Targeted Next-generation Sequencing Method Research Articles

Identifying the Pathogenic Variants in Heart Genes in Vietnamese Sudden Unexplained Death Victims by Next-Generation Sequencing.

In forensics, one-third of sudden deaths remain unexplained after a forensic autopsy. A majority of these sudden unexplained deaths (SUDs) are considered to be caused by inherited cardiovascular diseases. In this study, we investigated 40 young SUD cases (<40 years), with non-diagnostic structural cardiac abnormalities, using Targeted NGS (next-generation sequencing) for 167 genes previously associated with inherited cardiomyopathies and channelopathies. Fifteen cases identified 17 variants on related genes including the following: AKAP9, CSRP3, GSN, HTRA1, KCNA5, LAMA4, MYBPC3, MYH6, MYLK, RYR2, SCN5A, SCN10A, SLC4A3, TNNI3, TNNI3K, and TNNT2. Of these, eight variants were novel, and nine variants were reported in the ClinVar database. Five were determined to be pathogenic and four were not evaluated. The novel and unevaluated variants were predicted by using in silico tools, which revealed that four novel variants (c.5187_5188dup, p.Arg1730llefsTer4 in the AKAP9 gene; c.1454A>T, p.Lys485Met in the MYH6 gene; c.2535+1G>A in the SLC4A3 gene; and c.10498G>T, p.Asp3500Tyr in the RYR2 gene) were pathogenic and three variants (c.292C>G, p.Arg98Gly in the TNNI3 gene; c.683C>A, p.Pro228His in the KCN5A gene; and c.2275G>A, p.Glu759Lys in the MYBPC3 gene) still need to be further verified experimentally. The results of our study contributed to the general understanding of the causes of SUDs. They provided a scientific basis for screening the risk of sudden death in family members of victims. They also suggested that the Targeted NGS method may be used to identify the pathogenic variants in SUD victims.

Simultaneous genotyping for human platelet antigen systems and HLA-A and HLA-B loci by targeted next-generation sequencing.

In order to treat the alloimmunization platelet transfusion refractoriness (PTR), human leukocyte antigen (HLA)-type and/or human platelet antigen (HPA)-type matched platelets between donors and patients are usually used. Therefore, genotyping of HLA-A and HLA-B loci, as well as HPA systems, for donors and patients, is of great significance. However, there is a rare report of genotyping for HLA-A and HLA-B loci as well as HPA systems at the same time. In this study, a high-throughput method for simultaneous genotyping of HLA-A and HLA-B loci, as well as HPA genotyping, was developed. A RNA capture probe panel was designed covering all exon sequences of the GP1BA, GP1BB, ITGA2, CD109, ITGB3, and ITGA2B genes and HLA-A and HLA-B loci. The HLA-A, HLA-B, and 34 HPA systems were genotyped using a targeted next-generation sequencing (NGS) method. The genotypes of the HLA-A and HLA-B loci, as well as the HPA, were assigned based on the nucleotides in the polymorphism sites. Using the NGS method, 204 unrelated blood specimens were successfully genotyped for all 34 HPA systems as well as HLA-A and HLA-B loci. The accuracy of the NGS method was 100%. Only HPA-2, HPA-3, HPA-5, HPA-6w, HPA-15, and HPA-21w showed polymorphism with frequencies of 0.9412, 0.6863, 0.9853, 0.9779, 0.4314, and 0.9951 for a allele, respectively. Thirty-two single nucleotide variants (SNVs) were detected. Of them, 12 SNVs can lead to amino acid change. HLA-A*11:01 and HLA-B*46:01 are the most common alleles for HLA-A and HLA-B loci. A targeted next-generation sequencing method for simultaneously genotyping HPA systems and HLA-A and HLA-B loci was first established, which could be used to create a database of HLA-typed and/or HPA-typed unrelated donors.

Culture-Free Phylogenetic Analysis of Legionella pneumophila Using Targeted CRISPR/Cas9 Next-Generation Sequencing.

