Generate Sequencing Libraries Research Articles

Abstract C057: Fibrolamellar carcinoma single-nucleus RNA sequencing reveals alteration of mitochondrial energetic pathways

Abstract Background: Fibrolamellar carcinoma (FLC) is a primary liver cancer, which arises in a background of normal liver. The DNAJB1-PRKACA (DP) fusion gene is present in the majority of FLC and is an oncogenic driver in animal models. The oncogenic mechanism of DP is unclear. To date, transcriptomic analyses of FLC have been based on bulk RNA sequencing. Given the lack of cell-specific data in bulk transcriptomic analysis, we hypothesize that using single nucleus RNA sequencing (snRNA-Seq) will allow for identification of significantly dysregulated signaling pathways specific to tumor cells. Methods: Single nuclei were isolated from 7 FLC tumors and 4 normal liver (NL). The Chromium Next GEM Single Cell 3’ Reagent Kit was used to generate sequencing libraries. All samples passing QC were submitted for sequencing by GeneWiz with a read depth of 20,000 reads/cell. An R-based pipeline for the single-cell profiling was built using Seurat R toolkit. Cells exhibiting extremely low or high library sizes, gene features outside the 95% confidence interval, or displaying high mitochondrial content were filtered out. Clusters from snRNA-Seq analysis were annotated using singleR, enrichR, and publicly available datasets. Results: FLC and NL nuclei counts were 98664 and 56644, respectively. We generated thirty one clusters and annotated 21 cell types. The FLC tumor group was annotated based on previously published FLC bulk transcriptomic data. We identified increased expression of SLC16A14, TMEM163, PDE10A, OAT, and GALNTL6 in FLC tumor cells. The predominant cells types and percentage of cells (%NL/%FLC) per group were: hepatocytes (43/57), endothelial cells (18/82), FLC tumor cells (15/85), stellate cells (28/72), Kupffer cells (50/50), cholangiocytes (62/38), Other 1 (22/78), T cells (36/64), and fibroblasts (69/31). FLC tissue contained only 3% of the liver sinusoidal endothelial cells. Comparison of FLC tumor cells demonstrated differential upregulation of 1879 and downregulation of 1579 genes in tumor vs. NL. Gene ontology (GO) enrichment identified the following top processes in FLC tumor cells: ATP metabolic process, cellular respiration, and response to peptide hormone. GO analysis of hepatocytes identified: ribonucleotide metabolic process, energy derivation by oxidation of organic compounds, and response to peptide hormone as significant pathways. Conclusions: FLC tumor cells demonstrate alterations in mitochondrial processes and energetics. FLC cells are known to contain abundant mitochondria, which is not observed in other primary liver epithelial cancers. The PRKACA component of DP fusion protein is the catalytic subunit of protein kinase A (PKA). Since PKA is known to regulate mitochondrial function, these data support the possibility that DP may directly alter mitochondrial function. Another interesting finding is the presence of the FLC tumor cell type in NL group (15%), which may suggest its dedifferentiation status or its cell-of-origin as a subpopulation of liver cells distinct from mature hepatocytes or cholangiocytes. Citation Format: Nihal Bharath, Biju Isaac, Marc S. Sherman, Colton J. Smith, Michael Karski, Liang Sun, Brian J. Pepe-Mooney, Ina Kycia, Michael Rogers, Wolfram Goessling, Khashayar Vakili. Fibrolamellar carcinoma single-nucleus RNA sequencing reveals alteration of mitochondrial energetic pathways [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C057.

Facilitating accessible, rapid, and appropriate processing of ancient metagenomic data with AMDirT

Background: Access to sample-level metadata is important when selecting public metagenomic sequencing datasets for reuse in new biological analyses. The Standards, Precautions, and Advances in Ancient Metagenomics community (SPAAM, https://spaam-community.github.io) has previously published AncientMetagenomeDir, a collection of curated and standardised sample metadata tables for metagenomic and microbial genome datasets generated from ancient samples. However, while sample-level information is useful for identifying relevant samples for inclusion in new projects, Next Generation Sequencing (NGS) library construction and sequencing metadata are also essential for appropriately reprocessing ancient metagenomic data. Currently, recovering information for downloading and preparing such data is difficult when laboratory and bioinformatic metadata is heterogeneously recorded in prose-based publications.  Methods: Through a series of community-based hackathon events, AncientMetagenomeDir was updated to provide standardised library-level metadata of existing and new ancient metagenomic samples. In tandem, the companion tool 'AMDirT' was developed to facilitate automated metadata curation and data validation, as well as rapid data filtering and downloading.  Results: AncientMetagenomeDir was extended to include standardised metadata of over 5000 ancient metagenomic libraries. The companion tool 'AMDirT' provides both graphical- and command-line interface based access to such metadata for users from a wide range of computational backgrounds. We also report on errors with metadata reporting that appear to commonly occur during data upload and provide suggestions on how to improve the quality of data sharing by the community. Conclusions: Together, both standardised metadata and tooling will help towards easier incorporation and reuse of public ancient metagenomic datasets into future analyses.

Identifying osteosarcoma signatures in cell free DNA using 5-hydroxymethylcytosine profiling.

