Next-generation Sequencing Platforms Research Articles

Microfluidics-based EGFR mutation detection and its implication in the resource-limited clinical setting.

Management of lung cancer today obligates a mutational analysis of the epidermal growth factor receptor (EGFR) gene particularly when Tyrosine Kinase Inhibitor (TKI) therapy is being considered as part of prognostic stratification. This study evaluates the performance of automated microfluidics-based EGFR mutation detection and its significance in clinical diagnostic settings. Formalin-fixed, paraffin-embedded (FFPE) samples from NSCLC patients (n = 174) were included in a two-phase study. Phase I: Validation of the platform by comparing the results with conventional real-time PCR and next-generation sequencing (NGS) platform. Phase II: EGFR mutation detection on microfluidics-based platform as part of routine diagnostics workup. The microfluidics-based platform demonstrates 96.5% and 89.2% concordance with conventional real-time PCR and NGS, respectively. The system efficiently detects mutations across the EGFR gene with 88.23% sensitivity and 100% specificity. Out of 144 samples analysed in phase II, the platform generated valid results in 94% with mutation detected in 41% of samples. This microfluidics-based platform can detect as low as 5% mutant allele fractions from the FFPE samples. Therefore the microfluidics-based platform is a rapid, complete walkaway, with minimum tissue requirement (two sections of 5 μ thickness) and technical skill requirement. The method can detect clinically actionable EGFR mutations efficiently and can be considered a reliable diagnostic platform in resource-limited settings. From receiving samples to reporting the results this platform provides accurate data without much manual intervention. The study helped to devise an algorithm that emphasizes effective screening of the NSCLC cases for EGFR mutations with varying tumour content. Thus it helps in triaging the cases judiciously before proceeding with multigene testing.

Circulating tumor DNA- and cancer tissue-based next-generation sequencing reveals comparable consistency in targeted gene mutations for advanced or metastatic non-small cell lung cancer.

Molecular subtyping is an essential complementarity after pathological analyses for targeted therapy. This study aimed to investigate the consistency of next-generation sequencing (NGS) results between circulating tumor DNA (ctDNA)-based and tissue-based in non-small cell lung cancer (NSCLC) and identify the patient characteristics that favor ctDNA testing. Patients who diagnosed with NSCLC and received both ctDNA- and cancer tissue-based NGS before surgery or systemic treatment in Lung Cancer Center, Sichuan University West China Hospital between December 2017 and August 2022 were enrolled. A 425-cancer panel with a HiSeq 4000 NGS platform was used for NGS. The unweighted Cohen's kappa coefficient was employed to discriminate the high-concordance group from the low-concordance group with a cutoff value of 0.6. Six machine learning models were used to identify patient characteristics that relate to high concordance between ctDNA-based and tissue-based NGS. A total of 85 patients were enrolled, of which 22.4% (19/85) had stage III disease and 56.5% had stage IV disease. Forty-four patients (51.8%) showed consistent gene mutation types between ctDNA-based and tissue-based NGS, while one patient (1.2%) tested negative in both approaches. Advanced diseases and metastases to other organs would be fit for the ctDNA-based NGS, and the generalized linear model showed that T stage, M stage, and tumor mutation burden were the critical discriminators to predict the consistency of results between ctDNA-based and tissue-based NGS. ctDNA-based NGS showed comparable detection performance in the targeted gene mutations compared with tissue-based NGS, and it could be considered in advanced or metastatic NSCLC.

Rapid Detection and Quick Characterization of African Swine Fever Virus Using the VolTRAX Automated Library Preparation Platform

African swine fever virus (ASFV) is the causative agent of a severe and highly contagious viral disease affecting domestic and wild swine. The current ASFV pandemic strain has a high mortality rate, severely impacting pig production and, for countries suffering outbreaks, preventing the export of their pig products for international trade. Early detection and diagnosis of ASFV is necessary to control new outbreaks before the disease spreads rapidly. One of the rate-limiting steps to identify ASFV by next-generation sequencing platforms is library preparation. Here, we investigated the capability of the Oxford Nanopore Technologies’ VolTRAX platform for automated DNA library preparation with downstream sequencing on Nanopore sequencing platforms as a proof-of-concept study to rapidly identify the strain of ASFV. Within minutes, DNA libraries prepared using VolTRAX generated near-full genome sequences of ASFV. Thus, our data highlight the use of the VolTRAX as a platform for automated library preparation, coupled with sequencing on the MinION Mk1C for field sequencing or GridION within a laboratory setting. These results suggest a proof-of-concept study that VolTRAX is an effective tool for library preparation that can be used for the rapid and real-time detection of ASFV.

Fungi associated with fine roots of Fraxinus excelsior affected by ash dieback detected by next-generation sequencing

AbstractThe decline of European ash by dieback caused by Hymenoscyphus fraxineus together with stem collar necroses and rots caused by various fungi has been investigated intensively during the last years. Nevertheless, hitherto nearly nothing is known about the species diversity of the fungal rhizobiome of ash trees. Here we investigated the fine roots of affected ash trees on 15 sampling sites in 6 federal countries of Germany. Fine-root samples have been treated in three different sample regimes each as root-adhering soil, unsterilized fine roots and sterilized fine roots. The samples of trees in sampling sites were pooled to get an overview of the species-richness in the area. The next-generation sequencing platform Oxford Nanopore MinION was used to sequence the entire ITS of pooled probes. Most abundant phyla in all samples were the Basidiomycota and Ascomycota. Species richness in sterilized roots was significantly different from unsterilized roots and root-adhering soil. Surprisingly most abundant genera in sterilized roots were the genera Mycena, Mycenella and Delicatula, all of them agaricoids with saprophytic lifestyle. Eleven genera of Glomeromycota have been detected in various abundances, whereas the detection of H. fraxineus was neglectable.

