Genomic Next-generation Sequencing Research Articles

Abstract 100: Real-world clinical outcomes associated with first line treatments in patients with advanced non-small cell lung cancer without EGFR mutation or ALK rearrangement based on AACR GENIE

Abstract Background Patients with locally advanced or metastatic non-small cell lung cancer (aNSCLC) have a poor prognosis, with a historic five-year overall survival (OS) of less than 10%. Patients with aNSCLC without common actionable oncogenic drivers such as EGFR mutations or ALK rearrangements are often limited to immunotherapy and/or chemotherapy, leaving a group of patients with unmet medical need. This study explored OS and progression-free survival based on radiographic imaging (PFS-I) among aNSCLC patients with EGFR wild type (wt) and ALK wt treated in real-world academic setting using AACR GENIE data. Methods Data collected from 4 academic cancer centers in North America from AACR Project GENIE cancer registry was used for this study. The data included patients who underwent next-generation genomic sequencing (NGS) between 2014 and 2017 linked with clinical outcomes obtained during routine practice. Patients initially diagnosed with stage III or IV aNSCLC, underwent NGS prior to or within 60 days after starting their first line of therapy (LOT1), with NGS-confirmed ALK wt and EGFR wt were selected. Patients were then classified into four groups based on their LOT1 regimen: chemotherapy single agent or combinations (chemo), immunotherapy-monotherapy (IO), targeted therapy (TT), and bevacizumab plus chemo (bev+chemo). PFS-I and OS were measured from the start of LOT1 for each therapy group. As progression data were only available for stage IV patients in AACR GENIE, PFS-I was reported for this subgroup only. Baseline characteristics were not adjusted. Results 263 patients satisfied criteria for inclusion in the study: 175 (67%), 31 (12%), 14 (5%), and 38 (14%) initiated chemo, IO, TT, and bev+chemo respectively. Of the 263 patients: 57% were female, mean age was 64, and 85% were current or former smoker. 99% of patients initiated LOT1 in year 2017 or earlier. 68%, 97%, 93%, and 100% were diagnosed with stage IV among chemo, IO, TT, and bev+chemo groups respectively. Median PFS-I (mPFS-I) in stage IV patients was 5.0 (confidence interval [CI]: 3.9-5.7) months, and median OS (mOS) in stage III/IV patients was 20.4 (CI:14.8-25.3) months. Among stage IV patients, mPFS-I by regimen group was 4.2 (CI:3.1-5.7), 4.0 (CI:1.4-7.1), 6.2 (CI:3.3-15.0), and 6.8 (CI:4.4-10.3) months for chemo, IO, TT, and bev+chemo respectively. Among stage III/IV patients, mOS by regimen group was 19.7 (CI:12.7-25.4), 18.4 (7.1-not reached [NR]), NR (CI:6.2-NR), and 23.5 (CI:10.3-35.1) months for chemo, IO monotherapy, TT, and bev+chemo respectively. Conclusion This is the first study to assess outcomes among aNSCLC patients who are EGFR-wt and ALK-wt from the AACR Project GENIE registry. It provides a descriptive assessment of clinical outcomes associated with standard care treatment in the academic setting as more treatment options become available. Citation Format: Hemanth Kanakamedala, Beilei Cai, Sara Bruce Wirta, Rohit Borker, Nydia Caro, Wen-Hsing Wu, Ariadna Holynskyj. Real-world clinical outcomes associated with first line treatments in patients with advanced non-small cell lung cancer without EGFR mutation or ALK rearrangement based on AACR GENIE [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 100.

The complete chloroplast genome of Semenovia thomsonii (Tordylieae: Apiaceae), a new record from Xizang, China

Semenovia thomsonii is a perennial herb native to India. In this study, we assembled and annotated the complete chloroplast (cp) genome of a specimen collected from Xizang, China, using whole genome next-generation sequencing. The cp genome is circular in structure and 147,137 bp in length, consisting of one large single-copy (LSC) region of 92,885 bp, one small single-copy (SSC) region of 17,448 bp, and a pair of inverted repeat regions of 36,804 bp. The overall GC content of the genome is 37.6%. The cp genome was predicted to contain 129 genes, including 85 protein-coding, 36 tRNA, and eight rRNA. Phylogenetic analysis of S. thomsonii and 21 cp genomes in the Apiaceae fully resolved S. thomsonii in a clade with S. gyirongensis, and S. transiliensis. These genetic data represent the first confirmed report of S. thomsonii from Xizang, China and provide useful information to the phylogenetic history of the genus Semenovia.

