Targeted Sequencing Technology Research Articles

Clinical application of nanopore-targeted sequencing technology in bronchoalveolar lavage fluid from patients with pulmonary infections.

The rapid and effective identification of pathogens in patients with pulmonary infections has posed a persistent challenge in medicine, with conventional microbiological tests (CMTs) proving time-consuming and less sensitive, hindering early diagnosis of respiratory infections. While there has been some research on the clinical performance of targeted sequencing technologies, limited focus has been directed toward bronchoalveolar lavage fluid (BALF). This study primarily evaluates the pathogen detection capabilities of nanopore-targeted sequencing (NTS) in BALF, providing a comprehensive analysis. The retrospective study, spanning from January 2022 to November 2023, includes 223 patients exclusively sourced from a single center. We conducted a detailed comparative analysis among NTS, targeted next-generation sequencing (tNGS), and CMTs. Initially, we compared the detection capabilities of NTS and tNGS and found no significant differences in their sensitivity and specificity. Specifically, we observed that the sensitivity of NTS was significantly higher than that of CMTs (74.83% vs 33.11%, P < 0.001). Furthermore, NTS exhibited a higher positivity rate in common pulmonary infections (62.88% vs. 23.48%) and in clinically suspected tuberculosis patients compared to CMTs (87.18% vs. 48.72%). Additionally, NTS showed less susceptibility to antibiotic interference, indicating a more sensitive detection capability, especially in detecting fastidious organisms. It complements GeneXpert in tuberculosis diagnosis and offers excellent advantages in identifying pathogens challenging for CMTs, such as non-tuberculous mycobacteria and viruses. Moreover, NTS significantly shortens the reporting time and is only a quarter of the cost of metagenomic next-generation sequencing. Clearly, NTS can facilitate faster and more cost-effective early diagnosis of respiratory infections.IMPORTANCEThis study holds paramount significance in advancing the field of respiratory infection diagnostics. By assessing the pathogen detection capabilities in bronchoalveolar lavage fluid (BALF) of patients with pulmonary infections, we illuminate the promising potential of nanopore-targeted sequencing (NTS). The findings underscore NTS as a comparable yet distinct alternative to traditional methods like comprehensive conventional microbiological tests (CMTs). Notably, NTS demonstrates a pivotal edge, expanding the spectrum of identified pathogens, particularly excelling in the detection of challenging entities like non-tuberculous mycobacteria and viruses. The study also highlights the complementary role of NTS alongside GeneXpert in the identification of tuberculosis, providing a comprehensive overview of the diagnostic landscape for respiratory infections. This insight carries significant implications for clinicians seeking rapid, cost-effective, and accurate diagnostic tools in the realm of pulmonary infections.

The Application of Targeted Sequencing Technology in Clinical Practice

The Application of Targeted Sequencing Technology in Clinical Practice

Designing and evaluating a cost‐effective single nucleotide polymorphism liquid array for Chinese native chickens

AbstractGenotyping plays an important role in breeding and population studies. Currently available genotyping technologies, including solid‐phase chips and sequencing, often have several limitations in their application to local chickens in China, including high costs, insufficient diversity, and poor universality. In the present study, we developed a chicken 5 K single nucleotide polymorphism (SNP) array suitable for breeding and genetic analysis using genotyping by targeted sequencing technology. The chip design was based on genomic data from 33 local breeds, and 5847 SNPs were selected for the final chip design. Among these SNPs, 3427 sites were associated with economic traits in broiler chickens. Our chip contained 25,000 high‐quality SNP markers captured from 5 K regions with highly efficient target site capture. Population analyses of the eight breeds showed high detection rates and minor allele frequencies for SNP markers on the array, enabling clear differentiation of different populations and families within populations. The genetic diversity trend obtained using the chip was consistent with the results obtained from microsatellite analyses. Additionally, the 5 K array was applied to a genome‐wide association study of broilers, resulting in the identification of several growth‐ and meat quality‐related loci. Therefore, the newly developed chip is efficient, cost‐effective, and well suited for application in local Chinese chickens, which will accelerate poultry breeding improvements and enhance conservation efforts.