Abstract PS11-03: Comparison of next-generation sequencing (NGS) results from the cerebrospinal fluid, peripheral blood, and systemic metastatic tumor tissue of patients with metastatic breast cancer (MBC) and leptomeningeal disease (LMD)

Abstract Introduction. Leptomeningeal disease (LMD) is a devastating complication of advanced malignancies and occurs in 5-15% of patients (pts) with metastatic breast cancer (MBC). Molecular characterization of the primary tumor to identify actionable mutations is standard of care, and targeted therapies have significantly improved survival. In contrast, metastatic tumors to the central nervous system (CNS) are not routinely assessed for the presence of actionable mutations, so the frequency and concordance of actionable mutations centrally vs. peripherally is not well characterized. In this single-center prospective study, we collected cerebrospinal fluid (CSF) in pts with known or suspected LMD and performed next generation sequencing (NGS) to evaluate the status of actionable mutations in the CSF and compared these results with NGS from matched peripheral blood and systemic metastatic tumor samples. Methods. We enrolled sixteen (16) pts with MBC and suspected or confirmed LMD at a single academic center from 2021-2023 in this non-therapeutic prospective study and collected CSF, peripheral blood, and archival systemic tumor samples from each pt. We used NGS to evaluate the status of actionable biomarkers in the CSF, blood, and tissue using the following NGS platforms: CNSide (Biocept) for CSF; Guardant 360, Foundation One CDx and CNSide for blood; and Caris and the UCSF CLIA-validated UCSF500 panel for the systemic metastatic tumor samples. We also evaluated CSF tumor cell enumeration and HER2 status in a subset of pts (CNSide). Results. Of the sixteen pts with MBC and suspected or confirmed LMD, 81% (13/16) were ultimately determined to be LMD-positive based on positive CSF cytology and/or convincing MRI imaging, including 6 pts with HR+/HER2- MBC, 6 pts with metastatic triple negative breast cancer (mTNBC), and 1 pt with HR+/HER2+ MBC; 3 pts were determined to be LMD-negative based on similar criteria. Of the 6 pts with HR+/HER2- MBC and LMD, NGS detected actionable mutations in the CSF in 67% (4/6), including two pts with PIK3CA E545E, one pt with both PIK3CA E542K and ERBB2 amplification, and 1 pt with ESR1 Y537S. Matched NGS analysis of the peripheral blood and/or systemic metastatic tissue showed concordance with PIK3CA E542K detected across tissue types in two pts, but discordance across tissue types in the other pts, including ESR1 detected systemically in two pts but not in the CSF. Of the 6 pts with mTNBC and LMD, CSF NGS detected actionable mutations in the CSF in 17% (1/6) of pts, including one pt with PIK3CA E542K; analysis of blood NGS for this pt is pending. 3 pts (50%) had mutations in TP53 in the CSF (1 pt with R273H and two pts with K132R), which were concordant across tissue types in all 3 pts. NGS did not detect any variants in the CSF of the one pt with HR+/HER2+ MBC and LMD. CSF tumor cell enumeration and central HER2 status will also be reported. Conclusion. This study demonstrates that NGS can detect actionable mutations in the CSF of pts with MBC and LMD. We observed concordance and heterogeneity in the status of actionable mutations between the CSF, peripheral blood, and systemic metastatic tumor tissue. Larger studies are needed to assess the clinical utility of these observations, particularly with the development of several novel targeted agents that are CNS-penetrant. Citation Format: Laura Huppert, Lindy Her, Christine Hodgdon, Susie Brain, Carol Simmons, Jo Chien, Melanie Majure, Hope Rugo, Mark Jesus Magbanua, Ron Balassanian, Michelle Melisko. Comparison of next-generation sequencing (NGS) results from the cerebrospinal fluid, peripheral blood, and systemic metastatic tumor tissue of patients with metastatic breast cancer (MBC) and leptomeningeal disease (LMD) [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PS11-03.

Abstract PO2-04-06: Investigation of the genomic evolution of HER2-positive breast cancer following progression on dual HER2-targetted therapy with Trastuzumab and Tucatinib

