Total Nucleic Acid Research Articles

Effect of photoperiod on the DNA, RNA and protein concentration in the silkgland of tasar silkworm, Antheraea mylitta (D) (Lepidoptera, Saturniidae)

In Antheraea mylitta (D) (Lepidoptera, Saturniidae), the effect of photoperiod on total DNA, RNA, and protein concentration in the middle silk gland (MSG) and posterior silk gland (PSG) was studied. The photoperiod caused enhancement in the secretory activity and stimulates the sericogenesis process in MSG and PSG. The total nucleic acids and protein concentration of MSG and PSG under 24L:00D condition reached maximum level and then it decreased. Maximum nucleic acids and protein concentration were observed during long-day period and the minimum during short-day period. The photoperiod activated or inhibited the biological clock which acted upon the respective endocrine glands accordingly and control the process of sericogenesis. The larvae underwent significant changes with high DNA, RNA and protein concentration under 24L:00D, which is highly suitable for the silk protein synthesis.

Implementation of an ISO 15189 accredited next generation sequencing service for cell-free total nucleic acid (cfTNA) analysis to facilitate driver mutation reporting in blood: the experience of a clinical diagnostic laboratory

AimsNext generation sequencing (NGS) on tumour tissue is integral to the delivery of personalised medicine and targeted therapy. NGS on liquid biopsy, a much less invasive technology, is an emerging...

A Simple and Rapid Microscale Method for Isolating Bacterial Lipopolysaccharides.

Bacterial endotoxins (lipopolysaccharides (LPSs)) are important mediators of inflammatory processes induced by Gram-negative microorganisms. LPSs are the key inducers of septic shock due to a Gram-negative bacterial infection; thus, the structure and functions of LPSs are of specific interest. Often, highly purified bacterial endotoxins must be isolated from small amounts of biological material. Each of the currently available methods for LPS extraction has certain limitations. Herein, we describe a rapid and simple microscale method for extracting LPSs. The method consists of the following steps: ultrasonic destruction of the bacterial material, LPS extraction via heating, LPS purification with organic solvents, and treatment with proteinase K. LPSs that were extracted by using this method contained less than 2-3% protein and 1% total nucleic acid. We also demonstrated the structural integrity of the O-antigen and lipid A via the sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) methods, respectively. We demonstrated the ability of the extracted LPSs to induce typical secretion of cytokines and chemokines by primary macrophages. Overall, this method may be used to isolate purified LPSs with preserved structures of both the O-antigen and lipid A and unchanged functional activity from small amounts of bacterial biomass.

Use of a deep learning model on H&E slides to predict nucleic acid yield for NGS testing.

e13589 Background: Samples sent for NGS may fail testing, leading to treatment delays or missed opportunities to offer patients targeted therapies. A common reason for assay failure is insufficient total nucleic acid (TNA). We developed and validated a model based on a digitized H&E slide and readily available clinical data that predicts whether samples will yield sufficient TNA for NGS testing. This model could lead to faster turnaround times and lower failure rates by helping choose patient blocks most likely to complete sequencing, or routing likely-to-fail samples to specialized extraction methods or smaller panel assays with lower input TNA requirements. We also evaluated the model’s ability to identify samples that yield abundant TNA, which could help pathologists spare tissue for other testing. Methods: We developed our model on samples received for NGS testing from January 1 - June 30, 2023, and tested on a large, real-world cohort of 9,707 samples received from July 1 - September 30, 2023. Cell count, assessed by a previously developed deep learning model on digitized H&E slides, along with the number of slides scraped, sample age, procedure type, and tissue site were used as model inputs. We measured the model’s ability to predict whether a sample yielded less than 100 ng of TNA (the target mass for the NGS assay used) and whether it failed sequencing. We also evaluated the model’s ability to predict whether a sample would have abundant TNA (defined here as yielding more than 1000 ng of TNA, 10x the target mass for the assay). Results: On the validation set, the model predicted samples that yielded TNA < 100 ng with a specificity of 98%, sensitivity of 26% and PPV of 82% (prevalence = 24%). It predicted samples that failed DNA sequencing with a specificity of 96%, sensitivity of 30% and PPV of 58% (prevalence = 15%); and samples that failed either DNA or RNA sequencing with a specificity of 98%, sensitivity of 23%, and PPV of 80% (prevalence = 27%). On the validation set, 742 samples were flagged, 430 of which failed DNA sequencing, and 590 of which failed either DNA or RNA sequencing. When predicting abundant TNA, the model predicted samples yielding TNA > 1000 ng at a specificity of 97%, sensitivity of 54% and PPV of 88% (prevalence = 26%). On the validation set, the model predicted 1544 samples as abundant, only 185 of which yielded TNA < 1000 ng and only 16 with TNA < 100 ng. Conclusions: The developed model holds promise as a tool for use at the beginning of NGS assays. It can be utilized to optimize testing decisions including selection of which samples to sequence, routing likely-to-fail samples to alternate testing methods, and sparing tissue for future assays from samples with abundant tissue.

Pathogens Detected in Questing Ixodes ricinus Ticks in a Mountainous Area in Greece.

