Next-generation Sequencing Platforms Research Articles

The impact of misclassification errors on the performance of biomarkers based on next-generation sequencing, a simulation study

ABSTRACT The development of next-generation sequencing (NGS) opens opportunities for new applications such as liquid biopsy, in which tumor mutation genotypes can be determined by sequencing circulating tumor DNA after blood draws. However, with highly diluted samples like those obtained with liquid biopsy, NGS invariably introduces a certain level of misclassification, even with improved technology. Recently, there has been a high demand to use mutation genotypes as biomarkers for predicting prognosis and treatment selection. Many methods have also been proposed to build classifiers based on multiple loci with machine learning algorithms as biomarkers. How the higher misclassification rate introduced by liquid biopsy will affect the performance of these biomarkers has not been thoroughly investigated. In this paper, we report the results from a simulation study focused on the clinical utility of biomarkers when misclassification is present due to the current technological limit of NGS in the liquid biopsy setting. The simulation covers a range of performance profiles for current NGS platforms with different machine learning algorithms and uses actual patient genotypes. Our results show that, at the high end of the performance spectrum, the misclassification introduced by NGS had very little effect on the clinical utility of the biomarker. However, in more challenging applications with lower accuracy, misclassification could have a notable effect on clinical utility. The pattern of this effect can be complex, especially for machine learning-based classifiers. Our results show that simulation can be an effective tool for assessing different scenarios of misclassification.

B-355 Measurable Residual Disease Detection in Mantle Cell Lymphoma (MCL) Subjects Using NGS B-Cell MRD Assay

Abstract Background Mantle Cell Lymphoma (MCL) is an aggressive, rare form of non-Hodgkin B-cell lymphoma. Measurable Residual Disease (MRD) assessment can provide useful information when assessing and tracking response to therapy, refining treatment and predicting clinical outcome for subjects with B-cell lymphoproliferative diseases. One method of MRD detection is to identify and track specific clonal immunoglobulin heavy chain (IGH) gene rearrangement sequences on a next-generation sequencing (NGS) platform. Here, we present the NGS-based B-cell MRD Assay with 10−6 sensitivity for detecting MRD in malignancies in mantle cell lymphoma (MCL). Methods Residual DNA samples from different specimen types (44 BM and 77 PB) at different post-treatment time points were obtained from anonymized 47 MCL subjects that had been enrolled in a study approved by the Spanish Group of Lymphoma and Autologous Stem Cell Transplantation (clinical trial: NCT02682641, publication reference: 10.1200/JCO.21.02321). 121 of these follow up samples were tested by the B-cell MRD Assay by tracking the clonal sequences detected in corresponding baseline samples. Among them, 90 follow up samples were paired PB and BM samples. The B-cell MRD Assay workflow consists of DNA extraction and PCR based library preparation with proprietary multiplex master mixes (MMx) targeting either IGH Framework 1 (FR1) or 3 (FR3) regions, which both track the patient- and tumor-specific CDR3 sequence. Purified libraries are then equimolar pooled and sequenced on Illumina’s NextSeq Dx platform. The FASTQ output files from sequencing are analyzed using Invivoscribe’s B-Cell MRD Software-NextSeqDx. Results from Invivoscribe were compared to the results from Salamanca lab that were generated by qPCR. Results Based on the results from B-cell MRD Assay, 115/121 (95%) samples were valid, 42/115 (37%) valid samples had, “MRD Detected”, and 73/115 (63%) samples had, “MRD Not Detected”, with an “MRD Detection” rate of 28% in PB and 50% in BM specimen types. When comparing MRD detectable status between paired PB and BM samples from 36 MCL subjects, 30/44 (68%) paired samples had same MRD call and were concordant, whereas 14/44 (32%) calls were discordant. More specifically, 12/14 (86%) discordant samples had PB specimen type with a “MRD Not detected” call, whereas the paired BM specimen showed a “MRD Detected” call. Overall, in comparison with the MRD results (n = 84) provided by Salamanca lab using qPCR method, the B-cell MRD Assay demonstrated concordance of 80%, PPA of 79%, and NPA of 80% for MRD detection in MCL samples. Conclusion Good concordance was observed between the qPCR used at Salamanca and Invivoscribe’s B-cell MRD assay. It was important to note that the BM specimen type was determined to be more sensitive in detecting MRD when compared to PB in assessment of paired PB and BM samples. These results demonstrate that the B-cell MRD Assay can be utilized for MRD detection in follow-up sample types for MCL subjects.

