Ng Of DNA Research Articles

Versatile and Portable Cas12a-mediated Detection of Antibiotic Resistance Markers.

Antimicrobial resistance (AMR) is a global public health problem particularly accentuated in low-and middle-income countries, largely due to a lack of access to sanitation and hygiene, lack of awareness and knowledge, and the inadequacy of molecular laboratories for timely and accurate surveillance programs. This study introduces a versatile molecular detection toolbox (C12a) for antibiotic resistance gene markers using CRISPR/Cas12a coupled to PCR. Our toolbox can detect less than 3×10 -7 ng of DNA (100 attoMolar) or 10 2 CFU/mL. High concordance was observed when comparing the C12a toolbox with sequenced genomes and antibiotic susceptibility tests for the blaCTX-M-15 and floR antibiotic resistance genes (ARGs), which confer resistance to cefotaxime and other β- lactams, and amphenicols, respectively. C12a INT , designed to detect the Integrase 1 gene, confirmed a high prevalence of the integrase/integron system in E. coli containing multiple ARGs. The C12a toolbox was tested across a wide range of laboratory infrastructure including a portable setup. When combined with lateral flow assays (LFA), C12a exhibited competitive performance, making it a promising solution for on-site ARG detection. Altogether, this work presents a collection of molecular tools (primers, crRNAs, probes) and validated assays for rapid, versatile, and portable detection of antibiotic resistance markers, highlighting the C12a toolbox potential for applications in surveillance and ARG identification in clinical and environmental settings.

Abstract A014: A methylation state specific targeted background depletion technique for enrichment of ctDNA fraction

Abstract Liquid biopsy continues to be a focus of the cancer diagnostics industry, primarily due to the non-invasive nature of sample collection, however the low levels of circulating tumor DNA (ctDNA) remain a challenge. Methylation has emerged as a promising biomarker for detecting ctDNA, yet detecting the cancerous signal in an excess of non-cancerous, unmethylated cfDNA in the background has been proven difficult. Harbinger Health has developed a novel method that depletes unmethylated background at the CpG-level from specific genomic regions of interest, resulting in an enrichment of the ctDNA fraction. We developed a background cfDNA depletion method that utilizes the endonuclease activity and the specificity of CRISPR/Cas12a to remove unmethylated DNA fragments at ctDNA methylation biomarker sites. First, we identified cancer-informative regions that showed consistent contiguous elevated methylation states in cancer compared to normal samples. We then designed guide RNAs that specifically bind and cut unmethylated CpG sites within the target regions. Cas depletion of bisulfite-converted DNA enriches cancer informative signal for bioanalysis. To test validity of our Cas complex designs, we created model samples that mimic fully unmethylated and fully methylated species. We spiked these cfDNA models into a background of genomic DNA from 5% to 0.01%. In triplicate, we examined cutting efficiency, multiplexing and specificity of Cas to unmethylated species of model DNA. Results were assessed using qPCR by comparing differences in Ct with and without Cas treatment. Additionally, we applied this depletion step during our library preparation process to show utility in methylation analysis of low tumor content liquid biopsy samples. Using both the model sample dilution series and bisulfite converted cfDNA samples, which represent a range of cancerous and background methylation signal, we deplete unmethylated targets using Cas prior to indexing PCR. This rendered unmethylated targets non-amplifiable, removing them from analysis. Results were assessed using both qPCR and sequencing data, such as methylated read counts over the target region. We demonstrated our analytical approach efficiently depletes non-cancer cfDNA by on average 9-fold, which increases the fraction of ctDNA signal and can be multiplexed. This background signal depletion driven by CRISPR is sensitive and specific, maintaining methylated DNA signal detection down to 0.01% at inputs of 10ng DNA. Our Cas-mediated depletion of background signal method has proven useful for enriching rare methylated fragments over background by improving the signal to noise ratio. This technique is novel by harnessing the specificity of CRISPR to target DNA in both a sequence and methylation state specific manner. The depletion of precise methylation states at the CpG-level make it suitable for use in multiple bioassays. Citation Format: Caitlin Gilley, Miguel Williams, Emily Neaga, Sarah Falotico, Kade Pettie, Anthony Shuber. A methylation state specific targeted background depletion technique for enrichment of ctDNA fraction [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr A014.

High chromosomal instability is associated with higher 10-year risks of recurrence for hormone receptor-positive, human epidermal growth factor receptor 2-negative breast cancer patients: clinical evidence from a large-scale, multiple-site, retrospective study.

