Genomic Next-generation Sequencing Research Articles

Advancing clinical cohort selection with genomics analysis on a distributed platform.

The affordability of next-generation genomic sequencing and the improvement of medical data management have contributed largely to the evolution of biological analysis from both a clinical and research perspective. Precision medicine is a response to these advancements that places individuals into better-defined subsets based on shared clinical and genetic features. The identification of personalized diagnosis and treatment options is dependent on the ability to draw insights from large-scale, multi-modal analysis of biomedical datasets. Driven by a real use case, we premise that platforms that support precision medicine analysis should maintain data in their optimal data stores, should support distributed storage and query mechanisms, and should scale as more samples are added to the system. We extended a genomics-based columnar data store, GenomicsDB, for ease of use within a distributed analytics platform for clinical and genomic data integration, known as the ODA framework. The framework supports interaction from an i2b2 plugin as well as a notebook environment. We show that the ODA framework exhibits worst-case linear scaling for array size (storage), import time (data construction), and query time for an increasing number of samples. We go on to show worst-case linear time for both import of clinical data and aggregate query execution time within a distributed environment. This work highlights the integration of a distributed genomic database with a distributed compute environment to support scalable and efficient precision medicine queries from a HIPAA-compliant, cohort system in a real-world setting. The ODA framework is currently deployed in production to support precision medicine exploration and analysis from clinicians and researchers at UCLA David Geffen School of Medicine.

Assessment of Genetic Aspects of Non-alcoholic Fatty Liver and Premature Cardiovascular Events.

Recent evidence has demonstrated a strong interplay and multifaceted relationship between non-alcoholic fatty liver disease (NAFLD) and cardiovascular disease (CVD). CVD is the major cause of death in patients with NAFLD. NAFLD also has strong associations with diabetes and metabolic syndrome. In this comprehensive review, we aimed to overview the primary environmental and genetic risk factors of NAFLD, and CVD and also focus on the genetic aspects of these two disorders. NAFLD and CVD are both heterogeneous diseases with common genetic and molecular pathways. We have searched for the latest published articles regarding this matter and tried to provide an overview of recent insights into the genetic aspects of NAFLD and CVD. The common genetic and molecular pathways involved in NAFLD and CVD are insulin resistance (IR), subclinical inflammation, oxidative stress, and atherogenic dyslipidemia. According to an investigation, the exact associations between genomic characteristics of NAFLD and CVD and casual relationships are not fully determined. Different gene polymorphisms have been identified as the genetic components of the NAFLDCVD association. Some of the most documented ones of these gene polymorphisms are patatin-like phospholipase domain-containing protein 3 (PNPLA3), transmembrane 6 superfamily member 2 (TM6SF2), hydroxysteroid 17-beta dehydrogenase 13 (HSD17B13), adiponectin-encoding gene (ADIPOQ), apolipoprotein C3 (APOC3), peroxisome proliferator-activated receptors (PPAR), leptin receptor (LEPR), sterol regulatory element-binding proteins (SREBP), tumor necrosis factor-alpha (TNF-α), microsomal triglyceride transfer protein (MTTP), manganese superoxide dismutase (MnSOD), membrane-bound O-acyltransferase domain-containing 7 (MBOAT7), and mutation in DYRK1B that substitutes cysteine for arginine at position 102 in kinase-like domain. Further cohort studies with a significant sample size using advanced genomic assessments and next-generation sequencing techniques are needed to shed more light on genetic associations between NAFLD and CVD.

High-Quality Genome Assembly and Annotation of the Big-Eye Mandarin Fish (Siniperca knerii).

