Generation Sequencing Platforms Research Articles

Genetic Analyses of Primary Liver Cancer Cell Lines: Correspondence With Morphological Features of Original Tumors.

Advancements in genetic analysis technologies have led to establishment of molecular classifications systems for primary liver cancers. The correlation between pathological morphology and genetic mutations in hepatocellular carcinoma (HCC) is becoming increasingly evident. To construct appropriate experimental models, it is crucial to select cell lines based on their morphology and genetic mutations. In this study, we conducted comprehensive genetic analyses of primary liver cancer cell lines and examined their correlations with morphology. Thirteen primary liver cancer cell lines established in our Department were investigated. Eleven cell lines were HCC cell lines, whereas 2 were combined hepatocellular-cholangiocarcinoma (CHC) cell line characteristics. Whole exome sequencing and fusion gene analyses were conducted using a next generation sequencing platform. We also examined correlations between cell mutations and morphological findings and conducted experiments to clarify the association between morphological findings and genetic alterations. Mutations in TP53, HMCN1, PCLO, HYDIN, APOB, and EYS were found in 11, 5, 4, 4, 3, and 3 cell lines, respectively. CTNNB1 mutation was not identified in any cell line. The original tumor of four cell lines (KYN-1, KYN-2, KYN-3, and HAK-6) showed morphologically macrotrabecular massive patterns and these cell lines harbor TP53 mutations. Two cell lines (KYN-2 and KMCH-2) showed an extremely high tumor mutation burden. These two cell lines possess ultra-mutations associated with DNA repair and/or DNA polymerase. The study identified correlations between morphological findings and genetic mutations in several HCC cell lines. Cell lines with unique genetic mutations were found. This information will be a valuable tool for the selection of suitable experimental models in HCC research.

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 4004: Fine-tuning affinity/avidity of anti-CAIX CAR T cells to mitigate on-target off-tumor toxicity in clear cell renal cell carcinoma

Abstract On-target off-tumor toxicity (OTOT) is one of the major hurdles preventing tumor-associated antigen (TAA) targeted therapy from effectively treating patients with solid tumors due to the shared presence of the same TAA on normal tissues. We have previously demonstrated high expression of carbonic anhydrase IX (CAIX) in ccRCC patient samples and low-density expression on healthy bile duct epithelium. In 2006, a clinical trial was stopped due to OTOT bile duct toxicity observed in patients infused with anti-CAIX CAR T cell therapy. To address the issue of OTOT in the bile duct, we performed affinity/avidity fine-tuning of the anti-CAIX CAR G36 to specifically target ccRCC tumor cells with high CAIX expression while sparing cholangiocytes with low CAIX expression. We generated a complementarity-determining region (CDR) single variant mutagenesis library of G36 and displayed the library on yeast for screening. Using flow cytometry, we were able to identify six populations based on antigen-binding affinity: high, med-high, med, med-low, low, and loss of binding. These libraries were sequenced via PacBio, a next generation sequencing (NGS) platform that excels in long-read sequencing, providing a powerful tool to detect single mutations in the scFv. The results were analyzed to identify enriched mutations compared to the wild-type G36, and inspected using in silico modeling and docking. Furthermore, enriched mutants from med-low and low populations were tested in CAR-T format for their cytotoxic capacity on tumor cells and bile duct cholangiocytes, as well as their avidity. We demonstrate that mutations in CDR2 and CDR3 of the heavy chain successfully mitigate OTOT toxicity on cholangiocytes with physiological levels of CAIX expression while maintaining tumor-killing capacity, shedding light on CDR mutagenesis as a useful tool to mitigate OTOT toxicity. Citation Format: Stephen Hsien-Chi Yuan, Yufei Wang, Rabia Abbas, Peterson Mathenge, Charles Chien, Christian D. Coherd, Branson Bajoua, Matthew R. Chang, Nithyassree Murugan, Gabriella M. Kastrunes, Guilin Wang, Song-My Hoang, Wayne A. Marasco. Fine-tuning affinity/avidity of anti-CAIX CAR T cells to mitigate on-target off-tumor toxicity in clear cell renal cell carcinoma [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 4004.

