencing. The complete mitochondrial genome was assembled and compared to published A. cantonensis and Angiostrongylus malaysiensis sequences. To determine if the commonly sequenced partial cox1 can unequivocally identify A. cantonensis genetic lineages, the diversity of cox1 was re-evaluated in the context of the publicly available cox1 sequences and the entire mitochondrial genomes. Published experimental studies available in Web of Science were systematically reviewed to reveal published identities of A. cantonensis used in experimental studies.ResultsNew A. cantonensis mitochondrial genomes from Sydney (Australia), Hawaii (USA), Canary Islands (Spain) and Fatu Hiva (French Polynesia), were assembled from next-generation sequencing data. Comparison of A. cantonensis mitochondrial genomes from outside of Southeast Asia showed low genetic diversity (0.02–1.03%) within a single lineage of A. cantonensis. Both cox1 and cox2 were considered the preferred markers for A. cantonensis haplotype identification. Systematic review revealed that unequivocal A. cantonensis identification of strains used in experimental studies is hindered by absence of their genetic and geographical identity.ConclusionsLow coverage whole genome sequencing provides data enabling standardised identification of A. cantonensis laboratory strains and field isolates. The phenotype of invasive A. cantonensis, such as the capacity to establish in new territories, has a strong genetic component, as the A. cantonensis found outside of the original endemic area are genetically uniform. It is imperative that the genotype of A. cantonensis strains maintained in laboratories and used in experimental studies is unequivocally characterised.

Association between chromosomal translocation imbalances and aneuploidies in PGT-SR in reciprocal translocation carriers

Introduction Reciprocal translocation carriers are known to be at high risk of conceiving genetically unbalanced embryos, which may result in miscarriage or an affected neonate. Preimplantation genetic testing for chromosomal structural rearrangements (PGT-SR), a method for embryonic chromosomal analysis, can detect not only translocation chromosome imbalance but also aneuploidy via comprehensive chromosomal analysis with whole-genome amplification (WGA), thereby potentially decreasing miscarriage rate. In this study, we aimed to investigate the relationship between chromosomal abnormalities derived from translocation chromosomes and aneuploidies and the frequency of chromosomal abnormalities among stimulation cycles in reciprocal translocation carriers receiving multiple PGT-SR cycles. Material and Method Between 2013 and 2017, we assessed 20 cycles of 8 couples (reciprocal translocation carriers in 5 females and 3 males; mean women's age, 35.1 years) who received 2 or more PGT-SR cycles at our clinic.Ovarian stimulation was primarily controlled, and when chromosomally normal embryos could not be obtained, a different protocol for ovarian stimulation was adapted. All oocytes were fertilized via intracytoplasmic sperm injection (ICSI). All embryos were cultured to the blastocyst stage. Approximately 5-10 trophectoderm (TE) cells were subjected to a biopsy using a laser system. Chromosomal analysis was performed using the microarray-based comparative genomic hybridization method or next-generation sequencing with whole-genome amplification. Result In all 20 cycles, the number of oocytes retrieved was 432 and the number of blastocysts subjected to biopsy was 90. Among 88 analyzable blastocysts, 19 had normal chromosomes (21.6%) and 69 had abnormal chromosomes (78.4%). Fifty-six embryos (81.2%) contained chromosomal imbalances derived from reciprocal translocation chromosomes and 37 embryos (53.6%) had aneuploidy, 24 embryos (34.8%) had both types of abnormalities. The incidence of chromosomal translocation-derived chromosomal imbalance was 60.0% (12/20) for male carriers and 64.7% (44/68) for female carriers, with no significant difference.The incidence of chromosomally normal embryos per cycle was 0–67%. Normal embryos were not obtained in 9 cycles (45%); however, they were obtained at different stimulation cycles. In 9 PGT-SR cycles with three or more embryos of female carriers subjected to biopsy, there was no correlation between the incidence of the abnormality due to chromosome translocation and aneuploidy. Conclusion There is no association between the incidence of translocation chromosome imbalance and aneuploidy. However, approximately 80% of embryos with chromosomal translocation-derived chromosomal imbalance include aneuploidy. The incidence of chromosomal aberrations in embryos has a large difference among the PGT-SR cycles even in the same carrier. In translocation carriers without normal embryos, performing different ovarian stimulation methods enhances the possibility of obtaining normal embryos.