The big-eye mandarin fish (Siniperca knerii) is an endemic species of southern China. It belongs to the family Sinipercidae, which is closely related to the well-known North American sunfish family Centrarchidae. Determining the genome sequence of S. knerii would provide a foundation for better examining its genetic diversity and population history. A novel sequenced genome of the Sinipercidae also would help in comparative study of the Centrarchidae using Siniperca as a reference. Here, we determined the genome sequence of S. knerii using 10x Genomics technology and next-generation sequencing. Paired-end sequencing on a half lane of HiSeq X platform generated 56 Gbp of raw data. Read assembly using Supernova assembler resulted in two haplotype genomes with 732.1 Mb in size and an average GC content of 40.4%, which is consistent with genome size previously reported or estimated using k-mer counting. A total of 7,989 scaffolds with an N50 score of 12.64 Mb were obtained. The longest scaffold was 30.54 Mb. Evaluation of the genome completeness using BUSCO confirmed that 96.5% genes of the Actinopterygii Benchmarking Universal Single-Copy Orthologs were found in the assembled genome of S. knerii. Gene prediction using Maker annotation kit resulted in 28,440 genes, of which 25,899 genes had at least one hit comparing to the NCBI Nr database, KEGG or InterProScan5. Pairwise sequentially Markovian coalescent (PSMC) analysis of the genome showed that there was a bottleneck event of the population of S. knerii between 70 ka – 20 ka, which was concordant with the Tali glacier period, suggesting a population decline of S. knerii probably due to climate conditions.

ESHRE PGT Consortium good practice recommendations for the detection of structural and numerical chromosomal aberrations.

The field of preimplantation genetic testing (PGT) is evolving fast, and best practice advice is essential for regulation and standardisation of diagnostic testing. The previous ESHRE guidelines on best practice for PGD, published in 2005 and 2011, are considered outdated, and the development of new papers outlining recommendations for good practice in PGT was necessary. The current paper provides recommendations on the technical aspects of PGT for chromosomal structural rearrangements (PGT-SR) and PGT for aneuploidies (PGT-A) and covers recommendations on array-based comparative genomic hybridisation (aCGH) and next-generation sequencing (NGS) for PGT-SR and PGT-A and on fluorescence in situ hybridisation (FISH) and single nucleotide polymorphism (SNP) array for PGT-SR, including laboratory issues, work practice controls, pre-examination validation, preclinical work-up, risk assessment and limitations. Furthermore, some general recommendations on PGT-SR/PGT-A are formulated around training and general risk assessment, and the examination and post-examination process. This paper is one of a series of four papers on good practice recommendations on PGT. The other papers cover the organisation of a PGT centre, embryo biopsy and tubing and the technical aspects of PGT for monogenic/single-gene defects (PGT-M).Together, these papers should assist everyone interested in PGT in developing the best laboratory and clinical practice possible.

Challenges in molecular diagnosis of X-linked Intellectual disability.

Intellectual disability (ID) affects 1-3% of the Western population and is heterogeneous in origin. Mutations in X-linked genes represent 5-10% of ID in males. Fragile X syndrome, due to the silencing of the FMR1 gene, is the most common form of ID, with a prevalence of around 1:5000 males. Females are usually non- or mildly affected carriers, and in a few rare cases, the only gender affected. Array comparative genome hybridization (aCGH) and next-generation sequencing (NGS) have dramatically changed the nature of human genome analysis leading to the identification of new X-linked intellectual disability syndromes and disease-causing genes. Original papers, reviews, guidelines and experiences of the diagnostic laboratories. Family history and clinical examination still are essential to choose the appropriate diagnostic tests, including, a disease-specific genetic test, aCGH or FMR1 molecular analysis. If negative, NGS approaches like well-defined gene panels, whole exome, or even whole genome sequencing, are increasingly being used, improving diagnostics and leading to the identification of novel disease mechanisms. The main challenge in the era of NGS is filtering and interpretation of the data generated by the analysis of a single individual. In X-linked cases, assessing pathogenicity is particularly challenging, even more when the variant is found to be inherited from a healthy carrier mother or when a heterozygous X-linked mutation is found in an impaired female. At present, variant interpretation remains a challenging task, especially in X-linked disorders. We review the main difficulties and propose a comprehensive overview that might aid in variant interpretation. Establishing a genetic diagnosis facilitates counseling and allows better delineation of clinical phenotypes. To improve variant interpretation, there is need to refine in silico predictions with specific criteria for each gene, and to develop cost-effective functional tools, which can be easily transferred to diagnostics.

