The ENmix DNA methylation analysis pipeline for Illumina BeadChip and comparisons with seven other preprocessing pipelines.

BACKGROUND: Illumina DNA methylation arrays are high-throughput platforms for cost-effective genome-wide profiling of individual CpGs. Experimental and technical factors introduce appreciable measurement variation, some of which can be mitigated by careful "preprocessing" of raw data. METHODS: Here we describe the ENmix preprocessing pipeline and compare it to a set of seven published alternative pipelines (ChAMP, Illumina, SWAN, Funnorm, Noob, wateRmelon, and RnBeads). We use two large sets of duplicate sample measurements with 450 K and EPIC arrays, along with mixtures of isogenic methylated and unmethylated cell line DNA to compare raw data and that preprocessed via different pipelines. RESULTS: Our evaluations show that the ENmix pipeline performs the best with significantly higher correlation and lower absolute difference between duplicate pairs, higher intraclass correlation coefficients (ICC) and smaller deviations from expected methylation level in mixture experiments. In addition to the pipeline function, ENmix software provides an integrated set of functions for reading in raw data files from mouse and human arrays, quality control, data preprocessing, visualization, detection of differentially methylated regions (DMRs), estimation of cell type proportions, and calculation of methylation age clocks. ENmix is computationally efficient, flexible and allows parallel computing. To facilitate further evaluations, we make all datasets and evaluation code publicly available. CONCLUSION: Careful selection of robust data preprocessing methods is critical for DNA methylation array studies. ENmix outperformed other pipelines in our evaluations to minimize experimental variation and to improve data quality and study power.

High performance methylated DNA markers for detection of colon adenocarcinoma.

BACKGROUND: Colon cancer (CC) is treatable if detected in its early stages. Improved CC detection assays that are highly sensitive, specific, and available at point of care are needed. In this study, we systematically selected and tested methylated markers that demonstrate high sensitivity and specificity for detection of CC in tissue and circulating cell-free DNA. METHODS: Hierarchical analysis of 22 candidate CpG loci was conducted using The Cancer Genome Atlas (TCGA) COAD 450K HumanMethylation database. Methylation of 13 loci was analyzed using quantitative multiplex methylation-specific PCR (QM-MSP) in a training set of fresh frozen colon tissues (N = 53). Hypermethylated markers were identified that were highest in cancer and lowest in normal colon tissue using the 75th percentile in Mann-Whitney analyses and the receiver operating characteristic (ROC) statistic. The cumulative methylation status of the marker panel was assayed in an independent test set of fresh frozen colon tissues (N = 52) using conditions defined and locked in the training set. A minimal marker panel of 6 genes was defined based on ROC area under the curve (AUC). Plasma samples (N = 20 colorectal cancers, stage IV and N = 20 normal) were tested by cMethDNA assay to evaluate marker performance in liquid biopsy. RESULTS: In the test set of samples, compared to normal tissue, a 6-gene panel showed 100% sensitivity and 90% specificity for detection of CC, and an AUC of 1.00 (95% CI 1.00, 1.00). In stage IV colorectal cancer plasma versus normal, an 8-gene panel showed 95% sensitivity, 100% specificity, and an AUC of 0.996 (95% CI 0.986, 1.00) while a 5-gene subset showed 100% sensitivity, 100% specificity, and an AUC of 1.00 (95% CI 1.00, 1.00), highly concordant with our observations in tissue. CONCLUSIONS: We identified high performance methylated DNA marker panels for detection of CC. This knowledge has set the stage for development and implementation of novel, automated, self-contained CC detection assays in tissue and blood which can expeditiously and accurately detect colon cancer in both developed and underdeveloped regions of the world, enabling optimal use of limited resources in low- and middle-income countries.

High concordance in preimplantation genetic testing for aneuploidy between automatic identification via Ion S5 and manual identification via Miseq.

