Prep Methods Research Articles

B-229 Extraction-free Nucleic Acid Sample Preparation from Inhibitor Rich Samples via Hyperbaric Heating

Abstract Background Extraction-based nucleic acid sample preparation for PCR is limited by long processing times, liquid transfers, cost, and storage requirements. Extraction-free approaches enable streamlined sample prep but are severely impacted by inhibitors present in biological sample matrices. We previously reported the use of Hyperbaric Heating (HBH) for effective organism lysis and demonstrate here its effectiveness to prepare nucleic acids for PCR from various inhibitor-rich sample matrix types. Methods HBH exposes the sample to high temperature/high pressure environment for a short duration. This process effectively lyses organisms and denatures inhibitors within seconds. Samples are sealed within a pressure-tight metallic vessel, then rapidly heated using magnetic induction. Within the sealed metallic vessel, vaporization of the aqueous sample raises the internal pressure, enabling an internal temperature higher than 100°C. Proprietary sample processing beads containing chelating agents and other proteins protect nucleic acids from fragmentation during heating. Results The efficacy of HBH for PCR amplification was tested against commercial kits and extraction-free methods. HBH treatment of nasal swab samples spiked with inactivated SARS-CoV-2 elicited detection down to 125 viral copies/mL. Viruses such as RSV and Influenza spiked in nasal swab samples demonstrated comparable PCR results as extraction prepped samples. HBH treatment of microbe samples, including Candida albicans spiked in urine and Bacillus subtilis spiked in serum, had comparable PCR threshold cycles as microbe specific DNA extraction methods (Table 1). Conclusions HBH is a generalizable extraction-free nucleic acid sample prep technology affording detection capabilities comparable to commercial sample prep methods. We demonstrated the use of HBH sample prep for detection of several infectious microorganisms in common biological matrices (nasal swab, serum, urine) without additional sample purification. With its ease of use, speed, and lower cost, HBH will be a key tool in applications where nucleic acid amplification is pivotal for diagnostic and research use.

Comparison of characteristics of VZHL718 alloy metal powder compositions produced by prep and viga methods after selective laser melting

Comparison of characteristics of VZHL718 alloy metal powder compositions produced by prep and viga methods after selective laser melting

Factors associated with HIV pre-exposure prophylaxis use among Asian men who have sex with men in Sydney and Melbourne, Australia: a cross-sectional study

Asian-born MSM are a priority population as Australia aims to end HIV transmission, but they reported additional barriers to access PrEP and other HIV prevention methods. This study investigates factors associated with PrEP use among Asian MSM in Sydney and Melbourne, Australia, to inform strategies to improve PrEP uptake in this population. This was a sub-analysis of a community-based cross-sectional survey conducted from March to June 2021. We recruited participants online in Sydney and Melbourne, Australia. Univariable and multivariable logistic regression analyses were performed to identify the factors associated with PrEP use in the last six months and lifetime. Latent class analyses were used to identify subgroups of Asian MSM sharing similar characteristics related to their risk practices for HIV. Overall, 870 Asian MSM were included: 288 Oceanian-born Asian MSM and 582 Asian-born MSM. Three latent classes were identified: 1) Asian-born MSM who recently arrived in Australia with limited English, were less likely to use PrEP and at higher risk of HIV infection (e.g., had condomless anal sex with a casual sex partner in the last six months) (4.6%); 2) Asian MSM who were at lower risk of HIV infection and less likely to use PrEP (69.3%) and; 3) Asian MSM who were at substantial risk of HIV infection and more likely to use PrEP (26.1%). Compared to Oceanian-born Asian MSM, those who were born in Southeast Asia (adjusted odds ratio (aOR)=0.5, 95% confidence interval (CI) 0.3-0.7) and South Asia (aOR=0.4, 95% CI 0.2-0.8) were less likely to ever use PrEP. Compared to Oceanian-born Asian MSM, those who were born in Southeast Asia (aOR=0.4, 95% CI 0.3-0.7), Northeast Asia (aOR=0.5, 95% CI 0.3-0.8) and South Asia (aOR=0.4, 95% CI 0.2-0.7) were less likely to use PrEP in the last six months. To end HIV transmission in Australia, it will be necessary to develop strategies to improve PrEP access for the significant minority of Asian-born MSM who are at substantial risk of HIV infection. EPFC and JJO are supported by an Australian National Health and Medical Research Council (NHMRC) Emerging Leadership Investigator Grant (EPFC: GNT1172873 and JJO: GNT1193955). CKF is supported by an Australian National Health and Medical Research Council (NHMRC) Leadership Investigator Grant (GNT1172900).