Challenges in clinical interpretation of next-generation sequencing data: Advantages and Pitfalls

Next-generation sequencing (NGS) technologies have completely changed the field of genomics by making it possible to rapidly and inexpensively produce genome-scale sequence data with higher precision and resolution. In recent years, rapid technological advancements driven by companies and academic institutions have expanded the range of NGS applications, from research to the clinic. Latest scientific developments have highlighted the effects of NGS technology on complex and Mendelian diseases, particularly cancer. However, the rapid adoption of NGS poses significant challenges in data processing, storage, management, and interpretation, as well as sequencing quality control, making it challenging to integrate the sequence data into clinical practice. Here, we first outline the technical features and performance of the most recent NGS platforms. Furthermore, we summarize the applications of NGS technologies in clinical diagnostics, data interpretation, and valuable bioinformatic tools and databases for analysis. Common issues in NGS processes are also discussed.

P02.13.A THIRD-GENERATION SEQUENCING OF BRAIN TUMORS ON SINGLE-CELL LEVEL

Abstract BACKGROUND Intratumor heterogeneity (ITH) in brain tumors has been suggested to increase tumor adaptability and thus drive tumor progression and treatment resistance. Single cell sequencing has widened the range of possibilities in detecting clonal diversity and tumor evolution. As of now, most single cell experiments rely on next-generation sequencing (NGS) platforms. However, they detect only short RNA/DNA fragments that do not show the full picture of existing transcript isoforms or genetic variants. New, long-read sequencing methods such as Oxford Nanopore Technologies (ONT) or PacBio sequencing may now enable novel approaches to single cell experiments. We here set-out to complement existing single-cell analysis workflows and data pipelines with long-read sequencing approaches. We compare and integrate the data output in order to advance the understanding of ITH in brain tumors, paradigmatically demonstrated on meningioma. MATERIAL AND METHODS We set up a pipeline for single-cell/nuclei RNA long-read sequencing on ONT devices. First, libraries as derived from the 10x genomics workflow were employed, subsequently followed by independently generated libraries. To widen the insight into ITH on multiple levels, we next assessed single-cell/nuclei DNA for small somatic variants and methylation along a modified version of this platform. RESULTS Long-read RNA-sequencing yielded results comparable to short-read sequencing. Depending on sample quality, higher reads per cell and library were required for ONT sequencing to achieve similar discrimination of clusters by expression. On the other hand, cell-type specific isoforms could be identified. Specific small somatic variants known from bulk sequencing data were identified when the region was sufficiently covered in the RNA data, which was naturally more often achieved in long read than short-read data. Thus, cell-specific variant calling was enabled in a high-throughput pipeline. Maintaining the methylation information in DNA sequencing could so far only be set-up in a plate-based approach. CONCLUSION Using these approaches, we resolved features of ITH and clonal tumor evolution, aiding us in gaining a broader understanding of brain tumor biology. Furthermore, it showed novel approaches that could possibly be implemented in routine diagnostic procedures in the future. These novel techniques could be applied to other tumor entities and possibly further omics layers such as single cell proteomics and metabolomics could be added to gain a greater understanding of different tumor types and their clonal diversity.

Rare HER2 L796P missense mutation promotes the growth and oncogenic signaling in breast cancer cells.

This research aimed to find potential HER2 mutations that would have an impact on breast cancer and investigate the underlying mechanism. This study first investigated 238 pairs of breast cancer and para-cancerous tissue samples from patients on the targeted next-generation sequencing (tNGS) platform. CCK-8 and clone formation assay were used to investigate whether the mutation exerts proliferative effects on breast cancer cells. In addition, mass spectrometry-based comparative proteomic and phosphoproteomic analyses of the mutation types and wild types of MCF-7 cell lines were carried out. Among the identified mutations, a new mutation HER2 L796P promoted the proliferation of breast cancer cells and had resistance to lapatinib using CCK-8 cell proliferation assay and clone formation assay. The bioinformatic analysis showed that RAS family proteins and ERK phosphorylated proteins significantly increased in the L796P mutant cells. The Gene Ontology (GO) analysis revealed that L796P mutation affected the function of breast cancer at the level of upstream genes in the MAPK and PI3K-AKT-TOR pathways. This study demonstrated that a rare mutation HER2 L796P could be a potential therapeutic target for the clinical management of breast cancer.