The Ion S5 (Thermo Fisher Scientific) and Miseq (Illumina) NGS systems are both widely used in the clinical laboratories conducting PGT-A. Each system employs discrepant library preparation steps, sequencing principles, and data processing algorithms. The automatic interpretation via Ion Reporter software (Thermo Fisher Scientific) and the manual interpretation via BlueFuse Multi software (Illumina) for chromosomal copy number variation (CNV) represent very different reporting approaches. Thus, it is intriguing to compare their ability of ploidy detection as PGT-A/NGS system. In the present study, four aneuploid cell lines were individually mixed with a diploid cell line at different aneuploid ratios of 0% (0:5), 10% (1:9), 20% (1:4), 40% (2:3), 50% (3:3), 60% (3:2), 80% (4:1) and 100% (5:0) to assess the sensitivity and specificity for whole chromosomal and segmental aneuploidy detection. The clinical biopsies of 107 blastocysts from 46 IVF/PGT-A cycles recruited between December 2019 and February 2020 were used to calculate the concordance. Initially, the pre-amplified products were divided into two aliquots for different library preparation procedures of each system. Applying the same calling criteria, automatic identification was achieved through the Ion Reporter, while well-trained technicians manually identified each sample through the BlueFuse Multi. The results displayed that both systems reliably distinguished chromosomal CNV of the mixtures with at least 10% aneuploidy from karyotypically normal samples ([Ion S5] whole-chromosomal duplication: 2.14 vs. 2.05, p value = 0.009, segmental deletion: 1.88 vs. 2.05, p value = 0.003; [Miseq] whole-chromosomal duplication: 2.12 vs. 2.03, p value = 0.047, segmental deletion: 1.82 vs. 2.03, p value = 0.002). The sensitivity and specificity were comparable between the Ion S5 and Miseq ([sensitivity] 93% vs. 90%, p = 0.78; [specificity] 100% vs. 100%, p value = 1.0). In the 107 clinical biopsies, three displayed chaotic patterns (2.8%), which could not be interpreted for the ploidy. The ploidy concordance was 99.04% (103/104) per embryo and 99.47% (2265/2277) per chromosome pair. Since their ability of detection were proven to be similar, the automatic identification in Ion S5 system presents comparatively faster and more standardized performance.

Two genetic analyses to elucidate causality between body mass index and personality.

BACKGROUND/OBJECTIVES: Many personality traits correlate with BMI, but the existence and direction of causal links between them are unclear. If personality influences BMI, knowing this causal direction could inform weight management strategies. Knowing that BMI instead influences personality would contribute to a better understanding of the mechanisms of personality development and the possible psychological effects of weight change. We tested the existence and direction of causal links between BMI and personality. SUBJECTS/METHODS: We employed two genetically informed methods. In Mendelian randomization, allele scores were calculated to summarize genetic propensity for the personality traits neuroticism, worry, and depressive affect and used to predict BMI in an independent sample (N = 3 541). Similarly, an allele score for BMI was used to predict eating-specific and domain-general phenotypic personality scores (PPSs; aggregate scores of personality traits weighted by BMI). In a direction of causation (DoC) analysis, twin data from five countries (N = 5424) were used to assess the fit of four alternative models: PPSs influencing BMI, BMI influencing PPSs, reciprocal causation, and no causation. RESULTS: In Mendelian randomization, the allele score for BMI predicted domain-general (ß = 0.05; 95% CI: 0.02, 0.08; P = 0.003) and eating-specific PPS (ß = 0.06; 95% CI: 0.03, 0.09; P < 0.001). The allele score for worry also predicted BMI (ß = -0.05; 95% CI: -0.08, -0.02; P < 0.001), while those for neuroticism and depressive affect did not (P ≥ 0.459). In DoC, BMI similarly predicted domain-general (ß = 0.21; 95% CI:, 0.18, 0.24; P < 0.001) and eating-specific personality traits (ß = 0.19; 95% CI:, 0.16, 0.22; P < 0.001), suggesting causality from BMI to personality traits. In exploratory analyses, links between BMI and domain-general personality traits appeared reciprocal for higher-weight individuals (BMI > ~25). CONCLUSIONS: Although both genetic analyses suggested an influence of BMI on personality traits, it is not yet known if weight management interventions could influence personality. Personality traits may influence BMI in turn, but effects in this direction appeared weaker.

Use of a Standardized Tool to Identify Women at Risk for Hereditary Breast and Ovarian.

