Next-generation Deep Sequencing Research Articles

Vertebrate-Aedes aegypti and Culex quinquefasciatus (Diptera)-arbovirus transmission networks: Non-human feeding revealed by meta-barcoding and next-generation sequencing.

Aedes aegypti mosquito-borne viruses including Zika (ZIKV), dengue (DENV), yellow fever (YFV), and chikungunya (CHIKV) have emerged and re-emerged globally, resulting in an elevated burden of human disease. Aedes aegypti is found worldwide in tropical, sub-tropical, and temperate areas. The characterization of mosquito blood meals is essential to understand the transmission dynamics of mosquito-vectored pathogens. Here, we report Ae. aegypti and Culex quinquefasciatus host feeding patterns and arbovirus transmission in Northern Mexico using a metabarcoding-like approach with next-generation deep sequencing technology. A total of 145 Ae. aegypti yielded a blood meal analysis result with 107 (73.8%) for a single vertebrate species and 38 (26.2%) for two or more. Among the single host blood meals for Ae. aegypti, 28.0% were from humans, 54.2% from dogs, 16.8% from cats, and 1.0% from tortoises. Among those with more than one species present, 65.9% were from humans and dogs. For Cx. quinquefasciatus, 388 individuals yielded information with 326 (84%) being from a single host and 63 (16.2%) being from two or more hosts. Of the single species blood meals, 77.9% were from dogs, 6.1% from chickens, 3.1% from house sparrows, 2.4% from humans, while the remaining 10.5% derived from other 12 host species. Among those which had fed on more than one species, 11% were from dogs and humans, and 89% of other host species combinations. Forage ratio analysis revealed dog as the most over-utilized host by Ae. aegypti (= 4.3) and Cx. quinquefasciatus (= 5.6) and the human blood index at 39% and 4%, respectively. A total of 2,941 host-seeking female Ae. aegypti and 3,536 Cx. quinquefasciatus mosquitoes were collected in the surveyed area. Of these, 118 Ae. aegypti pools and 37 Cx. quinquefasciatus pools were screened for seven arboviruses (ZIKV, DENV 1-4, CHIKV, and West Nile virus (WNV)) using qRT-PCR and none were positive (point prevalence = 0%). The 95%-exact upper limit confidence interval was 0.07% and 0.17% for Ae. aegypti and Cx. quinquefasciatus, respectively. The low human blood feeding rate in Ae. aegypti, high rate of feeding on mammals by Cx. quinquefasciatus, and the potential risk to transmission dynamics of arboviruses in highly urbanized areas of Northern Mexico is discussed.

IsomiRs-Hidden Soldiers in the miRNA Regulatory Army, and How to Find Them?

Numerous studies on microRNAs (miRNA) in cancer and other diseases have been accompanied by diverse computational approaches and experimental methods to predict and validate miRNA biological and clinical significance as easily accessible disease biomarkers. In recent years, the application of the next-generation deep sequencing for the analysis and discovery of novel RNA biomarkers has clearly shown an expanding repertoire of diverse sequence variants of mature miRNAs, or isomiRs, resulting from alternative post-transcriptional processing events, and affected by (patho)physiological changes, population origin, individual’s gender, and age. Here, we provide an in-depth overview of currently available bioinformatics approaches for the detection and visualization of both mature miRNA and cognate isomiR sequences. An attempt has been made to present in a systematic way the advantages and downsides of in silico approaches in terms of their sensitivity and accuracy performance, as well as used methods, workflows, and processing steps, and end output dataset overlapping issues. The focus is given to the challenges and pitfalls of isomiR expression analysis. Specifically, we address the availability of tools enabling research without extensive bioinformatics background to explore this fascinating corner of the small RNAome universe that may facilitate the discovery of new and more reliable disease biomarkers.

Differential Effects of Fingolimod and Natalizumab on B Cell Repertoires in Multiple Sclerosis Patients.