Ixodes ricinus ticks are vectors of a plethora of pathogens. The purpose of this study was to screen 398 I. ricinus ticks for a variety of pathogens. Following the pooling, homogenization, and extraction of total nucleic acids, a real-time PCR was applied for the detection of a panel of tick-borne pathogens, while additional conventional PCRs combined with Sanger sequencing were applied for the detection of viruses and typing of Rickettsia and Borrelia species. At least one pathogen was detected in 60 of the 80 (75%) tick pools. Rickettsia spp. predominated, as it was detected in 63.75% of the pools (51/80; MIR 12.81%), followed by Borrelia spp. (35 pools (45%); MIR 8.79%), while Anaplasma phagocytophilum was detected in 2 pools (2.5%, MIR 0.5%). The ticks of six Rickettsia-positive pools were tested individually (from stored half-ticks); all sequences were identical to those of R. monacensis. Similarly, the ticks of six Borrelia-positive pools were tested individually, and it was shown that four belonged to the genospecies Borrelia garinii and two to Borrelia valaisiana. Phleboviruses were detected in 3 pools (3.75%; MIR 0.75%), with sequences clustering in the Ixovirus genus, while nairoviruses were detected in 7 pools (8.75%; MIR 1.76%), with one sequence clustering in the Orthonairovirus genus, and six clustering in the Norwavirus genus. Although a small number of ticks from only one area in Greece were tested, a variety of pathogens together with recently identified viruses were detected, prompting further studies in ticks and surveillance studies in humans.

Human benign Leydig cell tumor − Biochemical evaluation

Recently we reported expressional alterations in 219 genes and their transcripts in Leydig cell tumors but nowadays there is still a lack of full basic biochemical characteristics of these tumors. The discovery of potential biochemical markers for tumor management from early detection, treatments, and control of therapy results may markedly supplement genetic data. Leydig cell micronodules were obtained from patients with azoospermia who were qualified for testicular biopsy. The biochemistry of Leydig cell tumors was analyzed using histological staining and spectrophotometric measurements of total proteins, carbohydrates, lipids, and nucleic acids. In addition, the levels of calcium (Ca2 +), copper (Cu2 +), zinc (Zn2 +), and selenium (Se2 +) ions were measured. When compared to healthy testis we revealed, for the first time, that in the interstitial tissue with Leydig cell tumors, great amounts of proteins, carbohydrates, lipids, and acids were dislocated from the seminiferous tubules. Measurements of organic compounds showed a decrease (P < 0.05) only in the Cu2 + content in Leydig cell tumors which may be related to their altered biochemical structure. This specific result may be promising for designing further approaches to manage this tumor based on combining morphological and molecular data.

Abstract PO5-16-05: Integrating Circulating Tumor Cells (CTCs) and Cell-free DNA (cfDNA) Signatures for Monitoring Treatment Response in Stage III/IV Inflammatory Breast Cancer

Abstract Background: Inflammatory breast cancer (IBC) is a rare and aggressive form of breast cancer, comprising only between 1-5% of all breast cancer cases in the United States. Despite its low incidence, IBC accounts for 10% of breast cancer-related deaths and is often diagnosed with metastasis, resulting in shorter survival compared to non-IBC cases. The 5-year relative survival rate for all stages of IBC is 39%, dropping to 19% for patients with distant metastasis. Liquid biopsy-based biomarkers have emerged as potential analytes for predicting prognosis and monitoring response to therapies in IBC patients. We explored the prognostic role of liquid biopsy encompassing both CTCs and cfDNA in the blood of patients with stage III/IV IBC and their clinical outcomes. Methods: CTC and cfDNA analysis was performed on 90 blood samples from 47 patients with stage III and IV IBC. Samples were collected before (pre-treatment, n=43) initiation and after (post-treatment, n=47) completion of neoadjuvant chemotherapy. CTCs were enriched and enumerated using the CellSearch® platform (Menarini-Silicon Biosystems). CfDNA was extracted from plasma using MagMax™ Cell-Free Total Nucleic Acid Isolation Kit (Applied Biosystems™) and quantified using the Qubit™ dsDNA High Sensitivity kit (Invitrogen™). Differences in cfDNA concentrations were used to plot a receiver operating characteristic (ROC) curve which was then used to determine Area Under the Curve (AUC) and a cfDNA cut-off point. ROC cut-off was calculated based on Youden Index J. Survival analysis was performed by the Kaplan-Meier estimator and Cox regression models. P-values were measured using the Student t-test. Results: Prior to neoadjuvant treatment, CTC was detected in 51% of patient samples, and the median cfDNA concentration was 5.87 ng/mL (range 0.95-24.11 ng/mL). After completion of neoadjuvant treatment, CTC was detected in 25% of patient samples, and the median cfDNA concentration was 11.19 ng/mL (range 3.41-88.13 ng/mL). The cut-off points determined for pre-treatment and post-treatment cfDNA concentration was &amp;gt;4.2 ng/mL and &amp;gt;9.5 ng/mL respectively. At pre-treatment, CTC detection and cfDNA concentration &amp;gt;4.2 ng/mL were independently associated with disease relapse (P=0.007, 0.016, respectively). The combination of CTC detection and cfDNA concentration &amp;gt;4.2 ng/mL was significantly associated with disease relapse (P=0.0004), decreased relapse free survival (RFS, P=0.005) and overall survival (OS, P=0.007). At the post-neoadjuvant treatment time point, the combination of CTC detection and cfDNA concentration &amp;gt;9.5 ng/mL was significantly associated with relapse regardless of pCR status (P=0.02). Conclusion: The combined presence of CTCs and elevated cfDNA concentrations in patients with stage III/IV IBC after completion of neoadjuvant chemotherapy was associated with disease relapse regardless of pCR status. A combination of elevated cfDNA and CTC detection signatures after completion of neoadjuvant chemotherapy may be useful for risk stratification and adjuvant systemic therapy planning. Citation Format: Sridevi Addanki, Salyna Meas, Vanessa Sarli, Abdulkader Almosa, Wendy Woodward, Dhruvajyoti Roy, Anthony Lucci. Integrating Circulating Tumor Cells (CTCs) and Cell-free DNA (cfDNA) Signatures for Monitoring Treatment Response in Stage III/IV Inflammatory Breast Cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-16-05.

Detection of Hepatitis E Virus (HEV) in Pork Sold in Saint-Louis, the North of Senegal.