Application of a Multiplex Ligation-Dependent Probe Amplification-Based Next-Generation Sequencing Approach for the Detection of Pathogenesis of Duchenne Muscular Dystrophy and Spinal Muscular Atrophy Caused by Copy Number Aberrations.

Both Duchenne muscular dystrophy (DMD; OMIM no. 310200) and spinal muscular atrophy (SMA; OMIM no. 253300/253550/253400/271150) are genetic disorders characterized by progressive muscle degeneration and weakness. Genetic copy number aberrations in the pathogenetic genes DMD and SMN1 lead to alterations in functional proteins, resulting in DMD and SMA, respectively. Multiplex ligation-dependent probe amplification (MLPA) has become a standard method for the detection of common copy number aberrations (CNAs), including DMD and SMN1 deletions, both of which are associated with poor clinical outcomes. However, traditional MLPA assays only accommodate a maximum of 60 MLPA probes per test. To increase the number of targeted sequences in one assay, an MLPA-based next-generation sequencing (NGS) assay has been developed that is based on the standard MLPA procedure, allows high-throughput screening for a large number of fragments and samples by integrating additional indices for detection, and can be analyzed on all Illumina NGS platforms.

Cross-platform comparisons for targeted bisulfite sequencing of MGISEQ-2000 and NovaSeq6000

BackgroundAn accurate and reproducible next-generation sequencing platform is essential to identify malignancy-related abnormal DNA methylation changes and translate them into clinical applications including cancer detection, prognosis, and surveillance. However, high-quality DNA methylation sequencing has been challenging because poor sequence diversity of the bisulfite-converted libraries severely impairs sequencing quality and yield. In this study, we tested MGISEQ-2000 Sequencer’s capability of DNA methylation sequencing with a published non-invasive pancreatic cancer detection assay, using NovaSeq6000 as the benchmark.ResultsWe sequenced a series of synthetic cell-free DNA (cfDNA) samples with different tumor fractions and found MGISEQ-2000 yielded data with similar quality as NovaSeq6000. The methylation levels measured by MGISEQ-2000 demonstrated high consistency with NovaSeq6000. Moreover, MGISEQ-2000 showed a comparable analytic sensitivity with NovaSeq6000, suggesting its potential for clinical detection. As to evaluate the clinical performance of MGISEQ-2000, we sequenced 24 clinical samples and predicted the pathology of the samples with a clinical diagnosis model, PDACatch classifier. The clinical model performance of MGISEQ-2000’s data was highly consistent with that of NovaSeq6000’s data, with the area under the curve of 1. We also tested the model’s robustness with MGISEQ-2000’s data when reducing the sequencing depth. The results showed that MGISEQ-2000’s data showed matching robustness of the PDACatch classifier with NovaSeq6000’s data.ConclusionsTaken together, MGISEQ-2000 demonstrated similar data quality, consistency of the methylation levels, comparable analytic sensitivity, and matching clinical performance, supporting its application in future non-invasive early cancer detection investigations by detecting distinct methylation patterns of cfDNAs.

Malignant Proliferating Pilar Tumor: Clinicopathologic, Immunohistochemical, and Molecular Study of 17 Cases.

Proliferating pilar tumors are rare neoplasms that differentiate toward the outer sheath near the isthmus and can rarely undergo malignant transformation. We performed histopathologic evaluation on 26 benign proliferating pilar tumor (BPPT) and 17 malignant proliferating pilar tumor (MPPT). Ki-67 and p53 immunostains were performed on 13 BPPT and 10 MPPT. Six MPPT cases were successfully analyzed by a next-generation sequencing platform which surveyed exonic DNA sequences of 447 cancer genes and 191 regions across 60 genes for rearrangement detection. Patient demographics and clinical characteristics were similar between the BPPT and MPPT groups. Follow-up data of 16 of 17 MPPT (median, 25mo) showed metastasis in 1 MPPT. The histologic features associated with MPPT include size >2.5cm, adjacent desmoplastic stroma, small nests or cords of atypical epithelium in surrounding stroma, irregular infiltration or borders, abnormal keratinization, large hyperchromatic nuclei, prominent nucleoli, severe cytologic atypia, nuclear pleomorphism, necrosis, and increased mitotic figures. MPPT harbors copy number gains of 15q and losses of 6p and 6q, findings previously reported in BPPT. However, MPPT harbors frequent TP53 mutations as molecular markers of progression. Different from cutaneous squamous cell carcinoma, MPPT more frequently demonstrates low tumor mutational burden and typically lacks a UV signature, suggestive of a different etiologic pathway than squamous cell carcinoma. In summary, with a median follow-up of 25 months, this study shows that MPPT is a biologically indolent carcinoma with rare metastasis. Molecular analyses suggest a non-UV-related pathogenesis with frequent TP53 aberration.