Abstract Background: Advanced HER2-positive breast cancer remains an incurable and highly morbid condition. One of the key challenges has been treatment of central nervous system (CNS) metastases, commonly seen in those with HER2-positive disease. Tucatinib is a recently approved treatment HER2-targetting tyrosine kinase inhibitor with proven CNS activity. There has been little analysis of potential genomic mechanisms of drug resistance. Method: We retrospectively identified patients that were treated with tucatinib, capecitabine and trastuzumab. We collated baseline clinical data and treatment history and all tumour and plasma samples available were obtained for testing. DNA was extracted from plasma samples using the QIAamp DNA Mini Kit (Qiagen) on 4ml plasma samples. Targeted next generation sequencing (NGS) of serial tumour and plasma samples was performed using a custom hybrid capture assay that covered 180 genes known to be recurrently mutated in breast cancer focusing on the known genomic landscape of metastatic disease as well as the inclusion of specific actionable targets. Survival analyses was performed using Cox regression models. Results: We identified 11 patients at our institution. Plasma samples were available for all 11 patients with a total of 107 serial samples across multiple time points, with a median of 8 (range 2-19) samples per patient. All 11 patients had baseline and End of Treatment (EOT) samples. In addition, 6/11 (54%) patients had tumour sequenced on the same NGS platform prior to commencement of therapy, with 12 individual tumour samples tested. The median PFS of our cohort was 9.6 months (4.0 - NR) with a median OS of 36.2 months (10.4-NR). 7/11 (64%) patients had CNS disease at baseline with their median PFS being 13.5 months (3.9-24.4) and median OS of 36.2months (10.4-NR). Overall, we observed an average of 18.4 (4-35) mutations per patient in tumour, 5.1 (1-15) in baseline plasma and 5.8 (2-14) in EOT plasma. The most frequent mutations seen in baseline tumour samples were ERBB2 (92%), KMT2C (67%) and NCOR1 (58%) compared with KMT2C (64%), NOTCH4 (36%) and PIK3CA (27%) in baseline plasma. Patients with a lower number of ctDNA mutations at baseline showed a trend towards improved survival (PFS 14 months vs 7.5 months). In this small cohort of patients we identified a higher than expected rates of KMT2C mutations. A KMT2C mutation was identified in 67% of samples which is higher than previously described rates of 7-15% in TCGA and METABRIC cohorts respectively. The presence of a concordant ctDNA KMT2C mutation in baseline and progression samples was a poor prognostic indicator (mPFS 7.5 months) when compared with no KMT2C mutation (mPFS 15.5 months) p =0.14 suggesting a possible mechanism of resistance to tucatinib. Conclusions: In this retrospective analysis of patients treated with Tucatinib, Trastuzumab and Capecitabine we established the genomic landscape of heavily pre-treated, advanced HER2-positive disease is complex. We observed there may be a correlation with KMT2C mutations and poorer clinical outcomes, however given the very small number of patients this data remains hypothesis generating, requiring validation with larger datasets. Citation Format: Elizabeth Blackley, Courtney van Geelen, Yi-An Ko, Stephen Wong, Miriam Yeung, Stephen Luen, Sarah-Jane Dawson, Sherene Loi. Investigation of the genomic evolution of HER2-positive breast cancer following progression on dual HER2-targetted therapy with Trastuzumab and Tucatinib [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-04-06.

Simultaneous sequencing of 102 Y-STRs on Ion Torrent TM GeneStudio TM S5 System

The Precision ID NGS System from Thermo Fisher Scientific is a mainstream next-generation sequencing (NGS) platform used in forensic laboratories to detect almost all commonly used forensic markers, except for Y-chromosomal short tandem repeats (Y-STRs). This study aimed to: 1) develop a Y-STR panel compatible with the automatic workflow of the NGS system using Ion AmpliSeq Technology, 2) evaluate the panel performance following the SWGDAM guidelines, and 3) explore the possibility of using a combination workflow to detect autosomal STRs and Y-STRs (AY-STR NGS workflow). The GrandFiler Y-STR Panel was successfully designed using the ‘separating’ and ‘merging’ strategies, including 102 Y-STRs and Amelogenin with an average amplicon length of 133bp. It is a mega Y-STR multiplex system in which up to 16 samples can be sequenced simultaneously on an Ion 530 TM Chip. Developmental validation studies of the performance of the NGS platform, species specificity, reproducibility, concordance, sensitivity, degraded samples, case-type samples, and mixtures were conducted to unequivocally determine whether the GrandFiler Y-STR Panel is suitable for real scenarios. The newly developed Y-STR panel showed compelling run metrics and NGS performance, including 92.47% bases with ≥ Q20, 91.80% effective reads, 2106 × depth of coverage (DoC), and 97.09% inter-locus balance. Additionally, it showed high specificity for human males and 99.40% methodological and bioinformatical concordance, generated complete profiles at ≥ 0.1ng input DNA, and recovered more genetic information from severely degraded and diverse case samples. Although the outcome when used on mixtures was not as expected, more genetic information was obtained compared to that from capillary electrophoresis (CE) methods. The AY-STR NGS workflow was established by combining the GrandFiler Y-STR Panel with the Precision ID GlobalFiler TM NGS STR Panel v2 at a 2:1 concentration ratio. The combination workflow on NGS performance, reproducibility, concordance, and sensitivity was as stable as the single Y-STR NGS workflow, providing more options for forensic scientists when dealing with different case scenarios. Overall, the GrandFiler Y-STR Panel was confirmed as the first to effectively detect a large number of Y-STR markers on the Precision ID NGS System, which is compatible with 51 Y-STRs in commercial CE kits and 51 Y-STRs in commercial NGS kits and the STRBase. The panel is as robust, reliable, and sensitive as current CE/NGS kits, and is suitable for solving real cases, especially for severely degraded samples (degradation index > 10).

Genetic Diversity and Population Structure of Four Anatolian Sheep Revealed by Genome-wide ddRADseq Data

This is the first wide-scale study to reveal next-generation sequencing-based and comprehensive study to reveal genetic diversity, population structure, and phylogenetic relationships of some of Türkiye's indigenous fat-tailed sheep breeds. A total of 80 animals belonging to Akkaraman (AKR), Güney Karaman (GKR), Morkaraman (MKR), and Karakaş (KRK) sheep breeds were genotyped with genome-wide of 296.097 single nucleotide polymorphisms (SNPs) data recovered from double digest restriction site-associated DNA sequencing (ddRADseq) libraries processed by Illumina HiSeq X Ten instrument. Minor allele frequency (MAF) ranged from 0.311 (MKR) to 0.316 (AKR and GKR). Observed heterozygosity (HO) values were higher than expected values (HE) across all populations with a mean of 0.307. Negative inbreeding coefficient (FIS) values were observed in four sheep populations varying between -0.034 (AKR) to -0.060 (MKR). Cross-entropy criterion-based ADMIXTURE analysis demonstrated that four Anatolian sheep breeds were represented by two ancestral populations in which GKR and AKR were distinct, while KRK and MKR were mixed populations. Genetic distance-based tree, discriminant analysis of principal components (DAPC), and ADMIXTURE analyses confirmed that GKR, which is believed to be derived from the AKR breed, has become genetically different from other sheep populations. Moreover, no migration from and into the GKR breed was detected, while a migration edge was drawn from the KRK And MKR clade into the AKR breed by the TreeMix algorithm. This study confirmed that high-density SNP data obtained from advanced next-generation sequencing (NGS) platforms are efficient in genetically distinguishing animal breeds, even varieties. It is recommended that further NGS-based studies could be carried out to better characterize different indigenous livestock populations raised in Türkiye.