Reconciling advantages and difficulties: knowledge and perceptions of event-driven PrEP among young people.

This study aims to analyze the knowledge about the HIV event-driven pre-exposure prophylaxis (event-driven PrEP) scheme and the perception of its potential use among young men who have sex with men (MSM), travestis, and transgender women (TrTW) who were followed up in the cohort. This qualitative study included 50 interviews with participants from the municipalities of Salvador and São Paulo, aged 15 to 19 years, who made daily use of PrEP or other preventive methods. They were addressed by different demand creation strategies. The in-depth interviews covered topics such as sexual practices, event-driven PrEP knowledge, acceptability, and motivations for its use. A two-stage thematic analysis was carried out on Nvivo, version 12. Most participants were unaware of event-driven PrEP, and many questioned its effectiveness and safety when receiving information about it. However, on learning about the program, many young people saw advantages, such as not having to take daily medication and the possibility of using it only at times of greater risk. Participants also found barriers to using event-driven PrEP, such as the unpredictability of sexual relations and the difficulty in administering dosages in this modality. Limited knowledge and experiences with daily oral PrEP influenced interest in event-driven PrEP, which highlights the need for information strategies that enable young MSM and TrTW to read about event-driven PrEP. Young people valued the autonomy and management of preventive methods provides by this new modality, which is more in line with the dynamics of their sexual lives, but they face challenges in managing event-driven PrEP.

PrEP use and stigma among a sample of older adults in Columbus, Ohio.

Despite older adults (age ≥50years) continuing to account for 1 in 6 new HIV diagnoses, the majority of research has focused on young adults. Assessing PrEP use and stigma among this understudied population is key to achieving the U.S.'s goals of Ending the HIV Epidemic, a federal initiative focusing on reducing new HIV infections by at least 90% by 2030. Data for this analysis came from the Columbus Health Aging Project (N = 794). This study was designed to assess several domains of health among adults aged 50years and older in Columbus, Ohio. Multiple logistic and linear regression models were used to examine the associations between sociodemographic factors and past 6-month PrEP use, PrEP stigma, and concurrent use of PrEP and other prevention methods, adjusting for known confounders. Overall, 93 (11.7%) participants reported past 6-month PrEP use. Transgender women (aOR = 6.90; 95% CI: 2.19, 21.72), cisgender gay men (aOR = 5.58; 95% CI: 2.49, 12.50), cisgender lesbians (aOR = 2.24; 95% CI: 1.05, 4.80), and those living with family members or roommates (aOR = 6.59; 95% CI: 3.49, 12.45) were each more likely to report past 6-month PrEP use relative to cisgender women, heterosexuals, and those living with a spouse/partner, respectively. Relative to cisgender women, PrEP-related stigma was lower among transgender women (β = -5.05; 95% CI: -8.44, -1.66) and higher among cisgender men (β = 1.96; 95% CI: 0.46, 3.46). Future research should aim to continue developing a firm understanding of PrEP use and stigma among older adults to reduce HIV risk among this population and to understand unique needs of sub-populations of older adults.

S954 Experiences With Bowel Preparation for Colonoscopy in Crohn’s Disease

Introduction: Patients with inflammatory bowel disease (IBD) require frequent colonoscopies. Crohn’s disease (CD) may present unique challenges to bowel prep for colonoscopy due to the presence of strictures, fistulas, or prior bowel resections. Little is known about CD patients’ experiences with bowel preps. The purpose of this study was to develop an in-depth understanding of experiences with bowel prep for colonoscopy in CD, from the perspectives of patients and providers. Methods: We used a mixed methods approach, inviting CD patients ≥ 18 years of age scheduled for outpatient colonoscopy at two medical centers between July 2021-January 2022, and gastroenterologists, to participate in a survey and focus groups. Domains that were covered included experiences with existing bowel preps, including barriers to tolerance, and perceptions about ideal preps. Results: A total of 144 patients completed the survey and 7 participated in focus groups, with 50% between 18-39 years of age, 56% female, 80% Caucasian, and 65% reporting good/excellent health status. CD phenotypes included 32% with strictures, 20% with fistulas, 35% with prior bowel surgeries, and 34% with active disease. Reported challenges to bowel preps included the inability to eat, volume of liquid required, and disruptions to work and sleep. Ideal preps were described as lower volume, more palaTable, and without the need to fast. Worry about the prep was one of the leading reasons that patients delayed their colonoscopies. Despite these challenges, 91% of patients would undergo colonoscopy again if recommended by their physician. Six gastroenterologists participated in the study. Volume, taste, and safety concerns were reported as limitations to currently-available preps. All agreed that, if available, they would favor a prep that was tested in CD patients. Barriers to optimizing colonoscopy bowel prep for CD patients included limited knowledge about ideal volume required, lack of standardization of dietary modifications, and lack of bowel prep quality scoring methods. Conclusion: Bowel preparation is perceived as the most difficult part of undergoing colonoscopy among CD patients with worry about the prep as one of the leading causes of delaying colonoscopies. Given that CD patients may have altered anatomy due to their disease phenotypes and require frequent colonoscopies, future studies are needed to determine the optimal prep formulation, volume, and prep quality scoring methods for patients with CD.