ABSTRACTCurrently available methods for the laboratory investigation of Legionella pneumophila outbreaks require organism culture. The ability to sequence L. pneumophila directly from clinical samples would significantly reduce delays. Here, we develop a method for targeted next-generation sequencing (NGS) of selected L. pneumophila genes utilizing a CRISPR/Cas9-based target enrichment system. We determine the method’s utility by typing cultured L. pneumophila isolates and subsequently apply the method directly to patient samples. We sequenced 10 L. pneumophila isolates by 2 methods, (i) whole-genome sequencing (WGS) and (ii) targeted (CRISPR/Cas9-based) finding low-abundance sequences by hybridization (FLASH)-NGS, sequencing 57 selected genes. The targeted NGS of 57 genes was more efficient than WGS, and phylogenetic analysis of the 57 genes yielded the same classification of the L. pneumophila isolates as that based on analysis of whole-genome data. Furthermore, targeted NGS of L. pneumophila performed directly on patient respiratory samples correctly classified the patients according to their corresponding cultured isolates. This provides proof of concept that targeted NGS can be used to sequence L. pneumophila directly from patient samples. Studies on a larger number of patient samples will further validate this method. Nonetheless, CRISPR/Cas9 targeted NGS methods have the potential to be widely applicable to microbial-outbreak investigations in the future, particularly in the context of difficult and slow-growing organisms.IMPORTANCE The bacterium Legionella pneumophila is responsible for outbreaks of serious and life-threatening pneumonia called Legionnaires’ disease. There is a need for new molecular methods that allow investigation of Legionella outbreaks directly from patient samples, without the need for prior microbiological culture, which causes delays. Our study aims to address this problem. We have utilized a CRISPR/Cas9-based targeted next-generation sequencing (NGS) method that can be applied directly on human specimens. Furthermore, we show that analysis of the sequences of a small number of targeted genes offers the same classification of L. pneumophila as that based on data derived from the whole genome. Given the rising interest globally in sequencing pathogens directly from human samples, CRISPR/Cas9 targeted NGS methods have the potential to be widely applicable to microbial-outbreak investigations in the future, particularly in the context of difficult and slow-growing organisms.

16S rRNA Based Profiling of Bacterial Communities Colonizing Bakery-Production Environments.

Conventional culture-based techniques are largely inadequate in elucidating the microbiota contained in an environment, due to low recovery within a complex bacterial community. This limitation has been mitigated by the use of next-generation sequencing (NGS)-based approaches thereby facilitating the identification and classification of both culturable and uncultivable microorganisms. Amplicon targeted NGS methods, such as 16S ribosomal RNA (16S rRNA) and shotgun metagenomics, are increasingly being applied in various settings such as in food production environments to decipher the microbial consortium therein. Even though multiple food matrices/food production environments have been studied, the low-moisture environment associated with bakery food production remains to be investigated. To address this knowledge gap, in this study, we investigated the microbiome associated with two bakery production sites (designated as A and B) located in Ireland using 16S rRNA-amplicon-based sequencing. Amplicons corresponding to a hypervariable region contained within the 16S rRNA gene were amplified from DNA samples purified from environmental swabs and ingredients collected at both sites at various stages (preparation, production, postproduction, and storage) across the bakery production chain, over three seasons (winter, spring, and summer). These amplicons were sequenced, and data were analyzed using the mothur pipeline and visualized using MicrobiomeAnalyst and a series of R packages. The top seven bacterial phyla identified at both sites were composed of Proteobacteria, Firmicutes, Actinobacteria, Bacteroidetes, Deinococcus-Thermus, Patescibacteria, and Verrucomicrobia. In addition, the phyla Tenericutes (Mycoplasmatota) and Acidobacteria were observed only in samples taken at site B. Different bacterial compositions were identified at each stage of production. These same bacteria were also found to be present in the final processed food suggesting the influence of the environment on the food matrix. This study is the first demonstration of the utility of 16S rRNA amplicon-based sequencing to describe the microbiota associated with bakery processing environments.

Insights into mutational differences between upper tract urothelial carcinoma and urothelial carcinoma of the bladder.

e15125 Background: Upper tract urothelial carcinoma (UTUC) is histologically similar to bladder urothelial carcinoma (BUC), yet several clinical, biological and molecular features are unique to UTUC. UTUC may have unique genomic factors compared to BUC, while major knowledge gaps remain in our understanding of the biology and genomic landscape of UTUC. Methods: We retrospectively sequenced tumor samples and matched germline DNA using targeted next-generation sequencing methods and analyzed at least 642 cancer-associated genes. The cohort included 35 UTUC patients and 36 BUC patients. Results: Among all cases, KMT2D and TP53 were the most common mutations (account for 66% and 75% in UTUC and BUC, respectively). The most frequently mutated genes in UTUC were KMT2D (37%), TP53 (29%), ELF3 (26%), FGFR3 (23%), ASXL1 (20%), DKN2A (20%), DKN2B (17%), REBBP (17%), MT3B (17%), GNAS (17%) and PIK3CA (17%). The most frequently mutated genes in BUC were KMT2D (36%), TP53 (39%), MUC17 (14%), ARID1A (22%), GNAS (19%), RB1 (19%), BCL6 (19%), E2F3 (19%), EIF4A2 (19%) and FGF3 (19%). However significant differences in the prevalence of mutations in individual genes were observed. FGFR3 (23% vs. 8%) and ELF3 (26% vs. 11%) were more frequently altered, whereas RB1 (6% vs. 19%), E2F3 (3% vs. 19%) and BCL6 (3% vs. 19%) were less frequently altered in UTUC (P &lt; 0.05). FGFR3 mutations have been shown to be oncogenic and RB1 inactivation has been associated with genomic instability in urothelial cancer in previous researches, which represent potential therapeutic targets. Conclusions: Â UTUC and UCB exhibit significant differences in the prevalence of genomic landscape and carcinogenesis. A comprehensive understanding of the biology of UTUC and UCB is needed to identify new drug targets in order to improve clinical outcomes.