Genetic variation and structure of endemic and endangered wild celery (Kelussia odoratissima Mozaff.) quantified using novel microsatellite markers developed by next-generation sequencing.

Kelussia odoratissima Mozaff. (Apiaceae) is a native plant that has been traditionally consumed in Iran's food and pharmaceutical industries. Overharvesting of the taxon, especially at the beginning of the growing season, due to its considerable medicinal and economic value, is believed to be the main reason for the extirpating of this plant. The consequences of the severe anthropogenic impacts on the genetic diversity of populations are poorly known. In order to investigate the level of genetic variation and patterns of the genetic structure of K. odoratissima, we developed novel microsatellite markers using the 454 Roche next-generation sequencing (NGS) platform for the first time. Out of 1,165 microsatellite markers bioinformatically confirmed, twenty-five were tested, of which 23 were used to screen genetic variation across 12 natural populations. Our results showed that the average number of alleles per locus and the polymorphic information content (PIC) were 10.87 (range 7 to 27), and 0.81 (range 0.67 to 0.94), respectively. The mean observed and expected heterozygosities (± SD) across all populations were 0.80 ± 0.31 and 0.72 ± 0.14, respectively. The average pairwise FST among the populations was 0.37 (range 0.04 to 0.81). Bayesian and distance-based clustering, and principal coordinate analyses revealed at least four major genetic clusters. Although high level of structure can be explained by landscape topography and geographic distance, presence of admixed populations can be associated to seed or pollen dispersal. Contrary to expectations, the high level of genetic variation and lack of inbreeding suggest that overexploitation has not yet significantly purged the allelic variability within the natural populations in protected areas.

Abstract 5041: Next generation liquid biopsy reference material performance across NGS assays and platforms

Abstract Liquid biopsy testing has grown to support early disease diagnosis, therapy selection, and disease and treatment surveillance in cancer survivors. Recent advances in next generation sequencing (NGS) have enabled larger panel sizes, pan-cancer target sets, and wider ranges of genomic alterations. More inclusive analytical validation materials are needed to assess assay sensitivity, specificity, and limit of detection of all variant types to serve the expanded utility of liquid biopsy testing. Here we describe the development and multisite evaluation of expanded Seraseq® ctDNA Mutation Mix v4 reference materials. Genomic DNA from the GM24385 cell line was blended with 93 clinically relevant biosynthetic DNA variants including single nucleotide variants (SNVs), insertions and deletions (indels), structural variants (SVs), copy number variations (CNVs), and mono- and dinucleotide biomarkers for microsatellite instability (MSI). Variant allele frequency (VAF) was targeted to 0.5% and total copy number for CNVs was targeted to 2.5 and verified using the Bio-Rad QX-200 Droplet Digital PCR (ddPCR) System. The DNA mix was fragmented and purified to create ctDNA-like size profiles. Wild-type (WT) material was prepared for comparison and identification of background variants. Agilent Bioanalyzer High Sensitivity DNA and TapeStation Cell-free DNA ScreenTape analyses were used to assess fragment size. The ctDNA mixes were analyzed with the Illumina TruSight™ Oncology 500 (TSO 500) ctDNA assay, the Thermo Oncomine™ Precision assay, and additional assays. All biosynthetic SNVs, indels, SVs, and CNVs in the 0.5% ctDNA mix were detected near target values. The average fragment size was about 190 bp, similar to native ctDNA. The VAFs and CNVs observed in the TSO 500 data correlated well with those that were obtained by dPCR. Variants reported by all evaluation site assays showed good concordance of VAF values. The two sites that tested the materials with TSO 500 revealed consistency in both VAF and total copies with an average CV of 16.1% and 2.5%, respectively. The sequencing performance of the reference material was similar to patient samples and showed comparable levels of background noise. The performance of the Seraseq ctDNA v4 mutation mix across NGS assays, supported by robust digital PCR data, demonstrated that these materials will allow for assessment of complex and sensitive liquid biopsy tests. Comparing data measured at different sites highlights the variability in liquid biopsy assay performance and the importance of highly multiplexed and consistent reference materials. Remnant samples are typically not an option due to limited yields from a standard blood draw, thus contrived standards are especially needed. Expanded variant content and improvements in the methods used to produce the Seraseq ctDNA Mutation Mix v4 materials will enable advancements in the blood-based NGS clinical diagnostics space. Citation Format: Dana Ruminski Lowe, Sanchita Jamindar, Yves Konigshofer, Andrew Anfora, Krystyna Nahilk, Dianren Xia, Edward S. Davis, David Merriam, Catherine Huang, Russell Garlick, Bharathi Anekella. Next generation liquid biopsy reference material performance across NGS assays and platforms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5041.