Molecular residual disease detection in resected, muscle-invasive urothelial cancer with a tissue-based comprehensive genomic profiling-informed personalized monitoring assay.

Circulating tumor DNA (ctDNA) detection postoperatively may identify patients with urothelial cancer at a high risk of relapse. Pragmatic tools building off clinical tumor next-generation sequencing (NGS) platforms could have the potential to increase assay accessibility. We evaluated the widely available Foundation Medicine comprehensive genomic profiling (CGP) platform as a source of variants for tracking of ctDNA when analyzing residual samples from IMvigor010 (ClinicalTrials.gov identifier NCT02450331), a randomized adjuvant study comparing atezolizumab with observation after bladder cancer surgery. Current methods often involve germline sampling, which is not always feasible or practical. Rather than performing white blood cell sequencing to filter germline and clonal hematopoiesis (CH) variants, we applied a bioinformatic approach to select tumor (non-germline/CH) variants for molecular residual disease detection. Tissue-informed personalized multiplex polymerase chain reaction-NGS assay was used to detect ctDNA postsurgically (Natera). Across 396 analyzed patients, prevalence of potentially actionable alterations was comparable with the expected prevalence in advanced disease (13% FGFR2/3, 20% PIK3CA, 13% ERBB2, and 37% with elevated tumor mutational burden ≥10 mutations/megabase). In the observation arm, 66 of the 184 (36%) ctDNA-positive patients had shorter disease-free survival [DFS; hazard ratio (HR) = 5.77; 95% confidence interval (CI), 3.84-8.67; P < 0.0001] and overall survival (OS; HR = 5.81; 95% CI, 3.41-9.91; P < 0.0001) compared with ctDNA-negative patients. ctDNA-positive patients had improved DFS and OS with atezolizumab compared with those in observation (DFS HR = 0.56; 95% CI, 0.38-0.83; P = 0.003; OS HR = 0.66; 95% CI, 0.42-1.05). Clinical sensitivity and specificity for detection of postsurgical recurrence were 58% (60/103) and 93% (75/81), respectively. We present a personalized ctDNA monitoring assay utilizing tissue-based FoundationOne® CDx CGP, which is a pragmatic and potentially clinically scalable method that can detect low levels of residual ctDNA in patients with resected, muscle-invasive bladder cancer without germline sampling.

Genomic analysis of Plasmodium falciparum isolates across different altitudinal zones along the slope of Mount Cameroon

BackgroundMalaria elimination is threatened by the emergence and rapid spread of drug resistance. Understanding the demographic history of Plasmodium falciparum and the genetic basis of adaptation to antimalarial treatment and host immunity is critical to elimination efforts. This study sought to characterize the diversity of P. falciparum isolates across different altitudes along the slope of Mount Cameroon.MethodologyUsing the Illumina next-generation sequencing platform P. falciparum isolates from low, intermediate and high altitudes along the slope of Mount Cameroon were studied to determine their genetic diversity, population structures, and signatures of selection in known drug resistance alleles.ResultsA total of 77,253 single-nucleotide polymorphisms were identified from 220 quality P. falciparum clinical isolates from high (24,214), intermediate (24,426), and low (28,613) altitude. About 49%, 48.2% and 30% of the parasite isolates from high, intermediate, and low altitudes, respectively had FWS values &amp;gt; 0.95 indicative of dominant mixed genotype infections and low population sub-structure with high potential for out-crossing. No significant difference was observed in within-host diversity while population structure analysis did not separate the isolates in the three major altitudinal groups by PCA, FST and admixtures, suggesting bidirectional gene flow among the populations. A total of 94 antigenic genes under balancing selection were detected in the area including vaccine candidate gene ama1, eba175, msp1, trap, dblmsp, and clag2. Moreover, 17 of these genes were identified to be under both recent positive directional and positive balancing selection including the prominent host immune target genes surfin 8.2, trap, and ama1. Recent directional selection analysis using integrated standardized haplotype score (iHS) did not detect any selection signatures in the Pfdhfr, Pfdhps, Pfmdr1, and PfK13 genes. Furthermore, no PfKelch13 validated mutation associated with artemisinin resistance was identified in this study and no structural divergence was noticed among the P. falciparum parasite populations across different altitudes around the Mount Cameroon region.DiscussionFindings revealed high genetic diversity and low population structure suggesting that malaria transmission remain high in the study area and parasite populations circulating around the slope of Mount Cameroon are homogenous.