10022 Background: Osteosarcoma is the most common primary bone tumor in children and adolescents. Prognostic biomarkers are limited, and post-treatment surveillance relies heavily on radiologic imaging. Capture and analysis of cell-free DNA (cfDNA) from the bloodstream is an emerging technique that could be used to provide additional prognostic information and track residual disease following treatment. 5-hydroxymethylcytosine (5-hmC) is an epigenetic marker of open chromatin and active gene expression, and tumor specific patterns of global 5-hmC deposition are identifiable in several cancers and vary according to disease burden. We profiled 5-hmC using plasma-derived cfDNA from osteosarcoma patients and healthy children to assess its utility as a biomarker of disease status. Methods: cfDNA was isolated from patients diagnosed with osteosarcoma and well child controls. Nano-hmC-Seal, a technique developed to isolate 5-hmC-modified DNA fragments, was used to generate sequencing libraries of cfDNA. Genomic DNA from osteosarcoma cell lines was serially diluted with healthy donor-derived cfDNA. DESeq2 was used to identify genes with high 5-hmC deposition in osteosarcoma cell line DNA relative to serial dilutions. In patient cfDNA samples, the relative abundance of 5-hmC deposition on these osteosarcoma genes was determined using gene set variation analysis (GSVA). GSVA returns a score ranging from -1 to 1, with positive values indicating a relative abundance of 5-hmC deposition on osteosarcoma genes. Results: The cohort consisted of 33 healthy children and 17 patients with osteosarcoma including 53 serially collected samples from these patients. 139 osteosarcoma genes were identified and used for GSVA analysis. Patients with osteosarcoma had significantly higher GSVA scores than well children (0.26 vs -0.38; p < 0.001). Hierarchal clustering identified two clusters of samples from children with osteosarcoma. Cluster 1 contained samples with scores correlating with osteosarcoma-derived DNA compared to Cluster 2 (0.14 vs -0.18; p < 0.001). Furthermore, Cluster 1 contained 17 of 23 samples from patients with active disease on clinical imaging (sensitivity = 74%, specificity = 57%). High 5-hmC deposition on MYC was readily identified in one sample obtained at diagnosis from the only patient with a MYC amplified tumor. Additional samples from this patient during treatment showed decreasing 5-hmC deposition on MYC, consistent with a treated tumor shedding less DNA into the blood. Additionally, decreased disease burden following surgical resection was identifiable in 5-hmC profiles (presurgical GSVA score 0.22 vs postsurgical -0.08; p = 0.004). Conclusions: Osteosarcoma 5-hmC signatures can identify patients with active disease. Monitoring cfDNA in osteosarcoma using 5-hmC profiling is a promising technique that warrants further investigation with larger patient cohorts.

The BD Rhapsody&[trade] HT Xpress System supports dynamic and high-throughput single-cell capture enabling a seamless workflow for analysis of cellular immune responses

Abstract The BD Rhapsody™ Single-Cell Analysis System is an innovative platform that aids high-throughput sequencing of mRNA and proteins in thousands of single cells. Here we present a new eight-lane cartridge design concept, the BD Rhapsody™ HT Xpress System. This novel device architecture can reduce both hands-on and assay time by enabling the users to simultaneously load multiple cell samples while preserving the flexibility to use one lane at a time. To demonstrate the increased throughput capabilities and workflow flexibility of the system, we measured in vitro T cell activation over time. To do this, peripheral blood mononuclear cells from four different subjects were prepared for cell culture in IL-2 or IL-2/anti-CD3/anti-CD28. After one day in culture, cell samples from each culture condition were barcoded using the BD® Single-Cell Multiplexing Kit. Cells from the same subject were pooled and stained with the BD® AbSeq Immune Discovery Panel. Pooled cells from each subject were loaded into the cartridge (one subject per lane, four lanes total). Remaining cells were cultured for another day and similarly barcoded, stained and loaded into the other four lanes of the cartridge. Eight different next generation sequencing libraries were generated and analyzed with the BD Rhapsody™ Analysis Pipeline and SeqGeq™ Software. This strategy enabled us to assess dynamic changes in both T cell surface protein profiles and transcriptome during cell activation while using a streamlined workflow for concomitant analysis of multiple samples. For Research Use Only. Not for use in diagnostic or therapeutic procedures. BD, the BD Logo, BD Rhapsody and SeqGeq are trademarks of Becton, Dickinson and Company or its affiliates. © 2023 BD. All rights reserved.

Analysis of Small Non-coding RNAs as Signaling Intermediates of Environmentally Integrated Responses to Abiotic Stress.

Research to date on abiotic stress responses in plants has been largely focused on the plant itself, but current knowledge indicates that microorganisms can interact with and help plants during periods of abiotic stress. In our research, we aim to investigate the interkingdom communication between the plant root and the rhizo-microbiota. Our investigation showed that miRNA plays a pivotal role in this interkingdom communication. Here, we describe a protocol for the analysis of miRNA secreted by the plant root, which includes all of the steps from the isolation of the miRNA to the bioinformatics analysis. Because of their short nucleotide length, Next Generation Sequencing (NGS) library preparation from miRNAs can be challenging due to the presence of dimer adapter contaminants. Therefore, we highlight some strategies we adopt to inhibit the generation of dimer adapters during library preparation. Current screens of miRNA targets mostly focus on the identification of targets present in the same organism expressing the miRNA. Our bioinformatics analysis challenges the barrier of evolutionary divergent organisms to identify candidate sequences of the microbiota targeted by the miRNA of plant roots. This protocol should be of interest to researchers investigating interkingdom RNA-based communication between plants and their associated microorganisms, particularly in the context of holobiont responses to abiotic stresses.