Knowledge Is Power: Benefits, Risks, Hopes, and Decision-Making Reported by Parents Consenting to Next-Generation Sequencing for Children and Adolescents with Cancer.

To qualitatively describe parent perspectives of next-generation genomic sequencing (NGS) for their children with cancer, including perceived benefits, risks, hopes/expectations, and decision-making process when consenting or not consenting to NGS and prior to result disclosure. Qualitative interviews were used. Altruism is an important factor in parents consenting to NGS testing, as well as making sense of their child's cancer and legacy building. Parents described realistic hopes and expectations associated with NGS participation. Although parents endorsed the likelihood of no medical benefit, those consenting to NGS felt there was no reason not to participate. Parents declining participation expressed avoidance of worry and parent guilt if a germline variant were to be disclosed. As NGS evolves into a component of the routine diagnostic workup for pediatric cancer patients, genetic nurses play a role in conducting informed consent conversations and ensuring that patients and families have realistic hopes and expectations associated with NGS.

The Feasibility of Using Biomarkers Derived from Circulating Tumor DNA Sequencing as Predictive Classifiers in Patients with Small-Cell Lung Cancer

Background: Exploring predictive biomarkers to individualize patients with small cell lung cancer (SCLC) remains challenging in clinical practice, and current imaging techniques cannot fully meet the clinical needs due to their inherent limitations. Methods: A total of 127 longitudinal biological DNA samples from 35 SCLC patients treated with first-line standard therapy were analyzed by capture-based targeted (average coverage for tissue and circulating tumor DNA (ctDNA) samples were 841× and 2670.5×, respectively) genome next-generation sequencing, which was designed to cover coding sequencing or hot exons of 1,021 genes frequently mutated in solid tumors. PyClone was used to infer the molecular tumor burden index (mTBI). Pre-treatment tumor tissues [T1] and serial plasma samples were collected (pre-treatment [B1], after two [B2], six [B3] cycles of chemotherapy and at progression [B4]). Findings: In total, 38 SCLC patients were enrolled with 35 patients included in the analysis set. The overall concordance rate between tissue and plasma sequencing in 30 patients with matched T1 and B1 samples was 66.5%. The median progression-free survival (PFS) was 9.8 months (95% confidence interval [CI]: 6.3-13.3). Patients with persisted RB1 and/or TP53 mutations at B2 demonstrated inferior PFS compared with others (5.2 months [95%CI: 3.8-6.6] vs. 13.1 months [95%CI: 9.8-16.4], p=0.06). The median PFS for patients with persisted RB1 mutations at B2 was significantly worse than the other two groups (4.2 months [95%CI: 2.8-5.6] vs. not reached, p<0.0001 and vs. 10.2 months, [95%CI: 6.3-14.1], p=0.001). The performance of mTBI monitoring was highly consistent with tumor imaging, and earlier mTBI elevation was observed in 64.7% of patients with B3 samples. In addition, the dynamic changes of mTBI from B4 to B1 were sensitive to predict new metastases, with sensitivity and specificity of 100% and 85.7% (95%CI: 50.8-120.7), respectively. Interpretation: Monitoring ctDNA based RB1 mutations and mTBI provided a feasible tool to predict prognosis of first-line therapy in SCLC, which guaranteed the development of prospective stratification in future trials. Trial Registration: This study was registered at the Chinese Clinical Trial Registry as ChiCTR1900023956. Funding: This study was supported by the National Key Research and Development Project (2019YFC1315704, to ZW), CAMS Innovation Fund for Medical Sciences (2017-I2M-1-005, to ZW), the National Natural Sciences Foundation (81871889, 82072586 to ZW) and Beijing Natural Science Foundation (7212084 to ZW). Declaration of Interest: The authors declare no potential competing interests. Ethical Approval: All procedures were conducted in accordance with the Declaration of Helsinki. This study was approved by the ethics committee of National Cancer Center/Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College (Beijing, China; approved No. 18- 151/1729).