Progress and prospect of genome wide association study (GWAS) in forest trees

With the rapid development of next-generation genome sequencing technologies, the application of systems genetics based on genome-wide association study (GWAS) strategy is extensively increasing in revealing the genetic variation underlying complex traits and dissecting the genetic regulation mechanism of key genes in forest trees. Firstly, we introduced the core theories, research methods and progress of GWAS in the wood traits and adaptation of forest trees. Next, we proposed a joint use of high-throughput phenome platform, multiple genetic markers, multi-omics data and the models of additive, dominance and epistasis to resolve the problem of “missing heritability” of quantitative traits in forest trees. Finally, we provided an outlook of the GWAS strategy in the molecular breeding of forest trees.

There is no hiding if you Seq: recent breakthroughs in Pseudomonas aeruginosa research revealed by genomic and transcriptomic next-generation sequencing.

The advent of next-generation sequencing technology has revolutionized the field of prokaryotic genetics and genomics by allowing interrogation of entire genomes, transcriptomes and global transcription factor binding profiles. As more studies employing these techniques have been performed, the state of the art regarding prokaryotic gene regulation has developed from the level of individual genes to genetic regulatory networks and systems biology. When applied to bacterial pathogens, particularly valuable insights have been gained into the regulation of virulence-associated genes, their relative importance to bacterial survival in planktonic, biofilm or host infection scenarios, antimicrobial resistance and the molecular details of host-pathogen interactions. This review outlines some of the latest developments and applications of next-generation sequencing techniques that have used primarily Pseudomonas aeruginosa as a model system. We focus particularly on insights into Pseudomonas virulence and infection that have been gained from these approaches and the future directions in which this field could develop.

Embryos derived from donor or patient oocytes are not different for in vitro fertilization outcomes when PGT allows euploid embryo selection: a retrospective study

BackgroundAt our facilities, patients that received embryos using donor oocyte during in vitro fertilization (IVF), usually have had at least one failed attempt to produce at least one euploid embryo with their own oocytes; however, the current debate between using donor over patient oocytes remains inconclusive. We examined the aneuploidy rate and IVF clinical outcomes from embryos derived from either donor or patient oocytes.MethodsRetrospectively, 973 cycles were examined of patients who underwent a standard IVF protocol. Chromosomal content was determined using Pre-implantation Genetic Testing (PGT) by either microarray-comparative genomic hybridization or Next-generation sequencing from either Day 3 (blastocysts) or Day 5 (trophectoderm) embryo biopsies, respectively. Embryo implantation was confirmed by serum β-hCG (> 10 m IU/mL/Day 14), whereas clinical pregnancy by a fetal heartbeat (Week 6.5–8).ResultsEmbryos derived from donor oocytes presented with more monosomies than embryos derived from patient oocytes (41.2% vs. 25.4%, p < 0.05, respectively); however, only Trisomy 7 (0.4% vs. 2.3%, p < 0.05) and Trisomy in X (0.7% vs. 2.3%, p < 0.05) were significantly less present when compared to patient oocyte derived embryos. Interestingly, rates for embryo implantation (46.7% vs. 50.8%, p = 0.35), clinical pregnancy (38.5% vs. 43.1%, p = 0.30), and live birth (30.5% vs. 30.5%, p = 0.99) were similar for embryos derived from donor and patient oocytes. These results did not change when adjusted for the number of embryos implanted.ConclusionHere, we show no significant differences in achieving pregnancy when using donor oocytes. Taking into consideration that aneuploidy rates are > 30% in embryos, independent of the oocyte origin, PGT should be recommended with donor oocytes as well.

Identification of a SNP and development of a PCR-based allele-specific marker of the sporulation-deficient (sporeless) trait of the Tamogitake 108Y2D mutant using next-generation sequencing.

The mass scattering of basidiospores during the cultivation of edible mushrooms causes serious problems, such as allergic reactions in workers. Sporulation-deficient (sporeless) cultivars would be very useful for preventing these issues. We aimed to identify the single-nucleotide polymorphism (SNP) that is responsible for the single dominant sporeless mutation of the Tamogitake 108Y2D mutant using next-generation sequencing (NGS) and TILLING technology and to develop an allele-specific PCR marker for sporeless breeding. By comparing the sequences of the wild-type and its mutant genomes, we identified 685 mutation loci in gene regions and pinpointed one SNP only consistent with sporeless phenotype for 105 segregants, i.e., a C to T located at position 1,950 of the exonic region of a putative fungal transcription factor that generated a stop codon. We developed an allele-specific marker based on the identified SNP, and its high practicality was validated using tests against progenies from several hybrids and wild isolates from different geographical origins. Thus, the allele-specific PCR marker developed here will be useful for marker-assisted selection in the breeding of the sporeless trait of this mushroom. Furthermore, the technical success of SNP identification and marker development based on NGS genome data can help achieve efficient mutation breeding in mushrooms.