The hepatitis E virus (HEV) is a zoonotic pathogen with various hosts, including pigs, which act as reservoirs. In industrialized countries, sporadic cases caused by genotype 3, contracted by ingesting contaminated uncooked or undercooked meat, have been reported. However, in developing countries, HEV infection is mainly dominated by genotype 2 and often associated with poor hygiene conditions and drinking water supplies. HEV infection and its circulation in domestic fauna in West Africa are poorly documented. This study aimed to assess the presence of HEV in pork sold in Saint-Louis, Senegal. Meat products (250 g samples, n = 74) were purchased in August 2022 from three locations. Then, 2 g/sample was minced to extract total nucleic acids using the Purelink™ Viral DNA/RNA kit. RT-PCR reactions were performed using the One-Taq™ One-Step RT-PCR kit targeting the HEV ORF2 genomic region. The products obtained were visualized on a 1% agarose gel. Of a total of 74 samples, divided into pork meat (n = 65) and pork liver (n = 9), 5.4% (n = 4) tested positive for HEV. In both cases, two samples were positive, representing a rate of 3.1% and 22.2% for meat and pork liver, respectively. All new viral sequences were obtained from a monophyletic group within HEV genotype 3. This study is the first to report the presence of HEV in pork sold in Senegal and the results reveal a potential circulation of HEV in the pig population. The high proportion of contamination in the pork liver samples highlights a major risk associated with their consumption.

First report of sweepoviruses infecting Ipomeas batatas L. cultivars and landraces in Trinidad.

Root crops, referred to as ground provisions in the Caribbean, are traditional staples in Trinidad. One widely consumed example is sweet potato (Ipomeas batatas L.). The crop is mainly produced by subsistence farming which together with imports from neighboring Caribbean countries meet domestic demand (Singh et al. 2008). The Central Experiment Station, situated in the eastern part of Trinidad, maintains a sweet potato germplasm collection comprising both imported and locally-sourced landraces for cultivar development and distribution of propagules. In May 2017 chlorosis and leaf curling symptoms, typically associated with sweepoviruses, were observed on imported cultivars, Centennial, Jewel, 86 BM 31, TIB 313, TIB 8 21 1, and S128, and the landraces, Kick Up Jenny, John, and Carrot. Leaf samples from these nine symptomatic plants were collected for analysis, along with samples from the asymptomatic landrace, Chickenfoot. Total nucleic acids were extracted (Sharma et al. 2008) and the samples were assayed by PCR using degenerate primers SPG1 and SPG2 (Li et al. 2004) that target the replication associated protein gene (ORF C1), a highly conserved region of sweepoviruses. Amplicons of 912-bp were obtained from two of the nine symptomatic plants (TIB 8 21 1, Kick Up Jenny), but not from the asymptomatic Chickenfoot. The same samples were assayed by PCR amplification using primers SpvF and SpvR (Avelar 2015) which are specific to a highly conserved 632-bp region of the coat protein gene (ORF V1) of sweet potato leaf curl virus (SPLCV). All 10 samples tested positive for SPLCV, including the asymptomatic landrace, Chickenfoot. The ORF V1 PCR products from three of the 10 samples, namely Chickenfoot, TIB 8 21 1, and Kick Up Jenny, were cloned and sequenced (two clones per sample). Comparison of the sequences (GenBank accession nos. OR882007 [Chickenfoot], OR913125 [TIB 8 21 1] and OR913126 [Kick Up Jenny]) identified up to 4% nt sequence variability between samples. In BLASTn analysis, they were most closely related to the SPLCV isolate China:Sichuan (GenBank accession no. KJ013557), sharing 94 to 98% nt identity. Total nucleic extracts from one representative sample (TIB 8 21 1) was used as template for rolling circle amplification (RCA, TempliPhi Amplification Kit, GE Healthcare Life Sciences, Piscataway, NJ, USA). Digestion of the RCA product with StuI (Thermo Scientific, MA, USA) yielded ~2.8 kb DNA fragments indicative of monomeric full length genomes. Digested fragments were cloned, completely sequenced and deposited in GenBank under the accession nos. OR866202 (2,821 nts) and OR866203 (2,828 nts). Two species of sweepoviruses were detected. In BLASTn analysis, OR866202 showed 95% nt identity with sweet potato golden vein associated virus (SPGVaV) US:MS:1B-3 (GenBank accession no. HQ333143.1) which is a recombinant virus comprised of SPLCV and sweet potato leaf curl Georgia virus (SPLCGV) (Zhang and Ling 2011) and in BLASTx analysis OR866202 was most similar (92-99%) to SPLCV isolates from Brazil (GenBank accession nos. ACI23475.1, AGW16179.1, ACY79479.1), Peru (GenBank accession no. ACY79466.1) and China (GenBank accession nos. ACY79439.1). OR866203 shared 96% nt identity with SPLCV China:Henan25(8):2012 (GenBank accession no. KF040465.1) in BLASTn analysis and BLASTx analysis revealed ≥ 94% aa sequence identity with SPLCV from Brazil (GenBank accession nos. ACI23475.1, AGW16179.1, ADZ96559.1), Peru (GenBank accession no. ACY79479.1), China (GenBank accession no. ACY79466.1). and Spain (GenBank accession no. QWQ56365.1). Both Trinidad isolates also showed 90-96% nt identity with SPLCV from Korea (GenBank accession no.s KT992061.1, KT992064.1, unpublished). This is the first detection of sweepoviruses in Trinidad. SPGVaV has been reported in Brazil, the United States and Korea (Kil et al. 2014), while SPLCV has been described in other Caribbean islands, including Cuba, Jamaica, Puerto Rico, St. Vincent (Cuellar et al. 2015), and Barbados (Alleyne et al. 2019), as well as several countries in South America. Although Koch's postulates were not completed, our findings suggest that sweet potato crops in Trinidad harbor sweepoviruses, notwithstanding efforts to distribute pathogen-free materials and, in some instances, the apparent absence of visible symptoms on infected plants. Further studies on the management and impact of these viruses are necessary, including their prevalence in the sweet potato production regions of Trinidad.