HERCULES: A prostate cancer (PC) sequencing panel for parallel analysis of circulating tumor DNA (ctDNA) and circulating tumor cells (CTCs).

5036 Background: In metastatic PC, tracking genomic alterations over time and with treatment can predict disease outcomes, guide treatment, and identify novel therapeutic targets. Analysis of peripheral blood ctDNA offers a non-invasive liquid biopsy for genomic profiling, but ctDNA often comprises a small portion of total cell-free DNA (cfDNA) in the blood, requiring high depth of sequencing which may still miss PC-relevant variants. We and others have shown that single CTCs captured from the same blood sample yield additional PC-relevant genomic data when combined with ctDNA profiling, but currently there is no assay that can be applied uniformly to both analytes. Here, we present a new amplicon-based sequencing panel capable of detecting PC-relevant variants in both cfDNA and single CTCs. Methods: PC-relevant genes were curated by overlapping reviews of cBioPortal for cancer genomics, the catalogue of somatic mutations in cancer (COSMIC), and PC-relevant genomic literature. HERCULES targets 36 genes with 258 amplicons covering PC-associated mutations and copy number variations. AmpliSeq HD targeted sequencing technology was applied for analysis of both cfDNA and CTCs. Analytical validation was performed sequentially, first using AcroMetrix and Seracare DNA control samples, then single and pooled LNCap and PC3 PC cells and cfDNA, then 8 patient peripheral blood samples of matched plasma cfDNA and single CTCs captured using the RareCyte platform from participants in SWOG S1802, an ongoing CTEP/CIRB-approved phase 3 trial for men with metastatic PC. Results: In DNA control samples with allele frequencies of 1%, 0.25%, and 0.1%, HERCULES displayed sensitivity of 100%, 75%, and 50%, respectively. In matched single cells and cfDNA from cell lines, HERCULES detected 100% of database-annotated somatic variants in cfDNA and in pools of 2 or more cells, and it identified &gt;90% of variants in single cells. In 16 matched patient samples of cfDNA and single CTCs, with read coverage depths of &gt;50,000x for 20ng cfDNA and &gt;1,500x for single CTCs, HERCULES detected shared variants (mean N per sample = 6.1) as well as unique somatic variants (mean of 1.1 CTC variants not called in the cfDNA, and 4.1 cfDNA variants not called in the CTCs). Conclusions: HERCULES is, to our knowledge, the first PC-specific targeted sequencing panel capable of parallel genomic profiling of both ctDNA and CTCs. In our preliminary analytical validation, it detected somatic variants with high sensitivity in matched cfDNA and CTC samples. With additional validation, HERCULES has the potential to augment conventional ctDNA profiling by illuminating both cell-free and cellular genomic evolution during PC treatment and progression.

Mutation characteristics of osteosarcoma: a single center study of 64 cases using next-generation sequencing

Objective: To investigate the distribution and characteristics of gene mutations in osteosarcoma, and to analyze the frequency and types of detectable mutations, and to identify potential targets for individualized treatment of osteosarcoma. Methods: The fresh tissue or paraffin-embedded tissue samples of 64 cases of osteosarcoma that were surgically resected or biopsied and then subject to next generation sequencing, were collected from Beijing Jishuitan Hospital, China from November 2018 to December 2021. The tumor DNA was extracted to detect the somatic and germline mutations using targeted sequencing technology. Results: Among the 64 patients, 41 were males and 23 were females. The patient age ranged from 6 to 65 years with a median age of 17 years, including 36 children (under 18 years old) and 28 adults. There were 52 cases of conventional osteosarcoma, 3 cases of telangiectatic osteosarcoma, 7 cases of secondary osteosarcoma, and 2 cases of parosteosarcoma. The detection rate of gene mutations was overall 84.4% (54/64). There were 324 variations in 180 mutated genes, including 125 genes with copy number variations, 109 single nucleotide variants, 83 insertions or deletions, and 7 gene fusions. The most common mutated genes were TP53, VEGFA, CCND3, ATRX, MYC, RB1, PTEN, GLI1, CDK4 and PTPRD. Among them, TP53 had the highest mutation rate (21/64, 32.8%), single nucleotide variant was the main mutation type (14/23, 60.9%), and 2 cases carried the TP53 germline mutation. VEGFA and CCND3 showed copy number amplification simultaneously in 7 cases. Conclusions: The high-frequency mutation of TP53 suggests that it plays an important role in the pathogenesis and development of osteosarcoma. VEGFA, CCND3 and ATRX are mutated genes in osteosarcoma and worthy of further studies. Combination of pathologic diagnosis and next generation sequencing with clinical practice can guide individualized treatment for patients with refractory, recurrent and metastatic osteosarcoma.