Characterization of the complete chloroplast genome sequence of submerged macrophyte Stuckenia pectinata (Potamogetonaceae) and its phylogenetic position

Stuckenia pectinata is widely distributed submerged macrophyte in the world. Herein, the complete chloroplast genome of this species was assembled and characterized using whole genome next-generation sequencing. The complete chloroplast genome showed a circular genome of 156,669 bp size with 36.5% GC content. The genome is of typical structure and contain a pair of inverted repeat (IR) regions with 26,074 bp, separated by one large single-copy (LSC) with 86,285 bp, and one small single-copy (SSC) regions with 18,236 bp. De novo assembly and annotation showed the presence of 131 unique genes with 85 protein-coding genes, 38 tRNA genes, and eight rRNA genes. A maximum-likelihood phylogenomic tree reconstructed based on 15 chloroplast genomes reveals that S. pectinata is most closely related to Zostera marina.

Characterization of the complete chloroplast genome of a medicinal plant, Wikstroemia indica (Thymelaeaceae)

The complete chloroplast genome of Wikstroemia indica, a medicinal plant with a variety of medicinal functions in treatment for arthritis, whooping cough, cancer, and bacillosis, was generated by de novo assembly using whole genome next-generation sequencing. The plastome was a quadripartite circular with 151,731 bp in length; containing a large single-copy (LSC) region of 86,523 bp and a small single-copy (SSC) region of 12,384 bp; separated by 2 inverted repeat (IR) regions of 26,403 bp each. The chloroplast genome contained 124 genes, including 79 protein-coding genes, 37 tRNA genes, and 8 rRNA genes. The GC content in the whole cp genome, LSC region, SSC region, and IR region were 37.4, 34.9, 32.4, and 42.6%, respectively. The phylogenetic tree indicated that W. indica has a close relationship with Stellera chamaejasme with 100% support.

Exome Sequencing in Clinical Hepatology.

The clinical relevance of the Human Genome Project and next-generation sequencing technology was demonstrated for the first time in 2009, when whole-exome sequencing (WES) provided the definitive diagnosis of congenital chloride diarrhea in an infant with presumed renal salt-wasting disease. Over the past decade, numerous studies have shown the utility of WES for clinical diagnosis as well as for discovery of novel genetic disorders through analysis of a single or a handful of informative pedigrees. Hence, advances in improving the speed, accuracy, and computational analysis combined with exponential decrease in the cost of sequencing the human genome is transforming the practice of medicine. The impact of WES has been most noticeable in pediatric disorders and oncology, but its utility in the liver clinic is recently emerging. Here, we assess the current status of WES for clinical diagnosis and acceleration of translation research to enhance care of patients with liver disease.

Utilidad clínica de la secuenciación de nueva generación en el diagnóstico etiológico de la hipoacusia neurosensorial en una Unidad de Hipoacusia Infantil

IntroductionSensorineural hearing loss (SNL) is the most prevalent sensory deficit in our environment. Next generation genomic sequencing (NGS) enables an aetiological diagnosis in a high percentage of patients. Our pilot study shows the results of the systematic application of NGS in a Childhood Hearing Loss Unit, as well as its implications for the clinical management of patients and their families. Material and methodWe included 27 patients diagnosed with SNL between 2014 and 2017, in which an environmental cause was ruled out. The genetic test consisted of a panel of genes analyzed by NGS (OTOgenicsTM panel). This panel has been designed to include genes associated with sensorineural or mixed hearing loss, early onset or late, syndromic and non-syndromic, regardless of their inheritance pattern. ResultsA genetic diagnosis was obtained in 56% (15/27) of the patients (62% in the case of bilateral SNL). Of the patients, 5/27 (19%) presented pathogenic variants in the GJB2 gene and the rest pathogenic and / or probably pathogenic variants in other genes associated with isolated SNL (PR2X2, TECTA and STRC), with syndromic SNL (CHD7, GATA3, COL4A5, MITF and SOX10) or with syndromic and non-syndromic SNL (BSND, ACTG1 and CDH23). DiscussionThe aetiological diagnosis of SNL is a challenge in clinical practice. Our series demonstrates that it is possible to implement genetic diagnosis in the care routine and that this information has prognostic and therapeutic implications.