Bringing Cell Subpopulation Discovery on a Cloud-HPC Using rCASC and StreamFlow.

The idea behind novel single-cell RNA sequencing (scRNA-seq) pipelines is to isolate single cells through microfluidic approaches and generate sequencing libraries in which the transcripts are tagged to track their cell of origin. Modern scRNA-seq platforms are capable of analyzing up to many thousands of cells in each run. Then, combined with massive high-throughput sequencing producing billions of reads, scRNA-seq allows the assessment of fundamental biological properties of cell populations and biological systems at unprecedented resolution.In this chapter, we describe how cell subpopulation discovery algorithms, integrated into rCASC, could be efficiently executed on cloud-HPC infrastructure. To achieve this task, we focus on the StreamFlow framework which provides container-native runtime support for scientific workflows in cloud/HPC environments.

High-throughput primer design by scoring in piecewise logistic model for multiple polymerase chain reaction variants

Polymerase chain reaction (PCR) variants requiring specific primer types are widely used in various PCR experiments, including generic PCR, inverse PCR, anchored PCR, and ARMS PCR. Few tools can be adapted for multiple PCR variants, and many tools select primers by filtration based on the given parameters, which result in frequent design failures. Here we introduce PrimerScore2, a robust high-throughput primer design tool that can design primers in one click for multiple PCR variants. It scores primers using a piecewise logistic model and the highest-scored primers are selected avoiding the issue of design failure and the necessity to loosen parameters to redesign, and it creatively evaluates specificity by predicting the efficiencies of all target/non-target products. To assess the prediction accuracy of the scores and efficiencies, two next generation sequencing (NGS) libraries were constructed—a 12-plex and a 57-plex—and the results showed that 17 out of 19 (89.5%) low-scoring pairs had a poor depth, 18 out of 19 (94.7%) high-scoring pairs had a high depth, and the depth ratios of the products were linearly correlated with the predicted efficiencies with a slope of 1.025 and a coefficient of determination (R2) 0.935. 116-plex and 114-plex anchored PCR panels designed by PrimerScore2 were applied to 26 maternal plasma samples with male fetuses, the results showed that the predicted fetal DNA fractions were concordant with fractions measured in gold standard method (Y fractions). PrimerScore2 was also used to design 77 monoplex Sanger sequencing primers, the sequencing results indicated that all the primers were effective.

Altered pathways of keratinization, extracellular matrix generation, angiogenesis, and stromal stem cells proliferation in patients with systemic sclerosis.

Systemic sclerosis is characterized by endothelial dysfunction, autoimmunity abnormalities, and fibrosis of the skin and internal organs. The pathogenetic mechanisms underlying systemic sclerosis vasculopathy are still not clarified. A complex cellular and extracellular network of interactions has been studied, but it is currently unclear what drives the activation of fibroblasts/myofibroblasts and the extracellular matrix deposition. Using RNA sequencing, the aim of the work was to identify potential functional pathways implied in systemic sclerosis pathogenesis and markers of endothelial dysfunction and fibrosis in systemic sclerosis patients. RNA-sequencing analysis was performed on RNA obtained from biopsies from three systemic sclerosis patients and three healthy controls enrolled in our University Hospital. RNA was used to generate sequencing libraries that were sequenced according to proper transcriptomic analyses. Subsequently, we performed gene set enrichment analysis of differentially expressed genes on the entire list of genes that compose the RNA-sequencing expression matrix. Gene set enrichment analysis revealed that healthy controls were characterized by gene signatures related to stromal stem cells proliferation, cytokine-cytokine receptor interaction, macrophage-enriched metabolic network, whereas systemic sclerosis tissues were enriched in signatures associated with keratinization, cornification, retinoblastoma 1 and tumor suppressor 53 signaling. According to our data, RNA-sequencing and pathway analysis revealed that systemic sclerosis subjects display a discrete pattern of gene expression associated with keratinization, extracellular matrix generation, and negative regulation of angiogenesis and stromal stem cells proliferation. Further analysis on larger numbers of patients is needed; however, our findings provide an interesting framework for the development of biomarkers useful to explore potential future therapeutic approaches.

Abstract 1831: 9-ING-41, a glycogen synthase kinase 3B inhibitor targets TP53 in pediatric brain tumors ATRT and ETMR