Targeted Identification of Rice Grain-Associated Gene Allelic Variation Through Mutation Induction, Targeted Sequencing, and Whole Genome Sequencing Combined with a Mixed-Samples Strategy

BackgroundThe mining of new allelic variation and the induction of new genetic variability are the basis for improving breeding efficiency.ResultsIn this study, in total, 3872 heavy ion-irradiated M2 generation rice seeds and individual leaves were collected. The grain length was between 8 and 10.22 mm. The grain width was between 1.54 and 2.87 mm. The results showed that there was extensive variation in granulotype. The allelic variation in GS3 and GW5 was detected in 484 mixed samples (8:1) using targeted sequencing technology, and 12 mixed samples containing potential mutations and 15 SNPs were obtained; combined with Sanger sequencing and phenotype data, 13 key mutants and their corresponding SNPs were obtained; protein structural and functional analysis of key mutants screened out 6 allelic variants leading to altered grain shape, as well as the corresponding mutants, including long-grain mutants GS3-2 and GS3-7, short-grain mutants GS3-3 and GS3-5, wide-grain mutant GW5-1 and narrow-grain mutant GW5-4; whole genome sequencing identified new grain length gene allelic variants GS3-G1, GS3-G2 and GS3-G3.ConclusionBased on the above studies, we found 6 granulotype mutants and 9 granulotype-related allelic variants, which provided new functional gene loci and a material basis for molecular breeding and genotype mutation and phenotype analysis. We propose a method for targeted identification of allelic variation in rice grain type genes by combining targeted sequencing of mixed samples and whole genome sequencing. The method has the characteristics of low detection cost, short detection period, and flexible detection of traits and genes.

A Novel A > G Polymorphism in the Intron 1 of LCORL Gene Is Significantly Associated with Hide Weight and Body Size in Dezhou Donkey

Simple SummaryThe annual shortage of donkey hides is significant in China. Therefore, to meet the market demand, it is important to improve donkey hide production through molecular breeding. In this study, one SNP was identified in intron 1 of the LCORL gene, and polymorphism information content (PIC) was moderate in the population. The association analysis of g.112558859 A > G with body height, body length, chest circumference and hide weight was conducted and found to be significantly associated with these traits. The hide weight of donkeys with the GG genotype was 1.07 Kg heavier than those with AA genotype. This provides the foundation for breeding large donkey breeds with high hide production.Several studies have shown the association between the ligand-dependent nuclear receptor compression-like protein (LCORL) gene and body size in horses, pigs and donkeys. Based on previous studies, the LCORL gene was hypothesized to be associated with growth traits and hide weight in Dezhou donkeys. In this study, we aimed to reveal the variation of the LCORL gene in the Dezhou donkey and explore whether the gene is associated with hide weight and body size. In this study, genetic polymorphisms in the LCORL gene of the Dezhou donkey were studied using targeted sequencing technology, and single nucleotide polymorphisms (SNPs) of the LCORL gene were analyzed for association with hide weight and body size in Dezhou donkeys. The results showed that there was an SNP locus situated in intron 1 of the LCORL gene. Association analysis revealed that individuals with the GG genotype had significantly higher body height, body length, chest circumference and hide weight than those with the AA genotype (p < 0.05). Therefore, the g.112558859 A > G locus can be used as a potential candidate marker affecting body size and hide weight. This study provides the foundation for breeding high-quality donkeys with high hide yield.

The potential, analysis and prospect of ctDNA sequencing in hepatocellular carcinoma.