The complete chloroplast genome sequence of endangered plant Trachycarpus nanus (Arecaceae)

Trachycarpus nanus is an endangered plant that is endemic to southwest of China. In the present study, 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 158,713 bp size with 36.6% GC content. The genome is of typical structure and contain a pair of inverted repeat (IR) regions with 27,240 bp, separated by one large single-copy (LSC) with 86,395 bp, and one small single-copy (SSC) regions with 17,838 bp. The genome contained 132 genes, including 86 protein-coding genes, 8 rRNA genes and 38 tRNA genes. A phylogenetic tree reconstructed based on 21 chloroplast genomes reveals that Trachycarpus nanus is most related with Chamaerops humilis. The information provides important genetic basis for the species’ future studies on phylogenetic and utilization.

High Throughput Diagnostics and Dynamic Risk Assessment of Variants of Concern

Background: The rise of new SARS-CoV-2 variants worldwide requires global molecular surveillance strategies, in support of public health control. Early detection and evaluation of their risk of spreading within the population is pivotal. Methods: Between April 2020 and February 2021, the Lighthouse Laboratory at Alderley Park Network tested more than eight million nose and throat swab samples for the presence of SARS-CoV-2. The PCR assay used, targets three genomic regions of the virus N, Orf1ab and S. Whole genome next-generation sequencing was used to confirm PCR positive results. Positive sample results were mapped to postal code origin of samples to allow real-time tracking of the spread of the variant through the UK. Findings: In mid-November 2020 the assay identified an increasing number of S gene negative, N and Orf1ab positive samples. Whole genome sequencing demonstrated that the loss of S gene detection was due to the appearance of a SARS-CoV-2 lineage (B.1.1.7) designated as Variant of Concern (VOC) 202012/01. By the beginning of January 2021, the new SARS-CoV-2 VOC had risen to comprise 70% of daily positive samples tested at Alderley Park and ~98% by the end of February 2021. Interpretation: The timeline view identified the rapid spread of the variant across England during the first three weeks of December. Coupling high throughput diagnostics and molecular surveillance was pivotal to the early detection of the spread of this variant. The availability of real time tracking of an emerging variant is an important new tool to inform decision-making for risk mitigation. In a respiratory pandemic, a tool for the timely response to the emergence and spread of a novel variant is vital especially when a variant is associated with enhanced transmission, as has occurred with VOC 202012/01. Funding Statement: The Lighthouse laboratory network was generated using funding from DHSC. Declaration of Interests: None to declare. Ethics Approval Statement: The manuscript was reviewed by the Head of Approvals Support, NHS Health Research Authority (HRA-NHS, UK), who confirmed that the study, which used unlinked and anonymised data was health surveillance and did not require NHS Research ethics review.

Role of next-generation genomic sequencing in targeted agents repositioning for pancreaticoduodenal cancer patients

BackgroundPancreaticoduodenal cancer (PDC) is a group of malignant tumors arising in the ampullary region, which lack approved targeted therapies for their treatment. MethodsThis retrospective, observational study is based on Secondary Data Use (SDU) previously collected during a multicenter collaboration, which were subsequently entered into a predefined database and analyzed. FoundationOne CDx or Liquid, a next-generation DNA sequencing (NGS) service, was used to identify genomic alterations of patients who failed standard treatments. Detected alterations were described according to ESMO Scale of Clinical Actionability for molecular Targets (ESCAT). ResultsNGS analysis was performed in 68 patients affected by PDC. At least one alteration ranking tier I, II, III, or IV according to ESCAT classification was detected in 8, 1, 9, and 12 patients respectively (44.1%). Ten of them (33.3%) received a matched therapy. Patients with ESCAT tier I to IV were generally younger than the overall population (median = 54, range = 26–71 years), had an EGOG performance status score = 0 (83.3%), and an uncommon histological or clinical presentation. The most common mutations with clinical evidence of actionability (ESCAT tier I-III) involved genes of the RAF (10.3%), BRCA (5.9%) or FGFR pathways (5.9%). We present the activity of the RAF kinases inhibitor sorafenib in patients with RAF-mutated advanced PDC. ConclusionsIn advanced PDC, NGS is a feasible and valuable method for enabling precision oncology. This genomic profiling method might be considered after standard treatments failure, especially in young patients maintaining a good performance status, in order to detect potentially actionable mutations and offer molecularly targeted therapeutic approaches.