Rapid Denaturing Organic Digestion Method for Targeted Protein Identification and Characterization.

Bottom-up mass spectrometry-based protein analysis methods employing protease digestion are routinely used to identify and characterize proteins with high specificity and sensitivity. Method performance is generally measured by sequence coverage capability and the total number of characteristic peptides identified, when compared to predicted databases. Limitations to commonly used solvent-based digestion methods currently employed include long digestion times (18-24 h or more), leading to protease autolysis, which also precludes automation, decreases sensitivity, and increases both intra- and inter-day performance variability. This report describes the development and validation of a simple, 5 min tryptic denaturing organic digestion (DOD) method for use with tandem mass spectrometry in bottom-up protein identification and characterization. It has been evaluated across select protein toxins and diagnostic clinical protein targets, substantially improving digestion performance when compared to other solution-based and enzyme-immobilized methods. The method was compared to two currently used bottom-up methods, the 24 h filter-aided sample prep (FASP) and Flash Digest (1 and 4 h) methods. Single proteins used to compare the methods included the ricin light chain, ricin heavy chain, ricin holotoxin, serotype A Clostridium botulinum toxin, Staphylococcus enterotoxin B, ribonuclease A, and thyroglobulin. In tests, across the proteins investigated, the 5 min DOD digestion method resulted in sequence coverages ranging from 55 to 100%, with relatively high reproducibility and precision; results were better than or equal to FASP method results and were greatly enhanced when compared to Flash method results. Importantly, DOD method intra- and inter-day precision was much improved as compared to results for both FASP and Flash digestions. These data indicated that the DOD method, when compared to the FASP and Flash Digest methods, dramatically reduced digestion time, while maintaining or improving the ability to detect and characterize targeted proteins, and reduced analytical variability for tryptic digestion, resulting in markedly faster and more precise analyses.

MICROFLUIDIC DEVICE-BASED SEMEN PREPARATION INFLUENCES EUPLOIDY RATES OF HUMAN BLASTOCYSTS

The study has been conducted to assess preimplantation development of human embryos following ICSI with two different sperm separation techniques. Zymot, a microfluidic sperm separation device (MFSS), which was based on motility within a micro-environment, and density gradient centrifugation (DGS) which was based on centrifugal force. The study was carried out between 2018 and 2019. Retrospective A total of 371 ART-cycles were included in the study. Semen samples from patients with severe male pathology, TESE, MESA, and donor semen were excluded. Fresh ejaculated specimens from consenting men were collected for standard semen analysis in accordance with WHO 2015 guidelines. DGC (Control Group, n=220) and MFSS (Treatment Group, n=151) were used to isolate motile spermatozoa based on cell motility and fluid dynamics. 1802 blastocysts were biopsied and analyzed to determine ploidy status . Patient characteristics such as male and female age, number of retrieved oocytes, number of embryos were comparable between control and treatment groups. All oocytes were inseminated by ICSI and cultured following standard embryo culture protocols. Trophectoderm biopsies were performed on Day 5 or 6 of development and screened for euploidy using NGS. Experimental end points of the study were fertilization, blastocyst conversion and euploidy rates of preimplantation embryos. Ch-square and regression analyses were used to compare treatments and to identify factors affecting ploidy status of blastocysts. Fertilization and blastocyst conversion rates were comparable between the sperm prep methods tested. Regression analysis of the factors influencing the ploidy status of the blastocysts indicated that use of MFSS method, ZyMot increases the number of euploid embryos by 0.44/cycle, controlling for other known characteristics of patient sets (p: 0.05 *).Tabled 1Semen Prep MethodICSI Cycles (N)ICSI Oocyte (n)Fertilization %Blastocysts Biopsied (%)Euploid Blastocyst (%)DGS22020581667 (81.0)1050 (51.0)620 (59.0)MFSS15114191078 (76.0)752 (53.0)474 (63.0) * Open table in a new tab This study suggests that the use MFSS for sperm preparation for ICSI ccyles could improve the number of euploid embryos, modestly. Although the morphology and DNA integrity were not assessed in this study directly, widely reported improvements in these aspects by the MFSS may explain the improvements in PGT-A outcomes. The findings of our study should to be considered in the light of some limitations, such as a modest sample size and absence of morphology and DNA integrity data. These need to be addressed in future studies to provide a more complete assessment of the treatments.

