Highly accurate long reads are crucial for realizing the potential of biodiversity genomics.

BACKGROUND: Generating the most contiguous, accurate genome assemblies given available sequencing technologies is a long-standing challenge in genome science. With the rise of long-read sequencing, assembly challenges have shifted from merely increasing contiguity to correctly assembling complex, repetitive regions of interest, ideally in a phased manner. At present, researchers largely choose between two types of long read data: longer, but less accurate sequences, or highly accurate, but shorter reads (i.e., >Q20 or 99% accurate). To better understand how these types of long-read data as well as scale of data (i.e., mean length and sequencing depth) influence genome assembly outcomes, we compared genome assemblies for a caddisfly, Hesperophylax magnus, generated with longer, but less accurate, Oxford Nanopore (ONT) R9.4.1 and highly accurate PacBio HiFi (HiFi) data. Next, we expanded this comparison to consider the influence of highly accurate long-read sequence data on genome assemblies across 6750 plant and animal genomes. For this broader comparison, we used HiFi data as a surrogate for highly accurate long-reads broadly as we could identify when they were used from GenBank metadata. RESULTS: HiFi reads outperformed ONT reads in all assembly metrics tested for the caddisfly data set and allowed for accurate assembly of the repetitive ~ 20 Kb H-fibroin gene. Across plants and animals, genome assemblies that incorporated HiFi reads were also more contiguous. For plants, the average HiFi assembly was 501% more contiguous (mean contig N50 = 20.5 Mb) than those generated with any other long-read data (mean contig N50 = 4.1 Mb). For animals, HiFi assemblies were 226% more contiguous (mean contig N50 = 20.9 Mb) versus other long-read assemblies (mean contig N50 = 9.3 Mb). In plants, we also found limited evidence that HiFi may offer a unique solution for overcoming genomic complexity that scales with assembly size. CONCLUSIONS: Highly accurate long-reads generated with HiFi or analogous technologies represent a key tool for maximizing genome assembly quality for a wide swath of plants and animals. This finding is particularly important when resources only allow for one type of sequencing data to be generated. Ultimately, to realize the promise of biodiversity genomics, we call for greater uptake of highly accurate long-reads in future studies.

[Moving towards a personalized oncology: The contribution of genomic techniques and artificial intelligence in the use of circulating tumor biomarkers]. / En marche vers une oncologie personnalisée : l'apport des techniques génomiques et de l'intelligence artificielle dans l'usage des biomarqueurs tumoraux circulants.

Technological advances, in particular the development of high-throughput sequencing, have led to the emergence of a new generation of molecular biomarkers for tumors. These new tools have profoundly changed therapeutic management in oncology, with increasingly precise molecular characterization of tumors leading to increasingly personalized therapeutic targeting. Detection of circulating tumor cells and/or circulating tumor DNA in blood samples -so-called 'liquid biopsies'- can now provide a genetic snapshot of the patient's tumor through an alternative and less invasive procedure than biopsy of the tumor tissue itself. This procedure for characterizing and monitoring the disease in real time facilitates the search for possible relapses, the emergence of resistance, or emergence of a new therapeutic target. In the long term, it might also provide a means of early detection of cancer. These new approaches require the treatment of ever-increasing amounts of clinical data, notably, with the goal of calculating composite clinical-biological predictive scores. The use of artificial intelligence will be unavoidable in this domain, but it raises ethical questions and implications for the health-care system that will have to be addressed.

Trends in Use of Next-Generation Sequencing in Patients With Solid Tumors by Race and Ethnicity After Implementation of the Medicare National Coverage Determination.