The effect of endometrial thickness on live birth outcomes in women undergoing hormone-replaced frozen embryo transfer

ObjectiveTo determine the impact of endometrial thickness on live birth outcomes and obstetric complication rate after hormone-replaced frozen embryo transfer.DesignRetrospective cohort study.SettingLarge, urban, academic fertility center.Patient(s)All patients with a singleton live birth after single euploid embryo transfer (by array comparative genomic hybridization or next-generation sequencing) in a hormone-replaced frozen embryo transfer cycle between January 2017 and December 2018 were reviewed.Intervention(s)None.Main Outcome measure(s)The primary outcomes were birth weight and obstetric complication rate.Result(s)A total of 492 patients were included. The median endometrial thickness was 8.60 mm (range, 6.0–20.0). The median gestational age at live birth was 39.4 weeks with a median birth weight of 3,345.2 g. Endometrial thickness was significantly correlated with birth weight. When patients were dichotomized into groups (those with an endometrial thickness of <7 mm and those with an endometrial thickness of >7 mm), neonates born from endometria with a thickness of <7 mm were born earlier (37.3 vs. 39.4 weeks and born with lower birth weights (2,749.9 vs. 3,345.2 g). It should be noted that only seven patients had an endometrium measuring <7 mm. Moreover, 7.1% (n = 35) of patients had an obstetric complication. Endometrial thickness was not significantly associated with obstetric complications, even with adjustments for age and medical history.Conclusion(s)Endometrial thickness may be a valuable predictor of placental health and birth weight. Further study is required to examine the relationship with individual obstetric complications, as our study may not have been powered to observe differences in obstetric complication rate, as well as the relationship between endometrial thickness and outcomes in natural frozen embryo transfer cycles.

Receipt of Next-generation Genomic Sequencing among Patients with Metastatic Colorectal Cancer (mCRC) in a Real-World Cohort

Abstract Purpose of the Study: Disparities in genomic precision medicine approaches, through molecular profiling or next- generation sequencing (NGS), by race/ethnicity, insurance, and poverty have been identified in lung cancer, but not mCRC. Our goal was to examine disparities in receipt of NGS in patients with mCRC. Methods: We used all-payer electronic health record (EHR)-derived de-identified data from the Flatiron Health database generated from routine clinical care across the United States. Our study population included 26,524 patients with mCRC during the years 2013–2020. In addition to date of NGS testing, the FH-EHR data include demographics (age, sex, and race/ethnicity), payer type, and Eastern Cooperative Oncology Group (ECOG) performance status. We conducted descriptive analyses and multivariable logistic regression analysis to identify correlates of receipt of NGS within 6 months of metastatic diagnosis. Results: Among the 26,524 people with mCRC, 45% (n = 11,946) were women, 48% (n = 12,732) had a Commercial Health Plan, and the majority were seen in a community practice (92%) vs academic hospitals. Over 70% of the patients were White, 12% Black or African-American (AA), and 14% Other. Thirty-three percent (n = 8,821) of patients had documentation in the EHR of having received NGS. After simultaneously adjusting for other factors in the model, older age (ORper year increase: 0.97, 95% CI: 0.96–0.98) and Black/AA race (OR: 0.74, 95% CI: 0.68–0.81), compared to White, was associated with lower odds of receiving NGS testing. Conversely, female sex, better ECOG performance status, later calendar year, being seen in an academic practice, and having a Commercial Health Plan were associated with greater odds of receiving NGS. Conclusions: Our findings indicate that NGS is not received uniformly by all patients with mCRC. Future analyses will incorporate receipt of individual molecular biomarker tests, as recommended by professional societies, as well as their results (e.g., KRAS, NRAS, BRAF, MMR/MSI), treatment information, and survival.