Abstract 6469: The association of mismatch repair gene alterations with elevated tumor mutational burden in microsatellite stable gastrointestinal cancers

Abstract Background: Benefit from single-agent immune checkpoint inhibitor has been observed in 5-10% of the patients with microsatellite stable (MSS) gastrointestinal (GI) cancers. However, a reliable predictive marker to immune checkpoint inhibitor has not been discovered in this patient population. We sought to assess the incidence of mismatch repair (MMR) gene alterations and their association with tumor mutational burden (TMB) in MSS GI cancers. Methods: This is a retrospective cohort study of patients with advanced GI cancer who had molecular profiling of the tumor using either Guardant360 blood-based or Tempus tissue next generation sequencing (NGS) platforms between 2020 and 2023. We assessed four MMR genes including MLH1, MSH2, MSH6 and PMS2 for alterations. Continuous variables were summarized as median and interquartile range (IQR) and compared using Mann-Whitney test. Categorical variables were summarized as counts and proportions and compared using Fisher’s exact test. Results: A total of 2920 patients with Guardant360 were included with a median age of 65 (IQR: 56-73) years. 870 (30%) patients had pancreas cancer; 846 (29%) patients had colorectal cancer; 688 (24%) patients had biliary tract cancers; and the rest had other GI cancers. Among patients evaluable for microsatellite instability (MSI) status, 58 patients (2%) had MSI-high GI cancers, 31 (53%) of whom had MMR gene alterations. 2445 (98%) patients had MSS GI cancers, 130 (5.3%) of whom had MMR gene alterations. Patients with MSS GI cancers bearing MMR gene alterations had a median age of 70 (IQR: 62-75) years, significantly older than those without MMR gene alterations (p<0.001). In addition, patients with MMR gene alterations had significantly higher median blood TMB of 11 (IQR: 7.4-16) mut/Mb compared to 7.7 (IQR: 4.8-11) mut/Mb in patients without MMR gene alterations. In MSS GI cancers, MMR gene alterations are more commonly associated with the presence of alterations in APC (p=0.03), TP53 (p=0.01), CTNNB1 (p<0.001) and SMAD4 (p=0.01). 1453 patients with GI cancer and Tempus tissue NGS data were included as a validation cohort. They had comparable demographics with the Guardant360 cohort. 14 (1.6%) of 896 patients with MSS GI cancers had MMR gene alterations, significantly fewer (p<0.001) than those found in the Guardant360 cohort. Patients with MMR gene alterations and wild type POLE had significantly higher (p=0.001) median TMB of 9.8 (IQR: 4.8-14) mut/Mb compared to 4.7 (IQR: 3.2-6.3) mut/Mb in patients without MMR gene alterations. Conclusions: MMR gene alterations can be identified by clinical NGS platforms in a small proportion of patients with MSS GI cancers. They are associated with elevated TMB, which may suggest a hypermutated profile serving as a basis for potential role of immune checkpoint inhibitor in MSS GI cancers. Citation Format: Conor D. O'Donnell, Mojun Zhu, Hao Xie. The association of mismatch repair gene alterations with elevated tumor mutational burden in microsatellite stable gastrointestinal cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6469.

Abstract 5014: Analytical validation of a complete system for detection and characterization of genomic aberrations in circulating tumor DNA

Abstract Cell-free DNA (cfDNA) fragments are readily detected at a low concentration in the human blood stream. In healthy patients cfDNA is primarily derived from apoptosis of hematopoietic cells. In cancer patients circulating DNA derived from tumor cells (ctDNA) undergoing apoptosis, shedding, and necrosis contribute to total cfDNA. Next generation sequencing (NGS) of cfDNA isolated from patient plasma can be used to fully characterize cancer driven changes in the tumor DNA including the relative contribution of cfDNA, single nucleotide variants (SNVs), small insertions and deletions (Indels), larger deletions, regions of amplified DNA, and structural rearrangements. Frontage has developed a complete lab system for analysis of circulating cfDNA including automated sample extraction, preparation and sequencing of libraries targeting exon sequences of 293 oncology-related genes, and a bioinformatic reporting system. The Frontage sample extraction process uses a post-extraction size selection step to remove high molecular weight DNA derived from lysed blood cells in the plasma sample to increase the sensitivity of detection of ctDNA. The NGS platform couples hybridization-based selection of genomic DNA fragments derived from protein-coding exons with deep sequencing to ~4,000X gross coverage on the NextSeq 2000. The bioinformatic pipeline integrates unique molecular barcode-based generation of consensus reads for each template molecule, somatic variant calling with GATK Mutect2 and FreeBayes, and removal of false positive variants by comparison to a 60 samples healthy patient data set. Analytical validation was performed for each component of the system. For verification of the sample extraction process, cfDNA was extracted in duplicate from four lots of pooled K2 EDTA plasma of healthy patients as well as individual K2 EDTA plasma of colorectal cancer (CRC) patients in four independent extraction runs. The mean yields of cfDNA from healthy samples ranged from 2.3 - 4.2 nanograms per ml plasma and all 32 of the isolated cfDNA aliquots from healthy patient plasma showed a prominent peak around 167 nt with minimal contamination from high molecular weight DNA. For validation of the NGS and bioinformatics platform a set of 8 different reference cfDNAs were processed through four independent runs of library preparation, sequencing, and analysis. For each of the validation runs, greater than 97% of 208 SNVs and Indels with expected allele frequencies (AF) between 2-3% were detected by the platform with higher levels of accuracy for variants at above 3% AF. Two or less false positive variants per run were detected out of 278,000 nucleotides of verified non-mutated sequence that overlapped with the 293 gene panel. Four known DNA amplification events were detected in each of the validation runs. Data from characterization of CRC and other clinical samples with the system will be presented. Citation Format: Ericca Stamper, Weiming Shen, Aman Pruthi, Casey Nagel, Marcin Piechota, Katarzyna Tomala, Qi Wei, David Willoughby. Analytical validation of a complete system for detection and characterization of genomic aberrations in circulating tumor DNA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5014.