Profiling of Circulating Free DNA Using Targeted and Genome-wide Sequencing in Patients with SCLC

IntroductionSCLC accounts for approximately 250,000 deaths worldwide each year. Acquisition of adequate tumor biopsy samples is challenging, and liquid biopsies present an alternative option for patient stratification and response monitoring.MethodsWe applied whole genome next-generation sequencing to circulating free DNA (cfDNA) from 39 patients with limited-stage (LS) SCLC and 30 patients with extensive-stage SCLC to establish genome-wide copy number aberrations and also performed targeted mutation analysis of 110 SCLC associated genes. Quantitative metrics were calculated for copy number aberrations, including percent genome amplified (PGA [the percentage of genomic regions amplified]), Z-score (a measure of standard deviation), and Moran’s I (a measure of spatial autocorrelation). In addition CellSearch, an epitope-dependent enrichment platform, was used to enumerate circulating tumor cells (CTCs) from a parallel blood sample.ResultsGenome-wide and targeted cfDNA sequencing data identified tumor-related changes in 94% of patients with LS SCLC and 100% of patients with extensive-stage SCLC. Parallel analysis of CTCs based on at least 1 CTC/7.5 mL of blood increased tumor detection frequencies to 95% for LS SCLC. Both CTC counts and cfDNA readouts correlated with disease stage and overall survival.ConclusionsWe demonstrate that a simple cfDNA genome-wide copy number approach provides an effective means of monitoring patients through treatment and show that targeted cfDNA sequencing identifies potential therapeutic targets in more than 50% of patients. We are now incorporating this approach into additional studies and trials of targeted therapies.

SAAVpedia: Identification, Functional Annotation, and Retrieval of Single Amino Acid Variants for Proteogenomic Interpretation.

Next-generation genome sequencing has enabled the discovery of numerous disease- or drug-response-associated nonsynonymous single nucleotide variants (nsSNVs) that alter the amino acid sequences of a protein. Although several studies have attempted to characterize pathogenic nsSNVs, few have been confirmed as single amino acid variants (SAAVs) at the protein level. Here we developed the SAAVpedia platform to identify, annotate, and retrieve pathogenic SAAV candidates from proteomic and genomic data. The platform consists of four modules: SAAVidentifier, SAAVannotator, SNV/SAAVretriever, and SAAVvisualizer. The SAAVidentifier provides a reference database containing 18 206 090 SAAVs and performs the identification and quality assessment of SAAVs. The SAAVannotator provides functional annotation with biological, clinical, and pharmacological information for the interpretation of condition-specific SAAVs. The SNV/SAAVretriever module enables bidirectional navigation between relevant SAAVs and nsSNVs with diverse genomic and proteomic data. SAAVvisualizer provides various statistical plots based on functional annotations of detected SAAVs. To demonstrate the utility of SAAVpedia, the proteogenomic pipeline with protein-protein interaction network analysis was applied to proteomic data from breast cancer and glioblastoma patients. We identified 1326 and 12 breast-cancer- and glioblastoma-related genes that contained one or more SAAVs, including BRCA2 and FAM49B, respectively. SAAVpedia is a suitable platform for confirming whether a genomic variant is maintained in an amino acid sequence. Furthermore, as a result of the SAAV discovery of these positive controls, the SAAVpedia could play a key role in the protein functional study for the Human Proteome Project (HPP).

Genetic Analysis of Circulating Tumour Cells.