OBJECTIVE: To increase rates of identification and genetic counseling referral for women at risk of hereditary breast and ovarian cancer (HBOC). DESIGN: Evidence-based practice improvement initiative. SETTING/LOCAL PROBLEM: Private suburban obstetric and gynecologic (OB/GYN) practice in Tennessee with no standardized process for HBOC risk assessment or referral to genetic services. PARTICIPANTS: Provider-led women's health care teams delivering well-woman care for women ages 18 years and older. INTERVENTION/MEASUREMENTS: We implemented the use of a standardized familial risk assessment tool and clinical decision-making algorithm. Preimplementation and postimplementation risk identification and genetic services referral rates were measured, as was clinicians' compliance with using the risk assessment tool. The aim of the initiative was to increase identification and referral rates by 25 percentage points. RESULTS: Women at risk of HBOC in the postimplementation group were 25.9 times more likely to be identified as being at risk (OR = 25.88, 95% confidence interval [10.78, 62.14]) and 31.5 times more likely to be offered referral to genetic counseling (OR = 31.50, 95% CI [13.37, 74.22]) compared with those in the preimplementation group. Rates of risk identification and referral to genetic counseling for women at risk of HBOC improved by 58.2 and 69.3 percentage points, respectively, surpassing the aims of this initiative and showing statistical significance of p < .001 for both indices. CONCLUSION: The use of a standardized risk assessment tool and process for HBOC risk identification and genetic referral resulted in a significant increase in the identification and referral of women at risk in this setting. Early identification of women with HBOC is a crucial first step in increasing the use of enhanced screening and interventions that can reduce HBOC-associated cancer morbidity and mortality.

Genetic algorithm optimized node deployment in IEEE 802.15.4 potato and wheat crop monitoring infrastructure.

This proposal investigates the effect of vegetation height and density on received signal strength between two sensor nodes communicating under IEEE 802.15.4 wireless standard. With the aim of investigating the path loss coefficient of 2.4 GHz radio signal in an IEEE 802.15.4 precision agriculture monitoring infrastructure, measurement campaigns were carried out in different growing stages of potato and wheat crops. Experimental observations indicate that initial node deployment in the wheat crop experiences network dis-connectivity due to increased signal attenuation, which is due to the growth of wheat vegetation height and density in the grain-filling and physical-maturity periods. An empirical measurement-based path loss model is formulated to identify the received signal strength in different crop growth stages. Further, a NSGA-II multi-objective evolutionary computation is performed to generate initial node deployment and is optimized over increased coverage, reduced over-coverage, and received signal strength. The results show the development of a reliable wireless sensor network infrastructure for wheat crop monitoring.

Oncotype DX and Prosigna in breast cancer patients: A comparison study.

BACKGROUND: Oncotype Dx® (ODX) is the most used prognostic and predictive assay for ER + breast cancer (BCa) and is categorized into low (< 18), intermediate (18 to 30), or high (≥31) risk of recurrence. Prosigna® is a prognostic signature to estimate distant recurrence-free survival for stage I/II, ER+ cancer in postmenopausal women treated with adjuvant therapy. The goal of the study is to assess the agreement between ODX and Prosigna®. MATERIALS AND METHODS: 100 previously ODX classified peri and postmenopausal, early-stage (I or II) BCa patients were retrieved and Prosigna assay was performed on archived tumor blocks on a NanoString nCounter® DX Analysis System. RESULTS: ODX assay was assigned as follows: 57% low, 39% intermediate, and 4% high. There were 8% two-step disagreements (high to low or vice versa) between ODX and Prosigna®; and 42% one-step disagreement (low to intermediate or vice versa). 78% were classified by Prosigna as luminal A and 22% as luminal B. The majority of luminal A cases (67/78; 85.9%) had low ROR score whereas ODX classified almost two-thirds (50/78~ 64%) as low RS. An insignificant percentage of luminal B cases (1/22 - 4.5%) were classified as high RS by ODX, and a modest percentage were classified as high ROR by Prosigna (15/22 ~68%). According to our follow up results, recurrence was detected in three cases. In all three cases; Prosigna was a better indicator of recurrence. CONCLUSIONS: The agreement between ODX and Prosigna® is low, and this has management implications, especially when chemotherapy is needed.