Next-generation sequencing survey of acute febrile illness in Senegal (2020-2022).

Acute febrile illnesses (AFI) in developing tropical and sub-tropical nations are challenging to diagnose due to the numerous causes and non-specific symptoms. The proliferation of rapid diagnostic testing and successful control campaigns against malaria have revealed that non-Plasmodium pathogens still contribute significantly to AFI burden. Thus, a more complete understanding of local trends and potential causes is important for selecting the correct treatment course, which in turn will reduce morbidity and mortality. Next-generation sequencing (NGS) in a laboratory setting can be used to identify known and novel pathogens in individuals with AFI. In this study, plasma was collected from 228 febrile patients tested negative for malaria at clinics across Senegal from 2020-2022. Total nucleic acids were extracted and converted to metagenomic NGS libraries. To identify viral pathogens, especially those present at low concentration, an aliquot of each library was processed with a viral enrichment panel and sequenced. Corresponding metagenomic libraries were also sequenced to identify non-viral pathogens. Sequencing reads for pathogens with a possible link to febrile illness were identified in 51/228 specimens, including (but not limited to): Borrelia crocidurae (N = 7), West Nile virus (N = 3), Rickettsia felis (N = 2), Bartonella quintana (N = 1), human herpesvirus 8 (N = 1), and Saffold virus (N = 1). Reads corresponding to Plasmodium falciparum were detected in 19 specimens, though their presence in the cohort was likely due to user error of rapid diagnostic testing or incorrect specimen segregation at the clinics. Mosquito-borne pathogens were typically detected just after the conclusion of the rainy season, while tick-borne pathogens were mostly detected before the rainy season. The three West Nile virus strains were phylogenetically characterized and shown to be related to both European and North American clades. Surveys such as this will increase the understanding of the potential causes of non-malarial AFI, which may help inform diagnostic and treatment options for clinicians who provide care to patients in Senegal.

Occurrence of Praxelis clematidea Witches'-Broom Disease Association with 16SrI Group 'Candidatus Phytoplasma asteris' in Hainan Island of China.

Praxelis clematidea is an invasive herbaceous plant belonging to Asteraceae family. From August to November 2020, the plants showing severe witches'-broom symptoms were found in farms and roadsides from Ding'an of Hainan Province, a tropical island of China. The disease symptoms were suggestive of phytoplasma infection. For pathogen detection, P. clematidea samples consisting of six symptomatic and three asymptomatic plants were collected from the farms and roadsites of Ding'an with 40 % incidence by conducting surveys and statistics. Total nucleic acids were extracted using 0.10 g of fresh leaf tissues of the plant through CTAB DNA extraction method. Conserved gene sequences of 16S rRNA and secA genes from phytoplasma were amplified by direct PCR using primer pairs of R16mF2/R16mR1 and secAfor1/secArev3, respectively. R16mF2/R16mR1 PCR amplicons were obtained for all symptomatic samples but not from the symptomless plants. The amplicons were purified and sequenced by Biotechnology (Shanghai) Co., Ltd. (Guangzhou, China). Sequences of 16S rRNA gene (1323 bp) and secA (732 bp) were obtained and all the gene sequences were identical, designated as PcWB (Praxelis clematidea witches'-broom)-hnda. Representative sequencs were deposited in Genbank with accession numbers of PP098736 (16S rDNA) and PP072216 (secA). Nucleotide BLAST (Basic Local Alignment Search Tool) search based on 16S rRNA gene sequences indicated that PcWB-hnda had 100% sequence identity (1323/1323) with 'Candidatus Phytoplasma asteris'-related strains belonging to 16SrI group like Waltheria indica virescence phytoplasma (MW353909) and Capsicum annuum yellow crinkle phytoplasma (MT760793); had 99.62 % sequence identity (1321/1326) with the phytoplasma strains of 16SrI group such as Oenothera phytoplasma (M30790). RFLP (Restriction Fragment Length Polymorphism) pattern derived from 16Sr RNA gene sequences by iPhyClassifier showed identical (similarity coefficient=1.00) to the reference pattern of 16SrI-B subgroup (GenBank accession number: AP006628). The results obtained demonstrate that the phytoplasma strain PcWB-hnda under study is a member of 16SrI-B subgroup. A BLAST search based on secA gene sequences indicated that PcWB-hnda shares 100% sequence identity (732/732 bp) with Pericampylus glaucus witches'-broom phytoplasma (MT875200), 99% sequence identify (728/732 bp) with onion yellows phytoplasma OY-M(AP006628), and 99% sequence identify (729/732 bp) with rapeseed phyllody phytoplasma isolate RP166 (CP055264), among other phytoplasma strains that belong to 16SrI group. Previous studies demonstrated that P. clematidea can be infected by phytoplasmas affiliate to the 16SrII group (GenBank accession number: KY568717 and EF061924) in Hainan Island of China. To our knowledge, this is the first report of a natural infection of P. clematidea by a group 16SrI phytoplasma in the Island of China. 16SrI group can infect agronomic important species such as areca palm in the island and P. clematidea can be a reservoir of 16SrI phytoplasmas. Therefore, it is necessary to search of potential vectors of the pathogens, which would contribute to epidemiological monitoring and prevention of the related diseases.

Shigella Detection and Molecular Serotyping With a Customized TaqMan Array Card in the Enterics for Global Health (EFGH): Shigella Surveillance Study.