Natalizumab and fingolimod are effective multiple sclerosis (MS) therapies that disrupt lymphocyte migration but have differential effects on B cell maturation and trafficking. We investigated their effects on peripheral blood (PB) and cerebrospinal fluid (CSF) B cell repertoires using next-generation deep sequencing. Paired CSF and PB B cell subsets (naïve, CD27+ memory, and CD27-IgD- double-negative B cells and plasmablasts) were collected by applying flow cytometry at baseline and after 6months of treatment and their respective heavy-chain variable region repertoires assessed by Illumina MiSeq. Treatment with fingolimod contracted, whereas natalizumab expanded circulating PB B cells. CSF B cell numbers remained stable following fingolimod treatment but decreased with natalizumab therapy. Clonal overlap between CSF and PB B cells was reduced with natalizumab treatment but remained stable with fingolimod therapy. Lineage analyses of pre- and posttreatment CSF B cell repertoires revealed large, clonally expanded B cell clusters in natalizumab-treated MS patients but no intrathecal clonal expansion following fingolimod therapy. Our findings suggest that natalizumab diminishes the exchange of peripheral and intrathecal B cells without impacting intrathecal clonal expansion. In contrast, fingolimod treatment fails to alter blood-brain barrier B cell exchange but diminishes intrathecal clonal expansion. Sphingosine-1 phosphate receptor inhibition may alter intrathecal B cell biology in MS.

CTNI-69. PRECISION NEURO-ONCOLOGY TREATMENT GUIDED BY NEXT GENERATION SEQUENCING (NGS)-BASED COMPREHENSIVE GENOMIC PROFILING: REAL WORLD EXPERIENCE IN HONG KONG

Abstract BACKGROUND The development of next generation sequencing (NGS) based comprehensive genomic profiling (CGP) has enabled identification of druggable somatic mutations in brain tumours. This cohort reviewed the efficacy of CGP-guided precision treatment in a tertiary neuro-oncology centre. METHODOLOGY From May 2017 to May 2020, CGP were arranged for 43 patients. All patients had exhausted conventional treatments or received CGP for clinical trial screening. Targeted deep NGS was used to assess the mutational status, single nucleotide variant, small insertions and deletions and copy number variant of 440 cancer-related genes. RESULTS The diagnoses of the 43 patients were GBM (n=23), high grade glioma (n=11), brain metastases (n=4), chordoma (n=3), atypical choroid plexus papilloma (n=1) and meningioma (n=1). In most of the patients (42/43, 97.7%), CGP identified at least one druggable targets with a median of 3. Based on the CGP, 27 patients received precision treatment (62.7%). Among these, 14 were GBM and 6 were other high grade glioma. Treatment given included PARP inhibitors, immunotherapy, multi-kinase inhibitor, selective CDK4/6 inhibitor and mTOR inhibitor. Clinical benefit was achieved in 20 patients out of 27 (74%), including 2 complete response (7.4%), 9 partial response (33.3%) and 9 stable disease (33.3%). The median progression free survival (PFS) were 183 days [95% confident intervals (CI): 81–302 days]. For GBM/high grade glioma patients, median PFS was 125 days [95% CI: 52–215] and six-month PFS was 32.7%. Treatment toxicity was mild except two patients developed grade 3 complications and one grade 5 complication (fatal neutropenic fever). For the 16 patients who did not receive precision treatment, one had no druggable target identified, nine were still stable on standard therapies, 6 were too weak when CGP was available. CONCLUSION CGP guided precision treatment for selected, advanced neuro-oncological patients yielded modest clinical efficacy and satisfactory safety profile in real world setting.

Abstract 6468: Prognostic genomic alterations associated with recurrence after primary therapy for patients with luminal B breast cancer