Genetic Analysis of Pediatric Pancreatoblastoma: A Case Report.

ABSTRACT: Pediatric pancreatoblastoma (PBL) is a rare disease, and the treatment of which is diverse. The molecular alteration in pancreatoblastoma is not very clear. A 7-year-old female who presented with intermittent abdominal pain, anorexia, and abdominal mass was admitted in our hospital. Pancreaticoduodenectomy, cholecystectomy, and retroperitoneal lymph node dissection were conducted. Immunohistochemistry results after surgery showed creatine kinase +, clusters differentiation 56 -, clusters differentiation 99 +, carcinoembryonic antigen -, periodic acid-Schiff +, B- catenin +, Ki-67 + 70%, progesterone receptor +, neuron-specific enolase -, vimentin -, and insulin -. According to the cell shape and the results of immunohistochemistry, the patient was diagnosed with PBL. The tumor tissue, adjacent tissue, and blood were collected. Mutation profiles were detected using next-generation sequencing technique with a panel of 704 genes. The child recovered well without complications postoperatively. There were 261 genes mutated in her plasma or tumor tissue (mutant frequency ≥1%). The adjacent tissue and plasma harbored the echinoderm microtubule-associated protein-like 4 gene-anaplastic lymphoma kinase fusion and tumor tissue harbored proto-oncogene receptor tyrosine kinase 1-solute carrier family 34 member A2 fusion. The gene alteration profiles of PBL patients warrant further investigations, which may provide new insight into the treatment of this disease.

Comparative evaluation of autosomal STRs and X-chromosome STRs as a complement of autosomal STRs in kinship testing in Southern Han Chinese.

Nowadays, kinship testing is very common in forensic caseworks, but the power of autosomal short tandem repeats (A-STRs) may be limited in complex cases. X-Chromosome short tandem repeats (X-STRs), having a unique heritage mode, should be of special use in some deficient cases. To evaluate and compare the potential of A-STR and X-STR as supplement genetic markers in deficient kinship testing, we simulated 10,000 duos for each of 18 kinds of relationships involving full sibling, half-sibling, grandparent-grandchild, and uncle/aunt-nephew/niece. Loci from STRTyper10, PowerPlex 16, and Investigator Argus X-12 were studied in Southern Han Chinese and the distribution of likelihood ratio (LR) values was analysed. With the addition of the X-12 system, the distribution of LR values for the full sisters, paternal half-sisters, paternal grandmother-granddaughters, maternal aunt-nieces, and maternal aunt-nephews separated much more obviously from those of unrelated duos, and the effectiveness was 1.0000, 0.99865, 0.9991, 0.8996 and 0.9634, respectively, which was more efficient than A-STRs. For the individual duos with other relationships, the effects of adding X-STRs and A-STRs were similar. Therefore, for the Southern Han Chinese, X-STRs can be very useful in kinship testing involving full sisters, paternal half-sisters, paternal grandmother-granddaughters, and maternal aunt-nieces/nephews.

Case-Based Review and Clinical Guidance on the Use of Genomic Assays for Early-Stage Breast Cancer: Breast Cancer Therapy Expert Group (BCTEG).

In addition to classical clinicopathologic factors, such as hormone receptor positivity, human epidermal growth factor receptor 2 (HER2) status, and tumor size, grade, and lymph node status, a number of commercially available genomic tests may be used to help inform treatment decisions for early breast cancer patients. Although these tests improve our understanding of breast cancer and help to individualize treatment decisions, clinicians face challenges when deciding on the most appropriate test to order, and the advantages, if any, of one test over another. The Breast Cancer Therapy Expert Group (BCTEG) recently convened a roundtable meeting to discuss issues surrounding the use of genomic testing in early breast cancer, with the goal of providing practical guidance on the use of these tests by the community oncologist, for whom breast cancer may be only one of many tumor types they treat. The group recognizes that genomic testing can provide important prognostic (eg, risk for recurrence), and in some cases predictive, information (eg, benefit of chemotherapy, or extended adjuvant endocrine therapy), which can be used to help guide treatment decisions in breast cancer. The available tests differ in the types of information they provide, and in the patient populations and clinical trials that were conducted to validate them. We summarize the discussion of the BCTEG on this topic, and we also consider several patient cases and clinical scenarios in which genomic testing may, or may not, be useful to guide treatment decisions for the practicing community oncologist.