Abstract 3493: Read-level methylation pattern extraction for high-multiplex large-scale targeted NGS assay

Abstract The abnormal changes in DNA methylation are linked to the early stages of carcinogenesis. Identifying these epigenetic changes in circulating tumor DNA (ctDNA) can reveal potential biomarkers for the early diagnosis of various cancers. However, analyzing such data poses bioinformatics challenges due to the lack of sensitivity in detecting the low abundance ctDNA signals in biopsy samples, which are often overwhelmed by the complexity of libraries containing hundreds of targeted regions. Read-level methylation analysis holds the promise of more in-depth DNA methylation detection due to the wide coverage and high sensitivity of rare signals. However, this approach is hindered by the absence of a standardized workflow capable of generating interpretable reports suitable for both bench scientists and professional bioinformaticians. Here, we present a bioinformatics workflow that examines next-generation sequencing (NGS) data and characterizes the read-level methylation patterns of amplicons. Compared to other currently available tools, our method is designed to work with high-multiplex, large-scale targeted assays. It effectively eliminates the undesired noise derived from sequencing byproducts such as false CpG calls, dimers, and off-target alignments. Additionally, to accommodate the substantial volume of data generated by state-of-the-art NGS platforms, the workflow enables parallel processing of samples compatible with both cloud-based and on-premises computing resources. This workflow provides a comprehensive per-sample visualization of DNA methylation patterns and reports read-level methylation results in a “pattern-as-a-feature” table. In this table, the occurrence of an amplicon epiallelic haplotype (pattern) for every sample is represented as a “feature column” and is aggregated with all patterns discovered in the experiment. These read-level patterns, along with other information, can be used to develop machine learning algorithms to reiteratively harvest true predictive features and penalize confounding signals in predicting cancer diagnosis. Citation Format: Mingda Jin, Masatomo Kaneko, Steven Cen, Hongtao Li, Wei Guo, Xinyi Zhou, Atsuko Fujihara, Tsuyoshi Iwata, Lorenzo Storino Ramacciotti, Divyangi Paralkar, Giovanni E Cacciamani, Manju Aron, Osamu Ukimura, Inderbir S. Gill, Gangning Liang, Andre L. Abreu, Jeffrey Bhasin, Xiaojing Yang, Xi-Yu Jia. Read-level methylation pattern extraction for high-multiplex large-scale targeted NGS assay [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3493.

Abstract 1035: Identification of fibroblast growth factor receptors (FGFRs) alterations (alts) at DNA and RNA-level by one-step next-generation sequencing (NGS) panel

Abstract Background: FGFR inhibitors are currently in clinical development or approved for urothelial cancer and cholangiocarcinoma with FGFR fusion (FUS) or/and mutation (MUT). AmoyDx FGFR 1-4 NGS Panel (FGFR-P) was developed for FGFR alts detection based on both DNA and RNA. The goal of this study is to validate and assess the performance of FGFR-P in pan-cancer FGFR alts detection by comparing with an AmoyDx comprehensive genomic profiling test (CGP) and a health authority approved DNA-based NGS panel (DNA-P). Methods: Both FGFR-P and CGP are DNA (MUT) and RNA (FUS) based for detecting FGFR alts with optimized bioinformatics pipeline to eliminate baseline noise caused by deamination events, which is commonly found in aged FFPE samples. Cell lines, cell line/patient derived xenografts (CDXs/PDXs) and 397 samples of 26 cancer types (90 samples >10 years) were used for validation of FGFR-P. Total 382 and 50 pan-cancer samples were respectively tested to compare FGFR-P with CGP and DNA-P. Results: To validate accuracy, 36 FFPE samples from cell lines and CDXs/PDXs with known FGFR status (25 FUSs, 5 MUTs, 6 wild-types) were tested by FGFR-P with concordance rate 100%. In clinical sample testing, FGFR-P also showed high agreement with CGP (99.5%) and DNA-P (98.0%). Conflicting result confirmed by FISH revealed 1 FUS missed by DNA-P but detected by FGFR-P, which demonstrated advantage of FGFR alts detection by DNA+RNA. In addition, an optimized bioinformatics pipeline by mimic samples with deamination events effectively filtered out C:G>T:A false positive signals. FGFR-P achieved high success rate in testing pan-tumor samples (89.9%) including samples >10 years (75.6%). Conclusions: FGFR-P provides a novel opportunity to identify FGFR alt pan-cancer patients using a robust DNA+RNA NGS platform, which shows high success rate even in aged samples and will be a potent tool for sensitive and reliable detection of FGFR alts for clinical diagnostics. CGP FGFR-P MUT Positive Negative Total Positive 29 0 29 Negative 1 194 195 Total 30 194 224 FUS Positive Negative Total Positive 20 1 21 Negative 0 137 137 Total 20 138 158 The overall percent agreement 99.5% Citation Format: Min Qing, Xuejun Chen, Wenqing Su, Xiaofang Zhuo, Ting Shen, Chengjuan Xiong, Xuesong Lyu, Renee Tate, Qibiao Wu, Longen Zhou, Shibu Thomas, Wangwang Ning, Jianqing Wang, Huihui Yan, Zhiqiang Yin, Zhan Huang, Yaxin Xue, Changbin Zhu. Identification of fibroblast growth factor receptors (FGFRs) alterations (alts) at DNA and RNA-level by one-step next-generation sequencing (NGS) panel [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1035.