Abstract 3652: Spatial transcriptomic study of the tumor microenvironment in HNSCC

Abstract Background: The complex and dynamic nature of the tumor-immune microenvironment (TME) presents challenges for identification of robust and predictive biomarkers in immuno-oncology (IO). Standard multiplex immunohistochemistry (mIHC) or multiplex immuno-fluorescent assays (mIF) allow for the phenotyping on the cellular level but are limited by the number of markers available and the detection systems. Gene expression or next generation sequencing (NGS) platforms provide high-plex bulk data sets about the TME, but the spatial context of the tumor and immune cell interactions is lost. The NanoString GeoMx® Digital Spatial Profiler (DSP) combines spatial context, utilizing fluorescent morphological markers, with molecular profiling capabilities. This combination allows for the quantitative analysis of high-plex analyte abundance which can be traced back to a region of interest within a sample, providing biological inference in the region of interest. Methods: Formalin-fixed paraffin-embedded (FFPE) specimens from HNSCC patients were cut in 5µm sections for all analyses. Whole section tissue analysis was previously performed utilizing the NanoString nCounter® PanCancer IO 360™ Panel. Tumor inflammation signatures (TIS), an 18-gene signature measuring the adaptive immune response within tumors, was generated for each specimen and categorized each as immune-hot or immune-cold based on predefined parameters. Four specimens were selected based on their TIS score and two sets of cut sections were processed by either Labcorp lab in Morrisville, NC or Nanostring lab in Seattle, WA for precision evaluation, using the GeoMx Human Whole Transcriptome Atlas (WTA) panel (18,000 genes) with the NanoString GeoMx digital spatial profiling technology. The TME regions of interest (ROI) were identified using a set of morphology markers consisting of panCK, CD45 and Syto13; 12 ROIs were selected from each sectioned tumor material. The same ROIs were selected at the two testing sites with matching tissue location and morphology characters. Results: GeoMx WTA panel provided an unbiased view of 18,000 protein-coding genes at specific TME regions. These counts were successfully mapped to the 12 ROIs on the tissue. A pseudo-TIS score was generated using the same 18 gene signature from the WTA panel for each ROI. The pseudo-TIS scores from ROIs from the four specimens showed consistency with the specimen-wise TIS score, yet variations were observed across different TME/ROI within each specimen. Whole transcriptome expression results showed high correlation between the two testing sites, providing confidence in the reproducibility of the technology. High resolution fluorescent staining images were able to be fed to downstream quantitative digital analysis to generate complementary spatial data. Citation Format: Caitlin Schroyer, Shanshan Zhou. Spatial transcriptomic study of the tumor microenvironment in HNSCC [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 3652.

Metabarcoding mosquitoes: MinION sequencing of bulk samples gives accurate species profiles for vector surveillance (Culicidae)

Mosquitoes (Family: Culicidae) are dominant vectors of pathogens, and their surveillance has been incorporated into major disease control programs worldwide. However, routine, species-level identification of mosquitoes is often a bottleneck for management, and Next Generation Sequencing (NGS) platforms and DNA metabarcoding can revolutionize this process. MinION nanopore technologies promise on-site sequencing and rapid sample processing rates ideal for time-sensitive biosurveillance. Here, we benchmark the results of DNA metabarcoding on the MinION against the Illumina MiSeq platform, which is known for its higher sequencing accuracy. We used metazoan COI mini-barcode primers to carry out DNA metabarcoding of mosquito bulk samples caught during a real vector survey, then compared the mosquito species profiles recovered on each sequencing platform. We also tested the influence of using different trap lures, storage methods, and pooling different specimen body parts on the number of species recovered. We report that mosquito species-level identifications were highly congruent between MinION and Illumina (93% overlap). We also find that CO2 gas cylinders outperformed biogenic CO2 sources significantly, by two-fold. Notably, we demonstrated the feasibility of detecting zoonotic reservoirs and pathogen signals from mosquito bulk samples. We present the first use of DNA metabarcoding on the MinION for vector surveillance and discuss future applications.

Survival of patients with microsatellite instability-high and Lynch syndrome-associated pancreatic ductal adenocarcinomas.