Whole Exome Sequencing Identifies Novel Genetic Alterations in Patients with Pheochromocytoma/Paraganglioma.

BACKGROUND: Pheochromocytoma and paragangliomas (PPGL) are known as tumors with the highest level of heritability, approximately 30% of all cases. Clinical practice guidelines of PPGL recommend genetic testing for germline variants in all patients. In this study, we used whole exome sequencing to identify novel causative variants associated with PPGL to improve the detection of rare genetic variants in our cohort. METHODS: Thirty-six tested negative for pathogenic variants in previous Sanger sequencing or targeted gene panel testing for PPGL underwent whole exome sequencing. Whole exome sequencing was performed using DNA samples enriched using TruSeq Custom Enrichment Kit and sequenced with MiSeq (Illumina Inc.). Sequencing alignment and variant calling were performed using SAMtools. RESULTS: Among previously mutation undetected 36 patients, two likely pathogenic variants and 13 variants of uncertain significance (VUS) were detected in 32 pheochromocytoma-related genes. SDHA c.778G>A (p.Gly260Arg) was detected in a patient with head and neck paraganglioma, and KIF1B c.2787-2A>C in a patient with a bladder paraganglioma. Additionally, a likely pathogenic variant in BRCA2, VUS in TP53, and VUS in NFU1 were detected. CONCLUSION: Exome sequencing further identified genetic alterations by 5.6% in previously mutation undetected patients in PPGL. Implementation of targeted gene sequencing consisted of extended genes of PPGL in routine clinical screening can support the level of comprehensive patient assessment.

3D-printed Point-of-Care Platform for Genetic Testing of Infectious Diseases Directly in Human Samples Using Acoustic Sensors and a Smartphone.

The objective of this work is to develop a methodology and associated platform for nucleic acid detection at the point-of-care (POC) that is sensitive, user-friendly, affordable, rapid, and robust. The heart of this system is an acoustic wave sensor, based on a Surface Acoustic Wave (SAW) or Quartz Crystal Microbalance (QCM) device, which is employed for the label-free detection of isothermally amplified target DNA. Nucleic acids amplification and detection is demonstrated inside three crude human samples, i.e., whole blood, saliva, and nasal swab, spiked in with 10-100 Salmonella cells. To qualify for POC applications, a portable platform was developed based on 3D printing, integrating inside a single box: (i) simple fluidics based on plastic tubing and a mini peristaltic pump, (ii) a heating plate combined with disposable reaction tubes for isothermal amplification; (iii) a mini antenna analyzer operated through a tablet; and (iv) an acoustic wave device housing unit. The simplicity of the method combined with smartphone operation and detection, rapid sample-to-answer analysis time (30 min), and high performance (detection limit 4 × 103 CFU/ml) in three of the most important human samples in diagnostics suggest that the methodology could become a tool of choice for nucleic acid detection at the POC. In addition, the low cost of the platform and assay holds promise for its adoption in resource limited areas. The acoustic detection method is shown to give similar results with a standard colorimetric assay carried out in saliva and nasal swab but can also be used to detect nucleic acids inside whole blood, where a colorimetric assay failed to perform.

VarWatch-A stand-alone software tool for variant matching.

Massively parallel DNA sequencing of clinical samples holds great promise for the gene-based diagnosis of human inherited diseases because it allows rapid detection of putatively causative mutations at genome-wide level. Without additional evidence complementing their initial bioinformatics evaluation, however, the clinical relevance of such candidate genetic variants often remains unclear. In consequence, dedicated 'matching' services have been established in recent years that aim at the discovery of other, comparable case reports to facilitate individual diagnoses. However, legal concerns have been raised about the global sharing of genetic data, particularly in Europe where the recently enacted General Data Protection Regulation EU-2016/679 classifies genetic data as highly sensitive. Hence, unrestricted sharing of genetic data from clinical cases on platforms outside the national jurisdiction increasingly may be perceived as problematic. To allow collaborative data producers, particularly large consortia of diagnostic laboratories, to acknowledge these concerns while still practicing efficient case matching internally, novel tools are required. To this end, we developed VarWatch, an easy-to-deploy and highly scalable case matching software that provides users with comprehensive programmatic tools and a user-friendly interface to fulfil said purpose.