Abstract 939: Comprehensive genomic and transcriptomic analysis to guide therapy for patients with metastatic solid tumors

Abstract Introduction: Precision oncology, which aims to profile tumors for identifying actionable alterations to guide therapy has become mainstream in cancer clinics. While most approaches focus on DNA aberrancies on targeted panels, not all patients’ tumors show targetable genomic alterations in these limited gene sets, indicating a need for more comprehensive methodologies combining whole exome (WES) and transcriptome sequencing (RNAseq) to direct a larger fraction of patients to therapies. Here, we aimed to compare the feasibility and utility of BostonGene’s (BG) analytical platform with WES and RNAseq to two targeted exome panels in identifying actionable alterations and therapy selection for patients with metastatic solid tumors. Methods: Study enrolled patients with solid tumors who have had next-generation-sequencing (NGS) testing with one of two tissue-based targeted exome sequencing panels, Oncomine (ThermoFisher, 148 genes), or MDA-MAPP (615 genes) with identification of no College of American Pathologists Tier-1 actionable DNA alterations between August 2022 and November 2023. Patients’ tissue and blood/saliva samples were subjected to BG’s NGS platform. An alteration-level actionability analysis was done upon receipt of WES results to inform treatment decision making, based on functional and therapeutic effects of the alterations detected. Results: A total of 64 patients were enrolled; testing was cancelled for 7 patients (10.9%) with samples showing <10% tumor content, while 5 patients’ reports were pending at the time of data analysis. Median turn-around time from consent to sample acquisition (SA) and from SA to available genomic profile were 8 and 15.5 days, respectively. 52 patients had NGS results representing a diverse group of tumor types, including sarcoma (n=11, 21.2%), head and neck (n=9, 17.3%) and mesothelioma (n=5, 9.6%). BG reported DNA alterations (mean: 3.75; median: 4) not shown by prior testing for 94.2% (49/52) of patients, whereby the most common alteration types included SNVs (43.1%), deletions (23.6%) and amplifications (11.8%). BG identified additional actionable DNA alterations (referred to as “AAs”; mean: 1.9, median: 2) in 32 (61.5%) patients, whereby majority of AAs were deletions (59.7%) and amplifications (27.4%). BG detected AAs in 58.8% (10/17) vs 62.9% (22/35) of patients for whom the same vs different samples were used across different tests compared (P=0.8). Of 32 patients with AAs, 7 (21.9%) were directed to DNA-informed treatments within 6 months based upon the new genomic findings reported by BG, 14 (43.8%) are being followed for treatment matching, while 11 (34.4%) were deceased. Discussion: Our results show the feasibility and utility of comprehensive molecular testing to match patients to WES-informed treatments within a clinically relevant time frame. Assessment of the additional benefit of RNA expression analysis is forthcoming. Citation Format: Burak Uzunparmak, Fei Su, Amber M. Johnson, Krystle Nomie, Nathan Fowler, Lile Kontselidze, Aleksander Bagaev, Anna Butusova, Ecaterina E. Dumbrava, Jordi Rodon-Ahnert, Siqing Fu, David S. Hong, Timothy A. Yap, Aung Naing, Sarina A. Piha-Paul, Daniel D. Karp, Paula R. Pohlmann, Apostolia M. Tsimberidou, Keyur P. Patel, Funda Meric-Bernstam. Comprehensive genomic and transcriptomic analysis to guide therapy for patients with metastatic solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 939.

APPLICATION OF THE NEXT GENERATION SEQUENCING IN BIOLOGY AND MEDICINE

Next-Generation Sequencing (NGS), also known as high-throughput sequencing, refers to a set of modern DNA sequencing technologies that have revolutionized the field of genomics. Advantages of NGS techniques involving high speed (parallel sequencing is faster than traditional methods, allowing researchers to obtain results more quickly), cost-effectiveness (ability to sequence multiple fragments simultaneously reduces the cost per base compared to traditional sequencing), and scalability (platforms can be scaled to accommodate varying levels of throughput depending on experimental needs). NGS has significantly accelerated genomics research, enabling breakthroughs in fields such as personalized medicine, cancer genomics, and evolutionary biology. However, challenges such as data analysis complexity, error rates, and cost still exist and are areas of ongoing research and improvement within the field of sequencing technologies. Paper contains the brief explanation of the current NGS platforms and their features. NGS biomedical application is described with the main advantages and abilities of the analysed tools.