Abstract Purpose: Luminal type breast cancers account for the most common subtypes of breast cancers, among which luminal B subtype is relatively faster tumor growing and confers substantial risk of recurrence after primary treatment despite adjuvant endocrine therapy. Understanding the molecular alterations underlying the poorer survival outcome for luminal B tumors represents an unmet medical need. Experimental design: The archival samples of 16 recurrences within 5 years, 3 recurrences between 5-10 years and 24 non-recurrence tumor tissues from luminal B breast cancer patients were retrieved. Genomic alterations of these 43 breast cancer samples were detected using deep next-generation sequencing of a 440-gene panel. The mRNA levels of candidate genes in breast cancer patients were analyzed from The Cancer Genome Atlas (TCGA) database. Results: Overall, genomic alterations were found in 340 genes, including single nucleotide variants and copy number variation. Comparing to the non-recurrence tumors, NUP98 (31.3%), MAPK4 (18.8%), PTEN (18.8%), PER1 (18.8%) and TRIP11 (18.8%) showed higher population frequency in the recurrence tumors, and IGF2R (50.0%), PIM1 (45.8%) and ROS1 (37.5%) were dominate in the non-recurrence tumors. After a validation with the public clinical dataset, we identified three potential prognostic biomarkers, including NUP98, PER1, and TRIP11. We observed missense mutation or copy number deletion of NUP98, PER1 and TRIP11 in early recurrence tumors. Data from TCGA database exhibited patients with breast cancer harbored lower NUP98, PER1 and TRIP11 transcripts levels. Notably, lower NUP98, PER1 and TRIP11 expression levels correlated with worse recurrence-free survival in luminal B breast cancer. Interestingly, based on algorithm for visualization of protein interaction network, we found these 3 genes were all involved in androgen receptor signaling network. Conclusion: Our study identified NUP98, PER1, and TRIP11 which potentially served as biomarkers to predict recurrence in luminal B breast cancer. Further research is required to understand the association between genomic alterations and androgen receptor signaling as well as its clinical impact. Citation Format: Chun-Yu Liu, Ji-Lin Chen, Yi-Ting Yang, Yi-Fang Tsai, Ta-Chung Chao, Kien Thiam Tan, Wan-Lun Wang, Pei-Ju Lien, Shu-Jen Chen, Ling-Ming Tseng. Prognostic genomic alterations associated with recurrence after primary therapy for patients with luminal B breast cancer [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 6468.

Identification of olfactory genes of a forensically important blow fly, Aldrichina grahami (Diptera: Calliphoridae).

BackgroundThe time-length between the first colonization of necrophagous insect on the corpse and the beginning of investigation represents the most important forensic concept of minimum post-mortem inference (PMImin). Before colonization, the time spent by an insect to detect and locate a corpse could significantly influence the PMImin estimation. The olfactory system plays an important role in insect food foraging behavior. Proteins like odorant binding proteins (OBPs), chemosensory proteins (CSPs), odorant receptors (ORs), ionotropic receptors (IRs) and sensory neuron membrane proteins (SNMPs) represent the most important parts of this system. Exploration of the above genes and their necrophagous products should facilitate not only the understanding of their roles in forging but also their influence on the period before PMImin. Transcriptome sequencing has been wildly utilized to reveal the expression of particular genes under different temporal and spatial condition in a high throughput way. In this study, transcriptomic study was implemented on antennae of adult Aldrichina grahami (Aldrich) (Diptera: Calliphoridae), a necrophagous insect with forensic significance, to reveal the composition and expression feature of OBPs, CSPs, ORs, IRs and SNMPs genes at transcriptome level.MethodAntennae transcriptome sequencing of A. grahami was performed using next-generation deep sequencing on the platform of BGISEQ-500. The raw data were deposited into NCBI (PRJNA513084). All the transcripts were functionally annotated using Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. Differentially expressed genes (DEGs) were analyzed between female and male antennae. The transcripts of OBPs, CSPs, ORs, IRs and SNMPs were identified based on sequence feature. Phylogenetic development of olfactory genes of A. grahami with other species was analyzed using MEGA 5.0. RT-qPCR was utilized to verify gene expression generated from the transcriptome sequencing.ResultsIn total, 14,193 genes were annotated in the antennae transcriptome based on the GO and the KEGG databases. We found that 740 DEGs were differently expressed between female and male antennae. Among those, 195 transcripts were annotated as candidate olfactory genes then checked by sequence feature. Of these, 27 OBPs, one CSPs, 49 ORs, six IRs and two SNMPs were finally identified in antennae of A. grahami. Phylogenetic development suggested that some olfactory genes may play a role in food forging, perception of pheromone and decomposing odors.ConclusionOverall, our results suggest the existence of gender and spatial expression differences in olfactory genes from antennae of A. grahami. Such differences are likely to greatly influence insect behavior around a corpse. In addition, candidate olfactory genes with predicted function provide valuable information for further studies of the molecular mechanisms of olfactory detection of forensically important fly species and thus deepen our understanding of the period before PMImin.