Quantitative polymerase chain reaction (qPCR) targeting ipaH has been proven to be highly efficient in detecting Shigella in clinical samples compared to culture-based methods, which underestimate Shigella burden by 2- to 3-fold. qPCR assays have also been developed for Shigella speciation and serotyping, which is critical for both vaccine development and evaluation. The Enterics for Global Health (EFGH) Shigella surveillance study will utilize a customized real-time PCR-based TaqMan Array Card (TAC) interrogating 82 targets, for the detection and differentiation of Shigella spp, Shigella sonnei, Shigella flexneri serotypes, other diarrhea-associated enteropathogens, and antimicrobial resistance (AMR) genes. Total nucleic acid will be extracted from rectal swabs or stool samples, and assayed on TAC. Quantitative analysis will be performed to determine the likely attribution of Shigella and other particular etiologies of diarrhea using the quantification cycle cutoffs derived from previous studies. The qPCR results will be compared to conventional culture, serotyping, and phenotypic susceptibility approaches in EFGH. TAC enables simultaneous detection of diarrheal etiologies, the principal pathogen subtypes, and AMR genes. The high sensitivity of the assay enables more accurate estimation of Shigella-attributed disease burden, which is critical to informing policy and in the design of future clinical trials.

Abstract 5018: Evaluation of whole blood fractionation conditions for optimal liquid biopsy NGS applications

Abstract “For research use only. Not for diagnostic procedures.” Introduction: Liquid biopsy is a non-invasive alternative to tissue biopsies for cancer profiling using cell-free total nucleic acids (cfTNA; including cell free DNA (cfDNA) and cell free RNA (cfRNA)), the biological analytes derived from tumor cells floating in blood. However, it is crucial to optimize the whole blood fractionation workflows, since limited access to a suitable centrifuge at blood collection sites poses a challenge to this critical fractionation step, causing delays in processing the blood, leading to decreased sensitivity of variant detection and increased occurrence of false positives. Current study shows a systematic comparison of the effects of 8 different blood fractionation conditions for spin speeds, temperatures and duration on the levels of cfTNA derived from K2-EDTA-derived blood from multiple healthy and non-small cell lung cancer (NSCLC) donors, using next generation sequencing (NGS). Methods: CfTNA from K2-EDTA derived plasma was isolated using the MagMAXTM Cell-Free Total Nucleic Acid kit and the Genexus™ Cell free total nucleic acid kit, utilizing the KingFisher TM Flex and GenexusTMpurification systems. Yield and integrity of cfTNA isolated was evaluated by Qubit™, Agilent Tapestation and quantitative real time PCR (qRT-PCR). Furthermore, the impact of these test conditions on quality control specifications of DNA and RNA was assessed by performing sequencing on an Ion TorrentTM GenexusTM sequencing instrument using the AmpliSeqTM HD target amplification assay, that reports sequence variations in DNA for 42 cancer related genes and RNA in 7 cancer related genes. Results: Results showed comparable performance for all the 8 blood fractionation conditions tested, for yield and cfDNA profiles compared to control condition, indicating that there is no significant impact of these conditions on either yield or integrity of the cfDNA isolated. In addition, NGS data also showed that on target reads and median molecular coverage for DNA are comparable for all the conditions tested. However, sequencing data demonstrated that one test condition showed significantly better performance for RNA quality control specifications in terms of RNA mapped reads and molecular coverage for process controls used in the assay, compared to all other conditions tested. Conclusion: Data suggests that one of the whole blood fractionation conditions tested impact the cfRNA recovery from plasma without negatively affecting the yield, and integrity of isolated cfDNA and shows increased sensitivity for RNA process controls in NGS. Citation Format: MADHU JASTI, Sergio Hernandez, Nick Siepert, Lokesh Kodandaramaswamy, Thilanka Jayaweera, Jeoffrey Schageman, Angie Cheng, Susanne Lopez, Miao Der Chen. Evaluation of whole blood fractionation conditions for optimal liquid biopsy NGS applications [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 5018.

Natural occurrence of tomato chlorosis virus on tomatillo (Physalis philadelphica) in the United States.