Importance: In March 2018, Medicare issued a national coverage determination (NCD) for next-generation sequencing (NGS) to facilitate access to NGS testing among Medicare beneficiaries. It is unknown whether the NCD affected health equity issues for Medicare beneficiaries and the overall population. Objective: To examine the association between the Medicare NCD and NGS use by insurance types and race and ethnicity. Design, Setting, and Participants: A retrospective cohort analysis was conducted using electronic health record data derived from a real-world database. Data originated from approximately 280 cancer clinics (approximately 800 sites of care) in the US. Patients with advanced non-small cell lung cancer (aNSCLC), metastatic colorectal cancer (mCRC), metastatic breast cancer (mBC), or advanced melanoma diagnosed from January 1, 2011, through March 31, 2020, were included. Exposure: Pre- vs post-NCD period. Main Outcomes and Measures: Patients were classified by insurance type and race and ethnicity to examine patterns in NGS testing less than or equal to 60 days after diagnosis. Difference-in-differences models examined changes in average NGS testing in the pre- and post-NCD periods by race and ethnicity, and interrupted time-series analysis examined whether trends over time varied by insurance type and race and ethnicity. Results: Among 92 687 patients with aNSCLC, mCRC, mBC, or advanced melanoma, mean (SD) age was 66.6 (11.2) years, 51 582 (55.7%) were women, and 63 864 (68.9%) were Medicare beneficiaries. The largest racial and ethnic categories according to the database used and further classification were Black or African American (8605 [9.3%]) and non-Hispanic White (59 806 [64.5%]). Compared with Medicare beneficiaries, changes in pre- to post-NCD NGS testing trends were similar in commercially insured patients (odds ratio [OR], 1.03; 95% CI, 0.98-1.08; P = .25). Pre- to post-NCD NGS testing trends increased at a slower rate among patients in assistance programs (OR, 0.93; 95% CI, 0.87-0.99; P = .03) compared with Medicare beneficiaries. The rate of increase for patients receiving Medicaid was not statistically significantly different compared with those receiving Medicare (OR, 0.92; 95% CI, 0.84-1.01; P = .07). The NCD was not associated with statistically significant changes in NGS use trends by racial and ethnic groups within Medicare beneficiaries alone or across all insurance types. Compared with non-Hispanic White individuals, increases in average NGS use from the pre-NCD to post-NCD period were 14% lower (OR, 0.86; 95% CI, 0.74-0.99; P = .04) among African American and 23% lower (OR, 0.77; 95% CI, 0.62-0.96; P = .02) among Hispanic/Latino individuals; increases among Asian individuals and those with other races and ethnicities were similar. Conclusions and Relevance: The findings of this study suggest that expansion of Medicare-covered benefits may not occur equally across insurance types, thereby further widening or maintaining disparities in NGS testing. Additional efforts beyond coverage policies are needed to ensure equitable access to the benefits of precision medicine.

Formalin-Fixed and Paraffin-Embedded Samples for Next Generation Sequencing: Problems and Solutions.

Over the years, increasing information has been asked of the pathologist: we have moved from a purely morphological diagnosis to biomolecular and genetic studies, which have made it possible to implement the use of molecular targeted therapies, such as anti-epidermal growth factor receptor (EGFR) molecules in EGFR-mutated lung cancer, for example. Today, next generation sequencing (NGS) has changed the approach to neoplasms, to the extent that, in a short time, it has gained a place of absolute importance and diagnostic, prognostic and therapeutic utility. In this scenario, formaldehyde-fixed and paraffin-embedded (FFPE) biological tissue samples are a source of clinical and molecular information. However, problems can arise in the genetic material (DNA and RNA) for use in NGS due to fixation, and work is being devoted to possible strategies to reduce its effects. In this paper, we discuss the applications of FFPE tissue samples in the execution of NGS, we focus on the problems arising with the use of this type of material for nucleic acid extraction and, finally, we consider the most useful strategies to prevent and reduce single nucleotide polymorphisms (SNV) and other fixation artifacts.

Multi-Gene Testing Overview with a Clinical Perspective in Metastatic Triple-Negative Breast Cancer.

Next-generation sequencing (NGS) is the technology of choice for the routine screening of tumor samples in clinical practice. In this setting, the targeted sequencing of a restricted number of clinically relevant genes represents the most practical option when looking for genetic variants associated with cancer, as well as for the choice of targeted treatments. In this review, we analyze available NGS platforms and clinical applications of multi-gene testing in breast cancer, with a focus on metastatic triple-negative breast cancer (mTNBC). We make an overview of the clinical utility of multi-gene testing in mTNBC, and then, as immunotherapy is emerging as a possible targeted therapy for mTNBC, we also briefly report on the results of the latest clinical trials involving immune checkpoint inhibitors (ICIs) and TNBC, where NGS could play a role for the potential predictive utility of homologous recombination repair deficiency (HRD) and tumor mutational burden (TMB).