Implementation of an in-house real-time reverse transcription-PCR assay for the rapid detection of the SARS-CoV-2 Marseille-4 variant.

IntroductionThe SARS-CoV-2 pandemic has been associated with the occurrence since summer 2020 of several viral variants that overlapped or succeeded each other in time. Those of current concern harbor mutations within the spike receptor binding domain (RBD) that may be associated with viral escape to immune responses. In our geographical area a viral variant we named Marseille-4 harbors a S477 N substitution in this RBD.Materials and methodsWe aimed to implement an in-house one-step real-time reverse transcription-PCR (qPCR) assay with a hydrolysis probe that specifically detects the SARS-CoV-2 Marseille-4 variant.ResultsAll 6 cDNA samples from Marseille-4 variant strains identified in our institute by genome next-generation sequencing (NGS) tested positive using our Marseille-4 specific qPCR, whereas all 32 cDNA samples from other variants tested negative. In addition, 39/42 (93 %) respiratory samples identified by NGS as containing a Marseille-4 variant strain and 0/26 samples identified as containing non-Marseille-4 variant strains were positive. Finally, 2018/3960 (51%) patients SARS-CoV-2-diagnosed in our institute, 10/277 (3.6 %) respiratory samples collected in Algeria, and none of 207 respiratory samples collected in Senegal, Morocco, or Lebanon tested positive using our Marseille-4 specific qPCR.DiscussionOur in-house qPCR system was found reliable to detect specifically the Marseille-4 variant and allowed estimating it is involved in about half of our SARS-CoV-2 diagnoses since December 2020. Such approach allows the real-time surveillance of SARS-CoV-2 variants, which is warranted to monitor and assess their epidemiological and clinical characterics based on comprehensive sets of data.

5. Further evidence of GABRA4 and TOP3B as autism susceptibility genes

Copy number variants (CNVs) are a known contributor to neurodevelopmental conditions such as autism spectrum disorder (ASD). Both array comparative genomic hybridization and Next-Generation sequencing techniques have led to increased detection of small CNVs and the identification of many candidate susceptibility genes for ASD. We report familial inheritance of two CNVs that include genes with known involvement in neurodevelopment. These CNVs are found in various combinations among four siblings with autism spectrum disorder, as well as in their phenotypically normal parents. We describe a 2.4 Mb duplication of 4p12 to 4p11 that includes GABRA4 and other GABA receptor genes, as well as a 246 kb deletion at 22q11.22 involving the TOP3B gene. The maternally inherited 4p duplication was detected in three siblings, two of whom also had the paternally inherited 22q11.22 deletion. The fourth sibling only had the 22q11.22 deletion. These CNVs have rarely been reported in the literature. Upon review, a single publication was found describing a similar 4p duplication in three generations of a family with neurodevelopmental and neuropsychiatric disorders, as well as in an unrelated patient with autism. TOP3B falls within the distal 22q11.22 deletion syndrome and has been associated with schizophrenia, neurodevelopmental disorders including epilepsy, and cardiac defects. The identification of this family contributes to the understanding of specific genetic contributors to neurodevelopmental disorders and an emerging phenotype associated with proximal 4p duplication.

Endometrial Adenocarcinomas With No Specific Molecular Profile: Morphologic Features and Molecular Alterations of "Copy-number Low" Tumors.