Prevalence and dynamics of clonal hematopoiesis caused by leukemia-associated mutations in elderly individuals without hematologic disorders

Clonal hematopoiesis is frequently observed in elderly people. To investigate the prevalence and dynamics of genetic alterations among healthy elderly individuals, a cohort of 50 people >80 years was genotyped for commonly mutated leukemia-associated genes by targeted deep next-generation sequencing. A total of 16 somatic mutations were identified in 13/50 (26%) individuals. Mutations occurred at low variant allele frequencies (median 11.7%) and remained virtually stable over 3 years without development of hematologic malignancies in affected individuals. With DNMT3A mutations most frequently detected, another cohort of 160 healthy people spanning all age groups was sequenced specifically for DNMT3A revealing an overall mutation rate of 6.2% (13/210) and an age-dependent increase of mutation prevalence. A significant difference (p = 0.017) in the DNMT3A expression pattern was detected between younger and healthy elderly people as determined by qRT-PCR. To evaluate the selection of clonal hematopoietic stem cells (HSCs), bone marrow of two healthy individuals with mutant DNMT3A was transplanted in a humanized mouse model. Xenografts displayed stable kinetics of DNMT3A mutations over 8 months. These findings indicate that the appearance of low-level clones with leukemia-associated mutations is a common age-associated phenomenon, but insufficient to initiate clonal selection and expansion without the additional influence of other factors.

Human vtRNA1-1 Levels Modulate Signaling Pathways and Regulate Apoptosis in Human Cancer Cells.

Regulatory non-protein coding RNAs perform a remarkable variety of complex biological functions. Previously, we demonstrated a role of the human non-coding vault RNA1-1 (vtRNA1-1) in inhibiting intrinsic and extrinsic apoptosis in several cancer cell lines. Yet on the molecular level, the function of the vtRNA1-1 is still not fully clear. Here, we created HeLa knock-out cell lines revealing that prolonged starvation triggers elevated levels of apoptosis in the absence of vtRNA1-1 but not in vtRNA1-3 knock-out cells. Next-generation deep sequencing of the mRNome identified the PI3K/Akt pathway and the ERK1/2 MAPK cascade, two prominent signaling axes, to be misregulated in the absence of vtRNA1-1 during starvation-mediated cell death conditions. Expression of vtRNA1-1 mutants identified a short stretch of 24 nucleotides of the vtRNA1-1 central domain as being essential for successful maintenance of apoptosis resistance. This study describes a cell signaling-dependent contribution of the human vtRNA1-1 to starvation-induced programmed cell death.

What Genes Can Tell: A Closer Look at Vestibular Schwannoma.

Comprehensive molecular profiling of radioresistant and cystic vestibular schwannoma (VS) subtypes. Our study utilized whole-exome sequencing (WES), RNA-sequencing (RNAseq), and correlated clinical data from 12 samples (2 samples of solid sporadic subtype, 8 with cystic changes, and 2 previously irradiated). Academic medical center. Patients diagnosed with VS who required surgical treatment. Inclusion: Cystic and radioresistant tumors matched to age and tumor volume, with solid sporadic VS samples as control; Exclusion: NF-2 patients. WES using custom probes for copy number analysis. A modified version of the Agilent Human Whole Exome sequencing hybrid capture system was used to process samples. Recurrent variants were identified and compared between groups. Leukocyte-derived DNA was utilized as internal control to reduce false-positives. Analysis of genetic landscape of VS subtypes (naive solid VS, cystic VS, and previously irradiated VS) by performing deep next-generation sequencing. WES data achieved a mean coverage of 202X and RNAseq generated an average of 74 million total reads. As a group, 25% of samples had 22q loss. Somatic analysis identified previously reported genes and multiple novel mutations across samples. Differential expression analysis of RNAseq data found significantly mutated genes such as COL6A3, CLMP, ART4, Lumican that were shared by both cystic VS and irradiated VS, but not seen in sporadic VS. Using WES we were able to demonstrate that cystic and irradiated samples are subtypes of VS with an increased mutation burden and a unique genetic fingerprint. We identified differences between the genomic and molecular profile of cystic VS and radioresistant VS. Our results help advance the understanding of the pathophysiology of these tumor subtypes and suggest possible molecular targets for novel treatment strategies.

Genomic Underpinnings of Tumor Behavior in In Situ and Early Lung Adenocarcinoma.