Abstract Purpose: The study aims to determine the activity of the blood brain barrier permeable glycogen synthase kinase 3B (GSK3B) inhibitor, 9-ING-41, in pediatric brain tumors: embryonal tumor with multilayered rosettes (ETMR) and atypical rhabdoid tumor (ATRT). Methods: ETMR cell line, BT183, was grown in ultralow attachment flasks and ATRT cell lines: ATRT-7316-2187, ATRT-787197, and CHLA02 were grown in suspension flasks. Cells were treated with 9-ING-41 at dose range (0-20µM). After 24 and 48 hours of 9-ING-41 cells were analyzed by western blot. After 72 hours of 9-ING-41 cell viability was assessed with Cell Titer Glo 2.0 and IC50s were analyzed by GraphPad. RNA seq was performed on cells after 12, 24, and 48 hours of 9-ING-41 treatment. Total RNA was isolated with miRNeasy Mini Kit. RNA integrity was evaluated by an Agilent 2100 Bioanalyzer profile. rRNA was selectively depleted by employing RiboZero Globin. Next generation sequencing libraries were prepared using an Illumina TruSeq Stranded Total RNA kits. Read mapping was performed using STAR in BaseSpace. DESeq2 was used for normalization and differential expression analysis. Ingenuity Pathway Analysis was performed. Results: The IC50s of 9-ING-41 in the cell lines BT183, CHLA02-ATRT, ATRT-787197, and ATRT-2187 are 145.5nM, 481.2nM, 503.1nM, and 528.3nM, respectively. In BT183 cells treated with 150nM 9-ING-41 for 48 hours, XIAP decreased significantly by 55% and cleaved caspase 3 increased significantly by 29-fold, quantified by densitometry. In addition, western blot showed a decrease in LIN28A and an increase in TP53 protein levels. RNA-seq data show an increase in the p53 signaling pathway in the ETMR BT183 cell line with z-scores of 2.53 and 3 (p=3.73x10-13 and 2.35x10-14) after 12 and 24 hours of 150nM 9-ING-41, respectively. In ATRT-787197 cells p53 signaling increased with z-scores of 1.0 and 2.236 (p=2.62x10-8 and 2.89x10-5) after 24 and 48 hours of 400nM 9-ING-41, respectively. RNA seq data also show the ability of 9-ING-41 to significantly increase the expression of CDK inhibitor, CDKN1A, and pro-apoptotic genes BBC3, DRAM1, and BAX in ETMR and ATRT while significantly decreasing stemness associated genes: SMO and GLI1 in ETMR. Conclusions: 9-ING-41 decreases ETMR and ATRT cell viability at clinically relevant concentrations and these doses increase the p53 pathway and induce apoptosis while reducing the expression of stemness associated genes. Citation Format: Jason L. Haw, Elizabeth M. Moore, Kimberly McKinney, Karl Dykema, Abhinav Nagulapally, Nury Steuerwald, Giselle S. Sholler. 9-ING-41, a glycogen synthase kinase 3B inhibitor targets TP53 in pediatric brain tumors ATRT and ETMR [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1831.

Genetic alterations in tumor tissue and cell-free DNA in patients with papillary thyroid carcinoma at initial surgery.

e18083 Background: The value of sequencing cell-free DNA (cfDNA) from patients with papillary carcinoma of the thyroid (PTC) is controversial. Studies utilizing whole-exome sequencing (WES) or single-gene detection methods have reported lower mutational yield in cfDNA in PTC compared with other cancers. Alternatively, mutational analyses of anaplastic thyroid carcinoma (ATC), which is thought to evolve from PTC, demonstrate high levels of mutated cfDNA. We report on mutations in tumor and cfDNA at initial surgery for PTC, correlate with clinicopathologic status and further characterize the mutational landscape of these tumors in tissue and blood utilizing deep-sequencing panel testing. Methods: 50 patients were enrolled 2017-2020. Tempus xT (648 genes) and xF (105 genes) panels were used to prepare next generation sequencing libraries from tissue and blood samples, respectively. Cell-free DNA (cfDNA) sequencing depth average is 20,000x (raw reads)/5,000x (unique reads). Results: More than half of the patients had pT3-T4 tumors and/or local metastases and one had distant metastasis. 47/47 (100%) of processable tissue samples and 40/49 (81.6%) of blood samples yielded at least one relevant mutation. BRAF V600E was found in 77% of tissue and 8% of cfDNA, while mutated TERT was detected in 17% of tissue and none of the blood samples. The most frequently mutated genes in cfDNA were KMT2A (18% of blood samples, 6 % tissue), ATM (12% blood, 6% tissue), and TP53 (12% blood, 2 % tissue). There was marked mutational heterogeneity among samples, and a range of alterations representing multiple oncogenic pathways. Conclusions: Patients at initial surgery demonstrated highly mutated tissue DNA, including BRAF, TERT and other mutations known to be found in PTC. The most frequent tissue mutations were found at higher rates than previously reported by WES, which may reflect the sensitivity of targeted deep sequencing versus WES, and possibly a selection bias of more advanced PTC. A high percentage of cfDNA samples yielded mutations relevant to thyroid cancer, and the absence of TERT is consistent with prior studies. Mutations and co-mutations associated with de-differentiation and worse outcomes were demonstrated in both tissue and cfDNA. Interestingly, mutations more common in ATC than PTC, such as TP53, were detected in cfDNA, often without primary tumor correlate. Since mutations associated with aggressive behavior may be found in metastatic foci while not detected in the primary tumor, we conclude that cfDNA may reveal prognostic information important for the development of surveillance strategies in selected patients with residual PTC after surgery who may be at risk for poor outcomes.

Automation of hybridization and capture based next generation sequencing library preparation requires reduction of on-deck bead binding and heated wash temperatures.

Capture-based library preparation for next generation sequencing (NGS) offers a balance between sequencing depth and bioinformatics cost of analysis. Liquid handling automation enhances the reliability of the library preparation process by reducing sample-to-sample variation and substantially enhances throughput, particularly when it can be employed in a 'walk-away' fashion with limited hands-on interaction. This requires complex series of mixing and heating steps like those utilized in capture chemistries to happen on the liquid handler. While developing liquid handling automation for Integrated DNA Technologies (IDT) xGen Exome, Illumina TruSight Oncology 500, and Personal Genome Diagnostics (PGDx) elio Plasma Resolve chemistries on the PerkinElmer Sciclone liquid handler, we found that applying the capture temperatures recommended for manual library preparation results in low yield on automation. To restore the final library yield, we reduced bead binding and/or heated wash temperatures of the Peltier heaters on the liquid handlers by about 10°C. Since this applied across three unique capture-based chemistries, we consider this a generalizable principle of automating capture on the Sciclone. We hypothesize that this is driven by the very different thermodynamic environments represented by a sealed plate on a thermal cycler and a plate with a lid on a Peltier heater. This phenomenon should be considered when automating NGS library preparation on PerkinElmer Sciclone instruments.