BackgroundThe genome map of hepatocellular carcinoma (HCC) is complex. In order to explore whether circulating tumor cell DNA (ctDNA) can be used as the basis for sequencing and use ctDNA to find tumor related biomarkers, we analyzed the mutant genes of ctDNA in patients with liver cancer by sequencing.MethodsWe used next-generation targeted sequencing technology to identify mutations in patients with liver cancer. The ctDNA from 10 patients with hepatocellular carcinoma (including eight cases of primary hepatocellular carcinoma and two cases of secondary hepatocellular carcinoma) was sequenced. We used SAMtools to detect and screen single nucleotide polymorphisms (SNPs) and insertion deletion mutations (INDELs) and ANNOVAR to annotate the structure and function of the detected mutations. Screening of pathogenic and possible pathogenic genes was performed using American College of Medical Genetics and Genomics (ACMG) guidelines. GO analysis and KEGG analysis of pathogenic and possible pathogenic genes were performed using the DAVID database, and protein–protein interaction network analysis of pathogenic and possible pathogenic genes was performed using the STRING database. Then, the Kaplan–Meier plotter database, GEPIA database and HPA database were used to analyse the relationship between pathogenic and possible pathogenic genes and patients with liver cancer.ResultsTargeted capture and deep sequencing of 560 cancer-related genes in 10 liver cancer ctDNA samples revealed 8,950 single nucleotide variation (SNV) mutations and 70 INDELS. The most commonly mutated gene was PDE4DIP, followed by SYNE1, KMT2C, PKHD1 and FN1. We compared these results to the COSMIC database and determined that ctDNA could be used for sequencing. According to the ACMG guidelines, we identified 54 pathogenic and possible pathogenic mutations in 39 genes in exons and splice regions of 10 HCC patients and performed GO analysis, KEGG analysis, and PPI network analysis. Through further analysis, four genes significantly related to the prognosis of liver cancer were identified.ConclusionIn this study, our findings indicate that ctDNA can be used for sequencing. Our results provide some molecular data for the mapping of genetic variation in Chinese patients with liver cancer, which enriches the understanding of HCC pathogenesis and provides new ideas for the diagnosis and prognosis of HCC patients.

An improved molecular inversion probe based targeted sequencing approach for low variant allele frequency.

Deep targeted sequencing technologies are still not widely used in clinical practice due to the complexity of the methods and their cost. The Molecular Inversion Probes (MIP) technology is cost effective and scalable in the number of targets, however, suffers from low overall performance especially in GC rich regions. In order to improve the MIP performance, we sequenced a large cohort of healthy individuals (n = 4417), with a panel of 616 MIPs, at high depth in duplicates. To improve the previous state-of-the-art statistical model for low variant allele frequency, we selected 4635 potentially positive variants and validated them using amplicon sequencing. Using machine learning prediction tools, we significantly improved precision of 10–56.25% (P < 0.0004) to detect variants with VAF > 0.005. We further developed biochemically modified MIP protocol and improved its turn-around-time to ∼4 h. Our new biochemistry significantly improved uniformity, GC-Rich regions coverage, and enabled 95% on target reads in a large MIP panel of 8349 genomic targets. Overall, we demonstrate an enhancement of the MIP targeted sequencing approach in both detection of low frequency variants and in other key parameters, paving its way to become an ultrafast cost-effective research and clinical diagnostic tool.

Case study: Targeted RNA-sequencing of aged formalin-fixed paraffin-embedded samples for understanding chemical mode of action

Formalin-fixed paraffin-embedded (FFPE) samples are the only remaining biological archive for many toxicological and clinical studies, yet their use in genomics has been limited due to nucleic acid damage from formalin fixation. Older FFPE samples with highly degraded RNA pose a particularly difficult technical challenge. Probe-based targeted sequencing technologies show promise in addressing this issue but have not been directly compared to standard whole-genome RNA-Sequencing (RNA-Seq) methods. In this study, we evaluated dose-dependent transcriptional changes from paired frozen (FROZ) and FFPE liver samples stored for over 20 years using targeted resequencing (TempO-Seq) and whole-genome RNA-Seq methods. Samples were originally collected from male mice exposed to a reference chemical (dichloroacetic acid, DCA) at 0, 198, 313, and 427 mg/kg-day (n = 6/dose) by drinking water for 6 days. TempO-Seq showed high overlap in differentially expressed genes (DEGs) between matched FFPE and FROZ samples and high concordance in fold-change values across the two highest dose levels of DCA vs. control (R2 ≥ 0.94). Similarly, high concordance in fold-change values was observed between TempO-Seq FFPE and RNA-Seq FROZ results (R2 ≥ 0.92). In contrast, RNA-Seq FFPE samples showed few overlapping DEGs compared to FROZ RNA-Seq (≤5 for all dose groups). Modeling of DCA-dependent changes in gene sets identified benchmark doses from TempO-Seq FROZ and FFPE samples within 1.4-fold of RNA-Seq FROZ samples (93.9 mg/kg-d), whereas RNA-Seq FFPE samples were 3.3-fold higher (310.3 mg/kg-d). This work demonstrates that targeted sequencing may provide a more robust method for quantifying gene expression profiles from aged archival FFPE samples.