640 Background: Pancreatic ductal adenocarcinoma (PDAC) remains a challenging disease due to its aggressive nature, late-stage diagnosis, and limited treatment options. There is an increased interest in cancers which harbor microsatellite instability (MSI) due to their susceptibility to immune checkpoint inhibitors. MSI in PDAC is rare, and little is known about survival in this subtype of pancreas cancers. Our aim was to assess overall survival in surgically resected high microsatellite instability (MSI-H), mismatch repair deficient (MMRd), and Lynch Syndrome (LS) associated PDACs treated in our center. Methods: Patients with surgically resected PDACs from 1990 to 2023 were identified. A total of 936 cases were sequenced utilizing MSK-IMPACT, a targeted next generation sequencing platform. Patients with germline or sporadic pathogenic variants or likely pathogenic variants in one of the DNA MMR genes MLH1, MSH2, MSH6, PMS2, or 3’ end of EpCAM were identified. MSI-H, MMRd, and LS-associated cases were matched to patients with microsatellite stable or MMR proficient, non-LS-associated PDAC in a 1:2 ratio with a direct match on age (±5 years), gender, and year of surgery (±3 years). Kaplan-Meier curves were used to visualize the data. A generalized estimating equation (GEE) cox model with robust sandwich estimator was used to compare matched cohorts. Results: 17 patients with MSI-H, MMRd or LS-associated surgically resected PDACs were identified. Five patients were classified as MSI-H, 3 patients were MSI-indeterminate, and 9 patients were microsatellite stable. Eight patients were found to have pathogenic germline variants in LS-associated genes. The remaining nine patients had sporadic pathogenic DNA MMR gene variants ( MLH1 n=3, MSH6 n=6). Median age at surgery was 66 (interquartile range (IQR) 60-77), 41% of the patients were female, and median year of surgery was 2014. MSI-H, MMRd or LS-associated PDACs were matched to 34 control patients. The median survival time of the cases was 12 years vs. 1.9 years in the control group. Five-year survival rate was 81% (IQR 60-100%) and 18% (7.4%-43%), respectively. LS-associated, MSI-H or MMRd status in resectable PDAC was a strong predictor of good prognosis (hazard ratio 0.17, 95% CI 0.07-0.38, p value <0.001). Conclusions: MSI-H, MMRd or LS-associated PDACs displayed significantly better survival compared to their MSI-stable, MMR proficient, non-LS-associated counterparts, in the era before routine use of immunotherapy. It is expected that survival will be increased further with more frequent availability of immunotherapy.

Patient-derived biliary tract cancer organoid biobank: Is personalized medicine utilizing organoids feasible for patients with cholangiocarcinoma?

533 Background: Cholangiocarcinoma (CC) is an aggressive malignancy with an all stage 5-year survival rate of less than 15%. Preclinical research is of pivotal importance to acquire a deeper understanding of CC and improve outcomes. The establishment of CC organoids has previously been described to be difficult due to fewer driver mutations and limitations in the ability to mimic tumor microenvironment. The aim of this study was to develop a successful culturing system for CC organoids that preserves intratumoral heterogeneity, and in turn, to determine the feasibility of personalized medicine in patients with CC using organoids. Methods: From 2020-2023, 75 CC tumor samples were prospectively collected from 65 patients. The tumors were sequenced using MSK-IMPACT, a targeted next generation sequencing (NGS) platform. Samples were processed, and organoids were generated and cultured following a standardized protocol. The organoids were grown in three different types of enriched media (M1, M2, and M3). Once cultures were adequately expanded, organoid lines were sequenced using MSK-IMPACT and/or MiSeq, a benchtop NGS platform. Mutations of the organoid line were compared to that of the tumor of origin to determine tumor enrichment. To ascertain genotypic association with organoid formation, we examined frequencies of driver mutations of interest. Logistic regression was used to assess the univariate association between mutations of interest and organoid establishment. Two-sided p-values less than 0.05 were considered to be significant. Results: Of the 75 samples collected, 24 (32%) were extrahepatic CC tumors, and the remaining were of intrahepatic origin. 92% of the samples were collected from primary tumors, and 71% of the samples were chemotherapy naïve. 64 samples successfully grew organoids. Upon evaluation of MSK-IMPACT and MiSeq data, 11 of the 64 samples were noted to retain the mutations of the tumor of origin in organoid culture; the overall success rate of tumor organoid formation was 14.6% (11/75). Tumor enrichment of organoid lines was most commonly achieved using M3 (9 out of 11 samples), the median passage at which tumor enrichment occurred was 8 (interquartile range (IQR) 2-10), indicating that the approximate median time to tumor organoid formation was 16 weeks (IQR 4-20 weeks). There was no statistically significant association between driver mutations and organoid establishment. Conclusions: Intratumoral heterogeneity of cholangiocarcinoma is maintained in organoid culture. This preclinical model potentially recapitulates the clinical features of the disease seen in humans. However, prolonged duration and low success rate of tumor enrichment makes it a less appealing avenue for personalized medicine.

Understanding the bacterial community structure associated with the Eichhornia crassipes rootzone.