224 Ends of Endemics: Capturing Viral UTRs in Clinically Relevant Arbovirus Samples

OBJECTIVES/GOALS: Whole-genome viral sequencing is vital to inform public health and study evolution. Arboviruses evolve in vectors, reservoir hosts, and humans, and require surveillance at all points. We developed a new rigorous method of sequencing that captures whole viral genomes in field-collected and clinical samples. METHODS/STUDY POPULATION: ClickSeq is a novel method of Next Generation Sequencing (NGS) library synthesis using azido-nucleotides to terminate reverse transcription. The cDNA generated can be ligated to sequencing and indexing primers at room temperature using copper (Cu I) and vitamin C. With this approach, we designed primers located ~250 bp apart along the genomes of the arboviruses Chikungunya 37797, Zika Dakar, Yellow Fever Asibi, Dengue serotype 2, West Nile 385-99, and St. Louis Encephalitis Virus (SLEV) clade II. We tested this method with varying viral titers: lab-infected mosquito pools, field-collected mosquito pools from a Texas West Nile and SLEV outbreak, and patient isolates from a Pakistani CHIKV outbreak. The cDNA was sequenced in the UTMB NGS Core and aligned using bowtie. RESULTS/ANTICIPATED RESULTS: The use of a single protocol to capture whole viral genomes including UTRs for multiple viruses from different sample collection styles is ideal for arboviruses. Primers for multiple viruses were pooled and used to sequence mosquito pools. The Tiled ClickSeq method captured whole viral genomes without the need for host depletion. UTRs were captured even when the viral strain used for primer design differed from the resulting strain. Discreet variants were captured in both the hypervariable nsP3 region and the UTR in the patient isolates from the CHIKV outbreak compared to the 2017 outbreak. Texas WNV and SLEV outbreaks are now defined from the 2020 outbreak and can be further tracked to update public health measures and understand viral evolution. DISCUSSION/SIGNIFICANCE: UTRs impact both human and mosquito fitness, leading to further outbreaks. Tiled ClickSeq aims to capture whole viral genomes with a method and cost that can be implemented by public health researchers to understand disease evolution as it happens to update both public health and basic virology to the effects of evolution on arboviruses.

Impact of Early Feeding: Metagenomics Analysis of the Infant Gut Microbiome.

BackgroundDifferent feeding regimens in infancy alter the gastrointestinal (gut) microbial environment. The fecal microbiota in turn influences gastrointestinal homeostasis including metabolism, immune function, and extra-/intra-intestinal signaling. Advances in next generation sequencing (NGS) have enhanced our ability to study the gut microbiome of breast-fed (BF) and formula-fed (FF) infants with a data-driven hypothesis approach.MethodsNext generation sequencing libraries were constructed from fecal samples of BF (n=24) and FF (n=10) infants and sequenced on an Illumina HiSeq 2500. Taxonomic classification of the NGS data was performed using the Sunbeam/Kraken pipeline and a functional analysis at the gene level was performed using publicly available algorithms, including BLAST, and custom scripts. Differentially represented genera, genes, and NCBI Clusters of Orthologous Genes (COG) were determined between cohorts using count data and R (statistical packages edgeR and DESeq2).ResultsThirty-nine genera were found to be differentially represented between the BF and FF cohorts (FDR ≤ 0.01) including Parabacteroides, Enterococcus, Haemophilus, Gardnerella, and Staphylococcus. A Welch t-test of the Shannon diversity index for BF and FF samples approached significance (p=0.061). Bray-Curtis and Jaccard distance analyses demonstrated clustering and overlap in each analysis. Sixty COGs were significantly overrepresented and those most significantly represented in BF vs. FF samples showed dichotomy of categories representing gene functions. Over 1,700 genes were found to be differentially represented (abundance) between the BF and FF cohorts.ConclusionsFecal samples analyzed from BF and FF infants demonstrated differences in microbiota genera. The BF cohort includes greater presence of beneficial genus Bifidobacterium. Several genes were identified as present at different abundances between cohorts indicating differences in functional pathways such as cellular defense mechanisms and carbohydrate metabolism influenced by feeding. Confirmation of gene level NGS data via PCR and electrophoresis analysis revealed distinct differences in gene abundances associated with important biologic pathways.

A High-Throughput 3'-Tag RNA Sequencing for Large-Scale Time-Series Transcriptome Studies.

RNA sequencing (RNA-seq) has proven invaluable for exploring gene expression variation under complex environmental cues. However, the cost of standard RNA-seq (e.g., Illumina TruSeq or NEBNext) remains a barrier for high-throughput applications. 3'-Tag RNA-seq (3'-TagSeq) is a cost-effective solution that permits large-scale experiments. Unlike standard RNA-seq, which generates sequencing libraries for full-length mRNAs, 3'-TagSeq only generates a single fragment from the 3' end of each transcript (a tag read) and quantifies gene expression by tag abundance. Consequently, 3'-TagSeq requires lower sequencing depth (~5 million reads per sample) than standard RNA-seq (~30 million reads per sample), which reduces costs and allows increased technical and biological replication in experiments. Because 3'-TagSeq is considerably cheaper than standard RNA-seq while exhibiting comparable accuracy and reproducibility, researchers focusing on gene expression levels in large or extensive time-series experiments might find 3'-TagSeq to be superior to standard RNA-seq. In this chapter, we describe 3'-TagSeq sequencing library preparation and provide example bioinformatics and statistical analyses of gene expression data.