Genome-wide genetic variation and population structure of native and cosmopolitan cattle breeds reared in Türkiye

This is the first comprehensive study to reveal genetic variation and population structure at genome level in six Anatolian (Anatolian Black, East Anatolian Red, South Anatolian Red, South Anatolian Yellow, Turkish Grey Steppe, and Zavot) and two cosmopolitan (Brown Swiss and Holstein Friesian) cattle breeds reared in Türkiye. Being 20 samples from each population, a total of 160 blood samples retrieved from representative herds were utilized to generate genomic libraries by ddRADseq method. Genomic libraries sequenced by Illumina HiSeq X Ten instrument revealed a total of 211,119 bi-allelic SNPs with high call rate. Compared to cosmopolitan cattle breeds, a higher genetic variation was observed in native Turkish cattle with an average of 0.380 observed heterozygosity. Genetic distances were comparatively low between native cattle breeds, whereas the highest genetic distance (0.064) was detected between South Anatolian Yellow and Brown Swiss. Population structure analyses showed that the native Turkish and cosmopolitan cattle breeds were clearly different from each other according to their phylogenetic origin. Besides, a high level of genetic admixture was detected among Anatolian Black, Turkish Grey Steppe, South Anatolian Red, and South Anatolian Yellow, whereas East Anatolian Red and Zavot were distinct from the other native and cosmopolitan cattle breeds. TreeMix algorithm under the assumption of one and two migration events revealed a migration route from Anatolian clade to Anatolian Black, while a second migration edge was drawn from Brown Swiss to East Anatolian Red. This study demonstrates the importance of national conservation studies in the native breeds whose population size has dramatically decreased. In addition, SNP arrays and next-generation sequencing platforms are recommended for future studies to reveal the genetic variation of other local Turkish livestock species to arrange effective conservation programs.

Third-Generation Single-Molecule Sequencing for Preimplantation Genetic Testing of Aneuploidy and Segmental Imbalances.

Current strategies for preimplantation genetic testing for aneuploidy or structural rearrangements (PGT-A/SR) rely mainly on next-generation sequencing (NGS) and microarray platforms, which are robust but require expensive instrumentation. We explored the suitability of third-generation single-molecule sequencing as a PGT-A/SR screening platform for both aneuploidy and segmental imbalance. Single-cell and multicell replicates from aneuploid or segmentally unbalanced cell lines (n = 208) were SurePlex-amplified, randomized, and subjected to (a) Nanopore-based single-molecule sequencing (Oxford Nanopore Technologies) and (b) NGS using a leading commercial PGT-A solution (Illumina VeriSeq PGS). Archival SurePlex-amplified trophectoderm biopsy samples (n = 96) previously analyzed using the commercial kit were blinded and reanalyzed using Nanopore. Nanopore-based PGT-A identified the specific aberration in 95.45% (84/88) and 97.78% (88/90) of single-/multicells with an aneuploidy or segmental imbalance (10-30.5 Mb), respectively. Comparison against the commercial kit's results revealed concordances of 98.86% (87/88) and 98.89% (89/90) for the aneuploid and segmentally unbalanced (10-30.5 Mb aberration) samples, respectively. Detection sensitivity for smaller segmental imbalances (5-5.8 Mb aberration, n = 30) decreased markedly on both platforms. Nanopore-based PGT-A reanalysis of trophectoderm biopsy samples was 97.92% (94/96) concordant with the commercial kit results. Up to 24 SurePlex-amplified single-cell, multicell, or trophectoderm samples could be sequenced in a single MinION flow-cell for subsequent preimplantation genetic testing for aneuploidy or structural rearrangements (PGT-A/SR) analysis, with results obtainable in ≤3 days and at per-sample costs that are competitive with commercial offerings. Nanopore's third-generation single-molecule sequencing represents a viable alternative to current commercial NGS-based PGT-A solutions for aneuploidy and segmental imbalance (≥10 Mb) screening of single-/multicell or trophectoderm biopsy samples.