Abstract 1993: A comprehensive benchmarking of paired whole exome and transcriptome biomarker analysis of response to cancer immunotherapy

Abstract Recent advances in cancer immunotherapy have revolutionized cancer treatment. Notably, pembrolizumab - an anti-PD1 checkpoint inhibitor - has been approved as first-line treatment for metastatic non-small cell lung cancer. Clinical response to checkpoint inhibitors has been demonstrated in other cancer types; however, durable response is not observed in all patients. As a result, development of new biomarkers for response to checkpoint inhibitors has become an active area of research. Due to the complexity of the anti-tumor immune response, a single biomarker may not accurately predict patient response to immunotherapy. In parallel, dramatic decreases in the costs of next generation sequencing (NGS) have enabled broad whole exome sequencing (WES) and whole transcriptome sequencing (WTS)-based studies, enabling a new phase in both academic and clinical cancer research. Despite the proven utility of NGS-based biomarker analysis, the accuracy of combined WES/WTS results compared to other assays, specifically in clinical tumor tissues, has not been demonstrated. In this study, we perform comprehensive benchmarking of several immuno-oncology (IO) biomarkers on cell lines, fresh frozen tumor tissues, and formalin-fixed paraffin-embedded (FFPE) tumor tissues. WES and WTS libraries were generated using Illumina Nextera Flex for Enrichment, TruSight Oncology, and TruSeq Stranded Total RNA library prep methods, and sequenced on a NovaSeq 6000. Using an internally developed bioinformatics pipeline, the accuracy of several IO biomarker measurements was evaluated. Specifically, we investigated the performance of tumor-only (T) and/or paired tumor-normal (TN) WES/WTS in assessing tumor mutational burden (TMB), microsatellite instability (MSI), tumor purity, copy number variants (CNV), human leukocyte antigen (HLA) type, fusions, and tumor infiltrating lymphocytes (TILs) levels, among other features. Our benchmarking results demonstrated correlations of 0.99 and 0.98 for TMB as measured by TN and T, respectively (truth: FOCR reported TMB); 100.0% PPV and NPV for MSI status as measured by both TN and T (truth: MSI-PCR); 0.86 correlation for tumor purity estimates based on TN (0.64 based on T; truth: cell-line titration levels); 100% PPV for both TN and T CNV calling (truth: ddPCR); 92% accuracy for both TN and T HLA typing (truth: IHW HLA types); 92.9% sensitivity for fusion calling (truth: ddPCR); and R2 values of 0.87, 0.70, and 0.48 for CD19+, CD4+, and CD8+ TIL levels, respectively (truth: FACS). Taken together, this study demonstrates WES/WTS can not only be utilized for broad exome- and transcriptome-wide biomarker discovery, but can also be an accurate, comprehensive alternative to iteratively testing IO biomarkers. Future studies should further standardize assay and analysis approaches for NGS-based biomarker measurements to ensure consistent and accurate reporting. Citation Format: Mahdi Golkaram, Michael Salmans, Raakhee Vijayaraghavan, Shannon Kaplan, Robert Haigis, Joyee Yao, Kristina Kruglyak, Li Liu, Traci Pawlowski, Sven Bilke, Shile Zhang. A comprehensive benchmarking of paired whole exome and transcriptome biomarker analysis of response to cancer immunotherapy [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 1993.

MP84-02 BETADINE VERSUS CHLORHEXIDINE – A NEXT-GENERATION SEQUENCING ANALYSIS OF THE MICROBIOME PRESENT ON THE POST-PREP OPERATIVE FIELD DURING PENILE PROSTHESIS IMPLANTATION