P–534 A novel heterozygous mutation in the luteinizing hormone/choriogonadotropin receptor (LHCGR) gene in a patient with ‘genuine’ empty follicle syndrome

Abstract Study question Whether empty follicle syndrome (EFS) in a patient has a genetic basis. Summary answer Our findings would expand the mutational spectrum of LHCGR, in patients with GEFS What is known already The LHCGR gene (OMIM #52790) is located on chromosome 2p21 has 11 exons The LHCGRs present in gonadal cells; granulosa, theca and luteal cells in women and Leydig cells in menandplays a critical role in male sexual differentiation, female ovarian development and fertility (folliculogenesis, ovulation, corpus luteum formation and progesterone secretion) Inactivating mutations in males can lead to Leydig cell hypoplasia, which causes disorders in sexual development (MIM #238 320). Phenotype of women is less severe and variable and has no effect on the secondary sex characteristics, but it could cause amenorrhoea and infertility Study design, size, duration: In the context of clinical genetics, Next Generation Sequencing libraries were prepared in line with the manufacturer’s orders using QIASeq™ Targeted DNA Custom Panel (Qiagen) targeting exons and 20 bp exon-intron boundaries of selected genes (AR, BMP15, CATSPER1, CFTR, CYP21A2, FSHB, FSHR, HESX1, LHB, LHCGR, NR5A1, POU1F1, SRY, ZP1). The variant detected in NGS analysis was further confirmed using Sanger sequencing (MiSeq, Illumina, San Diego, CA). Participants/materials, setting, methods A 27 years old Turkish women with 6 year history of primary unexplained infertility underwent controlled ovarian hyperstimulation and IVF with an antagonist protocole in Ankara City Hospital. Although normal follicular development, E2 levels, and bioavailable β-hCG plasma levels, no oocytes or cumulus-corona complexes were retrieved by follicular aspiration. Main results and the role of chance A novel heterozygous mutation on Exon 5 of LHCGR (NM_000233.4):c.453C>G (p.Phe151Leu). This variant has not been reported in GnomAD database and is a novel variant as per controlled from ClinVar and HGMD mutation databases Also coagulation tests were done Patient was heterozygote for the prothrombin mutation (FXIII) and homozygote for MTHFR C677T mutation. The patient has normal pubertal development and female karyotype (46,XX). Gonadotropin and E2 levels were normal, nor any history of anosmia, primary amenorrhoea, polycystic ovaries, hyperandrogenism, systemic disorder, or neurologic defect. According to ACMG 2015 and HGMD detected variant was classified as unknown clinical significance (VUS) variant In 22nd World Congress COGI in 2015 we have presented another recurrent GEFS case with compound heterozygous frameshift mutations in exon 5 and 11 of LHCGRgene(c.1764_1765insT) and (c.430G>T, p.V144F) who developed premature ovarian failure at the end The different types of LHCGR mutations would lead to the different functional effects and clinical phenotypes in different ages; primary amenorrhea with high levels of LH, poor ovarian response (Poesidon group1), GEFS and secondary amenorrhea (premature ovarian failure) GEFS may be a gradual biological occurrence related to ovarian ageing. Long term follow up is important, since some clinical manifestations appear later in life. Limitations, reasons for caution LH resistance in females were always found due to their effected brothers and only 10 GEFS cases of 46, XX females with LHCGR gene defect reported in literature. Patient was heterozygous for indicated mutation; therefore segregation study should be done in family members and in vitro studies should be performed. Wider implications of the findings: Screening for mutations in the LHCGR gene in Poseidon group 1 and in patients GEFS especially the recurrent ones will provide valuable information and time for clinical management, corresponding treatment and give the opportunity of wise and cost effective counseling of patients about their future reproductive choices. Trial registration number it is a case report

A plate-based single-cell ATAC-seq workflow for fast and robust profiling of chromatin accessibility.

Profiling chromatin accessibility at the single-cell level provides critical information about cell type composition and cell-to-cell variation within a complex tissue. Emerging techniques for the interrogation of chromatin accessibility in individual cells allow investigation of the fundamental mechanisms that lead to the variability of different cells. This protocol describes a fast and robust method for single-cell chromatin accessibility profiling based on the assay for transposase-accessible chromatin using sequencing (ATAC-seq). The method combines up-front bulk Tn5 tagging of chromatin with flow cytometry to isolate single nuclei or cells. Reagents required to generate sequencing libraries are added to the same well in the plate where cells are sorted. The protocol described here generates data of high complexity and excellent signal-to-noise ratio and can be combined with index sorting for in-depth characterization of cell types. The whole experimental procedure can be finished within 1 or 2 d with a throughput of hundreds to thousands of nuclei, and the data can be processed by the provided computational pipeline. The execution of the protocol only requires basic techniques and equipment in a molecular biology laboratory with flow cytometry support.