Integrated routine workflow using next-generation sequencing and a fully-automated platform for the detection of KRAS, NRAS and BRAF mutations in formalin-fixed paraffin embedded samples with poor DNA quality in patients with colorectal carcinoma.

BACKGROUND: KRAS and NRAS mutations are identified resistance mutations to anti-epidermal growth factor receptor monoclonal antibodies in patients with metastatic colorectal cancer. BRAF status is also routinely assessed for its poor prognosis value. In our institute, next-generation sequencing (NGS) is routinely used for gene-panel mutations detection including KRAS, NRAS and BRAF, but DNA quality is sometimes not sufficient for sequencing. In our routine practice, Idylla platform is used for the analysis of samples that don't reach sufficient quality criteria for NGS assay. METHODS: In this study, data from mCRC samples analyzed from May 2017 to 2018 were retrospectively collected. All samples with a poor DNA quality for sequencing have been assessed using Idylla platform. First, KRAS Idylla assay cartridge has been used for the determination of KRAS mutational status. All KRAS wild-type samples have then been analyzed using NRAS-BRAF assay. Among 669 samples, 67 samples failed the DNA quality control and have been assessed on Idylla KRAS mutation test. RESULTS: Among 67 samples, 50 (75%) samples had a valid result with Idylla KRAS mutation test including 22 carrying a KRAS mutation. For 28 samples, NRAS and BRAF mutational statuses have been assessed using Idylla NRAS-BRAF mutation test. Among 28 samples, 27 (96%) had a valid result including 2 samples bearing a NRAS mutation and 3 samples bearing a BRAF mutation. CONCLUSIONS: Our study shows that an integrated workflow using NGS and Idylla platform allows the determination of KRAS, NRAS and BRAF mutational statuses of 651/669 (97.3%) samples and retrieve 49/67 (73.1%)samples that don't reach DNA quality requirements for NGS.

Interinstitutional variation in predictive value of the ThyroSeq v2 genomic classifier for cytologically indeterminate thyroid nodules.

BACKGROUND: The ThyroSeq v2 next-generation sequencing assay estimates the probability of malignancy in indeterminate thyroid nodules. Its diagnostic accuracy in different practice settings and patient populations is not well understood. METHODS: We analyzed 273 Bethesda III/IV indeterminate thyroid nodules evaluated with ThyroSeq at 4 institutions: 2 comprehensive cancer centers (n = 98 and 102), a multicenter health care system (n = 60), and an academic medical center (n = 13). The positive and negative predictive values of ThyroSeq and distribution of final pathologic diagnoses were analyzed and compared with values predicted by Bayes theorem. RESULTS: Across 4 institutions, the positive predictive value was 35% (22%-43%) and negative predictive value was 93% (88%-100%). Predictive values correlated closely with Bayes theorem estimates (r2 = 0.84), although positive predictive values were lower than expected. RAS mutations were the most common molecular alteration. Among 84 RAS-mutated nodules, malignancy risk was variable (25%, range 10%-37%) and distribution of benign diagnoses differed across institutions (adenoma/hyperplasia 12%-85%, noninvasive follicular thyroid neoplasm with papillary-like nuclear features 5%-46%). CONCLUSION: In a multi-institutional analysis, ThyroSeq positive predictive values were variable and lower than expected. This is attributable to differences in the prevalence of malignancy and variability in pathologist interpretations of noninvasive tumors. It is important that clinicians understand ThyroSeq performance in their practice setting when evaluating these results.

Rapid preimplantation genetic screening using a handheld, nanopore-based DNA sequencer.