BlaPSZ-1, a novel AmpC gene identified from a Pantoea isolate.

Pantoea species of the family Erwiniaceae are well-known plant pathogens and animal and human conditional pathogens. Due to the widespread and continuous use of antimicrobials, multidrug-resistant strains continue to emerge, making clinical treatment difficult; therefore, there is an increasing need to clarify the mechanisms of drug resistance. A rabbit anal fecal sample was collected by a swab and the streak plate method was used to isolate single colonies. The standard agar dilution method was used to determine the minimum inhibitory concentrations (MICs) against antimicrobials. The complete genome sequence of the bacterium was obtained using Next-Generation Sequencing platforms. The potential resistance gene was annotated based on the Comprehensive Antibiotic Resistance Database (CARD) and verified by molecular cloning. The β-lactamase PSZ-1 was expressed via the pCold I expression vector and its enzyme kinetic parameters were analyzed. The genetic environment and evolutionary process of the novel resistance gene-related sequences were analyzed by bioinformatic methods. The isolate Pantoea endophytica X85 showed some degree of resistance to penicillins as well as cephalosporins. A novel AmpC resistance gene, designated blaPSZ-1 in this research, was identified to be encoded in the plasmid (pPEX85) of P. endophytica X85. BlaPSZ-1 showed resistance to penicillins and several first-, second-and third-generation cephalosporins as well as aztreonam, but it did not show resistance to the fourth-generation cephalosporins or carbapenems tested. Enzyme kinetic assays revealed that it could hydrolyze amoxicillin, penicillin G, cephalothin, and cefazolin, and its hydrolytic activity could be strongly inhibited by the inhibitor avibactam, which was generally consistent with antimicrobial susceptibility testing results. No hydrolytic activity was observed for third-generation cephalosporins or aztreonam. In this study, a novel AmpC β-lactamase gene, designated blaPSZ-1, was characterized and it was encoded in the plasmid of the bacterium P. endophytica X85. It shows resistance to penicillins and several cephalosporins. The discovery of novel drug resistance mechanisms can help guide the scientific use of drugs in animal husbandry and clinical practice, effectively avoiding the abuse of antimicrobials and thus preventing the further development and spread of bacterial resistance.

Next-Generation Sequencing Technology: Current Trends and Advancements.

The advent of next-generation sequencing (NGS) has brought about a paradigm shift in genomics research, offering unparalleled capabilities for analyzing DNA and RNA molecules in a high-throughput and cost-effective manner. This transformative technology has swiftly propelled genomics advancements across diverse domains. NGS allows for the rapid sequencing of millions of DNA fragments simultaneously, providing comprehensive insights into genome structure, genetic variations, gene expression profiles, and epigenetic modifications. The versatility of NGS platforms has expanded the scope of genomics research, facilitating studies on rare genetic diseases, cancer genomics, microbiome analysis, infectious diseases, and population genetics. Moreover, NGS has enabled the development of targeted therapies, precision medicine approaches, and improved diagnostic methods. This review provides an insightful overview of the current trends and recent advancements in NGS technology, highlighting its potential impact on diverse areas of genomic research. Moreover, the review delves into the challenges encountered and future directions of NGS technology, including endeavors to enhance the accuracy and sensitivity of sequencing data, the development of novel algorithms for data analysis, and the pursuit of more efficient, scalable, and cost-effective solutions that lie ahead.