Genetic variants in Chinese patients with sporadic dilated cardiomyopathy: a cross-sectional study.

BackgroundMultiple genes have been associated with familial dilated cardiomyopathy (DCM). However, the role of genetic factors in sporadic DCM (SDCM) remains unclear. Therefore, we studied the genetic variations in Chinese patients with SDCM.MethodsSixty-six unrelated Chinese patients (mean age 49.1±17.0 years; 71% male) diagnosed with SDCM were enrolled. The clinical history and genomic DNA of the cohort were collected and examined. The exons of 24 genes closely associated with familial DCM (ABCC9, ACTC1, ACTN2, DES, LAMA4, LDB3, LMNA, MYBPC3, MYH6, MYH7, MYPN, PLN, PSEN1, PSEN2, RBM20, SCN5A, SGCD, TAZ, TCAP, TMPO, TNNI3, TNNT2, TPM1, and VCL) were sequenced using targeted next-generation sequencing method. All called nonsynonymous variants and their occurrence frequencies were compared against population data from public databases. And the nonsynonymous variants were also evaluated for pathogenicity by PolyPhen 2 (PP2) and Sorts Intolerant From Tolerant (SIFT) algorithms.ResultsEighty-five nonsynonymous variants were detected in 17 genes. The variants and their occurrence frequencies in the patients were compared against population data from the 1000 Genomes and NHLBI (National Heart, Lung, and Blood Institute) Go Exome Sequencing Project. Forty-nine nonsynonymous variants had occurrence frequencies that were significantly higher in the study patients than in the general population, indicating that they have the potential to increase the risk of DCM. The risk variants were distributed in 40 (61%) patients, among whom 25 carried a single variant, while the remaining patients carried multiple (2 to 4) variants. Risk variants occurred more frequently in MYBPC3 (14% of the patients), SCN5A (14%), MYH7 (12%), MYPN (9%), and LDB3 (8%), as verified by Poisson distribution analysis, which were considered “the five risky genes”.ConclusionsWe found that genetic variants with potential risk for DCM were commonly present in SDCM patients, indicating that genetic factors contribute to the pathogenesis, and (probably) the onset, of DCM in these patients.

Circulatory MicroRNAs in Plasma and Atrial Fibrillation in the General Population: The Rotterdam Study.

Background: MicroRNAs (miRNAs), small non-coding RNAs regulating gene expression, have been shown to play an important role in cardiovascular disease. However, limited population-based data regarding the relationship between circulatory miRNAs in plasma and atrial fibrillation (AF) exist. Moreover, it remains unclear if the relationship differs by sex. We therefore aimed to determine the (sex-specific) association between plasma circulatory miRNAs and AF at the population level. Methods: Plasma levels of miRNAs were measured using a targeted next-generation sequencing method in 1999 participants from the population-based Rotterdam Study. Logistic regression and Cox proportional hazards models were used to assess the associations of 591 well-expressed miRNAs with the prevalence and incidence of AF. Models were adjusted for cardiovascular risk factors. We further examined the link between predicted target genes of the identified miRNAs. Results: The mean age was 71.7 years (57.1% women), 98 participants (58 men and 40 women) had prevalent AF at baseline. Moreover, 196 participants (96 men and 100 women) developed AF during a median follow-up of 9.0 years. After adjusting for multiple testing, miR-4798-3p was significantly associated with the odds of prevalent AF among men (odds ratio, 95% confidence interval, 0.39, 0.24–0.66, p-value = 0.000248). No miRNAs were significantly associated with incident AF. MiR-4798-3p could potentially regulate the expression of a number of AF-related genes, including genes involved in calcium and potassium handling in myocytes, protection of cells against oxidative stress, and cardiac fibrosis. Conclusions: Plasma levels of miR-4798-3p were significantly associated with the odds of prevalent AF among men. Several target genes in relation to AF pathophysiology could potentially be regulated by miR-4798-3p that warrant further investigations in future experimental studies.

Current Methods and Role of Next-Generation Sequencing in the Diagnosis of Antimicrobial Resistance in Tuberculosis

Rapid diagnosis of tuberculosis (TB) and identification of antimicrobial resistance (AR) are critical to improving patient outcomes, especially for multidrug- and extensively drug-resistant TB (DR-TB) infections. Diagnosis can take weeks with traditional culture and growth-based antimicrobial susceptibility testing, delaying appropriate treatment, impacting transmission of resistant strains, and leading to additional health care costs. Next-generation sequencing (NGS) techniques are revolutionizing the diagnostic laboratory and provide rapid, comprehensive, and accurate detection of AR in one test. With NGS, the whole genome can be sequenced in as little as 4 days from identification of TB in a positive culture. Subsequent bioinformatic analysis of sequence data assesses nucleotide changes compared to a wild-type reference genome and can identify mutations known to cause AR. Additionally, data can be analyzed to determine the species and provide a genotype. Targeted NGS methods designed to detect DR-TB directly from clinical specimens hold promise for the future.