Plant microbiome acts as an interface between plants and their environment, aiding in the functioning of the ecosystem, such as protection against abiotic and biotic stress along with improving nutrient uptake. The rhizosphere is an essential interface for the interaction between plants and microbes and plays a substantial part in the removal as well as uptake of heavy metals and antibiotics from contaminated locations. Eichhornia crassipes is a promising plant that contains a rich community of microbes in its rhizosphere. Microorganism's association with plants embodies a crucial pathway via which humans can also be exposed to antibiotic-resistant genes and bacteria. In our earlier study enhanced removal of ciprofloxacin was observed by plant growth-promoting Microbacterium sp. WHC1 in the presence of E. crassipes root exudates. Therefore, the V3-V4, hypervariable region of the 16S rRNA gene was studied to assess the bacterial diversity and functional profiles of the microbiota associated with plant roots. Using the QIIME software program, 16S rRNA data from the Next Generation Sequencing (NGS) platform was examined. Alpha diversity including Chao1, Observed Shannon, and Simpson index denote significantly higher bacterial diversity. Proteobacteria (79%) was the most abundant phylum which was present in the root samples followed by Firmicutes (8%) and Cyanobacteria (8%). Sulfuricurvum (36%) is the most abundant genus belonging to the family Helicobacteraceae and the species kujiense in the genus Sulfuricurvum is the most abundant species present in the root sample. Also, the bacterial communities in the rhizoplane of Eichhornia crassipes harbor the genes conferring resistance to beta-lactams, tetracycline, fluoroquinolones, and penams. Metagenomic studies on the E. crassipes microbiome showed that the bacterial communities constituting the root exudates of the Eichhornia aid them to survive in a polluted environment.

Lesser prevalence of polyps/WNT-dysregulation and concomitant upregulation of gamma-catenin/MYC point to alternate pathways in colorectal cancer in India.

Colorectal cancer (CRC) is known to follow adenoma carcinoma sequence (ACS) in majority of the tumors and the driver variants and associated pathways are well delineated. However, most of the published data are from the west and information in other ethnicities is sparse. We therefore comprehensively evaluated the CRC tumors from Indian ethnicity for the prevalence of ACS. In this cohort study, clinical data of 100,497 patients who attended hospital between 2013 and 2018 were accessed. Tumors from patients (n = 130) with CRC who were treated primarily by surgery were included. DNA and RNA were isolated to assess variants (direct sequencing) and WNT-pathway dysregulation in genes related to ACS. Global gene expression was generated and analyzed on microarrays (Affymetrix; N = 10) and next generation sequencing platforms (Illumina; N = 25). Gene expression at mRNA (qRT-PCR) and protein level (IHC) of JUP/CTNNB1/MYC were assessed. Correlation between expression of JUP and MYC was evaluated by Karl Pearson's correlation coefficient. The prevalence of polyps was 16.75%, while 18.26% variants in APC/CTNNB1, 20.00% in KRAS, and 18.33% WNT dysregulation were noted. Interestingly, 29/60 (48.33%) tumors showed only MYC upregulation with normal APC/CTNNB1 expression. Global gene expression and validation in an independent tumor cohort confirmed concomitant upregulation of JUP (gamma-catenin) & MYC (r = 0.71; p = 0.001) at mRNA and protein in sizeable number of tumors (45/96; 46.88%). Our study provides evidence for limited prevalence of ACSin the Indian ethnicity. Preventive colonoscopies for early identification and management of CRC may not be an effective strategy in this ethnicity.

Mycobacterium tuberculosis complex molecular networks and their regulation: Implications of strain heterogeneity on epigenetic diversity and transcriptome regulation