Rationale: We have a limited understanding of the molecular underpinnings of early adenocarcinoma (ADC) progression. We hypothesized that the behavior of early ADC can be predicted based on genomic determinants.Objectives: To identify genomic alterations associated with resected indolent and aggressive early lung ADCs.Methods: DNA was extracted from 21 ADCs in situ (AISs), 27 minimally invasive ADCs (MIAs), and 54 fully invasive ADCs. This DNA was subjected to deep next-generation sequencing and tested against a custom panel of 347 cancer genes.Measurements and Main Results: Sequencing data was analyzed for associations among tumor mutation burden, frequency of mutations or copy number alterations, mutation signatures, intratumor heterogeneity, pathway alterations, histology, and overall survival. We found that deleterious mutation burden was significantly greater in invasive ADC, whereas more copy number loss was observed in AIS and MIA. Intratumor heterogeneity establishes early, as in AIS. Twenty-one significantly mutated genes were shared among the groups. Mutation signature profiling did not vary significantly, although the APOBEC signature was associated with ADC and poor survival. Subclonal KRAS mutations and a gene signature consisting of PIK3CG, ATM, EPPK1, EP300, or KMT2C mutations were also associated with poor survival. Mutations of KRAS, TP53, and NF1 were found to increase in frequency from AIS and MIA to ADC. A cancer progression model revealed selective early and late drivers.Conclusions: Our results reveal several genetic driver events, clonality, and mutational signatures associated with poor outcome in early lung ADC, with potential future implications for the detection and management of ADC.

Differences in the Inflammatory Response of White Adipose Tissue and Adipose-Derived Stem Cells.

The application of liposuctioned white adipose tissue (L-WAT) and adipose-derived stem cells (ADSCs) as a novel immunomodulatory treatment option is the currently subject of various clinical trials. Because it is crucial to understand the underlying therapeutic mechanisms, the latest studies focused on the immunomodulatory functions of L-WAT or ADSCs. However, studies that examine the specific transcriptional adaptation of these treatment options to an extrinsic inflammatory stimulus in an unbiased manner are scarce. The aim of this study was to compare the gene expression profile of L-WAT and ADSCs, when subjected to tumor necrosis factor alpha (TNFα), and to identify key factors that might be therapeutically relevant when using L-WAT or ADSCs as an immuno-modulator. Fat tissue was harvested by liposuction from five human donors. ADSCs were isolated from the same donors and shortly subjected to expansion culture. L-WAT and ADSCs were treated with human recombinant TNFα, to trigger a strong inflammatory response. Subsequently, an mRNA deep next-generation sequencing was performed to evaluate the different inflammatory responses of L-WAT and ADSCs. We found significant gene expression changes in both experimental groups after TNFα incubation. However, ADSCs showed a more homogenous gene expression profile by predominantly expressing genes involved in immunomodulatory processes such as CCL19, CCL5, TNFSF15 and IL1b when compared to L-WAT, which reacted rather heterogeneously. As RNA sequencing between L-WAT and ADSCS treated with TNFα revealed that L-WAT responded very heterogeneously to TNFα treatment, we therefore conclude that ADSCs are more reliable and predictable when used therapeutically. Our study furthermore yields insight into potential biological processes regarding immune system response, inflammatory response, and cell activation. Our results can help to better understand the different immunomodulatory effects of L-WAT and ADSCs.

Multicenter Evaluation of Circulating Cell-Free DNA Extraction and Downstream Analyses for the Development of Standardized (Pre)analytical Work Flows.

In cancer patients, circulating cell-free DNA (ccfDNA) can contain tumor-derived DNA (ctDNA), which enables noninvasive diagnosis, real-time monitoring, and treatment susceptibility testing. However, ctDNA fractions are highly variable, which challenges downstream applications. Therefore, established preanalytical work flows in combination with cost-efficient and reproducible reference materials for ccfDNA analyses are crucial for analytical validity and subsequently for clinical decision-making. We describe the efforts of the Innovative Medicines Initiative consortium CANCER-ID (http://www.cancer-id.eu) for comparing different technologies for ccfDNA purification, quantification, and characterization in a multicenter setting. To this end, in-house generated mononucleosomal DNA (mnDNA) from lung cancer cell lines carrying known TP53 mutations was spiked in pools of plasma from healthy donors generated from 2 different blood collection tubes (BCTs). ccfDNA extraction was performed at 15 partner sites according to their respective routine practice. Downstream analysis of ccfDNA with respect to recovery, integrity, and mutation analysis was performed centralized at 4 different sites. We demonstrate suitability of mnDNA as a surrogate for ccfDNA as a process quality control from nucleic acid extraction to mutation detection. Although automated extraction protocols and quantitative PCR-based quantification methods yielded the most consistent and precise results, some kits preferentially recovered spiked mnDNA over endogenous ccfDNA. Mutated TP53 fragments derived from mnDNA were consistently detected using both next-generation sequencing-based deep sequencing and droplet digital PCR independently of BCT. This comprehensive multicenter comparison of ccfDNA preanalytical and analytical work flows is an important contribution to establishing evidence-based guidelines for clinically feasible (pre)analytical work flows.