Noninvasive early diagnosis of intestinal diseases based on artificial intelligence in genomics and microbiome.

The maturing development in artificial intelligence (AI) and genomics has propelled the advances in intestinal diseases including intestinal cancer, inflammatory bowel disease (IBD), and irritable bowel syndrome (IBS). On the other hand, colorectal cancer is the second most deadly and the third most common type of cancer in the world according to GLOBOCAN 2020 data. The mechanisms behind IBD and IBS are still speculative. The conventional methods to identify colorectal cancer, IBD, and IBS are based on endoscopy or colonoscopy to identify lesions. However, it is invasive, demanding, and time-consuming for early-stage intestinal diseases. To address those problems, new strategies based on blood and/or human microbiome in gut, colon, or even feces were developed; those methods took advantage of high-throughput sequencing and machine learning approaches. In this review, we summarize the recent research and methods to diagnose intestinal diseases with machine learning technologies based on cell-free DNA and microbiome data generated by amplicon sequencing or whole-genome sequencing. Those methods play an important role in not only intestinal disease diagnosis but also therapy development in the near future.

Next-Generation Sequencing in the Field of Primary Immunodeficiencies: Current Yield, Challenges, and Future Perspectives.

Primary immunodeficiencies comprise a group of inborn errors of immunity that display significant clinical and genetic heterogeneity. Next-generation sequencing techniques and predominantly whole exome sequencing have revolutionized the understanding of the genetic and molecular basis of genetic diseases, thereby also leading to a sharp increase in the discovery of new genes associated with primary immunodeficiencies. In this review, we discuss the current diagnostic yield of this generic diagnostic approach by evaluating the studies that have employed next-generation sequencing techniques in cohorts of patients with primary immunodeficiencies. The average diagnostic yield for primary immunodeficiencies is determined to be 29% (range 10-79%) and 38% specifically for whole-exome sequencing (range 15-70%). The significant variation between studies is mainly the result of differences in clinical characteristics of the studied cohorts but is also influenced by varying sequencing approaches and (in silico) gene panel selection. We further discuss other factors contributing to the relatively low yield, including the inherent limitations of whole-exome sequencing, challenges in the interpretation of novel candidate genetic variants, and promises of exploring the non-coding part of the genome. We propose strategies to improve the diagnostic yield leading the way towards expanded personalized treatment in PIDs.

Clinical application of an NGS-based method in the preimplantation genetic testing for Duchenne muscular dystrophy.

PURPOSE: To determine whether next-generation sequencing (NGS) could be used to directly detect different mutations of Duchenne muscular dystrophy (DMD) during preimplantation genetic testing (PGT). METHODS: From Sep. 2016 to Aug. 2018, a total of six couples participated in this study. Four cases carried DMD exon deletions and two carried exon duplications. Trophectoderm cells were biopsied at day 5 or 6 and NGS was used in the genetic testing of the biopsied cells after whole-genome amplification. We developed a new method-DIRected Embryonic Cell Testing of Exon Deletion/Duplication (DIRECTED) to directly detect the single-gene mutation by NGS. Linage analysis based on single-nucleotide polymorphism (SNP) was used to validate the results from DIRECTED. RESULTS: In the four deletion cases, DIRECTED was used to detect DMD exon deletion in 16 biopsied embryos. All DIRECTED results were consistent with linkage analysis, indicating this method was reliable in detecting deletions around 1 Mb. In the two cases carrying exon duplications, no blastocyst was obtained for biopsy. Nonetheless, preliminary experiment results suggested that DIRECTED could also be used for direct detection of exon duplications in embryos. CONCLUSIONS: Exon deletions or duplications in DMD of preimplantation embryos could be detected directly by NGS-based methods during PGT.