Comparison of different platforms for preimplantation genetic testing in IVF cycle

Introduction Preimplantation genetic screening (PGS) is performed during in vitro fertilization (IVF) cycles and allows to reveal chromosomal aberrations before pregnancy. PGS is usually performed using either array comparative genomic hybridization (aCGH) or next-generation sequencing (NGS) technologies. The aim of this work is to compare data from three different platforms, two of which are based on the aCGH method and the third one is based on the NGS method. Materials & Methods 13 human blastocysts from IVF cycles were analyzed in this work. Biopsy material DNA was amplified using whole genomic amplification kit «Picoplex WGA kit» according to the instruction of the manufacturer company. Next step was performed with two different technologies: aCGH and NGS. For comparative genomic hybridization we used two different platforms. The first was GenetiSure kit (Agilent) and data analysis was performed using Agilent CytoGenomics software, the second one was CytoSure kit (OGT) and data analysis was performed using CytoSure Internet Software. Next-generation sequencing was done using VeriSeq PGS Kit (Illumina) on MiSeq System (Illumina) and sequencing data analysis was performed using BlueFuse Software (Illumina) Expert interpretation of results: For 6 of 13 samples we observed full agreement of the results between three platforms. For 4 of 13 cases the agreement was partial: VeriSeq PGS (Illumina) and CytoSure (OGT) yeilded similar results while results of the GenetiSure (Agilent) differed. For 3 of 13 cases results of all three platforms were distinct. Conclusions Our data do not allow us to draw conclusions about advantages of one of the methods over the other. Data obtained after automatic data processing contain considerable amount of algorithm specific misinterpretations. Therefore, it is necessary to perform an expert analysis of the automatic reports. Concordance of the data from different approaches significantly increases after expert interpretation but never reaches full agreement. Further studies with larger sample sizes may help to elucidate the reasons of discordant results.

Selecting single euploid blastocysts for transfer with NGS significantly improves IVF treatment outcomes: a randomized study

Introduction Recent advancements in whole genomic amplification (WGA) and next-generation sequencing (NGS) have provided new methods for preimplantation genetic testing for aneuploidy (PGT-A) of human embryos derived from in vitro fertilization (IVF) cycles. The present study aimed to investigate the effects of NGS on clinical and ongoing pregnancy outcomes of single embryo transfer (SET) for IVF patients with a good prognosis. Material and Methods A total of 247 IVF patients at mean age 31.4 ± 3.2 years who met the inclusion criteria (under 35 years old with normal uterine lining and normal karyotype) were randomized into two groups: 1) Group A (MA+NGS): patients (n=124) had blastocysts evaluated with morphological assessment (MA) and analyzed with NGS (Yikon Genomics, China; Illumina, USA) and 2) Group B (MA): patients (n=123) had blastocysts evaluated with morphological assessment (MA) only. One blastocyst was selected and transferred to individual patients based on NGS and morphological assessment (Group A) or morphological assessment alone (Group B). Clinical and ongoing pregnancy (≥20 weeks of gestation) rates were compared between the two groups. Results There were no significant differences in female patient's mean age, Day 3 FSH, AMH, E2, antral follicle number between the two groups (p>0.05). NGS identified single euploid blastocysts for transfer and resulted in significantly higher clinical pregnancy rates per transfer in Group A compared to morphological assessment alone in Group B (71.7% vs. 52.1%, respectively, p =0.001). The ongoing pregnancy rates per transfer also significantly increased in Group A compared to Group B (69.2% vs. 46.3%, respectively, p =0.002). There were no twin or multiple pregnancies in both groups. Conclusions While NGS has been recently introduced to IVF treatment, this is the first randomized clinical study showing that there is clinical benefit of NGS for selecting single blastocysts for transfer for good prognosis patients in comparison to morphology assessment alone. With the observed high accuracy of 24-chromosome testing and the improved clinical and ongoing pregnancy outcomes, NGS has demonstrated an efficient, robust high-throughput technology for selecting single blastocysts for transfer for IVF patients with a good prognosis to reduce multiple pregnancies.