Frequency of low-level and high-level mosaicism in sporadic retinoblastoma: genotype-phenotype relationships.

Somatic mutational mosaicism is a common feature of monogenic genetic disorders, particularly in diseases such as retinoblastoma, with high rates of de novo mutations. The detection and quantification of mosaicism is particularly relevant in these diseases, since it has important implications for genetic counseling, patient management, and probably also on disease onset and progression. In order to assess the rate of somatic mosaicism (high- and low-level mosaicism) in sporadic retinoblastoma patients, we analyzed a cohort of 153 patients with sporadic retinoblastoma using ultra deep next-generation sequencing. High-level mosaicism was detected in 14 out of 100 (14%) bilateral patients and in 11 out of 29 (38%) unilateral patients in whom conventional Sanger sequencing identified a pathogenic mutation in blood DNA. In addition, low-level mosaicism was detected in 3 out of 16 (19%) unilateral patients in whom conventional screening was negative in blood DNA. Our results also reveal that mosaicism was associated to delayed retinoblastoma onset particularly in unilateral patients. Finally we compared the level of mosaicism in different tissues to identify the best DNA source to identify mosaicism in retinoblastoma patients. In light of these results we recommended analyzing the mosaic status in all retinoblastoma patients using accurate techniques such as next-generation sequencing, even in those cases in which conventional Sanger sequencing identified a pathogenic mutation in blood DNA. Our results suggest that a significant proportion of those cases are truly mosaics that could have been overlooked. This information should be taking into consideration in the management and genetic counseling of retinoblastoma patients and families.

Whole-genome sequencing of human Pegivirus variant from an Egyptian patient co-infected with hepatitis C virus: a case report

BackgroundHuman pegivirus (HPgV) is structurally similar to hepatitis C virus (HCV) and was discovered 20 years ago. Its distribution, natural history and exact rule of this viral group in human hosts remain unclear. Our aim was to determine, by deep next-generation sequencing (NGS), the entire genome sequence of HPgV that was discovered in an Egyptian patient while analyzing HCV sequence from the same patient. We also inspected whether the co-infection of HCV and HPgV will affect the patient response to HCV viral treatment. To the best of our knowledge, this is the first report for a newly isolated HPgV in an Egyptian patient who is co-infected with HCV.Case presentationThe deep Next Generation Sequencing (NGS) technique was used to detect HCV sequence in hepatitis C patient’s plasma. The results revealed the presence of HPgV with HCV. This co-infection was confirmed using conventional PCR of the HPgV 5′ untranslated region. The patient was then subjected to direct-acting-antiviral treatment (DAA). At the end of the treatment, the patient showed a good response to the HCV treatment (i.e., no HCV-RNA was detected in the plasma), while the HPgV-RNA was still detected. Sequence alignment and phylogenetic analyses demonstrated that the detected HPgV was a novel isolate and was not previously published.ConclusionWe report a new variant of HPgV in a patient suffering from hepatitis C viral infection.

RNA-Seq analysis of peripheral blood mononuclear cells reveals unique transcriptional signatures associated with radiotherapy response of nasopharyngeal carcinoma and prognosis of head and neck cancer

ABSTRACTRadiotherapy is the main treatment for nasopharyngeal carcinoma (NPC); however, radioresistance limits the therapeutic efficacy and prognosis of patients with NPC. Here, we plan to identify the genes involved in radiotherapy response. Peripheral blood mononuclear cells (PBMC) from three paired NPC patients with pre-radiotherapy and post-radiotherapy were extracted. Next-generation deep sequencing was then performed to identify the PBMCs transcripts profiles in response to radiotherapy. Data of gene chip GSE48501 was obtained from Gene Expression Omnibus (GEO) database. The gene integration of differentially expressed genes identified from RNA-Seq data and gene chip was performed using “RobustRankAggreg” package. RNA-Seq data from 44 normal and 519 Head and neck squamous cell carcinoma (HNSCC) tissues (downloaded from TCGA) was integrated into the analysis to further support our study. Cox regression was used to identify risk factors impacting survival. Total of 45 genes were identified to be associated with radiotherapy response. Significantly enriched Gene Ontology (GO) terms and pathways were enriched. Univariate and multivariate analysis suggested the dysregulated genes, CHAC2, CLEC9A, GNG10, JCHAIN, KLRB1, NOG, OLR1, PRELID2, SYT1, VWCE, ZNF443 were associated with survival in HNSCC patients. Our data provide an overview of the profiles of radiotherapy-associated genes, which will facilitate future investigations into the function of radiotherapy resistance.