The study evaluated morphologic patterns, mutational profiles, and β-catenin immunohistochemistry (IHC) in copy-number low (CNL) endometrial adenocarcinomas (EAs). CNL EAs (n=19) with next-generation or whole genome sequencing results and available tissue for IHC were identified from our institutional database. Clinical data and histologic slides were reviewed. IHC for β-catenin was performed and correlated with mutation status. Images of digital slides of CNL EAs from The Cancer Genome Atlas (TCGA) database (n=90) were blindly reviewed by 4 pathologists, and morphology was correlated with mutation status. Categorical variables were analyzed using the Fisher exact test, and agreement was assessed using Fleiss κ. CTNNB1 mutations were present in 63% (12/19) of CNL EAs. β-catenin nuclear localization was present in 83% of CTNNB1-mutated tumors (10/12) and in 0% (0/7) of CTNNB1-wildtype tumors (sensitivity 0.83, specificity 1.00). Squamous differentiation (SD) was present in 47% (9/19) and was more often observed in CTNNB1-mutated tumors (P=0.02). Mucinous differentiation (MD) was associated with KRAS mutations (P<0.01). Digital image review of TCGA CNL EAs revealed that pathologist agreement on SD was strong (κ=0.82), whereas agreement on MD was weak (κ=0.48). Pathologists identified SD in 22% (20/90), which was significantly associated with the presence of CTNNB1 mutations (P<0.01). CNL EAs demonstrate several morphologies with divergent molecular profiles. SD was significantly associated with CTNNB1 mutations and nuclear localization of β-catenin in these tumors. Nuclear expression of β-catenin is a sensitive and specific IHC marker for CTNNB1 mutations in CNL EAs. CNL EAs with KRAS mutations often displayed MD.

Approaches for characterizing and tracking hospital-associated multidrug-resistant bacteria.

Hospital-associated infections are a major concern for global public health. Infections with antibiotic-resistant pathogens can cause empiric treatment failure, and for infections with multidrug-resistant bacteria which can overcome antibiotics of "last resort" there exists no alternative treatments. Despite extensive sanitization protocols, the hospital environment is a potent reservoir and vector of antibiotic-resistant organisms. Pathogens can persist on hospital surfaces and plumbing for months to years, acquire new antibiotic resistance genes by horizontal gene transfer, and initiate outbreaks of hospital-associated infections by spreading to patients via healthcare workers and visitors. Advancements in next-generation sequencing of bacterial genomes and metagenomes have expanded our ability to (1) identify species and track distinct strains, (2) comprehensively profile antibiotic resistance genes, and (3) resolve the mobile elements that facilitate intra- and intercellular gene transfer. This information can, in turn, be used to characterize the population dynamics of hospital-associated microbiota, track outbreaks to their environmental reservoirs, and inform future interventions. This review provides a detailed overview of the approaches and bioinformatic tools available to study isolates and metagenomes of hospital-associated bacteria, and their multi-layered networks of transmission.

What Is the Best Lens? Comparing the Resolution Power of Genome-Derived Markers and Standard Barcodes.

Fungal species delimitation was traditionally carried out with multicopy ribosomal RNA (rRNA) genes, principally for their ease of amplification. Since the efficacy of these markers has been questioned, single-copy protein-encoding genes have been proposed alone or in combination for Multi-Locus Sequence Typing (MLST). In this context, the role of the many sequences obtained with Next-Generation Sequencing (NGS) techniques, in both genomics and metagenomics, further pushes toward an analysis of the efficacy of NGS-derived markers and of the metrics to evaluate the marker efficacy in discriminating fungal species. This paper aims at proposing MeTRe (Mean Taxonomic Resolution), a novel index that could be used both for measuring marker efficacy and for assessing the actual resolution (i.e., the level of separation) between species obtained with different markers or their combinations. In this paper, we described and then employed this index to compare the efficacy of two rRNAs and four single-copy markers obtained from public databases as both an amplicon-based approach and genome-derived sequences. Two different groups of species were used, one with a pathogenic species of Candida that was characterized by relatively well-separated taxa, whereas the other, comprising some relevant species of the sensu stricto group of the genus Saccharomyces, included close species and interspecific hybrids. The results showed the ability of MeTRe to evaluate marker efficacy in general and genome-derived markers specifically.