Expert consensus on standardized management of tumor gene sequencing for cancer patients

AbstractBackgroundTumour gene detection by next‐generation sequencing (NGS) can comprehensively analyze the molecular characteristics of tumors, and predict drug efficacy and tumor resistance mechanisms through tumour cell models, thereby realizing individualized and precise treatment. Therefore, the whole process of standardized management is required. This consensus aims to refine the standardized management of tumor gene sequencing for cancer patients based on the NGS platform, hoping to provide help for the personalized treatment of cancer patients, and further promoting the development of precision medicine.Methods and resultsStandardized management for tumour gene detection of cancer patients includes patient management before gene testing, during gene testing, and after gene testing. Tumour patients should select appropriate test samples and test content before receiving genetic testing. There should be a clear informed consent statement during the test, a detailed report should be provided after the test, and counseling and monitoring follow‐up should be provided. The management of genetic testing of tumour patients should cover early screening and early diagnosis of tumors, molecular classification and companion diagnosis of tumours, and monitoring of tumour recurrence.ConclusionEffective management and standardized procedures for gene detection of cancer patients, covering the whole process before, during and after detection, will benefit the majority of patients from early screening, personalized diagnosis and treatment, prognosis evaluation and other aspects of cancer, and promote the healthy development of the precision medicine industry.

Circulating microbial cell–free DNA is increased during neutropenia after hematopoietic stem cell transplantation

AbstractWe used a next-generation sequencing platform to characterize microbial cell–free DNA (mcfDNA) in plasma samples from patients undergoing allogeneic hematopoietic stem cell transplantation (allo-HCT). In this observational study, we sought to characterize plasma mcfDNA in order to explore its potential association with the immunologic complications of transplantation. We compared serially collected patient samples with plasma collected from healthy control subjects. We observed changes in total mcfDNA burden in the plasma after transplantation, which was most striking during the early posttransplant neutropenic phase. This elevation could be attributed to a number of specific bacterial taxa, including Veillonella, Bacteroides, and Prevotella (genus level). For an additional cohort of patients, we compared the data of mcfDNA from plasma with 16s-ribosomal RNA sequencing data from stool samples collected at matched time points. In a number of patients, we confirmed that mcfDNA derived from specific microbial taxa (eg, Enterococcus) could also be observed in the matched stool sample. Quantification of mcfDNA may generate novel insights into mechanisms by which the intestinal microbiome influences systemic cell populations and, thus, has been associated with outcomes for patients with cancer.

Improved Diagnostics in Bacterial Neonatal Meningitis Using a Next-Generation Sequencing Platform.

Bacterial meningitis in infants is an infrequent but life-threatening condition. Empiric therapy should begin as soon as meningitis is thought likely. Consequently, the causative microorganisms may not always be detected using culturing techniques, as cerebrospinal fluid (CSF) cultures are influenced by antibiotics. Nucleic acid amplification tests, such as polymerase chain reaction (PCR) (multiplex panels), may overcome this limitation but require a priori knowledge of the likely pathogen present within the sample. With this in mind, we investigated to what extent a culture-free, broad-range 16S rRNA gene next-generation sequencing (NGS) platform (MYcrobiota) could add to the microbiological diagnosis of meningitis. Retrospective cohort study at level III neonatal intensive care unit. Included were all infants with suspected meningitis admitted between 10 November 2017 and 31 December 2020. A comparison was made of the bacterial pathogen detection rate between MYcrobiota and conventional bacterial culture. In a 3-year period, 37 CSF samples (diagnostic and follow-up) from 35 infants with proven or possible meningitis were available for MYcrobiota testing. MYcrobiota detected the presence of bacterial pathogens in 11 samples (30%), in contrast with the conventional CSF culture, which detected bacteria in 2 of 36 samples (5.6%). Addition of 16S rRNA sequencing to conventional culturing greatly improved the identification of the aetiology of bacterial meningitis compared to culturing of CSF samples alone.

Ramucirumab plus erlotinib versus placebo plus erlotinib in previously untreated EGFR-mutated metastatic non-small-cell lung cancer (RELAY): exploratory analysis of next-generation sequencing results