Next- generation sequencing is an effective method for diagnosing patients with different forms of monogenic diabetes

AimMonogenic diabetes (MD) represents 5–7% of antibody-negative diabetes cases and is a heterogeneous group of disorders. MethodsWe used targeted next-generation sequencing (NGS) on Illumina NextSeq 550 platform involving the SureSelect assay to perform genetic and clinical characteristics of a study group of 684 individuals, including 542 patients referred from 12 Polish Diabetes Centers with suspected MD diagnosed between December 2016 and December 2019 and their 142 family members (FM). ResultsIn 198 probands (36.5%) and 66 FM (46.5%) heterozygous causative variants were confirmed in 11 different MD-related genes, including 31 novel mutations, with the highest number in the GCK gene (206/264), 22/264 in the HNF1A gene and 8/264 in the KCNJ11 gene. Of the 183 probands with MODY1-5 diabetes, 48.6% of them were diagnosed at the pre-diabetes stage and most of them (68.7%) were on diet only at the time of genetic diagnosis, while 31.3% were additionally treated with oral hypoglycaemic drugs and/or insulin. ConclusionsIn summary, the results obtained confirm the efficacy of targeted NGS method in the molecular diagnosis of patients with suspected MD and broaden the spectrum of new causal variants, while updating our knowledge of the clinical features of patients defined as having MD.

Personalized circulating tumor DNA in patients with hepatocellular carcinoma: a pilot study.

Mutational analysis of circulating tumor DNA (ctDNA) can potentially be used for early detection of recurrence after resection for hepatocellular carcinoma (HCC). Mutations from tumor may be identified in plasma as an early sign of recurrence. We conducted a pilot study investigating if somatic mutations could be detected in plasma in patients undergoing liver resection for HCC and in patients with advanced non-resectable HCC. We prospectively included patients undergoing curative liver resection for HCC. Tumor tissue was investigated with whole exome sequencing and preoperative blood samples were evaluated for ctDNA using targeted next-generation sequencing (NGS) with TruSight Oncology 500 including 523 cancer-associated genes. Subsequently, the method was evaluated in patients with advanced HCC. We included eight patients curatively resected for HCC, where tumor tissue mutations were identified in seven patients. However, only in one patient tumor specific mutations were found in the preoperative blood sample. In all three patients with advanced HCC, tumor mutations were detected in the blood. In patients with resectable HCC, ctDNA could not be reliably detected using the applied targeted NGS method. In contrast, ctDNA was detected in all patients with advanced HCC. Small tumors, tumor heterogeneity and limited sequencing coverage may explain the lack of detectable ctDNA.

Canine Oncopanel: A capture-based, NGS platform for evaluating the mutational landscape and detecting putative driver mutations in canine cancers.

Canine cancer, a significant cause of mortality in domestic dogs, is a powerful comparative model for human cancers. Revealing genetic alterations driving the oncogenesis of canine cancers holds great potential to deepen our understanding of the cancer biology, guide therapeutic development, and improve cancer management in both dogs and people. Next generation sequencing (NGS) based-diagnostic panels have been routinely used in human oncology for the identification of clinically-actionable mutations, enabling tailored treatments based on the individual's unique mutation profiles. Here, we report the development of a comprehensive canine cancer gene panel, the Canine Oncopanel, using a hybridization capture-based targeted NGS method. The Canine Oncopanel allows deep sequencing of 283 cancer genes and the detection of somatic mutations within these genes. Vigorous optimization was performed to achieve robust, high-standard performance using metrics of similar cancer panels in human oncology as benchmarks. Validation of the Canine Oncopanel on reference tumour samples with known mutations demonstrated that it can detect variants previously identified by alternative methods, with high accuracy and sensitivity. Putative drivers were detected in over 90% of clinical samples, showing high sensitivity. The Canine Oncopanel is suitable to map mutation profiles and identify putative driver mutations across common and rare cancer types in dogs. The data generated by the Canine Oncopanel presents a rich resource of putative oncogenic driver mutations and potential clinically relevant markers, paving the way for personalized diagnostics and precision medicine in canine oncology.