Long-term survival varies among hormone receptor-positive (HR+) and human epidermal growth factor receptor 2-negative (HER2-) breast cancer patients and is seriously impaired by metastasis. Chromosomal instability (CIN) was one of the key drivers of breast cancer metastasis. Here we evaluate CIN and 10-year invasive disease-free survival (iDFS) and overall survival (OS) in HR+/HER2-- breast cancer. In this large-scale, multiple-site, retrospective study, 354 HR+/HER2- breast cancer patients were recruited. Of these, 204 patients were used for internal training, 70 for external validation, and 80 for cross-validation. All medical records were carefully reviewed to obtain the disease recurrence information. Formalin-fixed paraffin-embedded tissue samples were collected, followed by low-pass whole-genome sequencing with a median genome coverage of 1.86X using minimal 1 ng DNA input. CIN was then assessed using a customized bioinformatics workflow. Three or more instances of CIN per sample was defined as high CIN and the frequency was 42.2% (86/204) in the internal cohort. High CIN correlated significantly with increased lymph node metastasis, vascular invasion, progesterone receptor negative status, HER2 low, worse pathological type, and performed as an independent prognostic factor for HR+/- breast cancer. Patients with high CIN had shorter iDFS and OS than those with low CIN [10-year iDFS 11.1% versus 82.2%, hazard ratio (HR) = 11.12, p < 0.01; 10-year OS 45.7% versus 94.3%, HR = 14.17, p < 0.01]. These findings were validated in two external cohorts with 70 breast cancer patients. Moreover, high CIN could predict the prognosis more accurately than Adjuvant! Online score (10-year iDFS 11.1% versus 48.6%, HR = 2.71, p < 0.01). Cross-validation analysis found that high consistency (83.8%) was observed between CIN and MammaPrint score, while only 45% between CIN and Adjuvant! Online score. In conclusion, high CIN is an independent prognostic indicator for HR+/HER2- breast cancer with shorter iDFS and OS and holds promise for predicting recurrence and metastasis.

The employment of species-specific primer in combination with real-time polymerase chain reaction for analysis of pork in meatball products

In line with the implementation of Indonesian Act No. 33 year 2014 on Halal Products Assurance, the detection of a non-halal component in food products is critical to confirm the authenticity of halal products. The aim of this study was to employ real-time polymerase chain reaction (real-time PCR) using species-specific primer targeting D-loop mitochondria for analysis of pork DNA in meatballs. The real-time PCR technique was evaluated for the primer specificity, amplification efficiency, and sensitivity as determined by evaluating the limit of detection (LoD). The designed primer was specific to pork DNA, with an amplification efficiency of 82.7%. The developed method could detect DNA in meatballs as low as 10 ng DNA. The reproducibility of real-time PCR was acceptable, as indicated by the low relative standard deviation (RSD) of 1.81%. The developed method was successfully applied to analyze pork in meatballs available in the commercial market. Real-time PCR using species-specific primer targeting D-loop mitochondria offered a reliable method for the detection of pork DNA in meatball products and can be proposed as a standard method for supporting the authenticity of halal products.

Development of a 17-plex STR typing system for the identification of individuals and parentage testing in cattle

Accurate identification of animals and the verification of their parentage can be used to pedigree populations and support selective breeding. The International Society for Animal Genetics recommended 16 cattle STRs for individual identification and parentage testing in cattle, but no multiplex STR typing system contains these 16 STRs. Here, we develop an efficient 17-plex multiplex typing system for cattle that contains the 16 ISAG recommend STRs and a sex-determining marker. Compared to the Bovine Parenting Typing Kit (containing 11 of the 16 ISAG recommend STRs), our new typing system not only increases the number of molecular markers, but also simplifies the PCR operation and shortens the time for the typing procedure (from 4.5 h to 1 h 37 min). Profile can be generated from a single PCR reaction using as little as 1 ng of DNA. The combined probabilities of paternity exclusion CPEduo and CPEtrio were 0.999804697 and 0.999999260, respectively. These results indicate that our 17-plex typing system is a fast, sensitive and species-specific method for the identification of individuals and their parentage for cattle. The application of this system will improve the efficiency of the identification of cattle individuals and their paternity, supporting population genetic research and the selective breeding of cattle.