Mutations in key driver genes of pancreatic cancer: molecularly targeted therapies and other clinical implications.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers, with a minimal difference between its incidence rate and mortality rate. Advances in oncology over the past several decades have dramatically improved the overall survival of patients with multiple cancers due to the implementation of new techniques in early diagnosis, therapeutic drugs, and personalized therapy. However, pancreatic cancers remain recalcitrant, with a 5-year relative survival rate of <9%. The lack of measures for early diagnosis, strong resistance to chemotherapy, ineffective adjuvant chemotherapy and the unavailability of molecularly targeted therapy are responsible for the high mortality rate of this notorious disease. Genetically, PDAC progresses as a complex result of the activation of oncogenes and inactivation of tumor suppressors. Although next-generation sequencing has identified numerous new genetic alterations, their clinical implications remain unknown. Classically, oncogenic mutations in genes such as KRAS and loss-of-function mutations in tumor suppressors, such as TP53, CDNK2A, DPC4/SMAD4, and BRCA2, are frequently observed in PDAC. Currently, research on these key driver genes is still the main focus. Therefore, studies assessing the functions of these genes and their potential clinical implications are of paramount importance. In this review, we summarize the biological function of key driver genes and pharmaceutical targets in PDAC. In addition, we conclude the results of molecularly targeted therapies in clinical trials and discuss how to utilize these genetic alterations in further clinical practice.

Recent advancement of NGS technologies to detect active transposable elements in plants.

BACKGROUND: Unlike peoples' belief that transposable elements (TEs) are "junk DNAs" or "genomic parasites", TEs are essential genomic elements that bring about genetic diversity and enable evolution of a species. In fact, transposons are major constituent of chromosome in crop genomes, particularly in major cereal crops, the primary type of which is long terminal repeat (LTR) retrotransposon. Since TE mobilization can be controlled by specific environmental stimulation and as the result can generate novel genetic variations, it has been suggested that controlled mobilization of TEs can be a plausible method for crop breeding. To achieve this goal, series of sequencing techniques have been recently established to identify TEs that are active in mobility. These methods target and detect extrachromosomal DNAs (ecDNAs), which are final products of integration. The newly identified TEs by these methods exhibit strong transpositional activity which can generate novel genetic diversity and provide useful breeding resources. CONCLUSIONS: In this mini review, we summarize and introduce ALE-seq, mobilome-seq, and VLP DNA-seq techniques employed to detect active TEs in plants.

Potential of whole-genome sequencing-based pharmacogenetic profiling.

Pharmacogenetics represents a major driver of precision medicine, promising individualized drug selection and dosing. Traditionally, pharmacogenetic profiling has been performed using targeted genotyping that focuses on common/known variants. Recently, whole-genome sequencing (WGS) is emerging as a more comprehensive short-read next-generation sequencing approach, enabling both gene diagnostics and pharmacogenetic profiling, including rare/novel variants, in a single assay. Using the example of the pharmacogene CYP2D6, we demonstrate the potential of WGS-based pharmacogenetic profiling as well as emphasize the limitations of short-read next-generation sequencing. In the near future, we envision a shift toward long-read sequencing as the predominant method for gene diagnostics and pharmacogenetic profiling, providing unprecedented data quality and improving patient care.

The impact of patient, embryo, and translocation characteristics on the ploidy status of young couples undergoing preimplantation genetic testing for structural rearrangements (PGT-SR) by next generation sequencing (NGS).