Abstract PS16-02: Moving in the fast lane: Test design and validation to produce up-to-date hereditary breast and gynecologic cancer tests

Abstract Introduction Germline genetic testing is increasingly relevant to breast and gynecologic (GYN) cancer clinicians for monitoring and managing high-risk patients. In particular, multi-gene panels (MGPs) can identify unsuspected cancer syndromes and variants that may become clinically significant. Effective MGPs must be comprehensive and up-to-date. For the current study, we used a probe panel designed for targeted enrichment of 4,500 genes associated with various inherited diseases to develop and validate a 66-gene comprehensive hereditary cancer panel, including subsets of genes associated with breast and GYN cancers. Materials and Methods Genomic DNA was extracted and taken through next generation sequencing (NGS) library preparation to be sequenced on an Illumina NovaSeq instrument. Targeted capture-based enrichment with a long-range PCR (LR-PCR) component was used to interrogate all protein-coding exons, intron-exon splice sites (+/-10bp), as well as clinically relevant deep intronic, 5’UTR, and 3’UTR regions for single nucleotide variants (SNVs) and insertions/deletions (indels) of all genes of interest. Copy number variations (CNVs) were also interrogated for all applicable regions. Data analysis was performed using a proprietary in-house bioinformatics variant analysis pipeline. For validation, samples from the Coriell Repository and more than 100 unique de-identified genomic DNA specimens from whole blood and saliva were analyzed for 17,911 variants in 508 genes. Variants were previously identified by orthogonal methods (in-house Sanger sequencing, CLIA validated NGS assays, and microarray). The well-characterized Genome in a Bottle (GIAB) NA12878 and Ashkenazim Trio samples (NA24149, NA24385, and NA24143) were also included. The analytic sensitivity (Positive Percent Agreement, %PPA) and specificity (Technical Positive Predictive Value, %TPPV and Negative Percent Agreement, %NPA) were determined for each variant type (SNV, indel, and CNV). The 66-gene hereditary cancer panel includes genes that confer ≥2-fold increased risk or 5% lifetime risk for developing cancer (APC, ATM, AXIN2, BAP1, BARD1, BLM, BMPR1A, BRCA1, BRCA2, BRIP1, CDH1, CDK4, CDKN1B, CDKN2A (p16, p14), CHEK2, DICER1, EGFR, EPCAM, FANCA, FANCC, FANCM, FH, FLCN, GALNT12, GREM1, HOXB13, MAX, MEN1, MET, MITF, MLH1, MRE11 (MRE11A), MSH2, MSH3, MSH6, MUTYH, NBN, NF1, NTHL1, PALB2, PMS2, POLD1, POLE, POT1, PTCH1, PTEN, RAD50, RAD51C, RAD51D, RECQL, RET, SDHA, SDHAF2, SDHB, SDHC, SDHD, SMARCA4, SMAD4, STK11, SUFU, TMEM127, TP53, TSC1, TSC2, VHL, and XRCC2). Of these, 30 genes increase the lifetime risk of breast and/or GYN cancers and are also distributed among smaller phenotype-specific panels. Results The analytical sensitivity (%PPA) for SNVs and indels was 100.0% and 97.8% for CNVs. The overall specificity for SNVs, indels, and CNVs was >99.0%. The %TPPV for SNVs, Indels, and CNVs was 100.0%, 99.3%, and 100.0%, respectively. The %NPA for SNVs, Indels, and CNVs was 100.0%. The %PPA, %TPPV, and %NPA for LR-PCR was 100.0%. Conclusion Validation of the 66-gene hereditary cancer panel demonstrated high analytical sensitivity and specificity. As additional gene-cancer associations are established, using an already designed and developed comprehensive 4,500 gene panel will expedite the process of updating panel tests to include relevant candidate genes, allowing clinicians and patients to benefit from up-to-date and comprehensive testing. Citation Format: Taraneh E Angeloni, Anindya Bhattacharya, Linda L Cheng, Hansook K Chong, Kelli M Conlan, Christopher D Elzinga, Anna Gerasimova, David Grover, Andrew Grupe, Michael Hua, Domagoj Hodko, Krista Kazmierkiewicz, Rebecca E Nakles, Camille R Nery, Renius Owen, Dana M Goos-Root, Charles M Rowland, Alla Smolgovsky, Elaine C Weltmer, Ke Zhang, Felicitas L Lacbawan. Moving in the fast lane: Test design and validation to produce up-to-date hereditary breast and gynecologic cancer tests [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS16-02.

Library Preparation Based on Transposase Assisted RNA/DNA Hybrid Co-Tagmentation for Next-Generation Sequencing of Human Noroviruses.

Human noroviruses (HuNoVs) are one of the leading causes of foodborne illnesses globally. The viral genome is the most essential information for viral source tracing and viral transmission pattern monitoring. However, whole genome sequencing of HuNoVs is still challenging due to the sequence heterogeneity among different genotypes and low titer in samples. To address this need, in this study, the Transposase assisted RNA/DNA hybrid Co-tagmentation (TRACE-seq) method was established for next generation sequencing library preparation of HuNoVs. Our data demonstrated that almost the whole HuNoVs genome (>7 kb) could be obtained from all of the 11 clinical samples tested. Twelve genotypes including GI.3, GI.4, GI.5, GI.8, GII.2, GII.3, GII.4, GII.6, GII.12, GII.13, GII.14, and GII.21 were involved. Compared with the traditional method for viral metagenomics library preparation, optimized TRACE-seq greatly reduced the interference from the host’s and bacterial RNAs. In addition, viral genome sequences can be assembled by using less raw data with sufficient depth along the whole genome. Therefore, for the high versatility and reliability, this method is promising for whole viral genome attainment. It is particularly applicable for the viruses with a low titer that are mixed with a complicated host background and are unable to be cultured in vitro, like the HuNoVs utilized in this study.