Ramucirumab plus erlotinib (RAM+ ERL) demonstrated superior progression-free survival (PFS) over placebo+ ERL (PBO+ ERL) in the phase III RELAY study of patients with epidermal growth factor receptor (EGFR)-mutated metastatic non-small-cell lung cancer (EGFR+ mNSCLC; NCT02411448). Next-generation sequencing (NGS) was used to identify clinically relevant alterations in circulating tumor DNA (ctDNA) and explore their impact on treatment outcomes. Eligible patients with EGFR+ mNSCLC were randomized 1 : 1 to ERL (150 mg/day) plus RAM (10 mg/kg)/PBO every 2 weeks. Liquid biopsies were to be prospectively collected at baseline, cycle 4 (C4), and postdiscontinuation follow-up. EGFR and co-occurring/treatment-emergent (TE) genomic alterations in ctDNA were analyzed using Guardant360 NGS platform. In those with valid baseline samples, detectable activating EGFR alterations in ctDNA (aEGFR+) were associated with shorter PFS [aEGFR+: 12.7 months (n= 255) versus aEGFR-: 22.0 months (n= 131); hazard ratio (HR)= 1.87, 95% confidence interval (CI) 1.42-2.51]. Irrespective of detectable/undetectable baseline aEGFR, RAM+ ERL was associated with longer PFS versus PBO+ ERL [aEGFR+: median PFS (mPFS)= 15.2 versus 11.1 months, HR= 0.63, 95% CI 0.46-0.85; aEGFR-: mPFS= 22.1 versus 19.2 months, HR= 0.80, 95% CI 0.49-1.30]. Baseline alterations co-occurring with aEGFR were identified in 69 genes, most commonly TP53 (43%), EGFR (other than aEGFR; 25%), and PIK3CA (10%). PFS was longer in RAM+ ERL, irrespective of baseline co-occurring alterations. Clearance of baseline aEGFR by C4 was associated with longer PFS (mPFS= 14.1 versus 7.0 months, HR= 0.481, 95% CI 0.33-0.71). RAM+ ERL improved PFS outcomes, irrespective of aEGFR mutation clearance. TE gene alterations were most commonly in EGFR [T790M (29%), other (19%)] and TP53 (16%). Baseline aEGFR alterations in ctDNA were associated with shorter mPFS. RAM+ ERL was associated with improved PFS outcomes, irrespective of detectable/undetectable aEGFR, co-occurring baseline alterations, or aEGFR+ clearance by C4. aEGFR+ clearance by C4 was associated with improved PFS outcomes. Monitoring co-occurring alterations and aEGFR+ clearance may provide insights into mechanisms of EGFR tyrosine kinase inhibitor resistance and the patients who may benefit from intensified treatment schedules.

Factors influencing oral microbiome analysis: from saliva sampling methods to next-generation sequencing platforms

The exploration of oral microbiome has been increasing due to its relatedness with various systemic diseases, but standardization of saliva sampling for microbiome analysis has not been established, contributing to the lack of data comparability. Here, we evaluated the factors that influence the microbiome data. Saliva samples were collected by the two collection methods (passive drooling and mouthwash) using three saliva-preservation methods (OMNIgene, DNA/RNA shield, and simple collection). A total of 18 samples were sequenced by both Illumina short-read and Nanopore long-read next-generation sequencing (NGS). The component of the oral microbiome in each sample was compared with alpha and beta diversity and the taxonomic abundances, to find out the effects of factors on oral microbiome data. The alpha diversity indices of the mouthwash sample were significantly higher than that of the drooling group with both short-read and long-read NGS, while no significant differences in microbial diversities were found between the three saliva-preservation methods. Our study shows mouthwash and simple collection are not inferior to other sample collection and saliva-preservation methods, respectively. This result is promising since the convenience and cost-effectiveness of mouthwash and simple collection can simplify the saliva sample preparation, which would greatly help clinical operators and lab workers.

Verification of a loss of heterozygosity at the D8S1179 locus in a paternity case by the MiSeq FGx system.

Capillary electrophoresis is widely used to study short tandem repeats (STRs) in forensic genetics. However, next-generation sequencing platforms have become a new strategy for forensic DNA typing. In this study, we report a false four-step STR mutation between an alleged father (AF) and child in a paternity case. A total of 23 autosomal STR loci were evaluated using the Huaxia™ Platinum and Goldeneye™ 20A kits, revealing a single mismatch in D8S1179 between the AF (10/10) and the male child (14/14). Additional Y-STR typing of the AF and child was performed, and the results were consistent with those based on 27 Y-STR loci. To further confirm the experimental results, we sequenced the individuals using the MiSeq FGx system and detected 10/15 unbalanced alleles in the D8S1179 locus of the AF and 14/15 unbalanced alleles in the D8S1179 locus of the child. Sanger sequencing revealed that both the AF and child had the C→G point mutation in the primer binding region of D8S1179 resulting in allelic dropout. Therefore, the verification of STR typing by different sequencing systems is helpful for the interpretation of results in cases of multistep STR mutations.