Targeted microscopic rare cell retrieval yields sufficient deoxyribonucleic acid for molecular testing in fixed tissue with low tumor fraction

Abstract Introduction/Objective DNA extracted from formalin-fixed paraffin-embedded tissue (FFPE) blocks is often utilized for molecular oncologic testing. Cases with low tumor fraction often fail to obtain adequate tumor for molecular testing. We hypothesized that targeted retrieval of visualized tumor cells from FFPE sections, including those with low tumor fraction, could yield sufficient DNA for molecular testing. Methods/Case Report Unstained sections of various thicknesses were cut from low tumor fraction blocks and visualized by light microscopy and the RareCyte CyteFinder II (Seattle, WA). Microscopic cell retrieval was performed on the RareCyte using 40 µm diameter needles from identified regions of interest. Samples were deparaffinized using Zymo Research (Tustin, CA) solution, and digested and purified using Zymo-Spin Column technology. Sample DNA was quantified with NanoDrop Spectrophotometer (Chino, CA). Results (if a Case Study enter NA) Regions of interest were morphologically identifiable on 4-10 µm thick sections and harder to visualize on 12 µm thick sections. Targeted microscopic cell retrieval was performed with 40 µm diameter needles. Retrieval was performed from 26 to 34 regions of interest per sample and pooled for molecular testing. Samples (n=4) yielded a mean of 81.0 ng of DNA ± 18.4 ng (1.8 ng/µL ± 0.4 ng/µL) and a median of 87.75 ng (1.95 ng/µL). Conclusion Targeted microscopic cell retrieval from low tumor fraction FFPE slides using the RareCyte CyteFinder II can provide sufficient DNA material for molecular testing. Application of this technology has the possibility of recovering sufficient tumor DNA for molecular testing from specimens that may be deemed unsuitable for molecular testing according to current standards. We aim to focus on increasing sample size and completing comprehensive molecular analysis as we continue to expand this work.

Screening for Japanese Black cattle herds at risk of bovine leukemia virus transmission based on the presence of persistent lymphocytosis

This study aimed to develop a screening method to identify Japanese Black (JB) cattle farms at high risk of bovine leukemia virus (BLV) transmission. We introduced a recently established lymphocyte count (LC) cut-off to detect cattle with persistent lymphocytosis (PL). To identify high-risk farms, we examined the relationship between the proportion of cattle with PL and the mean blood proviral loads (PVL) per farm. The results showed a strong correlation between the proportion of cattle with high LC, higher than the LC cut-off values for JB cattle, and the mean blood PVL per farm. This indicates that the mean blood PVL on a herd basis of JB cattle can be estimated from the proportion of cattle with high LC. Specifically, the mean blood PVL on farms with >30 % cattle having high LC was estimated to be greater than 100 copies/10 ng DNA. The higher the proportion of cattle with high LC per farm, the higher the proportion of cattle at risk as sources of BLV infection. The study demonstrated that the proportion of cattle with high LC, based on the new LC cut-off, serves as a practical index for screening high-risk farms with PL cattle that have high PVL. This finding is meaningful for prioritizing farms with a high proportion of cattle with high LC, necessitating BLV infection prevention measures, such as voluntary culling and segregation, in order to develop a regional, stepwise BLV eradication strategy.

Unravelling red deer (Cervus elaphus) meat adulteration in gourmet foods by quantitative real-time PCR

Red deer (Cervus elaphus) meat is highly regarded as an exclusive gourmet food due to its sensory quality, nutritional value and premium price, thus being susceptible to adulteration practices by its substitution with inferior meat species. This study proposes a novel real-time PCR method targeting the troponin I gene for detecting and quantifying red deer meat in processed food products. The assay showed acceptable performance parameters, achieving absolute and relative limits of quantification of 0.01 ng of DNA and 0.5% (w/w) of red deer meat in both raw and thermally processed pâté. The assay parameters, using raw and autoclaved reference mixtures, regarding PCR efficiency (89.3% and 100.0%, respectively), slope of the calibration curve (−3.6067 and −3.3224, respectively) and R2 (0.9889 and 0.9893, respectively) highlight the high performance of the calibration models for the determination of red deer meat. The method was successfully validated using blind mixtures and applied to 46 commercial meat products from Poland, Portugal and Spain. Data suggested the adulteration of 48% of the samples by the total or partial substitution of red deer species and the suitability of the method for the routine testing of game meat products in quality control laboratories.