RNAseq in addition to next generation sequencing in advanced genitourinary cancers reveals transcriptomic silencing of DNA mutations: Implications for resistance to targeted therapeutics.

583 Background: Next generation sequencing (NGS) for advanced tumors is becoming more routine. However, not all patients respond to precision matched treatments. We hypothesized that one potential reason for treatment failure with targeted therapy could be discrepancies between DNA alterations and RNA expression. Methods: Tumor samples from patients with metastatic kidney, bladder, and prostate cancer were analyzed by whole exome or whole genome NGS and RNA sequencing (CLIA-certified laboratory; NantOmics LLC, Santa Cruz, CA). Only known pathogenic driver alterations were analyzed in the current study. Results: Of 45 patients, 10 had kidney cancer, 18 had bladder cancer; and 17 had prostate cancer. Median age was 66 years (range, 28 - 86). The most commonly altered genes were TP53 (35.6% [16/45]), PIK3CA (15.6% [7/45]), FGFR3 (11.1% [5/45]), ALK (8.9% [4/45]), and ATM (8.9% [4/45]). In total, 86 pathogenic DNA alterations were identified; 17 of these (19.8%) were not observed at the RNA level. Among 45 patients, 31.1% (14/45) had ≥1 DNA alteration that was not expressed at the RNA level. Discordance between DNA and RNA was seen in 40% of patients with kidney cancer (4/10), 28% of patients with bladder cancer (5/18), and 29% with prostate cancer (5/17). Examples of genes that had pathogenic DNA alterations not seen at the RNA level included ALK (four discordant cases), KDR (three discordant cases) and GNAS (one discordant case). On the other hand, alterations involving certain genes showed 100% concordance between DNA and RNA: TP53 [N = 16], PIK3CA [N = 7], and FGFR3 [N = 5]). Conclusions: A significant number of patients with genitourinary tumors had DNA alterations that are silenced at the RNA level (19.8%). Transcriptomic silencing merits additional investigation as a mechanism that could mediate resistance to therapeutics targeted at cognate alterations.

HIV controllers suppress viral replication and evolution and prevent disease progression following intersubtype HIV-1 superinfection.

The aim of this study was to investigate the impact of intersubtype HIV-1 superinfection on viremia, reservoir reseeding, viral evolution and disease progression in HIV controllers (HIC). A longitudinal analysis of two Brazilian HIC individuals (EEC09 and VC32) previously identified as dually infected with subtypes B and F1 viruses. Changes in plasma viremia, total HIV-1 DNA levels, CD4+ T-cell counts and HIV-1 quasispecies composition were measured over time. HIV-1 env diversity in peripheral blood mononuclear cell (PBMC) and plasma samples was accessed by single genome amplification and next-generation sequencing approaches, respectively. Viral evolution was evaluated by estimating nucleotide diversity and divergence. Individual EEC09 was probably initially infected with a CCR5-tropic subtype B strain and sequentially superinfected with a CXCR4-tropic subtype B strain and with a subtype F1 variant. Individual VC32 was infected with a subtype B strain and superinfected with a subtype F1 variant. The intersubtype superinfection events lead to a moderate increase in viremia and extensive turnover of viral population in plasma but exhibited divergent impact on the size and composition of cell-associated HIV DNA population. Both individuals maintained virologic control (<2000 copies/ml) and presented no evidence of viral evolution or immunologic progression for at least 2 years after the intersubtype superinfection event. These data revealed that some HIC are able to repeatedly limit replication and evolution of superinfecting viral strains of a different subtype with no signs of disease progression.

New voyages to explore the natural product galaxy.