Detection of MYD88 L265P mutation by next-generation deep sequencing in peripheral blood mononuclear cells of Waldenström's macroglobulinemia and IgM monoclonal gammopathy of undetermined significance.

We investigated the feasibility of using next-generation sequencing (NGS) technique using molecular barcoding technology to detect MYD88 L265P mutation in unselected peripheral blood mononuclear cells (PBMCs) in 52 patients with Waldenström’s macroglobulinemia [1] and 21 patients with IgM-monoclonal gammopathy of undetermined significance (MGUS). The NGS technique successfully detected the MYD88 L265P in unselected PBMCs at a sensitivity of 0.02%, which was ×5 higher than that of AS-PCR. All the results between paired BM and PB samples from 2 IgM MGUS and 4 untreated WM patients matched completely. MYD88 L265P mutation was detected in 14/21 (66.7%), 14/19 (73.7%), and 10/33 (30.3%) with the median mutant allele burden of 0.36% (range, 0.06–2.85%), 0.48% (range, 0.02–32.3%), and 0.16% (range, 0.02–33.8%), in IgM-MGUS, untreated WM, and previously treated WM, respectively. Multiple linear regression analysis identified an absolute peripheral lymphocyte count as the positive predictor of PB mutant allele burden (R2 = 0,72, P<0.0001). Our non-invasive, simple NGS method has the potential to detect MYD88 L265P mutations in PBMCs of IgM MGUS and WM patients, which may especially utilized for monitoring minimal residual tumor burden after treatment.

BEAT: A Python Program to Quantify Base Editing from Sanger Sequencing.

Through fusing CRISPR-Cas9 nickases with cytidine or adenine deaminases, a new paradigm-shifting class of genome-editing technology, termed "base editors," has recently been developed. Base editors mediate highly efficient, targeted single-base conversion without introducing double-stranded breaks. Analysis of base editing outcomes typically relies on imprecise enzymatic mismatch cleavage assays, time-consuming single-colony sequencing, or expensive next-generation deep sequencing. To overcome these limitations, several groups have recently developed computer programs to measure base-editing efficiency from fluorescence-based Sanger sequencing data such as Edit deconvolution by inference of traces in R (EditR), TIDER, and ICE. These approaches have greatly simplified the quantitation of base-editing experiments. However, the current Sanger sequencing tools lack the capability of batch analysis and producing high-quality images for publication. Here, we provide a base editing analysis tool (BEAT) written in Python to analyze and quantify the base-editing events from Sanger sequencing data in a batch manner, which can also produce intuitive, publication-ready base-editing images.

In Vivo Exon Replacement in the Mouse Atp7b Gene by the Cas9 System.

The application of CRISPR/Cas9 has opened a new era in gene therapy, making it possible to correct mutated genomes in vivo. Exon replacement can correct many mutations and has potential clinical value. In this study, we used a lentivirus-delivered transgene to obtain transgenic mice in which Cas9 and green fluorescent protein (GFP) were driven by the hTBG promoter and were specifically expressed in the liver. In Cas9-positive mice, only ∼11.6% of hepatocytes were GFP positive. The newborn Cas9-positive F1 mice were injected via the temporal vein with rAAV carrying a modified homologous replacement sequence for exon 8 of Atp7b and a pair of single-strand guide RNAs targeting the introns surrounding exon 8. When the Cas9-positive hepatocytes were sorted and analyzed by PCR and next-generation deep sequencing with different labels, ∼16.34 ± 4.02% to 19.37 ± 6.50% of the analyzed copies of exon 8 were replaced by the donor template in the genome of GFP-positive hepatocytes, that is, 1.81 ± 0.29% to 2.09 ± 0.54% replacement occurred in all liver genomes. However, when rAAV carrying a modified homologous replacement sequence was injected into the adult spCas9 mice, a double-cut deletion ratio of up to 99%, only about 1.10-1.13% of the exon 8 replacement rate was detected in Cas9-positive hepatocytes. This study is the first to achieve exon replacement via CRISPR/Cas9, which will benefit research on CRISPR/Cas9 technology for gene therapy.