Enzyme-Mediated Duplex Exponential Amplification: A New Platform for Rapid Screening of &amp;lt;i&amp;gt;Hylurgus ligniperda&amp;lt;/i&amp;gt;

As the world&amp;apos;s second largest timber importer, wood demand in China has been growing extremely rapidly, leading to an increase of 163% from 2009 to 2018. The plant quarantine pest &amp;lt;i&amp;gt;H. ligniperda Fabricius, 1787&amp;lt;/i&amp;gt; is an invasive species frequently intercepted at ports. &amp;lt;i&amp;gt;H. ligniperda&amp;lt;/i&amp;gt; causes damage mainly to pine and spruce. To improve the efficiency of on-site inspection and the efficacy of early detection, tight quarantine in ports, time-effective identification, and a national surveillance program for high-risk invasive bark beetles are in urgent need. In this study, a simple, fast and accurate classification method for &amp;lt;i&amp;gt;H. ligniperda &amp;lt;/i&amp;gt;is established based on the enzyme-mediated duplex exponential amplification (EmDEA) technique. Partial region from &amp;lt;i&amp;gt;inhibitor of apoptosis 2&amp;lt;/i&amp;gt; (&amp;lt;I&amp;gt;IAP2)&amp;lt;/I&amp;gt; gene was selected as the target and 6 primer/probe combinations were designed. Through selection, the combination of 3-HY-F3, 3-HY-R2 and 3-HY-RNA5 was chosen as the final primer-probe set, as it showed the lowest Ct with highest final fluorescence signal. Method validation and specificity test using 6 other beetle species living on coniferous wood showed that this method is result reliable and specific. Through parameter analysis with positive plasmid, the detection limit was calculated to be 13.6 copies/μL (9×10&amp;lt;sup&amp;gt;-7&amp;lt;/sup&amp;gt; ng DNA/ reaction), much higher than conventional molecular methods such as PCR. The whole process including isothermal amplification, data analysis, and result output can be finished in 30 min, which is highly time-effective. Besides, the operation is simple and little training is needed for non-professionals. The application prospects of this rapid screening system include customs screening in ports, wild survey in non-lab situations and early warning system development. The new analysis platform EmDEA, can also be implemented in rapid detection and identification of other forestry pests.

Cyt b gene as a valid molecular authentication marker of cow leathers

Authentication of true (genuine) cow leathers is in high demand to promote merchandise and economic growth. The present study employs RT-PCR-based TaqMan assay to facilitate the identification. Species-specific primers and probes were designed utilizing the existing NCBI data on mitochondrial DNA (mtDNA) genes, particularly the cytochrome b region (Cyt b). Mitochondrial DNA extracted from leather samples of both Bos taurus and Bos indicus and analyzed following the appropriate procedures. The RT-PCR results showed the designed primers and probes are exceptionally precise for cow leather samples. The established detection limit for the assay is estimated as 0.1 ng of DNA. In summary, the amplifiable mtDNA extracted from finished leather enables the identification of authentic cow leathers using the RT-PCR TaqMan assay, representing a pioneering report in this field.

Half-volume validation of the NGM Detect™ PCR Amplification Kit and its application on degraded casework samples.

The NGM Detect™ PCR Amplification Kit was designed particularly for genotyping degraded casework samples. This study aimed to validate the half-volume amplification of the kit and to present its successful long-term application. The validation was performed in accordance to the corresponding guidelines of the Scientific Working Group on DNA analysis methods and the European Network of Forensic Science Institutes. For validation parameters, such as sensitivity, reproducibility, and repeatability, polymerase chain reactions (PCR) were set up both manually and robotically, applying 29 cycles. For PCRs with sub-optimal DNA input (≤0.5 ng) the cycle numbers were increased to 31. Regardless of the PCR preparation method, the optimal 0.5 ng DNA input produced optimal allelic peak heights with no allelic dropout. The first alleles that failed to amplify started to appear at the level of 0.0375 ng input DNA, although the manually prepared PCRs produced fewer missing alleles. In this case, the raised cycle number produced 1.9% and 4.4% of dropout for manually and for robotically set up PCRs, respectively. In the case of 84 degraded casework samples, PCRs were prepared only by hand. The kit was able to provide informative profiles for 78.57%, 70.37%, and 69.77% for lowly, moderately, and highly degraded samples, respectively. Allelic dropouts were 26.05%, 44.88%, and 51.23% for the same groups. According to our results, we strongly recommend using the NGM Detect™ Kit in half-volume PCR system and encourage the usage of the kit in the particular cases when other kits fail to produce a complete DNA profile.

DNA Isolation From Six Spotted Ladybird Beetle &lt;i&gt;Cheilomenes Sexmaculata&lt;/i&gt; (F)

DNA extraction is a routine step in many insect molecular studies and extraction methods need to be evaluated for their efficiency, cost, and side effects, such as DNA degradation. Individuals of six spotted ladybird beetle, Cheilomenes sexmaculata (F.) were subjected to DNA extraction by the CTAB method and PureLink® Kit. The methods were compared in terms of DNA quantity and quality, cost of materials, and time consumed. The CTAB method resulted in higher DNA yield (ng DNA) at a much lower price and less degradation, as revealed on agarose gels. The PureLink® Kit was the most time-efficient but costliest, and degradation was observed on agarose gels. The DNA samples obtained were tested on agarose gel PCR for six SSRs located in various positions of the beetle’s genome. The results revealed that DNA isolated from the CTAB method showed successful amplifications, but the PureLink® Kit method did not show any amplification. These evaluations guide the choice of DNA extraction methods from C. sexmaculata beetles.