Tuberculosis has been a public health crisis since the 1900, which has caused the highest mortalities due to a single bacterial infection worldwide, that was recently further complicated by the Coronavirus disease 2019 pandemic. The causative agent of Tuberculosis, Mycobacterium tuberculosis, belongs to a genetically well-characterized family of strains known as the Mycobacterium tuberculosis complex, which has complicated progress made towards eradicating Tuberculosis due to pathogen-specific phenotypic differences in the members of this complex. Mycobacterium tuberculosis complex strains are genetically diverse human- and animal-adapted pathogens belonging to 7 lineages (Indo-Oceanic, East-Asian, East-African Indian, Euro-American, M. africanum West Africa 1, M. africanum West Africa 2 and Ethopia), respectively and the recently identified Lineage 8 and M. africanum Lineage 9. Genomic studies have revealed that Mycobacterium tuberculosis complex members are ∼99 % similar, however, due to selective pressure and adaptation to human host, they are prone to mutations that have resulted in development of drug resistance and phenotypic heterogeneity that impact strain virulence. Furthermore, members of the Mycobacterium tuberculosis complex have preferred geographic locations and possess unique phenotypic characteristics that is linked to their pathogenicity. Due to the recent advances in development next generation sequencing platforms, several studies have revealed epigenetic changes in genomic regions combined with “unique” gene regulatory mechanisms through non-coding RNAs that are responsible for strain-specific behaviour on in vitro and in vivo infection models. The current review provides up to date epigenetic patterns, gene regulation through non-coding RNAs, together with implications of these mechanisms in down-stream proteome and metabolome, which may be responsible for “unique” responses to infection by members of the Mycobacterium tuberculosis complex. Understanding lineage-specific molecular mechanisms during infection may provide novel drug targets and disease control measures towards World Health organization END-TB strategy.

Enhanced clinical assessment of hematologic malignancies through routine paired tumor and normal sequencing

Genomic profiling of hematologic malignancies has augmented our understanding of variants that contribute to disease pathogenesis and supported development of prognostic models that inform disease management in the clinic. Tumor only sequencing assays are limited in their ability to identify definitive somatic variants, which can lead to ambiguity in clinical reporting and patient management. Here, we describe the MSK-IMPACT Heme cohort, a comprehensive data set of somatic alterations from paired tumor and normal DNA using a hybridization capture-based next generation sequencing platform. We highlight patterns of mutations, copy number alterations, and mutation signatures in a broad set of myeloid and lymphoid neoplasms. We also demonstrate the power of appropriate matching to make definitive somatic calls, including in patients who have undergone allogeneic stem cell transplant. We expect that this resource will further spur research into the pathobiology and clinical utility of clinical sequencing for patients with hematologic neoplasms.

Quantum analysis of squiggle data

Squiggle data is the numerical output of DNA and RNA sequencing by the Nanopore next generation sequencing platform. Nanopore sequencing offers expanded applications compared to previous sequencing techniques but produces a large amount of data in the form of current measurements over time. The analysis of these segments of current measurements require more complex and computationally intensive algorithms than previous sequencing technologies. The purpose of this study is to investigate in principle the potential of using quantum computers to speed up Nanopore data analysis. Quantum circuits are designed to extract major features of squiggle current measurements. The circuits are analyzed theoretically in terms of size and performance. Practical experiments on IBM QX show the limitations of the state of the art quantum computer to tackle real life squiggle data problems. Nevertheless, pre-processing of the squiggle data using the inverse wavelet transform, as experimented and analyzed in this paper as well, reduces the dimensionality of the problem in order to fit a reasonable size quantum computer in the hopefully near future.

Comparison of nine extraction methods for bacterial identification using the ONT MinION sequencer.

The Anthrax mailings bioterrorism attack in 2001 revealed the need for universal and rapid microbial forensic analyses on unknown biological evidence. However, the gold standard for bacterial identification includes culturing isolates, which is laborious. Molecular approaches for bacterial identification revolve around 16S ribosomal gene sequencing using Sanger or next generation sequencing (NGS) platforms, but these techniques are laboratory-based and can also be time-consuming. The Oxford Nanopore Technologies (ONT) MinION sequencer can generate long read lengths that span the entire bacterial 16S rRNA gene and accurately identify the species level. This platform can be used in the field, allowing on-site evidence analysis. However, it requires higher quantities of pure DNA compared to other sequencing platforms; thus, the extraction method for bacterial DNA is critical for downstream analysis, which to date are tailored toward a priori knowledge of the species' taxonomic grouping. During an attack, the investigative team may not know what species they are handling; therefore, identifying an extraction method that can handle all bacterial groups and generate clean DNA for the MinION is useful for microbial forensic analysis. The purpose of this study was to identify a "universal" extraction method that can be coupled with ONT MinION sequencing for use in forensic situations for rapid identification. It also evaluated the cloud-based data analysis software provided by ONT, EPI2ME. No "universal" extraction method was identified as optimal for downstream MinION sequencing. However, the DNeasy PowerSoil Kit and Noda et al. Chelex-100 method gave comparable sequencing results and could be used as rapid extraction techniques. This study showed that the ONT 16S Barcoding Kit 1-24 coupled with the 16S FASTQ workflow might not be the best for use in forensic situations where species-level identification needs to be obtained, as most alignments were approximately 89% accurate. In all seven test organisms and nine extraction methods, accurate species identification was onlyobtained in 63% of the cases.