Tomatillo (Physalis philadelphica L.) is an annual plant native to Mexico and Guatemala, and cultivated in other tropical and subtropical regions. In October 2023, tomatillo plants with interveinal yellowing of leaves, marginal chlorosis, leaf thickening, and leaf rolling symptoms (Figure 1) were observed at Colquitt and Tift County, Georgia, US. The disease incidence ranged from 80-100 % which reduced fruit quality and marketability. Twenty tomatillo leaves exhibiting severe symptoms were collected, and, sub-sampled of the leaves were pooled into microcentrifuge tubes. Further, MagMAX 96 viral RNA isolation kit (Thermo Fisher Scientific, US), was used for the extraction of (n=4) total nucleic acid (TNA) (Kavalappara et al. 2021). Symptomatic leaves were tested for the presence of insect-transmitted viruses such as begomovirus (tomato yellow leaf curl virus, TYLCV), potyvirus (turnip mosaic virus, TuMV), crinivirus (tomato infectious chlorosis virus, TICV; tomato chlorosis virus, ToCV), and tospovirus (orthotospovirus tomatomaculae, TSWV). Polymerase chain reaction (PCR) was performed for detecting TYLCV, using gene-specific primers (Kumar et al., 2023). However, for ToCV, TuMV and TICV detection, cDNA was prepared using 100 ng of TNA as a template, followed by the PCR ( Liu et al., 2012). Moreover, the detection of TSWV was conducted using immuno-strips (Adgia, US) following the manufacturer's instructions. ToCV was detected from all the tested samples, while TuMV, TICV, TYLCV and TSWV were not detected in any symptomatic tissues. In addition, RT-PCR was performed using gene-specific primers targeting the RNA-dependent RNA polymerase (RdRP) gene and the heat-shock protein 70 (Hsp70) gene of ToCV. The PCR amplicon of 439 bp encoding Hsp70 and 643 bp corresponding to RdRP was gel-purified and Sanger sequenced (Azenta Life Sciences, US). BLASTn analysis shows RdRP gene from ToCV-tomatillo (OR905600) has 100 % identity with ToCV of RNA1 segment (RdRP, GenBank accession no. AY903447, Florida, US), while Hsp70 gene (OR900219) has 100 % identity with ToCV of RNA2 segment (Hsp70, GenBank accession no. LC778246, Cairo, Egypt). In addition, the symptomatic tomatillo leaves were studied for transmission assay using tomato, employing non-viruliferous whiteflies (Bemisia tabaci) with 48 h of acquisition access period. Further, two weeks post-infection, the presence of ToCV was detected from the test plants while other whitefly-transmitted viruses remins undetected. In 2023, ToCV is widespread in tomato-growing counties, infecting commercially grown tomato cultivars with intermediate resistance against TYLCV-IL (Israel strain). However, tomatillo plants infected with TuMV in California (Liu et al., 2012), TSWV in Georgia, (Díaz-Pérez and Pappu 2000) and TYLCV in Mexico (Gámez-Jiménez et al. 2009) were reported. This study suggests that tomatillo could be a permissive host for ToCV while restrictive to other prevalent viruses in the region. A recent investigation speculates a potential synergistic interaction between ToCV and TYLCV-IL, exacerbating the breakdown of host resistance in tomato (Fiallo-Olivé et al. 2019, Kumar et al. 2023). To the best of our knowledge, this is the first report for the natural incidence of ToCV on tomatillo within the US. The findings will contribute to developing more effective management strategies against emerging viral threats.

Abstract 5023: Analytical validation of the Oncomine™ Dx Express Test using liquid biopsy samples as an IUO assay in a CAP/CLIA lab

Abstract Introduction: The Oncomine™ Dx Express Test (ODxET) is a next-generation sequencing (NGS) test. It can be used as an investigational use only (IUO**) test to detect somatic DNA and RNA alterations in human cell-free total nucleic acid (cfTNA) isolated from plasma samples. This study outlines the analytical validation of the ODxET for relevant single nucleotide variants (SNV) and insertion/deletions (INDEL) in liquid biopsy specimens. The Ion Torrent Genexus™ platform has a short turnaround time with minimal operator input. This study was performed in a College of American Pathologists (CAP™) accredited and Clinical Laboratory Improvement Act (CLIA) licensed laboratory. Methods: Forty-seven unique samples (39 clinical, 3 derived, 2 reference and 3 commercially available DNA controls) were analyzed. All experiments began with sample extraction, except the lower input limit of detection (LLOD). We evaluated limit of detection, variant calling accuracy, and precision. Two control NIST™ samples were sequenced with 3 technical replicates each for INDEL and SNVs analytical accuracy. The LOD was evaluated at both the target input of 2ng/µl (40ng), and the minimum input,0.33ng/µl (6.6ng) with allele frequency (AF) levels at 1%, 0.5%, and 0.25%. The 39 clinical plasma samples were used for variant calling accuracy. Inter-assay reproducibility was assessed by sequencing DNA controls across four runs by different operators over multiple days/systems. Intra-assay repeatability was assessed by sequencing 4 samples in replicate on the same run by one operator on the same day/system. Results: The analytical accuracy and NPV was 100% for SNV/INDEL variants. The 40ng input LOD sensitivity was 96% for SNVs at 0.5% AF and 100% for INDELs at 0.25% Minimum AF. The lower input LOD sensitivity was 89% for SNVs and 100% for INDEL at 1% AF. Variant calling accuracy on samples with pre-characterized data was 100% for the 13 positive samples. Specificity was &amp;gt;99% for all samples including both healthy donor and positive samples. Repeatability was 100% for INDELs and &amp;gt;96% for SNVs, reproducibility was 100% for both variant classes. Conclusion: This validation study to estimate analytical accuracy, limit of detection, variant calling accuracy and precision has demonstrated that the ODxET assay is highly robust in detecting SNV and INDEL mutations in plasma samples and is suitable for use in a CAP/CLIA laboratory as an IUO assay.**=In Vitro Diagnostic use only. Not available in all regions including North America. Citation Format: Juan C. Espinoza, Rajendra Ramsamooj, Akemi Yuki, John Williamson, Diarra Hassell, Madhu Jasti, Suzanne Salazar, David Ginzinger. Analytical validation of the Oncomine™ Dx Express Test using liquid biopsy samples as an IUO assay in a CAP/CLIA lab [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 5023.

Abstract 2943: Investigation of microsatellite instability in RNA compared to DNA, using microsatellite targeted, anchored multiplex PCR and NGS