Mapping causal genes and genetic interactions for agronomic traits using a large F2 population in rice.

Dissecting the genetic mechanisms underlying agronomic traits is of great importance for crop breeding. Agronomic traits are usually controlled by multiple quantitative trait loci (QTLs) and genetic interactions, and mapping the underlying causal genes is still labor-intensive and time-consuming. Here, we present a genetic tool for directly targeting the specific causal genes by using a single-gene resolution linkage map that was constructed from 3756 F2 rice plants via targeted sequencing technology and Tukey-Kramer multiple comparisons test. Three large- and moderate-effect QTLs, qHD6-2, qGL3-1, and qGW5-2, were successfully mapped to their specific causal genes, Hd1, GS3, and GW5, respectively. A complex genetic interaction network containing 30 QTL–QTL interactions was constructed, revealing that the alternative allele of hub QTL, qHD6-2, can hide or release the genetic contributions of the alleles at interacting loci. Moreover, arranging genetic interactions in the models lead to more accurate phenotypic predictions. These results provide a community resource and new feasible strategy for deciphering the genetic mechanisms of complex agronomic traits and accelerating crop breeding.

Bordetella avium-associated endophthalmitis: case report

BackgroundBordetella avium, an aerobic bacterium that rarely causes infection in humans, is a species of Bordetella that generally inhabits the respiratory tracts of turkeys and other birds. It causes a highly contagious bordetellosis. Few reports describe B. avium as a causative agent of eye-related infections.Case presentationWe report a case of acute infectious endophthalmitis associated with infection by B. avium after open trauma. After emergency vitrectomy and subsequent broad-spectrum antibiotic treatment, the infection was controlled successfully, and the patient’s vision improved.ConclusionsB. avium can cause infection in the human eye, which can manifest as acute purulent endophthalmitis. Nanopore targeted sequencing technology can quickly identify this organism. Emergency vitrectomy combined with lens removal and silicone oil tamponade and the early application of broad-spectrum antibiotics are key for successful treatment.

Oncogenic Mutations and Gene Fusions in CD30-Positive Lymphoproliferations and Clonally Related Mycosis Fungoides Occurring in the Same Patients

The emergence of a common progenitor cell has been postulated for the association of CD30-positive lymphoproliferative disease (LPD) and mycosis fungoides (MF) within the same patient. Up to now, no comprehensive analysis has yet addressed the genetic profiles of such concurrent lymphoma subtypes. We aimed to delineate the molecular alterations of clonally related CD30-positive LPD and MF occurring in the same two patients. We analyzed the molecular profile of 16 samples of two patients suffering both from CD30-positive LPD and MF being obtained over a time course of at least 5 years. To detect oncogenic mutations, we applied targeted sequencing technologies with a hybrid capture-based DNA library preparation approach, and for the identification of fusion transcripts, an anchored multiplex PCR enrichment kit was used. In all samples of CD30-positive LPD and MF, oncogenic fusions afflicting the Jak/signal transducer and activator of transcription signaling pathway were present, namely NPM1‒TYK2 in patient 1 and ILF3‒JAK2 in patient 2. Additional signal transducer and activator of transcription 5A gene STAT5A mutations exclusively occurred in lesions of CD30-positive LPD in one patient. CD30-positive LPD and MF may share genetic events when occurring within the same patients. Constitutive activation of the Jak/signal transducer and activator of transcription signaling pathway may play a central role in the molecular pathogenesis of both entities.