Natural products are a large family of diverse and complex chemical molecules that have roles in both primary and secondary metabolism, and over 210,000 natural products have been described. Secondary metabolite natural products are of high commercial and societal value with therapeutic uses as antibiotics, antifungals, antitumor and antiparasitic products and in agriculture as products for crop protection and animal health. There is a resurgence of activity in exploring natural products for a wide range of applications, due to not only increasing antibiotic resistance, but the advent of next-generation genome sequencing and new technologies to interrogate and investigate natural product biosynthesis. Genome mining has revealed a previously undiscovered richness of biosynthetic potential in novel biosynthetic gene clusters for natural products. Complementing these computational processes are new experimental platforms that are being developed and deployed to access new natural products.

Comparative Genomics, Siderophore Production, and Iron Scavenging Potential of Root Zone Soil Bacteria Isolated from 'Concord' Grape Vineyards.

Iron (Fe) deficiency in crop production is a worldwide problem which often results in chlorosis in grapevines, particularly in calcareous soils. Siderophores secreted by microorganisms and Strategy II plants can chelate Fe and other metals in soil solution, and siderophore-Fe complexes can then be utilized by plants and microbes. Plants may also shift rhizosphere conditions to favor siderophore-producing microbes, which can increase plant available Fe. Between-row cover crops (barley, rye, wheat, wheat/vetch) were planted as living mulch to address grapevine chlorosis by enhancing soil health in two vineyards in central Washington. The objectives of the current study were to (1) enrich for siderophore-producing organisms fromwithin the indigenous rooting zone community of 'Concord' grapevines, and (2) perform comparative genomics on putative siderophore producing organisms to assess potentially important Fe acquisition-related functional domains and protein families. A high-throughput, chrome azurol S (CAS)-based enrichment assay was used to select siderophore-producing microbes from 'Concord' grapevine root zone soil. Next-generation whole genome sequencing allowed the assembly and annotation of ten full genomes. Phylogenetic analysis revealed two distinct clades among the genomes using the 40 nearest neighbors available in the public database, all of which were of the Pseudomonas genus. Significant differences in functional domain abundances were observed between the clades including iron acquisition and metabolism of amino acids, carbon, nitrogen, phosphate, and sulfur. Diverse mechanisms of Fe uptake and siderophore production/uptake were identified in the protein families of the genomes. The sequenced organisms are likely pseudomonads which are well-suited for iron scavenging, suggesting a potential role in Fe turnover in vineyard systems.

Reduction of HIV-1 Reservoir Size and Diversity After 1 Year of cART Among Brazilian Individuals Starting Treatment During Early Stages of Acute Infection.

The aim of early combined antiretroviral therapy (cART) of HIV is to limit the seeding of the viral reservoir during the initial phase of infection and, consequently, decrease intrahost viral diversity. Here, we assessed the effect of early cART on size and complexity of the proviral reservoir. Peripheral blood mononuclear cell (PBMC) and plasma samples were obtained from ten HIV-infected Brazilian individuals diagnosed at the acute phase of infection, before (PREART) and 12 months (M12ART) after suppressive cART. HIV proviral reservoir size was determined by quantitative real-time PCR; intrahost viral diversity of the env C2-V3 region was assessed by single genome amplification or next-generation sequencing in PBMC and plasma, respectively. Mean nucleotide diversity (π) and normalized Shannon entropy (HSN) were used to infer the complexity of the viral population. Compared to PREART, M12ART saw an immunological recovery with a gain of ∼200 CD4+ T cells (P = 0.008) and a normalization of the CD4/CD8 ratio [1.0 (IQR: 0.88–1.18), P = 0.016], as well as a significant decrease in HIV-1 RNA (∼4 log, P = 0.004) and DNA (∼1 log, P = 0.002) levels. The median time to achieve viral suppression was 3 months (IQR: 2.8–5.8 months). The high intermixing between sequences from both visits suggests that the HIV-1 DNA reservoir remained remarkably stable under cART. After 1 year of cART, there was a minor reduction in proviral π (PreART = 0.20 vs. M12ART = 0.10; P = 0.156) but a significant decrease in HSN (PreART = 0.41 vs. M12ART = 0.25; P = 0.019). We found no correlation between π or HSN at PreART and the rate of HIV DNA decay, T CD4+ counts, or CD4/CD8 ratio at M12ART. Based on a small cohort of Brazilian infected individuals under early cART and analyses of the env region, 1 year of follow-up suggested a reservoir size reduction, allowed a significant decrease of HIV-1 complexity, and achieved immunological restoration regardless of the initial HIV-1 plasma viral load, CD4+ T cell counts, or HIV-1 subtype. However, further studies in the Brazilian setting aiming a longer follow-up and larger cohort are required in this field.