Abstract MSI measurements are typically made on DNA, but it would be useful to make similar measurements from RNA or total nucleic acid inputs (TNA), so that MSI determinations could be made in parallel to interrogation of the RNA. Doing so requires characterizations of microsatellite lengths from those which are transcribed and untranscribed, because both will be detected following cDNA creation. Microsatellite instability (MSI) occurs when small unit repeats of DNA undergo incorrect replication resulting in the growth or contraction of the number of repeats in the newly synthesized DNA. This accumulation of errors at microsatellite DNA is usually mitigated by the mismatch repair (MMR) pathway which can correct these errors. MSI is of interest because it serves as a phenotypic readout of the status of the MMR machinery, and MSI status can correlate to tumor progression and response to agents that regulate these cellular mechanisms. We investigated the status of microsatellites using Anchored Multiplex PCR (AMP™) targeted panels (RUO) and compared results from TNA inputs using an AMP MSI module and both VARIANTPlex™ chemistry to interrogate DNA, and FUSIONPlex™ chemistry to interrogate both RNA + DNA. Our results indicate differences in the diversity of lengths of microsatellites present in RNA, and the resulting stability calls which would be made at those sites. Microsatellite length diversity was highly dependent on the genomic context of the microsatellite. Microsatellites located within transcripts showed increased length diversity when examining the RNA + DNA results compared to DNA alone. Microsatellites located in intergenic regions, which are unlikely to be transcribed, showed no increase in diversity of lengths when comparing the RNA + DNA results to DNA alone. While these results suggest that MSI determinations could be made from RNA or RNA + DNA NGS data, the unique behavior of microsatellites in RNA is likely to require additional investigations and unique data analysis techniques to correctly categorize microsatellites as stable or unstable. Confidential - Company Proprietary Citation Format: Ryan Rogge, Taylor Patterson, Alicia Navetta, Dhruti Legare. Investigation of microsatellite instability in RNA compared to DNA, using microsatellite targeted, anchored multiplex PCR and NGS [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 2943.

Abstract 5030: Evaluation of different blood collection tubes for liquid biopsy NGS applications

Abstract Intro: Liquid biopsy is fast becoming a standard practice in oncology research due to recent advancements in Next Generation Sequencing (NGS). To detect genetic variants, whole blood samples are collected in blood collection tubes (BCTs) and fractionated to obtain plasma which is then used to isolate cell free nucleic acid (cfNA) for analysis. While the limited availability of cfNA is a challenge in liquid biopsy, storage condition and duration between blood collection and fractionation have a significant impact on the quantity and quality of the cfNA due to degradation and genomic DNA and RNA contamination. There are several BCTs available on the market offering stabilization of cfNA profiles to overcome the above challenges. Methods: Here we compare the NGS performance of cfNA derived from three commonly used BCTs on the market, BD Vacutainer™ K2EDTA Tubes, Streck™ Cell-Free DNA, and PAXgene® Blood ccfDNA tubes. Matched whole blood samples collected in the above-mentioned tube types were obtained from commercial vendors which were derived from 38 healthy donors as well as 6 cancer research samples. Whole blood samples were fractionated as per manufacturer recommendations and cfNA was isolated and quantified using Genexus™ Cell-Free Total Nucleic Acid Purification Kit on Genexus™ purification system. Isolated cfNA was then sequenced using Ion Ampliseq™ HD target amplification assay on Genexus™ Integrated sequencer. Results: Data shows that K2EDTA collected samples contain slightly higher amounts of cfNA compared to PAXgene® and Streck™ samples. When the cfDNA profiles were analyzed by Agilent™ BioAnalyzer™, K2EDTA derived cfDNA showed varying degrees of genomic DNA contamination which can inflate the cfDNA concentration. However, both Streck™ and PAXgene® derived cfDNA showed little to no detectable genomic DNA contamination. As a result, a higher percentage of PAXgene® and Streck™ samples did not meet the minimum input requirement (5ng) for the sequencing assay. Sequencing data for DNA showed that K2EDTA samples had higher total reads and median molecular coverage compared to the other two BCT types. However, all samples had passing QC metrics, and mean read length, on target reads, and uniformity were comparable between the three tube types. When DNA variant detection was evaluated using cancer research samples, no significant difference was observed in performance between K2EDTA and PAXgene® samples. Interestingly, sequencing data for RNA shows that PAXgene® samples had slightly higher mapped reads on average, and significantly higher internal process control coverage compared to both K2EDTA and Streck™ samples. Whereas Streck™ samples had significantly lower internal process control coverage. Conclusion: In summary, this study demonstrates the advantages and disadvantages of using different BCTs for liquid biopsy applications, in terms of cfNA yields, isolated DNA profiles, and sequencing performance. Citation Format: Nicholas Siepert, Emilia Ostrowska, Luming Qu He, Madhuri Jasti, Kris Lea, Thilanka Jayaweera, Angie Cheng. Evaluation of different blood collection tubes for liquid biopsy NGS applications [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 5030.

Abstract 3671: Analytical performance of the Oncomine™ Dx Express Test, a CE-IVD NGS liquid biopsy assay for identification of clinically relevant variants

Abstract For in vitro Diagnostic use only. Not available in all regions including the United States. Introduction Minimally invasive blood-based liquid biopsy using cell-free total nucleic acid (cfTNA) and next generation sequencing (NGS) has substantially evolved in the field of clinical diagnostics for oncology, for detection of molecular therapeutic targets in non-small cell lung cancer (NSCLC). Here we describe analytical validation results for cfTNA using the CE-IVD Oncomine™ Dx Express Test (ODxET) assay, a qualitative in vitro diagnostic test that uses targeted NGS technology, the Ion Torrent Genexus™ Dx Integrated Sequencer to detect SNV, indel and copy number gain present in 42 genes and fusions in 7 genes from cfTNA isolated from NSCLC plasma samples. Methods Plasma samples from NSCLC patients were screened for variants of interest on several important gene loci. Screening involved isolation of cfTNA from K2-EDTA-derived plasma using the Genexus™ Cell-Free Total Nucleic Acid Purification kit on an automated purification system and sequencing was performed on the Genexus™ Dx integrated sequencer using the ODxET assay. Plasma samples having clinically relevant variants from the set of screened plasma samples were used to perform the validation studies. Results Analytical sensitivity and specificity of NSCLC plasma samples were characterized through determination of limit of detection (LOD) and limit of blank (LOB), respectively. The LOD level tested for DNA SNVs, and indels at 5 ng (minimum) input level ranged from 0.62% to 1.82% allelic frequency (AF), while 30 ng (maximum) input shows the LOD level ranging from 0.23% to 0.42% AF. The LOD for the tested RNA fusions ranged from 4.2 to 19.6 molecular counts for 5ng input and 4.9 to 8.0 molecular counts for 30ng input. Furthermore, LOB for the ODxET assay was determined by testing wild type (WT) cfTNA extracted from 30 healthy donor blood plasma samples, which were confirmed to be negative at all variant locations. The analytical accuracy study shows the false positive rate at 0.20% for SNVs, 0% for indel locations and fusion targets. In addition, the analytical reproducibility study shows 99.64% intra-run repeatability call rate for DNA variants and 98.75% for RNA variants. Conclusion In conclusion, we establish that the ODxET assay on Ion Torrent Genexus Dx Integrated Sequencer, is fast and efficient while demonstrating high sensitivity, specificity, and reproducibility for testing NSCLC liquid biopsy samples. Citation Format: Madhuri Jasti, Swasti Raut, Diarra Hassell, Nader Ezzedine, Daniela Garcia, Stephen Wunsch, Nick Siepert, Elliott Martinez, Emilia Ostrowska, Jeff Schagemann, Jian Gu, Thilanka Jayaweera, Luming He, Angie Cheng. Analytical performance of the Oncomine™ Dx Express Test, a CE-IVD NGS liquid biopsy assay for identification of clinically relevant variants [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 3671.