You have accessJournal of UrologySexual Function/Dysfunction: Basic Research & Pathophysiology (MP84)1 Apr 2020MP84-02 BETADINE VERSUS CHLORHEXIDINE – A NEXT-GENERATION SEQUENCING ANALYSIS OF THE MICROBIOME PRESENT ON THE POST-PREP OPERATIVE FIELD DURING PENILE PROSTHESIS IMPLANTATION Kirtishri Mishra*, Laura Bukavina, Amr Mahran, Austin Fernstrum, Al Ray, Mauricio Retuerto, Dane Winner, Shubham Gupta, Nannan Thirumavalavan, Mahmoud Ghannoum, and Aram Loeb Kirtishri Mishra*Kirtishri Mishra* More articles by this author , Laura BukavinaLaura Bukavina More articles by this author , Amr MahranAmr Mahran More articles by this author , Austin FernstrumAustin Fernstrum More articles by this author , Al RayAl Ray More articles by this author , Mauricio RetuertoMauricio Retuerto More articles by this author , Dane WinnerDane Winner More articles by this author , Shubham GuptaShubham Gupta More articles by this author , Nannan ThirumavalavanNannan Thirumavalavan More articles by this author , Mahmoud GhannoumMahmoud Ghannoum More articles by this author , and Aram LoebAram Loeb More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000000976.02AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: The risk of infection after penile prosthesis implantation is one of the main concerns for urologic surgeons. Various pre-operative prep methods have been investigated, with a plethora of clinical literature in support or against each of the techniques. In this study, we aimed to take a more objective look at he microbiome present on the operative field before and after prepping the skin with betadine (BET) versus chlorhexidine (CXD). METHODS: Males between the ages of 18-99, who were undergoing an elective penile prosthesis placement were eligible for the study. A swab was performed in a standardized way at the penoscrotal junction before the prep. Following DNA extraction form the swabs, amplification of the 16S and 5.8S rRNA genes were performed. Bioinformatics was performed for taxonomic identification. A total of 11 patients were included in the study. RESULTS: Figure 1 shows the bacterial amplicons identified before the prep (left) and after (right). Similarly, the fungal data is shown in Figure 2. There was no significant difference between the DNA amplicons identified in the bacterial of fungal studies, before or after the prep. The most predominant bacterial and fungal organisms are identified on the figures. CONCLUSIONS: Increased understanding of the microbiome present on the skin before and after the prep provides practitioners an insight into the possible infectious pathogens and tailor prep and antibiotics to optimize post-operative outcomes. In this review, we describe the flora present in these patients, and demonstrate no significant difference in the flora after the two preps. Source of Funding: None © 2020 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 203Issue Supplement 4April 2020Page: e1264-e1265 Advertisement Copyright & Permissions© 2020 by American Urological Association Education and Research, Inc.MetricsAuthor Information Kirtishri Mishra* More articles by this author Laura Bukavina More articles by this author Amr Mahran More articles by this author Austin Fernstrum More articles by this author Al Ray More articles by this author Mauricio Retuerto More articles by this author Dane Winner More articles by this author Shubham Gupta More articles by this author Nannan Thirumavalavan More articles by this author Mahmoud Ghannoum More articles by this author Aram Loeb More articles by this author Expand All Advertisement PDF downloadLoading ...

Acceptability of Long-Acting Injectable Cabotegravir (CAB LA) in HIV-Uninfected Individuals: HPTN 077

Long-acting injectable PrEP could offer an alternative to daily oral PrEP, improve adherence and protection, if found acceptable, safe and effective. HPTN 077 evaluated injectable cabotegravir safety, tolerability and pharmacokinetics among HIV-uninfected males and females in sequentially-enrolled cohorts of two dosing strategies. We compared acceptability of product attributes, prevention preferences and future interest in injectable PrEP (FIIP) by region, sex-at-birth, arm and cohort and used multivariable analysis to identify FIIP determinants. Baseline injectable PrEP preferences were higher in non-U.S. sites and increased in both regions over time. In multivariable models, FIIP was most strongly associated with acceptability of product attributes, was higher in non-U.S. sites and more altruistic participants. Treatment arm and report of pain were not associated with FIIP. Injectable acceptability was highest in non-U.S. sites. Preferences for injectable versus other PrEP methods were higher among U.S. males than females, but higher among males and females in non-U.S. settings.

Epidemiological impact and cost-effectiveness of providing long-acting pre-exposure prophylaxis to injectable contraceptive users for HIV prevention in South Africa: a modelling study.