Abstract 3538: Analysis of error profiles in deep next-generation sequencing data

Abstract Background Sequencing errors are a key confounding factor for detecting low frequency genetic variants for cancer screening, testing, treatment and surveillance through deep next-generation sequencing (NGS). However, there is a lack of comprehensive understanding of errors introduced at various steps of a conventional NGS workflow, such as sample handling, library preparation, enrichment PCR, and sequencing. In this study we systematically investigated the above question by using the current NGS technology. Results We discovered that the substitution error rate can be computationally suppressed to 10-5~10-4, which is 10~100-fold lower than current reports. We then quantified substitution errors attributable to sample handling, library preparation, enrichment PCR, and sequencing using multiple deep sequencing datasets from multiple sequencing centers. We show that 1) error rate differs by nucleotide substitution types, ranging from 10-5 for A&amp;gt;C/T&amp;gt;G, C&amp;gt;A/G&amp;gt;T, and C&amp;gt;G/G&amp;gt;C changes to 10-4 for A&amp;gt;G/T&amp;gt;C changes; 2) C&amp;gt;T/G&amp;gt;A errors exhibit strong sequence context dependency; 3) sample-specific effects dominate elevated C&amp;gt;A/G&amp;gt;T errors; 4) target enrichment PCR lead to ~6-fold increase of overall error rate; 5) more than 70% of hotspot variants, such as BRAF V600E, can be detected at 0.1%~0.01% frequency with the current NGS technology by applying in-silico error suppression. Conclusions We present the first comprehensive analysis of error sources in conventional NGS workflows. The error profiles revealed by our study highlight new directions for further improving NGS accuracy both experimentally and computationally which will lead to enhanced precision for deep sequencing. Citation Format: Xiaotu Ma, Jinghui Zhang. Analysis of error profiles in deep next-generation sequencing data [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3538.

Abstract 1822: A mitochondrial pfeRNA associates with Far Upstream Element Binding Protein 1 (FUBP1) to promote lung adenocarcinoma tumorigenesis

Abstract Background: Protein Functional Effector RNAs (pfeRNAs) are novel small non-coding RNA molecules with great prognostic and diagnostic potential for NSCLC. They feature direct binding to, and regulation of phosphorylated proteins involved in lung cancer tumorigenesis pathways. Using next-generation sncRNA deep sequencing, we have identified a mitochondrial pfeRNA (mtpfeRNA) that could play a key role in tumorigenesis and cancer development of lung adenocarcinoma (ADC). Methods: Using next-generation sncRNA deep sequencing, we analysed ADC and human normal bronchial epithelial (HBE) cell lines; and biospecimens including matched biopsy-proven ADC tissue, histologically normal adjacent lung tissue, benign lymph node, and (iv) malignant lymph nodes; Identifying a mitochondrial pfeRNA that could play critical roles in lung cancer tumorigenesis. Next, we examined the impact of this mtpfeRNA on lung ADC cell line viability. Liquid chromatography-tandem mass spectrometry analysis and reverse immunoprecipitation assays were used to identify critical interacting protein partners known to drive tumorigenesis. We further correlated our findings to clinical staging of ADC tumor cores by IHC staining. Results: The expression levels of mtpfeRNA were upregulated in ADC compared to that of squamous cell carcinoma and normal human bronchial epithelial cells. We also noted that blocking the upregulated mtpfeRNA resulted in significant decreases in cell viability in ADC cells. Next, we determined that mtpfeRNA binds to Far Upstream Element Binding Protein 1 (FUBP1). Furthermore, we confirmed that both mtpfeRNA and FUBP1 expression correlated to stages and lymph node metastasis in patients with ADC. Conclusion: We revealed novel functions of a mtpfeRNA in ADC, providing potential molecules for developing diagnostics and targeted therapy to improve the dismal survival rate of patients with ADC. Citation Format: Nadege T. Fackche, Yuping Mei, Tomoaki Ito, Matthew Garner, Malcolm Brock. A mitochondrial pfeRNA associates with Far Upstream Element Binding Protein 1 (FUBP1) to promote lung adenocarcinoma tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1822.