OBJECTIVE: To determine if a handheld, nanopore-based DNA sequencer can be used for rapid preimplantation genetic screening (PGS). DESIGN: Laboratory study. SETTING: Academic medical center. PATIENT(S): Amplified genomic DNA from euploid and aneuploid trophectoderm biopsy samples (n=9) that was also tested using traditional next generation sequencing (NGS). INTERVENTION(S): Short-read DNA library preparation and nanopore-based sequencing using a hand-held MinION sequencer. MAIN OUTCOME MEASURE(S): Comparison of cytogenetic testing result from NGS and nanopore-based sequencing and the time required for library preparation and sequencing. RESULT(S): Multiplexed short-read DNA library preparation was completed in 45 minutes. Sequencing on a single sample was completed within 20 minutes and 5 samples were simultaneously sequenced in under 2 hours. Whole-chromosome aneuploidy screening results obtained from nanopore-based sequencing were identical to those obtained using NGS. CONCLUSION(S): Here we report the first application of nanopore-based sequencing for PGS on trophectoderm biopsy samples using a novel rapid multiplxed short-read nanopore sequencing library preparation protocol. Sequencing for aneuploidy screening could be performed on a single sample in 20 minutes and on 5 samples, simultaneously, within 2 hours. Overall, nanopore sequencing is a promising tool to perform rapid PGS onsite, enabling same day testing and embryo transfer, thus obviating the need for complex, large and expensive DNA sequencers or embryo freezing.

[A novel quantitative JAK2V617F detection kit: prospective clinical performance study comparing MPN patients and healthy subjects].

The JAK2V617F mutation is the commonest major genetic mutation of myeloproliferative neoplasms (MPNs) and has been defined in the WHO diagnostic criteria for MPNs. However, there is still no approved in vitro diagnostic test kit available in Japan. We evaluated a JAK2V617F allele quantification kit (test method) in a prospective, multicenter clinical performance study involving patients with MPNs who were diagnosed with polycythemia vera, essential thrombocythemia, and primary myelofibrosis; healthy volunteers were also included in the analysis. Good correlation was observed between the allele burden determined using the test method vs. that determined using next-generation sequencing (NGS) in the patient group (r=0.998, y=1.071x-0.069; n=156). Furthermore, all allele burdens in the healthy group (n=54) were below the lower limit of the measurement range of the test method (0.042%). Our results confirmed that the test method could quantitatively measure the JAK2V617F allele burden in patients with MPN. Thus, the novel JAK2V617F allele quantification kit can be considered useful for the diagnosis of MPNs.

Minimally invasive skin tape strip RNA sequencing identifies novel characteristics of the type 2-high atopic dermatitis disease endotype.

BACKGROUND: Expression profiling of skin biopsy specimens has established molecular features of the skin in patients with atopic dermatitis (AD). The invasiveness of biopsies has prevented their use in defining individual-level AD pathobiological mechanisms (endotypes) in large research studies. OBJECTIVE: We sought to determine whether minimally invasive skin tape strip transcriptome analysis identifies gene expression dysregulation in AD and molecular disease endotypes. METHODS: We sampled nonlesional and lesional skin tape strips and biopsy specimens from white adult patients with AD (18 male and 12 female patients; age [mean ± SE], 36.3 ± 2.2 years) and healthy control subjects (9 male and 16 female subjects; age [mean ± SE], 34.8 ± 2.2 years). AmpliSeq whole-transcriptome sequencing was performed on extracted RNA. Differential expression, clustering/pathway analyses, immunostaining of skin biopsy specimens, and clinical trait correlations were performed. RESULTS: Skin tape expression profiles were distinct from skin biopsy profiles and better sampled epidermal differentiation complex genes. Skin tape expression of 29 immune and epidermis-related genes (false discovery rate < 5%) separated patients with AD from healthy subjects. Agnostic gene set analyses and clustering revealed 50% of patients with AD exhibited a type 2 inflammatory signature (type 2-high endotype) characterized by differential expression of 656 genes, including overexpression of IL13, IL4R, CCL22, CCR4 (log2 fold change = 5.5, 2.0, 4.0, and 4.1, respectively) and at a pathway level by TH2/dendritic cell activation. Both expression and immunostaining of skin biopsy specimens indicated this type 2-high group was enriched for inflammatory, type 2-skewed dendritic cells expressing FcεRI. The type 2-high endotype group exhibited more severe disease by using both the Eczema Area and Severity Index score and body surface area covered by lesions. CONCLUSION: Minimally invasive expression profiling of nonlesional skin reveals stratification in AD molecular pathology by type 2 inflammation that correlates with disease severity.