IntroductionAlthough pre‐exposure prophylaxis (PrEP.) is an efficacious HIV prevention strategy, its preventive benefit has not been shown among young women in sub‐Saharan Africa, likely due to non‐adherence. Adherence may be improved with the use of injectable long‐acting PrEP methods currently being developed. We hypothesize that providing long‐acting PrEP to women using injectable contraceptives, the most frequently used contraceptive method in South Africa, could improve adherence to PrEP, result in a reduction of new HIV infections, and be a relatively easy‐to‐reach target population. In this modelling study, we assessed the epidemiological impact and cost‐effectiveness of providing long‐acting PrEP to injectable contraceptive users in Limpopo, South Africa.MethodsWe developed a deterministic mathematical model calibrated to the HIV epidemic in Limpopo. Long‐acting PrEP was provided to 50% of HIV negative injectable contraceptive users in 2018 and scaled‐up over two years. We estimated the number of HIV infections that could be averted by 2030 and the drug price of long‐acting PrEP for which this intervention would be cost‐effective over a time horizon of 40 years, from a healthcare payer perspective. In the base‐case scenario we assumed long‐acting PrEP is 75% effective in preventing HIV infections and 85% of infected individuals are on antiretroviral drug therapy (ART) by 2030. In sensitivity analyses we adjusted PrEP effectiveness and ART coverage. Costs between $519 and $1119 per disability‐adjusted life‐year (DALY) averted were considered potentially cost‐effective, and <$519 as cost‐effective.ResultsWithout long‐acting injectable PrEP, 224,000 (interquartile range 176,000 to 271,000) new infections will occur by 2030; use of long‐acting injectable PrEP could prevent 21,000 (17,000 to 26,000) or 9.8% (8.9% to 10.6%) new HIV infections by 2030 (including 6000 (4000 to 7000) in men). Long‐acting PrEP would prevent 34,000 (29,000 to 39,000) or 12,000 (8000 to 15,000) at 75% and 95% ART coverage by 2030 respectively. To be considered potentially cost‐effective the annual long‐acting PrEP drug price should be <$16, and/or ART coverage remains at <85% in 2030.ConclusionsProviding long‐acting PrEP to injectable contraceptive users in Limpopo is only potentially cost‐effective when long‐acting PrEP drug prices are low. If low prices are not feasible, providing long‐acting PrEP only to women at high risk of HIV infection will become important.

Abstract 5093: Clinical-grade NGS analytic platform with explicit negative calling to improve variant reporting across different laboratory protocols: Accuracy study

Abstract Introduction: Research-grade bioinformatic pipelines share a common workflow with a logical shortcut that infers where no variant was called, no variant exists. The complexity of NGS protocols makes this inference clinically unsound. Incomplete enrichment, or a sample with deletions in the region of interest can result in false negative errors. Clinical-grade NGS analytics should explicitly evaluate each region of clinical interest to distinguish regions with no informative variants from those with too little evidence to make a call. An ideal platform would produce reliable results from sequencers and library prep in clinical use without protocol-specific tuning. This study evaluates the performance of a novel Clinical-grade Analytic Platform designed to meet these challenges. Study Design: 189 samples from the NA12878 cell line, processed by 13 different laboratories, using 18 different protocols and 11 different instruments were sourced as raw sequencing data from public repositories. Each sample file was run through the Clinical-grade Analytic Platform to produce a cVCF result, a detailed accounting of variants present, regions with no variant present and regions with insufficient coverage across 14,626 exons in the 1043-gene PanCancer TestPanel. High Confidence TRUTH, obtained from the GIAB consortium, established that NA12878 DNA contains 598 variants and 14,230 exons with no variant across the 1043 genes. The cVCFs were compared to TRUTH to quantify performance. Results: The Clinical-Grade NGS Analytic Platform ran all 189 samples without protocol or laboratory-specific tuning, with an overall sensitivity of 95% and specificity of 99%. This performance includes samples with average read depth ranging from 18x to 10,000x, although regions with local read depths above 30x performed better. Hybridization capture-based methods generally outperformed amplicon-based methods at all read depths. Full presentation will discuss performance stratified by instrument, library prep method, local read depth, and variant type. Conclusion: The Clinical-grade Analytic Platform, Farsight Correo, showed strong performance across a broad range of read depths, instruments and library prep methods. The platform’s unique ability to distinguish between “No Variant Present” and “Insufficient Coverage” contributed significantly to this performance. Robust, cross-protocol performance is clinically valuable. It can enable clinical laboratories to expand or change NGS protocols without replacing analytics. It can enable companion diagnostic programs to include multiple sequencing protocols and partners. And finally, explicit negative variant calling can help AI and machine learning mirror the underlying biology; integrating variant presence and absence into new pathway-based models to significantly advance genomics-guided medicine. Citation Format: Edward P. Tang, Charlie C. Kim, Kristine C. Mechem, Glenda G. Anderson. Clinical-grade NGS analytic platform with explicit negative calling to improve variant reporting across different laboratory protocols: Accuracy study [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 5093.

Individual Variability of Protein Expression in Human Tissues.