Abstract 5363: A direct amplicon-based targeted sequencing assay for mutation analysis of single circulating tumor cells and correlation with circulating tumor DNA

Abstract There is increasing interest in sequence analysis of circulating tumor cells (CTCs) to identify actionable mutations that may complement circulating tumor DNA (ctDNA) findings. Because the amount of DNA present in a single cell is miniscule (~6 pg), whole genome amplification (WGA) is typically performed prior to next generation sequencing (NGS) library preparation. Existing WGA methods have inherent amplification biases leading to non-uniform genome coverage that can cause dropout of desired targets, as well as replication errors that can lead to false positive results. Targeted sequencing is an alternative approach that does not require WGA. We modified the CleanPlex® OncoZoom® Cancer Hotspot Panel protocol to perform targeted sequencing of single CTCs isolated by the RareCyte platform using A549 cell line spike-in and patient samples. We also used the CleanPlex panel to evaluate ctDNA from plasma isolated by blood collection tubes from two manufacturers (RareCyte and Streck). In both cases we compared OncoZoom results with and without prior WGA. Increased uniformity of coverage with decreased target dropout was observed when CleanPlex NGS libraries were prepared directly from cell lysates without WGA. Median read depth increased 48-fold when compared to the WGA method. On average, 23 out of 29 (79%) variants present in bulk A549 genomic DNA were observed without WGA in single model CTCs, while 15 of 29 (52%) were observed after WGA. Additionally, the false positive error frequency of non-WGA samples was 8-fold lower than in the WGA samples. CTCs and cell-free DNA from two metastatic breast cancer patients were also sequenced using OncoZoom. In one patient, PIK3CA E542K, a well-documented oncogenic mutation, was observed in 3 of 5 CTCs. In the other, ERBB2 L755S, a known HER2-reactivating mutation associated with chemoresistance, was found in 3 of 5 CTCs. Both patients' variants were present at similar allelic frequencies in plasma isolated from RareCyte and Streck cell-free DNA BCT tubes. Single-cell CleanPlex amplicon-based sequencing without prior WGA resulted in libraries with more complete and consistent coverage and lower error frequencies, enabling efficient and accurate assessment of somatic mutations in CTCs. This approach shows promise for cell-based liquid biopsy diagnostic applications. Citation Format: Nolan G. Ericson, Vidushi Kapoor, Guoying Liu, Alisa C. Clein, Daniel E. Sabath, Eric P. Kaldjian, Tad George. A direct amplicon-based targeted sequencing assay for mutation analysis of single circulating tumor cells and correlation with circulating tumor DNA [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5363.

Abstract A23: Novel approaches to Ewing sarcoma therapy

Abstract Ewing family of sarcomas (EwS) comprises the fourth most common highly malignant childhood cancer. The driver translocation in these tumors, EWSR1-FLI1, introduces DNA damage and replication stress. These dysregulations render EwS cells reliant on the ATR/CHK1 axis to maintain genome stability. High sensitivity of EwS cells to PARP1 inhibition (PARPi) was reported and attributed to a positive PARP1/EWSR1-FLI1 feedback loop. Further, it has been shown that with EWSR1-FLI1 expression, BRCA1 is trapped with the active transcription complex unable to go to DNA breaks. PARPi impairs the repair of single-strand DNA breaks and alkylated bases leading to double-strand DNA breaks, which cannot be repaired efficiently in BRCA-deficient cancers (synthetic lethality) (1). Several groups including ours previously identified the combination of PARP1i talazoparib (TLZ) with DNA-damaging agent, temozolomide (TMZ), to cause regressions of ~50% of EwS xenograft models. However, both in mice and in children’s trial, the combination causes toxicity and necessitates reduction of the dose of TMZ to ~13% of its single-agent maximum tolerated dose. Combined inhibition of PARP1 and ATR has been shown to synergize in suppressing growth of BRCA-mutant patient-derived xenografts models (2). The long-term goal of the proposed study is to develop more effective EwS therapy based on EWSR1-FLI1-induced vulnerabilities. EwS cells represent heterogeneity in expressing EWSR1-FLI1 fusion protein. As sequencing is generally performed in “bulk” with results being interpreted as average of gene expression patterns derived from vast populations of cells, the biologically relevant differences between cells may not be represented. Single-cell RNA-seq therefore offers new possibilities of overcoming such challenges. By studying single cells, their transcripts and generating sequencing libraries of separate cells, we are able to study fundamental biologic properties of cell populations and biologic systems at a single-cell level. Our studies demonstrate positive correlation between the expression of EWSR1-FLI1 and DNA replication genes at single-cell resolution. Single-cell studies may provide deep understanding of time-dependent behaviors of individual cells in response to dynamic changes of EWSR1-FLI1 expression. We propose that EWSR1-FLI1-induced DNA replication stress leads to reliance of EwS cells on PARP1 and ATR DNA repair pathways in order to maintain genome stability. We have selected number of metal-conjugated antibodies to identify changes in DNA damage response genes in subpopulations of EwS cells using CyTOF. Latest findings on changes of DNA damage/repair genes upon downregulation of EWSR1-FLI1 at a single-cell level will be presented. Citation Format: Dauren Alimbetov, Yidong Chen, Peter Houghton, Raushan Kurmasheva. Novel approaches to Ewing sarcoma therapy [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr A23.