B-240 Development of AcroMetrixTM Human Microbiome Quality Control Materials

Abstract Background The human microbiome is receiving significant attention given its critical roles in the maintenance of human health. The compositional and functional alterations of the human microbiota have been associated with various pathological conditions including colorectal cancer (CRC). As the number of human metagenomic data increases, the interest to utilize these metagenomic biomarkers for diagnosis of colorectal cancer and other diseases has also elevated. Studies that evaluated the defined fecal microbial markers for CRC detection showed the test accuracy could vary from 73% to 98% depending on the detection panels of microbiome biomarkers. The measurement inconsistencies between laboratories due to the use of different detection platforms, the coverage of various assay panels, the biases in different steps of microbiome workflow all indicated the critical need for reliable quality control materials. Currently, the commercially available microbial standards usually contain limited species and also mixed with fixed ratios which may not reflect the microbial composition in the clinical samples. There is no commutable reference material that recapitulates the dysbiotic microbial composition under healthy or pathological conditions (e.g., CRC). Therefore, in this study, we developed DNA quality control materials (QCMs) that recapitulate the dysbiotic microbial composition for colorectal cancer to assess the technical and non-technical variation for part of the microbiome workflow. Methods We first identified the specific bacterial species that either are enriched in CRC patients or healthy human gut based on comprehensive literature search and the other available dataset. We then created microbiome QCMs to recapitulate their dysbiotic microbial composition in comparison to the compositions in healthy condition. The QCM consists of a mixture of genomic DNA from the selected bacterial species, the copy number of DNA was determined by Bio-RadTM droplet digital PCR (ddPCR) with the assays that target to species-specific regions to reflect the microbial composition. In addition, the microbiome controls are further evaluated and validated by Microbiome Health Research Assay on Ion Ampliseq and GeneStudio S5 platforms. Results The AcroMetrixTM human microbiome QCMs have been carefully formulated to mimic dysbiotic microbial composition for colorectal cancer or healthy human gut. This CRC QCM covers 29 different species with a wide range of guanine-cytosine content sequences, belonging to 10 phyla and 20 genera. 25 out of these 29 species (86.2%) were covered by Microbiome Health Research Assay. To mimic the trends of dysbiotic microbial composition, the DNA frequency among these 29 species was targeted from 0% to 15% according to the microbial composition. Droplet digital PCR results confirmed the target frequencies within the accepted tolerance range and the results were compared with the data from the Next Generation Sequencing platform. Conclusions The human microbiome QCMs that cover 29 bacterial species and mimic dysbiotic microbial composition for colorectal cancer or healthy human gut have been designed and developed. These QCMs could provide essential elements to verify, validate and monitor test performance, technical and non-technical variation of microbiome workflows, as well as product QC releases and method development for ddPCR & NGS assays.

B-210 Spectrum of Germline Mutation with Characteristic MLH1 Hotspots and High Concordance with MMR Deficiency Phenotype in Lynch Syndrome Patients in Taiwan

Abstract Background Patients carried defects on cancer susceptibility genes or had cancer family history have at least twice the risk of developing cancer than the general population, with both early onset and multiple cancers features. For elucidating the carrier rate of cancer susceptibility genes in patients with inherited colorectal cancer (CRC) and endometrial cancer (EnCa) in Taiwan, we established a NCCN guided 30-gene panel which included four DNA Mismatch Repair (MMR) genes to detect the germline mutation of patients with Lynch Syndrome. Correlation of genotype and phenotype of MMR enzymes are under investigation as well. Methods Sixty-eight blood samples were collected from unrelated cancer patients, 42 of them were from patients with inherited colon cancer, 13 from patients with diagnosis of both CRC and endometrial cancer and 13 from patients with early onset EnCa. 30-gene panel was performed by next generation sequencing (NGS) platform and variants were annotated following ACMG guideline. MMR protein expression in tumor tissue sections were identified by conventional immunohistochemistry (IHC) stain. Loss of nuclear staining were classified as MMR deficiency. Results Average age of first tumor onset of 68 patients were 49.2 years old. 54% (37/68) of the subjects had at least one germline mutations with pathogenic effect or deleterious variant predicted by in silico protein function software. Five of them carried two heterozygous mutations. Total 42 mutations appeared majority on three MMR genes, MLH1 (17/42, 40%), MSH2 (10/42, 24%) and MSH6 (6/42, 11%) with notified three hotspots on MLH1 p.R265C (7/17, 41%); p.T117M (2/17, 12%) and p.K618del(2/17, 12%). The rest mutations spread sporadically on nine genes (PMS2, TP53, CHEK2, POLE, POLD1, ATM, EPCAM, MLH3 and RNF43). For exploring the effect of germline mutation on MMR protein expression status on tissue, we compared the MMR IHC results and the germline mutation pattern. As high as 57% concordance between phenotype and genotype were found. We also collected six blood samples from relatives of our CRC cohort to demonstrate the inheritance and pathogenesis of the germline mutation. Exactly the same germline mutations can be found in diseased relatives among different generation or siblings. Conclusion In spite of having founder mutations on MLH1 gene in Taiwanese Lynch syndrome, most of the germline mutations randomly occurred in MMR-related genes. Comprehensive cancer susceptible gene screening panel by NGS is a cost-effective way to identify the inherited status of individuals with family cancer history and serves as a surrogate tool to predict MMR protein expression. Genetic counseling and prophylactic therapeutic information can be provided for positive cases for better cancer prevention.