Abstract LB059: Dual methylation and mutation analysis from a single input of plasma cfDNA without sample splitting

Abstract Genomic and epigenetic changes are known to be the underlying mechanism of cancer development. Assessing the genomic (mutation) and epigenetic (methylation) changes has become increasingly important for cancer detection and treatment selection, especially in the field of cfDNA-based liquid biopsy. Targeted Next-Generation Sequencing (NGS) enables in-depth analysis of specific genomic regions that is essential for the clinical application of liquid biopsy. However, current targeted NGS assays are complicated, lengthy, and inefficient. Two major hurdles of existing targeted NGS methods make them impractical for the clinical setting. First, there is no comprehensive, end to end, kit solution available for targeted methylation sequencing (TMS) from sample to sequencing ready library. Second, the low yield of cfDNA from clinical blood samples presents a major challenge for multi-modal analyses on one sample. Thus, an assay that is capable of both methylation and mutation analysis would be optimal for clinical research and future diagnostic assays.Here, we report the performance of Point-n-Seq, a kit solution that can provide in-depth DNA analysis with highly flexible and customizable focused panels to enable both methylation and mutation analysis without sample splitting. Custom panels of 10 to 1000s of markers can be designed with &amp;gt;99% first-pass success rate. We conducted both performance validation and multi-center, multi-operator, reproducibility studies. With spike-in titration of cancer cell-line gDNA with known mutation and methylation profile, we achieved a reliable detection level down to 0.003% of tumor DNA with a linear relationship between the measured and expected fractions.In a pilot study, we designed a colorectal cancer (CRC) TMS panel covering 560 methylation markers and a mutation panel with &amp;gt;350 hotspot mutations in 22 genes. From only 1ml of plasma from late stage CRC patients, we detected cancer-specific methylation signals in all samples tested, and hotspot mutations with clinical implications. Next, a tumor-informed mutation panel covering approximately 100 personalized mutations for each patient was compared to the tumor-independent CRC methylation panel to analyze 23 stage I and II CRC patient plasma samples. The initial results showed that tumor-independent TMS assay achieved a comparable or superior limit of detection compared to the personalized tumor-informed approach. Moreover, to further improve assay sensitivity, metrics for cfDNA size can be integrated into the analysis of the same workflow without sample splitting. The Point-n-Seq kit, with a highly streamlined workflow and dual methylation and mutation analysis, provides a highly sensitive and robust non-invasive method for cancer detection, monitoring and treatment guidance. Citation Format: Grace Q. Zhao, Yun Bao, Heng Wang, Vanita S. Natu, Jayashree Joshi, Yingmin Wang, Elena Arzumanyan, Wanping Wang, Jianmin Wang, John Coller, Shengrong Lin. Dual methylation and mutation analysis from a single input of plasma cfDNA without sample splitting [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 LB059.

Abstract 547: Urine tumor DNA MRD detection and correlation with pathologic complete response in muscle-invasive bladder cancer treated with curative-intent radical cystectomy

Abstract Objective: Standard-of-care for muscle-invasive bladder cancer (MIBC) is neoadjuvant chemotherapy followed by radical cystectomy. The inability to assess molecular residual disease (MRD) noninvasively limits our ability to offer bladder-sparing treatment. We seek to develop a liquid biopsy solution via urine tumor DNA (utDNA) analysis. Methods: We applied uCAPP-Seq, a targeted next-generation sequencing method for detecting utDNA, to urine cell-free DNA samples acquired on the day of curative-intent radical cystectomy from 42 patients with non-metastatic bladder cancer, 30 of whom had a confirmed diagnosis of MIBC and 19 of whom received neoadjuvant chemotherapy. utDNA mutational calling was performed noninvasively without tumor tissue sequencing knowledge. The overall utDNA level for each patient was represented by the non-silent mutation with the highest duplex-supported variant allele fraction after removing germline variants. Urine was similarly analyzed from 15 healthy donors. The concordance between urine cell-free DNA and tumor tissue was determined for a subset of patients. Tumor mutational burden (TMB) in utDNA MRD-positive patients was inferred from the number of mutations detected in urine cell-free DNA by applying a linear relationship derived from TCGA whole-exome sequencing of 409 MIBC tumors. Results: utDNA analysis revealed a median utDNA level of 0% in healthy donors and 2.4% in non-metastatic bladder cancer patients. Concordance between urine- and tumor-derived mutations, determined in 5 MIBC patients, was 87%. When patients were classified as those who had residual disease detected in their surgical sample (n = 16) compared to those who achieved a pathologic complete response (n = 26), median utDNA levels were 4.3% vs. 0%, respectively (P = 0.002). The lack of utDNA MRD detection was highly correlated with pathologic complete response by Fisher's exact test (P = 0.0001) with Youden's index-determined sensitivity of 81% and specificity of 81%. Moreover, utDNA MRD-positive patients exhibited significantly worse progression-free survival compared to utDNA MRD-negative patients (HR = 7.2; P = 0.03) with a median follow-up time of 200 days. Leveraging data from TCGA, the median inferred TMB in utDNA MRD-positive patients was 198 mutations per exome. We suggest that 58% of these patients with inferred TMB ≥ this level might have been candidates for early immune checkpoint blockade. Conclusion: The lack of utDNA MRD detection prior to curative-intent radical cystectomy for muscle-invasive bladder cancer correlated significantly with pathologic complete response. utDNA MRD detection status also correlated significantly with progression-free survival. Furthermore, utDNA results can be used to noninvasively infer TMB, which may facilitate the targeted use of adjuvant immunotherapy. Citation Format: Kevin Chen, Pradeep S. Chauhan, Ramandeep K. Babbra, Wenjia Feng, Peter K. Harris, Katherine Dienstbach, Andrew Atkocius, Lenon Maguire, Faridi Qaium, Armaan Nallicheri, Brian C. Baumann, Melissa A. Reimers, Eric H. Kim, Zachary L. Smith, Vivek K. Arora, Aadel A. Chaudhuri. Urine tumor DNA MRD detection and correlation with pathologic complete response in muscle-invasive bladder cancer treated with curative-intent radical cystectomy [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 547.