Human tissues are known to exhibit interindividual variability, but a deeper understanding of the different factors affecting protein expression is necessary to further apply this knowledge. Our goal was to explore the proteomic variability between individuals as well as between healthy and diseased samples, and to test the efficacy of machine learning classifiers. In order to investigate whether disparate proteomics data sets may be combined, we performed a retrospective analysis of proteomics data from 9 different human tissues. These data sets represent several different sample prep methods, mass spectrometry instruments, and tissue health. Using these data, we examined interindividual and intertissue variability in peptide expression, and analyzed the methods required to build accurate tissue classifiers. We also evaluated the limits of tissue classification by downsampling the peptide data to simulate situations where less data is available, such as clinical biopsies, laser capture microdissection or potentially single-cell proteomics. Our findings reveal the strong potential for utilizing proteomics data to build robust tissue classifiers, which has many prospective clinical applications for evaluating the applicability of model clinical systems.

Characterisation of a landslide by its fracture system using Electric Resistivity Tomography and Pressure Probe methods

We have studied a slowly moving loess landslide along the River Danube in South Hungary. In contrast with other efforts, we aimed to determine its fracture system. Due to the homogeneous composition of the loess, it seems to be the only possibility to get information about the landslide and its further evolution. Beside of the well-known Electrical Resistivity Tomography (ERT) the so-called Pressure Probe (PreP) method was applied to characterise the supposedly dense fracture system. This method was developed to detect and characterise mechanically weak zones, which may not visible from the surface, and may occur e.g. due to landslides. Fracture zones had been especially well localised by the ERT, enabling the prediction of the positions of future rupture surfaces and thus also the delineation of the endangered zones. PreP was able to give a very detailed image about the surface projection of the fractures. Both methods proved to be good to characterise the fracture system of such a landslide area. Geophysical predictions have been verified also in reality: the mass movements occurred about 1½ years after the measurements. Therefore, to provide early risk warnings and to avoid damage to constructions or endangering human life, the application of the ERT and PreP methods is highly recommended.

Webinar | Combating the "garbage-in-garbage-out" paradigm:The importance of library prep in next-gen sequencing workflows

In the context of next-generation sequencing, the old saying "garbage in, garbage out" translates to "poor sample preparation results in poor data"—sometimes even unusable data. As those poor results often become apparent only after expensive, time-consuming sequencing, it behooves researchers to pay extra attention to the preparation of their DNA samples at every step, from isolation through library preparation to loading the samples onto their sequencer of choice. This webinar will focus particularly on best practices for library preparation, including whole-genome library prep and shearing methods for PCR-free vs. PCR-plus prep, which can substantially impact bias and coverage, among other variables. Attendees will also hear about best practices for quality control of the library preparation process, as well as the impact of library quality on downstream data quality. View the Webinar

Abstract 3623: Comparison of dPCR versus hybridization capture methods for next generation sequencing of ctDNA for a SNV, copy number amplification, and fusion liquid biopsy assay

Abstract Circulating tumor DNA can be found in the plasma of cancer patients, and can be utilized for non-invasive cancer detection and genotyping through Next Generation Sequencing. However, unique characteristics of ctDNA pose technical challenges that limit technical sensitivities and robustness. ctDNA are fragmented, short, rare in abundance, and found in high background of normal DNA from leukocytes. Cancer variants range as low as 0.1-1% minor allele fractions, within the noise of NGS methods. Furthermore, clinical interest includes large number of genes and mutation types including single nucleotide variants, copy number amplification, and fusions. We compared two popular target enrichment categories, digital PCR and a hybridization-based capture panel for target enrichment. We assessed the assay performance and detection limitations, using a diverse sample set to test the range of input material that reflects ctDNA found in plasma. We titrated important cancer mutations (BRAF, EGFR, KRAS, PI3KCA, etc.) testing minor allele fractions ranging from 33% down to 0.1%, MET amplification ranges of 14x to 5x, and CCDC6-RET fusions of 50% to 2%. We also assessed input DNA ranges of 1ng and 10ng, well below manufacturer recommend input of library prep methods. Testing the performance of a 29kb amplicon panel and a 114kb hybridization-probes panel, we found superior on-target rates with digital PCR compared to the hybridization capture method across all samples. However, despite higher depths of coverage with droplet PCR, hybridization based methods had superior read mapping, uniformity (1.4x better), error rate (0.16% vs 0.02%), and greater linearity of SNV variants. Hybridization methods had greater accuracy of variant detection and were able to accurately identify the RET fusion. We demonstrated the feasibility of utilizing target enrichment methods for next generation sequencing for ctDNA variant detection. Citation Format: Jonathan Choi. Comparison of dPCR versus hybridization capture methods for next generation sequencing of ctDNA for a SNV, copy number amplification, and fusion liquid biopsy assay. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3623.