PURPOSE: To evaluate the factors that affect the incidence of euploid balanced embryos and interchromosomal effect (ICE) in carriers of different structural rearrangements. METHODS: This retrospective study includes 95 couples with reciprocal translocations (RecT) and 36 couples with Robertsonian translocations (RobT) undergoing Preimplantation Genetic Testing for Structural Rearrangements (PGT-SR) between March 2016 and July 2019. Next-generation sequencing (NGS) was the technique used coupled with trophectoderm (TE) biopsy. Only cases with females under 38 years were included. A total of 532 blastocysts were evaluated. RESULTS: The euploidy rate was similar in RobT when compared with RecT carriers [57/156 (36.5%) vs. 112/376 (29.8%), p = 0.127]. The pure ICE rate was significantly higher in RobT carriers [48/156 (30.8%) vs. 53/376 (14.1%), p < 0.001] than it was in RecT carriers. Female age was the independent factor for the probability of obtaining a euploid embryo in RecT and RobT carriers, and increasing female age decreases the probability of obtaining a euploid embryo. In RecT carriers, no significant differences were observed in euploidy rates, pure ICE, or combined ICE according to the length of the translocated fragment and the chromosome group. However, total ICE was significantly lower when there was a breakpoint in the short chromosome arm together with a breakpoint in the long arm [(44/158 (27.8%) for pq or qp, 51/155 (32.9%) for pp and 30/63 (47.6%) for qq; p = 0.02]. CONCLUSION: The incidence of euploid/balanced blastocysts was similar in both types of translocations. However, there was a significant increase in pure ICE in RobT compared to RecT carriers. In RecT carriers, the presence of the breakpoints in the long arm of the chromosomes involved in the rearrangement resulted in a higher total ICE.

Application of Next-Generation Sequencing in Neurodegenerative Diseases: Opportunities and Challenges.

Genetic factors (gene mutations) lead to various rare and prevalent neurological diseases. Identification of underlying mutations in neurodegenerative diseases is of paramount importance due to the heterogeneous nature of the genome and different clinical manifestations. An early and accurate molecular diagnosis are cardinal for neurodegenerative patients to undergo proper therapeutic regimens. The next-generation sequencing (NGS) method examines up to millions of sequences at a time. As a result, the rare molecular diagnoses, previously presented with "unknown causes", are now possible in a short time. This method generates a large amount of data that can be utilized in patient management. Since each person has a unique genome, the NGS has transformed diagnostic and therapeutic strategies into sequencing and individual genomic mapping. However, this method has disadvantages like other diagnostic methods. Therefore, in this review, we aimed to briefly summarize the NGS method and correlated studies to unravel the genetic causes of neurodegenerative diseases including Alzheimer's disease, Parkinson's disease, epilepsy, and MS. Finally, we discuss the NGS challenges and opportunities in neurodegenerative diseases.

Third-generation sequencing: any future opportunities for PGT?

PURPOSE: To investigate use of the third-generation sequencing (TGS) Oxford Nanopore system as a new approach for preimplantation genetic testing (PGT). METHODS: Embryos with known structural variations underwent multiple displacement amplification to create fragments of DNA (average ~ 5 kb) suitable for sequencing on a nanopore. RESULTS: High-depth sequencing identified the deletion interval for the relatively large HBA1/2--SEA alpha thalassemia deletion. In addition, STRs were able to be identified in the primary sequence data for potential use in conventional PGT-M linkage confirmation. Sequencing of amplified embryo DNA carrying a translocation enabled balanced embryos to be identified and gave the precise identification of translocation breakpoints, offering the opportunity to differentiate carriers from non-carrier embryos. Low-pass sequencing gave reproducible profiles suitable for simple identification of whole-chromosome and segmental aneuploidies. CONCLUSION: TGS on the Oxford Nanopore is a possible alternative and versatile approach to PGT with potential for performing economical workups where the long read sequencing information can be used for assisting in a traditional PGT workup to design an accurate and reliable test. Additionally, application of TGS has the possibility of providing combined PGT-A/SR or in selected stand-alone PGT-M cases involving pathogenic deletions. Both of these applications offer the opportunity for simultaneous aneuploidy detection to select either balanced embryos for transfer or additional carrier identification. The low cost of the instrument offers new laboratories economical entry into onsite PGT.

A primer for generating and using transcriptome data and gene sets.

Transcriptomic approaches have provided a growing set of powerful tools with which to study genome-wide patterns of gene expression. Rapidly evolving technologies enable analysis of transcript abundance data from particular tissues and even single cells. This Primer discusses methods that can be used to collect and profile RNAs from specific tissues or cells, process and analyze high-throughput RNA-sequencing data, and define sets of genes that accurately represent a category, such as tissue-enriched or tissue-specific gene expression.