The classification of human cancers has traditionally relied on the tissue of origin, the histologic appearance and anatomical extent of disease, otherwise referred to as grade and stage. However, this system fails to explain the highly variable clinical behaviour seen for any one cancer. Molecular characterization through techniques such as next-generation sequencing (NGS) has led to an appreciation of the extreme genetic heterogeneity that underlies most human cancers. Because of the difficulties associated with fresh tissue biopsy, interest has increased in using circulating tumour material, such as circulating tumour cells (CTCs), as a non-invasive way to access tumour tissue. CTC enumeration has been demonstrated to have prognostic value in metastatic breast, colon and prostate cancers. Recent studies have also shown that CTCs are suitable material for molecular characterization, using techniques such as reverse transcription-polymerase chain reaction (RT-PCR), fluorescence in situ hybridization (FISH), array comparative genomic hybridization (aCGH) and NGS. Furthermore, genetic analysis of CTCs may be more suitable to study tumour heterogeneity and clonal evolution than fresh tissue biopsy. Whether blood-based biopsy techniques will be accepted as a replacement to fresh tissue biopsies remains to be seen, but there is reason for optimism. While significant barriers to this acceptance exist, blood-based biopsy techniques appear to be reliable and representative alternatives to fresh tissue biopsy.

Pharmacogenetics and the treatment of epilepsy: what do we know?

Seizure control with antiepileptic drugs (AEDs) as well as susceptibility to adverse drug reactions varies among individuals with epilepsy. This interindividual variability is partly determined by genetic factors. However, genetic testing to predict the efficacy and toxicity of AEDs is limited and genetic variability is, as yet, largely unexplainable. Accordingly, genetic testing can only be advised in a very limited number of cases in clinical routine. Currently, by applying different methodologies, many trials have been undertaken to evaluate cost benefits of preventive pharmacogenetic analysis for patients. There is significant progress in sequencing technologies, and focus is on next-generation sequencing-based methods, like exome and genome sequencing. In this review, an overview of the current scientific knowledge considering the pharmacogenetics of AEDs is given.

Cytogenetics and Cytogenomics Evaluation in Cancer.

The availability of cytogenetics and cytogenomics technologies improved the detection and identification of tumor molecular signatures as well as the understanding of cancer initiation and progression. The use of large-scale and high-throughput cytogenomics technologies has led to a fast identification of several cancer candidate biomarkers associated with diagnosis, prognosis, and therapeutics. The advent of array comparative genomic hybridization and next-generation sequencing technologies has significantly improved the knowledge about cancer biology, underlining driver genes to guide targeted therapy development, drug-resistance prediction, and pharmacogenetics. However, few of these candidate biomarkers have made the transition to the clinic with a clear benefit for the patients. Technological progress helped to demonstrate that cellular heterogeneity plays a significant role in tumor progression and resistance/sensitivity to cancer therapies, representing the major challenge of precision cancer therapy. A paradigm shift has been introduced in cancer genomics with the recent advent of single-cell sequencing, since it presents a lot of applications with a clear benefit to oncological patients, namely, detection of intra-tumoral heterogeneity, mapping clonal evolution, monitoring the development of therapy resistance, and detection of rare tumor cell populations. It seems now evident that no single biomarker could provide the whole information necessary to early detect and predict the behavior and prognosis of tumors. The promise of precision medicine is based on the molecular profiling of tumors being vital the continuous progress of high-throughput technologies and the multidisciplinary efforts to catalogue chromosomal rearrangements and genomic alterations of human cancers and to do a good interpretation of the relation genotype—phenotype.