'PrEP is not ready for our community, and our community is not ready for PrEP': pre-exposure prophylaxis for HIV for people who inject drugs and limits to the HIV prevention response.

Pre-exposure prophylaxis for HIV, or 'PrEP', is the use of antiretroviral medicines by people who are HIV-negative to protect themselves against acquiring HIV. PrEP has shown efficacy for preventing HIV acquisition. Despite the potential, many concerns have been voiced by people who inject drugs (PWID) and their organizations. There is a need to engage with these views and ensure their integration in to policy and strategy. This paper presents PWID views on PrEP to foster the uptake of these opinions into scientific and policy debate around PrEP METHODS: Critical analysis of a report of a community consultation led by the International Network of People who Use Drugs (INPUD). The INPUD report highlights enthusiasm from PWID for PrEP, but also three main concerns: the feasibility and ethics of PrEP, its potential use as a substitute for other harm reduction strategies and how a focus on PrEP heralds a re-medicalization of HIV. Each concern relates to evidenced gaps in essential services or opposition to harm reduction and PWID human rights. People who use drugs have fundamental concerns about the potential impacts of pre-exposure prophylaxis for HIV which reflect a 'fault line' in HIV prevention: a predominance of biomedical approaches over community perspectives. Greater community engagement in HIV prevention strategy is needed, or we risk continuing to ignore the need for action on the underlying structural drivers and social context of the HIV epidemic.

Abstract 4884: Highly sensitive fusion transcript detection and quantification in cancer

Abstract Gene fusion detection in cancer samples can provide tumor-specific information for cancer research, clinical diagnosis and targeted treatment. Common fusion detection methods such as qPCR and FISH are restricted to known fusion junctions and limited in the number of genes that can be detected in parallel. In contrast, RNA sequencing is a powerful approach for simultaneous discovery of all possible fusion junctions in a single reaction. But, the sequencing depth required for sensitive detection of fusions from whole-transcriptome libraries can be cost-prohibitive. Here we describe a cancer-specific capture-based approach for fusion detection by RNA sequencing that requires only a fraction of the sequencing depth of whole-transcriptome methods. We designed oligo probes that densely target coding regions of over 200 clinically relevant gene fusions and cancer-associated genes. This oligo panel was used to capture cancer-specific fusions from total RNA-Seq libraries. We used commonly studied cancer cell lines including MCF-7, K562, PC-3, LnCAP, A431 and Universal Human Reference RNA (UHRR) to compare the sensitivity of fusion detection across three RNA-Seq library prep methods: (1) cancer panel library capture (2) whole-transcriptome library capture and (3) PolyA selection. We show that probes targeting individual exons can robustly capture well-characterized cancer gene fusions such as BCR-ABL and BCAS4-BCAS3, as well as translocations where fusion junctions are unknown. Furthermore, these comparisons demonstrate the enhanced sequencing efficiency of the targeted cancer panel, while maintaining highly accurate quantitation of gene expression. We show that selective enrichment of RNA-Seq libraries with cancer-specific capture probes enables high-resolution mapping of genomic rearrangements in patient cancer samples, even those derived from FFPE, facilitating sequencing studies that were not previously possible. Citation Format: Lisa C. Watson, Stephen M. Gross, Irina Khrevtukova, Smita Pathak, Claire Attwooll, Jason Goode, Anthony Mai, Gary P. Schroth. Highly sensitive fusion transcript detection and quantification in cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4884. doi:10.1158/1538-7445.AM2015-4884

Automated workflow for RNA-Seq analysis: application and testing with various types of RNA-Seq protocols

► Protocols for creating libraries for RNA-Seq using Illumina’s nextgeneration sequencing platforms include preps that utilize either poly-A selected mRNA or Total RNA. ► In addition the preps can also be performed with different types of cDNA synthesis chemistries, some of which are non-stranded and some that preserve the strandedness of the mRNA during cDNA creation, like RNA ligation methods or the incorporation of modified bases like dUTP. ► There are total RNA prep methods that use the RiboZeroTM kit (Epicentre) or the Duplex Specific Nuclease (DSN, from Evrogen) for rRNA reduction. Both of these approaches allow one to make full-length cDNA libraries that are completely independent of the integrity and degradation of the starting RNA material. Since these Total RNA-Seq methods do not use a poly-A selection procedure they allow us to sequence all of the RNA in the library including many non-coding RNAs that can be missed in other gene expression assays. Automated Workflow for RNA-Seq Data Analysis