Current Applications of Digital PCR in Veterinary Parasitology: An Overview

Digital PCR (dPCR) is an emerging technology that enables the absolute quantification of the targeted nucleic acids. The body of research on the potential applications of this novel tool is growing in human and veterinary medicine. Most of the research on dPCR applications in veterinary parasitology is concentrated on developing and validating new assays to detect and quantify parasites of great financial impact in the food-producing animal industry. Several studies describe the utility of dPCR for individualized medicine in companion animals. Most frequently, dPCR performance is assessed compared to quantitative PCR or Next Generation Sequencing platforms, while others also compare the accuracy of dPCR with traditional parasitological techniques considered gold standard methods. Other researchers describe dPCR assays for surveillance purposes, species identification, and quantification in mixed parasitic infections, the detection of mutations indicative of anthelmintic resistance, and the identification of new targets for drug development. This review provides an overview of the studies that employed dPCR in investigating animal parasites and parasitic diseases from a veterinary perspective and discusses how this novel technology could advance and facilitate diagnosis, surveillance, and the monitoring of response to treatment, or shed light on current gaps in our knowledge of the epidemiology of significant veterinary parasitic diseases.

Features of the rumen microbiome in reindeer with necrobacteriosis

Reindeer are a unique animal species that have adapted to the harsh Arctic conditions. Adaptive features of the structure of the gastrointestinal tract of reindeer allows you to eat actively digest and assimilate lichens, foliage, shrubs, snowy greens. Along with beneficial microflora, pathogenic and conditionally pathogenic microorganisms live in the reindeer rumen. The purpose of our research is to study and compare the features of the rumen microbiome in healthy and sick reindeer with necrobacteriosis. The subject of the study was samples of cicatricial contents taken from 38 reindeer (Rangifer tarandus) of the Nenets breed of different age groups. Microbiome studies were performed by NGS sequencing on the MiSeq next generation sequencing platform (Illumina, USA) using primers for the V3-V4 region of 16S rRNA.Based on the studies, it was possible to establish that the microbiome of the reindeer rumen in summer and winter is characterized by certain differences. During the summer grazing period, the presence of a greater number of pathogenic bacteria such as Fusobacterium sp., Parvimonas sp. and Porphyromonas endodontalis, while in the spectrum of pathogenic microorganisms, bacteria of the genus Fusobacterium, which are considered the main infectious agent in necrobacteriosis, were most often isolated. In the same period, the presence of bacteria of the genus Sharpea, belonging to the phylum Erysipelotrichaceae, which ferment sugars with the formation of lactic acid and can cause the development of lactic acidosis, was established in deer rumen samples. In calves, during the disease, the number of chytridiomycete fungi, which are involved in the fermentation of polysaccharides, decreases.

Metagenomic Profile of Endophytic Bacteria in Acacia mearnsii.