Nine residues in HLA-DQ molecules determine with susceptibility and resistance to type 1 diabetes among young children in Sweden

HLA-DQ molecules account over 50% genetic risk of type 1 diabetes (T1D), but little is known about associated residues. Through next generation targeted sequencing technology and deep learning of DQ residue sequences, the aim was to uncover critical residues and their motifs associated with T1D. Our analysis uncovered (αa1, α44, α157, α196) and (β9, β30, β57, β70, β135) on the HLA-DQ molecule. Their motifs captured all known susceptibility and resistant T1D associations. Three motifs, “DCAA-YSARD” (OR = 2.10, p = 1.96*10−20), “DQAA-YYARD” (OR = 3.34, 2.69*10−72) and “DQDA-YYARD” (OR = 3.71, 1.53*10−6) corresponding to DQ2.5 and DQ8.1 (the latter two motifs) associated with susceptibility. Ten motifs were significantly associated with resistance to T1D. Collectively, homozygous DQ risk motifs accounted for 43% of DQ-T1D risk, while homozygous DQ resistant motifs accounted for 25% protection to DQ-T1D risk. Of the identified nine residues five were within or near anchoring pockets of the antigenic peptide (α44, β9, β30, β57 and β70), one was the N-terminal of the alpha chain (αa1), one in the CD4-binding region (β135), one in the putative cognate TCR-induced αβ homodimerization process (α157), and one in the intra-membrane domain of the alpha chain (α196). Finding these critical residues should allow investigations of fundamental properties of host immunity that underlie tolerance to self and organ-specific autoimmunity.

Genetic investigation of 211 Chinese families expands the mutational and phenotypical spectra of hereditary retinopathy genes through targeted sequencing technology

BackgroundHereditary retinopathy is a significant cause of blindness worldwide. Despite the discovery of many mutations in various retinopathies, a large number of patients remain genetically undiagnosed. Targeted next-generation sequencing of the human genome is a suitable approach for the molecular diagnosis of retinopathy.MethodsWe describe a cohort of 211 families from central China with various forms of retinopathy; 95 patients were investigated using multigene panel sequencing, and the other 116 with suspected Leber hereditary optic neuropathy (LHON) were tested by Sanger sequencing. The detected variation of targeted sequencing was verified by PCR-based Sanger sequencing. We performed a comprehensive analysis of the cases using sequencing data and ophthalmologic examination information.ResultsPotential causal mutations were identified in the majority of families with retinopathy (57.9% of 95 families) and suspected LHON (21.6% of 116 families). There were 68 variants of a certain significance distributed in 31 known disease-causing genes in the 95 families; 37 of the variants are novel and have not been reported to be related to hereditary retinopathy. The NGS panel solution provided a 45.3% potential diagnostic rate for retinopathy families, with candidate gene mutations of undefined pathogenicity revealed in another 12.6%of the families.ConclusionOur study uncovered novel mutations and phenotypic aspects of retinopathy and demonstrated the genetic and clinical heterogeneity of related conditions. The findings show the detection rate of pathogenic variants in patients with hereditary retinopathy in central China as well as the diversity and gene distribution of these variants. The significance of molecular genetic testing for patients with hereditary retinopathy is also highlighted.

P88.05 A Recommended one-step Targeted Sequencing Technology for Identification of a Dual CD74-ROS1 in NSCLC

P88.05 A Recommended one-step Targeted Sequencing Technology for Identification of a Dual CD74-ROS1 in NSCLC

Identification of a novel &lt;i&gt;MYH9&lt;/i&gt; mutation (p. V782Y) in a Chinese patient with thrombocytopenia: a case report

MYH9-related diseases (MYH9-RD) are a group of autosomal dominant diseases caused by mutations in the MYH9 gene, which are featured by thrombocytopenia, giant platelets and granulocyte cytoplasmic inclusion bodies. MYH9-RD patients generally suffer from bleeding syndromes, progressive kidney disease, deafness, or cataracts. Here, we reported on a case of MYH9-RD. A novel heterozygous mutation of MYH9 (c.2344-2345delGTinsTA, p.T782Y) was discovered by targeted sequencing technology. Immunofluorescence analysis of neutrophils confirmed abnormal aggregation of MYH9 protein. The results of this study should expand the MYH9 gene mutation spectrum and provide reference for subsequent researchers and genetic counseling.