A dual target sequencing solution to assess genomic and epigenomic alterations in cell-free DNA with no sample splitting.

3043 Background: Assessing the genomic and epigenomic changes on plasma cell-free DNA (cfDNA) using next-generation sequencing (NGS) has become increasingly important for cancer detection and treatment selection guidance. However, two major hurdles of existing targeted NGS methods make them impractical for the clinical setting. First, there is no comprehensive, end to end, kit solution available for targeted methylation sequencing (TMS), let alone one that analyzes both mutation and methylation information in one assay. Second, the low yield of cfDNA from clinical blood samples presents a major challenge for conducting multi-omic analysis. Thus, an assay that is capable of both genomic and epigenomic analysis would be advantageous for clinical research and future diagnostic assays. Methods: Here, we report the performance of Point-n-SeqTM dual analysis, a kit solution that can provide in-depth DNA analysis with highly flexible and customizable focused panels to enable both genomic and epigenomic analysis without sample splitting. With custom panels of tens to thousands of markers designed with &gt; 99% first-pass success rate, we conducted both performance validation and multi-center, multi-operator, reproducibility studies. Using spike-in titration of cancer cell-line gDNA with known mutation and methylation profiles, Point-n-Seq assay achieved a reliable detection level down to 0.003% of tumor DNA with a linear relationship between the measured and expected fractions. Benchmarked with conventional targeted sequencing and methylation sequencing, Point-n-Seq solution also demonstrated improved performance, speed and shortened hands-on time. Results: In a pilot clinical study, a colorectal cancer (CRC) TMS panel covering 560 methylation markers and a mutation panel with &gt; 350 hotspot mutations in 22 genes were used in the dual assay. Using 1ml of plasma from late-stage CRC patients, cancer-specific methylation signals were detected in all samples tested, and oncogenic mutations. In an early-stage cohort (33 stage I/II CRC patient ), comparison of the analysis between tumor-informed, personalized-mutation panels (̃100 private SNVs) for each patient and the tumor-independent CRC methylation panels were conducted. The initial results showed that tumor-independent TMS assay achieved a comparable detection compared to the personalized tumor-informed approach. Moreover, cfDNA size information (fragmentome) is also integrated into the analysis of the same Point-n-Seq workflow to improve the assay sensitivity. Conclusions: Point-n-Seq dual analysis is poised to advance both research and clinical applications of early cancer detection, minimal residual disease (MRD), and monitoring.

A Novel FLCN Intragenic Deletion Identified by NGS in a BHDS Family and Literature Review.

Birt–Hogg–Dubé syndrome (BHDS, MIM #135150), caused by germline mutations of FLCN gene, is a rare autosomal dominant inherited disorder characterized by skin fibrofolliculomas, renal cancer, pulmonary cysts and spontaneous pneumothorax. The syndrome is considered to be under-diagnosed due to variable and atypical manifestations. Herein we present a BHDS family. Targeted next generation sequencing (NGS) and multiplex ligation-dependent probe amplification (MLPA) revealed a novel FLCN intragenic deletion spanning exons 10-14 in four members including the proband with pulmonary cysts and spontaneous pneumothorax, one member with suspicious skin lesions and a few pulmonary cysts, as well as two asymptomatic family members. In addition, a linkage analysis further demonstrated one member with pulmonary bullae to be a BHDS-ruled-out case, whose bullae presented more likely as an aspect of paraseptal emphysema. Furthermore, the targeted NGS and MLPA data including our previous and present findings were reviewed and analyzed to compare the advantages and disadvantages of the two methods, and a brief review of the relevant literature is included. Considering the capability of the targeted NGS method to detect large intragenic deletions as well as determining deletion junctions, and the occasional false positives of MLPA, we highly recommend targeted NGS to be used for clinical molecular diagnosis in suspected BHDS patients.