Development of a novel mycobiome diagnostic for fungal infection

BackgroundAmplicon-based mycobiome analysis has the potential to identify all fungal species within a sample and hence could provide a valuable diagnostic assay for use in clinical mycology settings. In the last decade, the mycobiome has been increasingly characterised by targeting the internal transcribed spacer (ITS) regions. Although ITS targets give broad coverage and high sensitivity, they fail to provide accurate quantitation as the copy number of ITS regions in fungal genomes is highly variable even within species. To address these issues, this study aimed to develop a novel NGS fungal diagnostic assay using an alternative amplicon target.MethodsNovel universal primers were designed to amplify a highly diverse single copy and uniformly sized DNA target (Tef1) to enable mycobiome analysis on the Illumina iSeq100 which is a low cost, small footprint and simple to use next-generation sequencing platform. To enable automated analysis and rapid results, a streamlined bioinformatics workflow and sequence database were also developed. Sequencing of mock fungal communities was performed to compare the Tef1 assay and established ITS1-based method. The assay was further evaluated using clinical respiratory samples and the feasibility of using internal spike-in quantitative controls was assessed.ResultsThe Tef1 assay successfully identified and quantified Aspergillus, Penicillium, Candida, Cryptococcus, Rhizopus, Fusarium and Lomentospora species from mock communities. The Tef1 assay was also capable of differentiating closely related species such as A. fumigatus and A. fischeri. In addition, it outperformed ITS1 at identifying A. fumigatus and other filamentous pathogens in mixed fungal communities (in the presence or absence of background human DNA). The assay could detect as few as 2 haploid genome equivalents of A. fumigatus from clinical respiratory samples. Lastly, spike-in controls were demonstrated to enable semi-quantitation of A. fumigatus load in clinical respiratory samples using sequencing data.ConclusionsThis study has developed and tested a novel metabarcoding target and found the assay outperforms ITS1 at identifying clinically relevant filamentous fungi. The assay is a promising diagnostic candidate that could provide affordable NGS analysis to clinical mycology laboratories.

Microbial diversity in dairy manure environment under liquid-solid separation systems

ABSTRACT In dairy manure, a wide array of microorganisms, including many pathogens, survive and grow under suitable conditions. This microbial community offers a tremendous opportunity for studying animal health, the transport of microbes into the soil, air, and water, and consequential impacts on public health. The aim of this study was to assess the impacts of manure management practices on the microbial community of manure. The key novelty of this work is to identify the impacts of various stages of manure management on microbes living in dairy manure. In general, the majority of dairy farms in California use a flush system to manage dairy manure, which involves liquid-solid separations. To separate liquid and solid in manure, Multi-stage Alternate Dairy Effluent Management Systems (ADEMS) that use mechanical separation systems (MSS) or weeping wall separation systems (WWSS) are used. Thus, this study was conducted to understand how these manure management systems affect the microbial community. We studied the microbial communities in the WWSS and MSS separation systems, as well as in the four stages of the ADEMS. The 16S rRNA gene from the extracted genomic DNA of dairy manure was amplified using the NovoSeq Illumina next-generation sequencing platform. The sequencing data were used to perform the analysis of similarity (ANOSIM) and multi-response permutation procedure (MRRP) statistical tests, and the results showed that microbial communities among WWSS and MSS were significantly different (p < 0.05). These findings have significant practical implications for the design and implementation of manure management practices in dairy farms.

Clinical value of multi-gene testing in distinguishing benign and malignant thyroid nodules.

The newly released 2022 WHO Classification of Neuroendocrine Neoplasms (version 5) and a recent update on thyroid tumor classifications have emphasized genetic testing to an unprecedented level. Fine needle aspiration (FNA) has been widely applied for the preoperative diagnosis of thyroid nodules. However, it is limited mainly to testing for a single gene-BRAFV600E, whereas multi-gene testing data are scarce, especially in the Asian population. This study aimed to explore the clinical value of multi-gene testing in the differential diagnosis of benign and malignant thyroid nodules based on the 2023 Bethesda System for Reporting Thyroid Cytopathology (BSRTC). A total of 615 thyroid nodules underwent ultrasound-guided fine-needle aspiration cytology (FNAC) were collected from Sir Run Run Shaw Hospital, Zhejiang University School of Medicine. The next-generation sequencing platform was applied for multi-gene testing. A panel of well-recognized commonly mutated genes in thyroid cancer were analyzed, including BRAFV600E, KRAS, NRAS, HRAS, TERT, TP53, PAX8/PPARG, CCDC6/ RET and NCOA4/ RET. Gene mutations were identified in 324 nodules (52.7%), with BRAFV600E being the most prevalent driver gene alteration observed in this cohort (233/324; 79.1%), followed by RAS (77/324, 23.8%). The overall malignancy rate of gene mutations was 89.7% in our cohort, of which the lymph node metastasis rate was 45.3%. The combination of multi-gene testing and cytology resulted in 89.3% sensitivity, 95.2% specificity, 98.9% positive predictive value, 64.5% negative predictive value and 90.3% accuracy, which were significantly higher than those from mere cytology (sensitivity 68.6%, specificity 87.5%, positive predictive value 95.9%, negative predictive value 39.8%, accuracy 72.2%). Multi-gene testing could substantially enhance the detection rate of malignant thyroid nodules and protect patients with benign nodules from unnecessary surgeries. Multi-gene testing provides a valuable reference for individualized preoperative decision-making, which may serve as a crucial method for postoperative treatment and prognosis assessment.

Human mtDNA-Encoded Long ncRNAs: Knotty Molecules and Complex Functions.

Until a few decades ago, most of our knowledge of RNA transcription products was focused on protein-coding sequences, which were later determined to make up the smallest portion of the mammalian genome. Since 2002, we have learnt a great deal about the intriguing world of non-coding RNAs (ncRNAs), mainly due to the rapid development of bioinformatic tools and next-generation sequencing (NGS) platforms. Moreover, interest in non-human ncRNAs and their functions has increased as a result of these technologies and the accessibility of complete genome sequences of species ranging from Archaea to primates. Despite not producing proteins, ncRNAs constitute a vast family of RNA molecules that serve a number of regulatory roles and are essential for cellular physiology and pathology. This review focuses on a subgroup of human ncRNAs, namely mtDNA-encoded long non-coding RNAs (mt-lncRNAs), which are transcribed from the mitochondrial genome and whose disparate localisations and functions are linked as much to mitochondrial metabolism as to cellular physiology and pathology.