Using molecular markers in breeding: ornamentals catch up

Thanks to advances in next generation sequencing it is now straightforward to develop tens or even hundreds of thousands of SNP markers. Advances in genotyping technology have made it feasible to genotype progenies of crosses, panels of genotypes, or even a complete breeding program, by using arrays with tens of thousands of SNPs, or by random or targeted sequencing technologies. Recently software has been developed for dosage scoring and linkage mapping in polyploid crops. This means that advanced genetic analyses can now also be performed in many polyploid ornamentals. A DNA marker, such as a single nucleotide polymorphism (SNP), linked to a trait enables following a gene or allele during crosses and in a breeding program. Association of a SNP marker to a trait or a component of a trait may be done through QTL analysis in segregating populations, by genome-wide association analysis (GWAS) in a set of accessions, or through an analysis across a pedigree. In these analyses, a dense linkage map is a very important tool, to delineate and possibly narrow down the QTL interval and to filter away false positive SNPs. New developments in linkage mapping include paying attention to even marker coverage, and new ways to use markers to infer haplotypes. The latter is especially important in polyploids, in populations with multiple parents, or in wide panels used for association studies. In such cases multiple functional alleles may segregate simultaneously, that cannot all be tagged uniquely by single biallelic SNP markers.

Recurrent Oncogenic JAK and STAT Alterations in Cutaneous CD30-Positive Lymphoproliferative Disorders

The group of cutaneous CD30-positive lymphoproliferative disorders (LPD) comprises two different entities, namely lymphomatoid papulosis (LyP) and cutaneous anaplastic large T-cell lymphoma (cALCL). LyP constitutes a benign lymphoproliferation with spontaneously regressing papules, whereas cALCL presents with solitary or multiple skin tumors with a low propensity to disseminate. To elucidate the hitherto largely unknown molecular pathogenesis of these entities, we performed comprehensive next-generation sequencing in a well-characterized cohort of 12 patients. Considering the low tumor cell content of LyP, we applied targeted sequencing technologies with a hybrid capture-based DNA library preparation approach and for the identification of fusion transcripts an anchored multiplex PCR enrichment kit. As the major finding, we detected, in 50% of LPD, genetic events that implied a constitutively activated Janus kinase-signal transducer and activator of transcription signaling (JAK-STAT) pathway in these entities. The identified molecular aberrations comprised either pathogenic STAT mutations or oncogenic fusion transcripts comprising effector domains of JAK. With respect to LyP, we report to our knowledge such previously unreported genetic aberrations in this specific entity. The detection of these convergent aberrations within the JAK-STAT signaling pathway deciphers common potential driving mechanisms of lymphomagenesis within LPD being shared between LyP and cALCL. Moreover, the presence of these oncogenic alterations paves the way to develop novel personalized treatment strategies.

Identification of six novel variants in Waardenburg syndrome type II by next-generation sequencing.

BackgroundWaardenburg syndrome (WS) is a dominantly inherited, genetically heterogeneous auditory‐pigmentary syndrome characterized by nonprogressive sensorineural hearing loss and iris discoloration. This study aimed to investigate the underlying molecular pathology in Chinese WS families.MethodsA total of 13 patients with Waardenburg syndrome type II (WS2) from six unrelated Chinese families were enrolled. We investigated the mutation profile of genes related to congenital deafness in these families through a targeted sequencing technology and validated the candidate variants by Sanger sequencing.ResultsWe identified six novel variants in microphthalmia‐associated transcription factor (MITF) and SRY‐box 10 (SOX10), which were predicted to be disease causing by in silico analysis. Our results showed that mutations in SOX10 and MITF are two major causes of deafness associated with WS, and de novo mutations were frequently found in probands with SOX10 mutations but not in those with MITF mutations.ConclusionResults showed that targeted next‐generation sequencing (NGS) enabled us to detect disease‐causing mutations with high accuracy, stability, speed and throughput. Our study extends the pathogenic mutation spectrum of MITF and SOX10.