Sperm DNA integrity status is associated with DNA methylation signatures of imprinted genes and non-imprinted genes.

To evaluate the association between the DNA methylation of specific genes and sperm DNA integrity status in human sperm samples. A total of 166 semen samples were evaluated (86 controls and 80 cases with impaired sperm DNA integrity). We detected the methylation status of 257 CpG sites among two imprinted genes (H19 and SNRPN) and four non-imprinted genes related to male infertility (MTHFR, GSTM1, DAZL, and CREM) by using a targeted next-generation sequencing method. Differential methylation was found in 43 CpG sites of the promoters of the six candidate genes. H19, SNRPN, MTHFR, DAZL, GSTM1, and CREM contained 22, 12, 1, 4, 0, and 4 differentially methylated CpG sites (P<0.05), respectively. The imprinting genes were associated with relatively higher rates of differentially methylated CpG sites (28.21% in H19 and 41.38% in SNRPN) than the non-imprinting genes. One CpG site in H19 remained significant after performing strict Bonferroni correction. In this study, we found that different site-specific DNA methylation signatures were correlated with sperm DNA integrity status. Further studies are needed to investigate the specific mechanisms leading to the epigenetic modifications.

Chronic Thrombocytopenia in Children: What Could It Hide?

Immune thrombocytopenic purpura (ITP) is one of the most common hemorrhagic disorders in childhood, often caused by an acute self-limiting event. However, 30% of these children develop chronic ITP. Identification of the underlying causes in ITP is an important challenge. Inherited thrombocytopenia (IT) is a rare, underdiagnosed disease, included among the chronic platelet disorders. Next-Generation-Sequencing (NGS) could be an efficient way of discovering potential IT-associated mutations in children with chronic ITP. The purpose of this retrospective study was to investigate children with chronic ITP using a targeted NGS, in order to identify IT-associated mutations. Between June 2017 and April 2020, mutational screening by a targeted NGS was performed on 19 children, either with a familial history of IT [4 unrelated patients (pts)], or with chronic ITP (15 pts), after all other causes of thrombocytopenia were excluded. Nineteen relatives were also investigated. This study was carried out in collaboration with the Laboratory of Genetics, IRCCS Burlo Garofolo in Trieste, that developed a targeted NGS method for the simultaneous analysis of 28 IT genes. The cost of the NGS tests were supported by the public healthcare service. We retrospectively divided our cohort of 19 pts, into three subgroups: Group I included 4 unrelated pts with familial IT; Group II consisted of 6 pts with chronic ITP and a clinical history and/or laboratory features associated with familial IT; and, Group III included 9 pts with chronic ITP refractory to several treatments (Table 1). The median age at the initial diagnosis of thrombocytopenia was lower in Group I than in Groups II and III (19/12 years vs 1310/12 years and 9 years, respectively, p=0.33). The median time between the diagnosis of thrombocytopenia, and the time of the study, was shorter in Group I compared to Groups II and III (11.7 months vs 45.3 and 51.7 months, respectively, p=0.16). Median platelet count at the disease onset was lower in Group III than in Groups I and II (21 x 109/L vs 99 x 109/L and 38 x 109/L, respectively, p=0.28). The median MPV values were 12.5 fL, 9.85 fL and 8.8 fL in Groups II, III, and I respectively. Bleeding symptoms requiring treatment were present at diagnosis in 1/6 (16%) and in 5/9 (55%) children of Groups II and III, respectively. Genetic variants, usually detected in IT, were found in heterozygosity in all children in Groups I and II, and in 7/9 (78%) in Group III. Two out of 4, 2/6 and 2/9 children in Groups I, II, and III, respectively, presented ≥2 variants. Among the 4 children of Group I, ANKDR26 variant was found in 2 pts, together with GP1BA and NBEAL2 (pt#1) and TUBB1 (pt#2). ANKDR26 variant was also recorded as a single mutation in their relatives. Two different variants involving GP1BA (c.98T&amp;gt;A and c.515C&amp;lt;T) were detected in the remaining 2 children of Group I and in their relatives. Pt #4 with GP1BA c.515C&amp;gt;T mutation with mild macrothrombocytopenia had relatives with a previous diagnosis of monoallelic Bernard-Souliers syndrome. As shown in table 1, ABCG8, ACTN1, ETV6, GP1BA, MYH9, SLFN14, or WAS variants, found in combination in 2 pts (pt#5, pt#8), were also detected in the children in Group II, as well as, at least one of the relatives (for a total of 7 cases). ABCG5, ABCG8, ETV6, FLNA, GP1BA, NBEAL2, or SLFN14 were found as variants in patients of Group III. The peripheral blood smear evaluation confirmed the diagnosis of grey platelet syndrome with two NBEAL2 mutations in pt #16. SLFN14, as a single variant, was associated with macrothrombocytopenia in one pt (#10). Three pts (#5, #6, #17), with variants of ABCG8 had hypercholesterolemia. In the cohort of pts with chronic ITP, 4 (#12, #13, #14, #15) had relatives with thrombocytopenia, and 2 (#11, #13) had a familial history of hematological malignancies. Segregation analysis in families, and functional studies to evaluate the pathogenic role of the variants reported, are still in progress. The clinical significance of IT-associated mutations in chronic ITP is uncertain, and yet to be clarified. However, our experience has shown that an in-depth clinical history, and accurate peripheral blood smear examinations, are important to better characterize chronic ITP in children. A targeted NGS method for the simultaneous analysis of different IT genes, has demonstrated to be an effective approach to explore in-depth the IT-associated mutations in children with chronic ITP, refractory to treatment. Table 1. Disclosures Giona: Novartis: Research Funding; Takeda: Speakers Bureau; Sanofi Genzyme: Research Funding, Speakers Bureau.