The effect of body mass index on the implantation potential of euploid embryos

Obesity has been associated with higher miscarriage rates after natural conception and IVF. However, the underlying mechanism, whether obesity affects egg quality or endometrial receptivity, is not well understood. Here we aim to determine the impact of body mass index (BMI) on the outcomes of frozen-thawed euploid embryo transfer cycles. Retrospective cohort study. Frozen-thawed embryo transfer (FET) cycles of euploid embryos between 2013 and 2017 were included. Embryos were cultured in time-lapse incubators. Preimplantation genetic testing for aneuploidy was performed using array comparative genomic hybridization or next-generation sequencing. Cycles were divided into three groups according to the female patients’ BMI: <25 kg/m2, 25-29.9 kg/m2 (overweight), and ≥ 30 kg/m2 (obese). The miscarriage rate and live birth rate (LBR) were compared between the three groups. χ2 and Fisher’s exact tests were used for categorical variables. Student’s t test and ANOVA were used for parametric data. Values were expressed as mean ± standard deviation. A total of 1011 FET of euploid embryo(s) were included: 758 with a BMI <25 kg/m2, 174 with a BMI 25-29.9 kg/m2, and 79 with a BMI ≥30 kg/m2. The women were of comparable age between the three groups (P=0.9) (Table 1). There was a trend toward a lower LBR in women with a BMI ≥30 kg/m2 compared to women with a BMI <25 kg/m2 (48.1% vs. 57.5%, respectively; P=0.1), but it did not reach statistical significance. The LBR for women with a BMI 25-29.9 kg/m2 (54%) was comparable with the other two groups (Table 1). There was no significant difference in miscarriage rates between the three groups (8.8% for BMI <25 kg/m2, 9.6% for BMI 25-29.9 kg/m2, and 11.6% for BMI ≥ 30 kg/m2; P=0.8). Overweight and obesity do not significantly affect the implantation potential of euploid embryos.Tabled 1BMI (kg/m2)<2525-29.9≥ 30P valueAge (years)36.3 ± 4.137.0 ± 4.336.7 ± 4.00.09Live birth rate (%)57.554.048.10.1Miscarriage rate (%)8.89.611.60.8 Open table in a new tab

Viral Genome Sequencing for Ultrasensitive Detection of Circulating Tumor DNA

Accurate detection of circulating tumor DNA (ctDNA) has the potential to guide personalized cancer therapy. DNA viruses such as Epstein-Barr virus (EBV) and human papillomavirus (HPV) are major oncologic drivers of nasopharyngeal cancer (NPC) and cervical cancer (CC), respectively, and provide a convenient etiological marker for measurement of ctDNA. Polymerase chain reaction (PCR)-based methods have been widely studied, but more accurate tests are needed. We hypothesized that a next generation sequencing (NGS) approach could outperform PCR-based methods for viral ctDNA detection. We developed a novel NGS method for viral ctDNA detection. Viral genome sequencing for EBV (EBV-seq) or HPV (HPV-seq) was achieved by hybrid capture NGS. Unique molecular identifiers were incorporated for accurate copy number quantification. We implemented a custom bioinformatics workflow to (1) quantify viral sequences, (2) report viral subtype, and (3) measure ctDNA fragment length. The lower limit of detection (LLOD) was compared with published PCR-based methods in cell line dilution series. To test performance on clinical samples, we applied EBV-seq to a cohort of 19 newly diagnosed EBV-associated NPC patients for whom the clinical EBV quantitative PCR test had provided a false negative result and to a control cohort of 20 healthy volunteers. We applied HPV-seq to 40 plasma samples from a cohort of 18 CC patients treated with chemoradiotherapy and to a control cohort of 5 healthy volunteers. The LLOD of EBV-Seq was ≤0.01 EBV genome-equivalents (GE), 100X lower than quantitative PCR. The LLOD of HPV-seq was ≤0.2 HPV GE, 10X lower than digital PCR. For EBV-seq, 19/19 NPC patients had detectable ctDNA (sensitivity=100%, range=0.08-141 EBV GE); 10/20 controls had detectable ctDNA (specificity=50%, range=0.02-0.71 EBV GE). 17/19 (89%) patients, including 6/6 with stage I-II disease, had EBV-seq levels that were >20X above the highest level seen in the control cohort (i.e., 89% sensitivity with 100% specificity). For HPV-seq, 6/6 baseline CC samples, 10/17 (59%) end-of-treatment samples, 1/14 (7%) 3-month post-treatment samples, and 3/3 samples taken upon recurrence had positive HPV-seq results (sensitivity=100%, range=0.02-559 HPV GE) that were highly correlated with digital PCR (Pearson R=0.97) but with 60X higher signal than digital PCR; 0/5 controls had detectable ctDNA (specificity=100%). EBV-seq and HPV-seq were able to report viral subtype in 49/49 positive samples. Viral ctDNA was consistently shorter than non-cancer-derived plasma DNA (median difference=15bp). For DNA virus-associated cancers, viral genome NGS provides dramatically greater sensitivity than PCR-based methods and enables viral subtyping and ctDNA fragment length analysis. Further evaluation is warranted to test the clinical utility of ultrasensitive ctDNA detection using this approach.