Acacia mearnsii is one of the invasive plants belonging to the Mimosaceae family. The family is known to be able to symbiosis with some endophytic nitrogen-fixing soil bacteria. Such ability will significantly contribute to plant growth and development by influencing nutrient uptake and stress tolerance and thus to improve soil conditions for a better and more fertile. Comprehensive identification of bacterial population dynamic in that zone should elucidate symbiosis mechanism that in turn can be used for soil improvement. Metagenomic-based profiling are now commonly used in studying whole total microbial communities in nature. Sampling of the root was collected from A. mearnsii young trees grown in Alahan Panjang at 60 and 120 days after transplanting, sterilized and stored at -80°C in a falcon tube before being subjected to metagenome analysis. The dynamics of growth and diversity of endophytic microbes were checked through amplicon sequence of the 16S rRNA gene in the V3-V4 region using Next Generation Sequencing (NGS) platform. Data analysis was performed using the QIIME2 application. The metagenomics analysis of endophytic bacteria on root A. mearnsii elucidates that the Proteobacteria is a dominant phyla in A. mearnsii root and analysis at the genera level shows that most of the Proteobacteria is dominated by the Bradyrhizobium which increase significantly during early growth phase.

Cell free DNA (cfDNA) assessment of esophagogastric (EG) cancer using MSK-ACCESS.

4036 Background: The incidence of EG cancer is rising, the treatment paradigm is increasingly complex, and the role of predictive biomarkers is critical in selecting optimal therapies. Therefore, longitudinal, non-invasive methods to characterize dynamic tumor molecular profiles, such as cfDNA, are needed. Methods: Patients with EG cancer receiving care at Memorial Sloan Kettering (MSK) had cfDNA analysis with an ultrasensitive capture based liquid biopsy for detection of somatic alterations in 129 cancer associated genes (MSK ACCESS). Tumor samples were analyzed with MSK-IMPACT, a capture based next generation sequencing (NGS) platform. Results: 100 patients with EG adenocarcinoma underwent cfDNA analysis to evaluate for molecular residual disease with MSK ACCESS. 85 (85%) had tissue NGS with MSK IMPACT. Median TMB was 5.3 mut/Mb (IQR 2.6-7.4) and median number of alterations per sample was 11 (IQR 7-17). 71% had TP53 mutations and 22% had an ERBB2 amplification (amp). 79 (79%) patients had cfDNA obtained prior to treatment (50 [63%] metastatic disease; 29 [37%] localized disease) and 21 (21%) at the time of progression. Alterations were detectable on cfDNA more frequently in patients with later stage disease (p=0.031) and fewer alterations per sample were detected compared to tissue NGS (mean difference 14, 95% CI 10-18, p<0.001). The most common were TP53 (62%), CDKN2A (12%), PIK3CA (10%), ARID1A (10%), and ATM (10%). TP53 was detected with increasing frequency in patients with later stage disease (p=0.004). However, amplifications, which included ERBB2, MYC, EGFR, and FGFR2, were detected exclusively in those with stage IV disease (p=0.005) and cfDNA was less sensitive in detecting amplifications compared to tissue NGS (p<0.001). This is notable as HER2 positivity is determined in part by ERBB2amp. 25 of 30 HER2+ patients had both tissue NGS and cfDNA. 19 (76%) had ERBB2amp on tissue NGS and only 8 (32%) had ERBB2amp on cfDNA (p=0.004). Additionally, copy number was lower on cfDNA compared to tissue NGS (mean difference 13, 95% CI 1.3-24.9, p=0.02) and IHC 3+ tumors were more likely to have ERBB2amp detected than IHC 2+ tumors (p=0.049). However, alterations along the MAPK, PI3K, WNT, and TGF-β pathways, which are associated with trastuzumab resistance and poor survival, were detected with similar frequency on both platforms (40 v 32%, p=0.77). Conclusions: Our findings in this large prospective analysis highlight the utility of cfDNA with MSK ACCESS as a complimentary tool for molecular characterization in patients with EG cancer. cfDNA in EG cancer. [Table: see text]

Integrated transcriptome catalog of Tenualosa ilisha as a resource for gene discovery and expression profiling

The silver pride of Bangladesh, migratory shad, Tenualosa ilisha (Hilsa), makes the highest contribution to the total fish production of Bangladesh. Despite its noteworthy contribution, a well-annotated transcriptome data is not available. Here we report a transcriptomic catalog of Hilsa, constructed by assembling RNA-Seq reads from different tissues of the fish including brain, gill, kidney, liver, and muscle. Hilsa fish were collected from different aquatic habitats (fresh, brackish, and sea water) and the sequencing was performed in the next generation sequencing (NGS) platform. De novo assembly of the sequences obtained from 46 cDNA libraries revealed 462,085 transcript isoforms that were subsequently annotated using the Universal Protein Resource Knowledgebase (UniPortKB) as a reference. Starting from the sampling to final annotation, all the steps along with the workflow are reported here. This study will provide a significant resource for ongoing and future research on Hilsa for transcriptome based expression profiling and identification of candidate genes.