Highly multiplexed quantifications of 299 somatic mutations in colorectal cancer patients by automated MALDI-TOF mass spectrometry

BackgroundDetection of somatic mutations in tumor tissues helps to understand tumor biology and guide treatment selection. Methods such as quantitative PCR can analyze a few mutations with high efficiency, while next generation sequencing (NGS) based methods can analyze hundreds to thousands of mutations. However, there is a lack of cost-effective method for quantitatively analyzing tens to a few hundred mutations of potential biological and clinical significance.MethodsThrough a comprehensive database and literature review we selected 299 mutations associated with colorectal cancer. We then designed a highly multiplexed assay panel (8-wells covering 299 mutations in 109 genes) based on an automated MADLI-TOF mass spectrometry (MS) platform. The multiplex panel was tested with a total of 319 freshly frozen tissues and 92 FFPE samples from 229 colorectal cancer patients, with 13 samples also analyzed by a targeted NGS method covering 532 genes.ResultsMultiplex somatic mutation panel based on MALDI-TOF MS detected and quantified at least one somatic mutation in 142 patients, with KRAS, TP53 and APC being the most frequently mutated genes. Extensive validation by both capillary sequencing and targeted NGS demonstrated high accuracy of the multiplex MS assay. Out of 35 mutations tested with plasmid constructs, sensitivities of 5 and 10% mutant allele frequency were achieved for 19 and 16 mutations, respectively.ConclusionsAutomated MALDI-TOF MS offers an efficient and cost-effective platform for highly multiplexed quantitation of 299 somatic mutations, which may be useful in studying the biological and clinical significance of somatic mutations with large numbers of cancer tissues.

Banking with precision: transfusion medicine as a potential universal application in clinical genomics.

To summarize the most recent scientific progress in transfusion medicine genomics and discuss its role within the broad genomic precision medicine model, with a focus on the unique computational and bioinformatic aspects of this emergent field. Recent publications continue to validate the feasibility of using next-generation sequencing (NGS) for blood group prediction with three distinct approaches: exome sequencing, whole genome sequencing, and PCR-based targeted NGS methods. The reported correlation of NGS with serologic and alternative genotyping methods ranges from 92 to 99%. NGS has demonstrated improved detection of weak antigens, structural changes, copy number variations, novel genomic variants, and microchimerism. Addition of a transfusion medicine interpretation to any clinically sequenced genome is proposed as a strategy to enhance the cost-effectiveness of precision genomic medicine. Interpretation of NGS in the blood group antigen context requires not only advanced immunohematology knowledge, but also specialized software and hardware resources, and a bioinformatics-trained workforce. Blood transfusions are a common inpatient procedure, making blood group genomics a promising facet of precision medicine research. Further efforts are needed to embrace transfusion bioinformatic challenges and evaluate its clinical utility.

Pooled clone collections by multiplexed CRISPR-Cas12a-assisted gene tagging in yeast

Clone collections of modified strains (“libraries”) are a major resource for systematic studies with the yeast Saccharomyces cerevisiae. Construction of such libraries is time-consuming, costly and confined to the genetic background of a specific yeast strain. To overcome these limitations, we present CRISPR-Cas12a (Cpf1)-assisted tag library engineering (CASTLING) for multiplexed strain construction. CASTLING uses microarray-synthesized oligonucleotide pools and in vitro recombineering to program the genomic insertion of long DNA constructs via homologous recombination. One simple transformation yields pooled libraries with >90% of correctly tagged clones. Up to several hundred genes can be tagged in a single step and, on a genomic scale, approximately half of all genes are tagged with only ~10-fold oversampling. We report several parameters that affect tagging success and provide a quantitative targeted next-generation sequencing method to analyze such pooled collections. Thus, CASTLING unlocks avenues for increasing throughput in functional genomics and cell biology research.