Evaluation of analytical protocols of alignment mapping tools using high throughput next-generation genome sequencing data

Background: Ever since the development of first next-generation genome sequencer (NGS) in 2005, there are rapid developments of high throughput next-generation genome sequencing (HT-NGS) techniques and tools used in genetics and genomics has become much more comfortable and cheaper. The result is the generation of a massive amount of data sets, requiring detailed analysis, which becomes impossible without the use of appropriate bioinformatics tools. One of the crucial steps in the analysis of NGS data is to map readings to a reference sequence. Although the dominance of Illumina synthesis by sequencing (SBS) technology has been noticeable in recent years, the choice of the tools is hampered and the variety of input data and reference genomes. Moreover, the tools used are crucial for result files and further analysis.Methods: The subject of this paper is the three most frequently used alignment mapping programs, which have functions to allow working with many platforms: BWA, Bowtie2 and SMALT. The task of the tested aligners is to match short sequences coming from NGS with reference sequences. The most popular: BWA and Bowtie2 use for this purpose the Burrows-Wheeler transformation and SMALT maps the sequences using hashing and dynamic programming. The presented paper aimed to compare the quality and efficiency of the alignment mapping programs under examination, due to three criteria: i) the quality of the compared sequences of different lengths and from different platforms; ii) coefficient of wrongly compared sequences; iii) the computational resources used.Results: By comparing the results of the mapping analyses for all the programs used, the least popular SMALT is the best. Obtaining the highest percentage of mapped readings for each platform and maintaining the lowest computational memory usage, turns out to be the most optimal choice.Conclusions: The results presented in this paper can be used to verify and rebuild data analysis pipelines from NGS based so far on other tools. We conclude that by using the tools under appropriate conditions, it is possible to improve the quality of the analyses, speed them up and reduce their cost.

Decoding Genetics of Congenital Heart Disease Using Patient-Derived Induced Pluripotent Stem Cells (iPSCs).

Congenital heart disease (CHD) is the most common cause of infant death associated with birth defects. Recent next-generation genome sequencing has uncovered novel genetic etiologies of CHD, from inherited and de novo variants to non-coding genetic variants. The next phase of understanding the genetic contributors of CHD will be the functional illustration and validation of this genome sequencing data in cellular and animal model systems. Human induced pluripotent stem cells (iPSCs) have opened up new horizons to investigate genetic mechanisms of CHD using clinically relevant and patient-specific cardiac cells such as cardiomyocytes, endothelial/endocardial cells, cardiac fibroblasts and vascular smooth muscle cells. Using cutting-edge CRISPR/Cas9 genome editing tools, a given genetic variant can be corrected in diseased iPSCs and introduced to healthy iPSCs to define the pathogenicity of the variant and molecular basis of CHD. In this review, we discuss the recent progress in genetics of CHD deciphered by large-scale genome sequencing and explore how genome-edited patient iPSCs are poised to decode the genetic etiologies of CHD by coupling with single-cell genomics and organoid technologies.

Whole plastomes are not enough: phylogenomic and morphometric exploration at multiple demographic levels of the bee orchid clade Ophrys sect. Sphegodes.