A semi-automated and high-throughput approach for the detection of honey bee viruses in bee samples.

Deformed wing virus (DWV) was first detected in dead honey bees in 1982 but has been in honey bees for at least 300 years. Due to its high prevalence and virulence, they have been linked with the ongoing decline in honey bee populations worldwide. A rapid, simple, semi-automated, high-throughput, and cost-effective method of screening colonies for viruses would benefit bee research and the beekeeping industry. Here we describe a semi-automated approach that combines an RNA-grade liquid homogenizer followed by magnetic bead capture for total virus nucleic acid extraction. We compare it to the more commonly applied nucleic acid column-based purification method and use qPCR plus Oxford Nanopore Technologies sequencing to evaluate the accuracy of analytical results for both methods. Our results showed high reproducibility and accuracy for both approaches. The semi-automated method described here allows for faster screening of viral loads in units of 96 samples at a time. We developed this method to monitor viral loads in honey bee colonies, but it could be easily applied for any PCR or genomic-based screening assays.

Occurrence of 'Candidatus Phytoplasma asteris'-Related Strains infecting Chinaberry (Melia azedarach) Showing Chlorotic Leaf Symptoms on Hainan Island of China.

Chinaberry (Melia azedarach), belonging to the family of Meliaceae, is an ornamental tree distributes across southern of China. In the autumn of 2021, In an area of 400 acres located in Wanning city of Hainan Province, a tropical island in China, with coordinates of 110°28'42.72″E, 19°2'9.96″N, about 20 % (100) of the chinaberry trees showed disease symptoms included chlorotic leaves. The disease symptoms were consistent with infections by a phloem-limited prokaryotic pathogen phytoplasma. The samples of six symptomatic and three asymptomatic were collected for pathogen detection. To identify the pathogen, total nucleic acids were extracted from 0.10 g fresh leaf tissues from the diseased and healthy plant using CTAB DNA extraction method based on Doyle and Doyle. Three primer pairs of R16mF2/R16mR1, secAfor1/secArev3 and fTuf1/rTuf1 were used for specific identification of phytoplasma conserved gene fragments of 16S rDNA, secA and tuf, PCR amplification. Target PCR bands were amplified from the DNA of six diseased chinaberry samples, but not from the DNA of the healthy samples. The products of amplified were cloned and sequenced by Biotechnology (Shanghai) Co., Ltd. (Guangzhou, China). The phytoplasma gene sequences of 16S rRNA, secA and tuf were obtained and all the sequences were identical with the length of 1336 bp, 710 bp and 955 bp, respectively. Representative sequence data for strain MaCL-hn were deposited in Genbank under accession Nos. OR438638 (16S rDNA), OR513089 (secA) and OR860415 (tuf). The phytoplasma strain identified in the study was described as chinaberry chlorotic leaf (MaCL) phytoplasma, MaCL-hn strain. BLAST search based on 16S rRNA genes showed that 43 strains in 16SrI group 'Candidatus Phytoplasma asteris' showed 100% similarity with the 16SRNA sequence of MaCL-hn. BLAST search based on secA genes showed that 9 strains in the phytoplasma group showed 100% similarity with the 16SRNA sequence of MaCL-hn. BLAST search based on tuf genes showed that 21 strains in the phytoplasma group showed 100% similarity with the 16SRNA sequence of MaCL-hn. RFLP analysis based on iPhyClassifier indicated that the MaCL-hn strain was a member of 16SrI-B subgroup with a similarity coefficient 1.00 to the reference phytoplasma strain (AP006628). Phylogenetic tree was constructed based on 16S rRNA by MEGA 11.0 using neighbor-joining (NJ) method with 1000 bootstrap value. The results showed that the MaCL-hn strains were clustered into one clade with 16SrI group 'Ca. Phytoplasma asteris' related strains with 99 % bootstrap value. Multilocus sequence analysis (MLSA) based on the concatenated sequences with the length of 3001 bp including the sequences of 16S rRNA, secA and tuf showed that the MaCL-hn strains were clustered into one clade with the phytoplasma strains in the group with 100 % bootstrap value. To our knowledge, this is the first report that chinaberry can be infected by 'Ca. Phytoplasma asteris'-related strains belonging to 16SrI-B subgroup on Hainan Island of China. This finding in the study will contribute to the epidemic monitoring and the preventive management of the phytoplasmas and their related diseases.