Overview of Genomic Tools for Circular Visualization in the Next-generation Genomic Sequencing Era.

After human genome sequencing and rapid changes in genome sequencing methods, we have entered into the era of rapidly accumulating genome-sequencing data. This has derived the development of several types of methods for representing results of genome sequencing data. Circular genome visual-ization tools are also critical in this area as they provide rapid interpretation and simple visualization of overall data. In the last 15 years, we have seen rapid changes in circular visualization tools after the de-velopment of the circos tool with 1-2 tools published per year. Herein we have summarized and revisited all these tools until the third quarter of 2018.

Characterization of the complete chloroplast genome of an endangered aquatic macrophyte, Ottelia cordata (Hydrocharitaceae)

Ottelia cordata (Wallich) Dandy (Hydrocharitaceae) is an endangered aquatic macrophyte. The complete chloroplast genome sequence of O. cordata was generated by de novo assembly using whole genome next-generation sequencing. The complete chloroplast genome was 157,879 bp in length and constructed out of four parts – a large single-copy (LSC) region of 87,672 bp, a small single-copy (SSC) region of 20,265 bp, and two inverted repeat (IRa and IRb) regions of 24,971 bp each. The genome annotation predicted a total of 117 genes, including 72 protein-coding genes, 37 tRNA genes, and 8 rRNA genes. Phylogenetic analysis of 20 selected chloroplast genomes revealed that sampled species of Hydrocharitaceae formed a monophyletic clade, in which O. cordata and O. acuminata var. songmingensis formed a sister clade to Elodea canadensis.

The complete chloroplast genome of Isoetes yunguiensis (Isoetaceae), a rare and endangered fern endemic to China

Isoetes yunguiensis is a rare and endangered fern endemic to Yunnan–Guizhou Plateau, China. The complete chloroplast genome sequence of I. yunguiensis was generated by de novo assembly using whole genome next-generation sequencing. The cp genome of I. yunguiensis was a circular structure and 145,355 bp in length, comprising a pair of inverted repeat (IR) regions of 26,130 bp, a large single copy (LSC) of 91,923 bp, and a small single copy (SSC) of 27,302 bp. The genome encoded 124 unique genes consisting of 82 protein-coding genes, 32 transfer RNA, four ribosomal RNA genes, and six pseudogenes. A maximum-likelihood phylogenetic analysis revealed that I. yunguiensis was closely related to I. butleri, I. engelmannii, I. flaccida, I. melanospora, and I. valida, with strong support values.

The complete chloroplast genome of Semenovia gyirongensis (Tribe Tordylieae, Apiaceae)

Semenovia gyirongensis is an endemic species in China. The complete chloroplast (cp) genome sequence of S. gyirongensis was generated by de novo assembly using whole genome next-generation sequencing. The cp genome was a circular structure and 147,246 bp in length, composed of four distinct regions such as large single-copy (LSC) region of 92,833 bp, small single-copy (SSC) region 17,545 bp, and a pair of inverted repeat regions of 36,868 bp. The cp genome contained 129 genes, including 85 protein-coding genes, 36 tRNA genes, and 8 rRNA genes. The GC content was 37.6%. Phylogenetic analysis of 21 representative plastomes indicated that the S. gyirongensis was close to Pastinaca pimpinellifolia, Cnidium officinale, and Ligusticum sinense in the family Apiaceae.