Plastid sequences have long dominated phylogeny reconstruction at all time depths, predicated on a usually untested assumption that they accurately represent the evolutionary histories of phenotypically circumscribed species. We combined detailed in situ morphometrics (124 plants) and whole-plastome sequencing through genome skimming (71 plants) in order to better understand species-level diversity and speciation in arguably the most challenging monophyletic group within the taxonomically controversial, pseudo-copulatory bee orchid genus Ophrys. Using trees and ordinations, we interpreted the data at four nested demographic levels-macrospecies, mesospecies, microspecies, and local population-seeking the optimal level for bona fide species. Neither morphological nor molecular discontinuities are evident at any level below macrospecies, the observed overlap among taxa suggesting that both mesospecies and microspecies reflect arbitrary division of a continuum of variation. Plastomes represent geographic location more strongly than taxonomic assignment and correlate poorly with morphology, suggesting widespread plastid capture and possibly post-glacial expansion from multiple southern refugia. As they are rarely directly involved in the speciation process, plastomes depend on extinction of intermediate lineages to provide phylogenetic signal and so cannot adequately document evolutionary radiations. The popular 'ethological' evolutionary model recognizes as numerous 'ecological species' (microspecies) lineages perceived as actively diverging as a result of density-dependent selection on very few features that immediately dictate extreme pollinator specificity. However, it is assumed rather than demonstrated that the many microspecies are genuinely diverging. We conversely envisage a complex four-dimensional reticulate network of lineages, generated locally and transiently through a wide spectrum of mechanisms, but each unlikely to maintain an independent evolutionary trajectory long enough to genuinely speciate by escaping ongoing gene flow. The frequent but localized microevolution that characterizes the Ophrys sphegodes complex is often convergent and rarely leads to macroevolution. Choosing between the contrasting 'discontinuity' and 'ethology' models will require next-generation sequencing of nuclear genomes plus ordination of corresponding morphometric matrices, seeking the crucial distinction between retained ancestral polymorphism-consistent with lineage divergence-and polymorphisms reflecting gene flow through 'hybridization'-more consistent with lineage convergence.

Obesity in Children with Leptin Receptor Gene Polymorphisms.

The study of single nucleotide polymorphisms (SNPs) of the leptin receptor gene (LEPR) based on next generation genomic sequencing (NGS) data is becoming an increasingly important aspect of diagnosis, treatment and prevention of both metabolically healthy (MHO) and metabolically unhealthy obesity (MUO) phenotypes. 35 obese children 6-18 years old were examined by the NGS method with bioinformatic analysis. The main group (n = 18) was formed by children with MUO, according to the recommendations of the expert group of the National Heart, Lung, and Blood Institute. The control group (n = 17) was represented by children with MHO. Statistical methods were used: analysis of variance, Wald's sequential analysis, Spearman's correlation analysis, analysis of nominal data and multiple discriminant analysis. 10 types of non-synonymous SNPs (rs3790435, rs1137100, rs2186248, rs70940803, rs79639154, rs1359482195, rs1137101, rs1805094, rs13306520, rs13306522) of the LEPR gene in obese children have been identified. Multiple discriminant analysis demonstrated that the following LEPR SNPs are of greatest importance in the development of MUO: rs3790435, rs13306522, rs13306520. Analysis of nominal data revealed significant differences in the groups for Copy number variation (CNV) rs3790435 of the LEPR gene. Wald's analysis allowed us to identify 6 important predictors of MUO (І ≥ 0.5): 2 CNV rs3790435 (Relative Risk, RR = 2, Prognostic coefficient, PC = +2.76); male gender of the child (RR = 1.3, PC = +1.35); rs3790435 (RR = 1.9, PC = +2.76); hyperleptinemia more than 40.56 ng/ml (RR = 2, PC = +3); CNV rs1359482195 ≥ 3 (RR = 1.9, PC = +5.8); SNP of the LEPR gene ≥4 (RR = 3.8, PC = +5.8). Children with the genotype rs3790435 gene LEPR had signs of MUO more often.

Generation of Sequencing Libraries for Building Immune Cell Methylomes.

The comparison of methylomes from immune cells enables the identification of differentially methylated regions and thereby region-associated gene loci. Those regions can be used to discriminate one immune cell population from the other, as well as help to identify key molecules and major pathways determining the unique phenotypes of immune cell lineages. The combination of bisulfite treatment of genomic DNA and next-generation sequencing provides the basis for studying epigenetic changes in different immune cell populations. Further development of whole-genome bisulfite sequencing resulted in a protocol for sequencing libraries that accept both single- or double-stranded DNA from fixed or nonfixed cells, respectively. Therefore, researchers can include immune cell populations in their methylation studies whose isolation depends on the staining of intracellular molecules.

Congenital Hypothyroidism: A Review of 2020 European Society for Pediatric Endocrinology and the European Society for Endocrinology Consensus Guidelines

Congenital Hypothyroidism: A Review of 2020 European Society for Pediatric Endocrinology and the European Society for Endocrinology Consensus Guidelines