Direct transposition of native DNA for sensitive multimodal single-molecule sequencing

Concurrent readout of sequence and base modifications from long unamplified DNA templates by Pacific Biosciences of California (PacBio) single-molecule sequencing requires large amounts of input material. Here we adapt Tn5 transposition to introduce hairpin oligonucleotides and fragment (tagment) limiting quantities of DNA for generating PacBio-compatible circular molecules. We developed two methods that implement tagmentation and use 90–99% less input than current protocols: (1) single-molecule real-time sequencing by tagmentation (SMRT-Tag), which allows detection of genetic variation and CpG methylation; and (2) single-molecule adenine-methylated oligonucleosome sequencing assay by tagmentation (SAMOSA-Tag), which uses exogenous adenine methylation to add a third channel for probing chromatin accessibility. SMRT-Tag of 40 ng or more human DNA (approximately 7,000 cell equivalents) yielded data comparable to gold standard whole-genome and bisulfite sequencing. SAMOSA-Tag of 30,000–50,000 nuclei resolved single-fiber chromatin structure, CTCF binding and DNA methylation in patient-derived prostate cancer xenografts and uncovered metastasis-associated global epigenome disorganization. Tagmentation thus promises to enable sensitive, scalable and multimodal single-molecule genomics for diverse basic and clinical applications.

Detection of Ochyromera ligustri (Coleoptera: Curculionidae: Curculioninae) in Ligustrum spp. (Oleaceae) Using Newly Developed PCR Primers.

Ligustrum spp. (Oleaceae) have become invasive species in the US and negatively affect native plant diversity and richness in forests. Ochyromera ligustri (Coleoptera: Curculionidae) is considered a potential biological control agent in the US because adults feed on the foliage and larvae are seed-feeders of Ligustrum spp. To discover the relationships between O. ligustri and Ligustrum spp., fruit dissections or rearing and field observations are required. In the current research project, novel PCR primers were developed to rapidly detect the DNA of O. ligustri in molecular analyses without rearing and observation. The developed PCR primers worked even with 0.01 ng of DNA and did not amplify the DNA of the other five curculionid species tested. When the novel primers were tested with three Ligustrum spp. species common in the southeastern US, the DNA of O. ligustri was detected from all three species. We expect that the novel primers will be utilized to find out the presence and impact of O. ligustri on Ligustrum spp rapidly and accurately.

Droplet based whole genome amplification for sequencing minute amounts of purified Mycobacterium tuberculosis DNA

Implementation of whole genome sequencing (WGS) for patient care is hindered by limited Mycobacterium tuberculosis (Mtb) in clinical specimens and slow Mtb growth. We evaluated droplet multiple displacement amplification (dMDA) for amplification of minute amounts of Mtb DNA to enable WGS as an alternative to other Mtb enrichment methods. Purified genomic Mtb-DNA (0.1, 0.5, 1, and 5 pg) was encapsulated and amplified using the Samplix Xdrop-instrument and sequenced alongside a control sample using standard Illumina protocols followed by MAGMA-analysis. The control and 5 pg input dMDA samples underwent nanopore sequencing followed by Nanoseq and TB-profiler analysis. dMDA generated 105-2400 ng DNA from the 0.1-5 pg input DNA, respectively. Followed by Illumina WGS, dMDA raised mean sequencing depth from 7 × for 0.1 pg input DNA to ≥ 60 × for 5 pg input and the control sample. Bioinformatic analysis revealed a high number of false positive and false negative variants when amplifying ≤ 0.5 pg input DNA. Nanopore sequencing of the 5 pg dMDA sample presented excellent coverage depth, breadth, and accurate strain characterization, albeit elevated false positive and false negative variants compared to Illumina-sequenced dMDA sample with identical Mtb DNA input. dMDA coupled with Illumina WGS for samples with ≥ 5 pg purified Mtb DNA, equating to approximately 1000 copies of the Mtb genome, offers precision for drug resistance, phylogeny, and transmission insights.

Development and validation of a new multiplex panel using SNaPshot-based DIP-TriSNP markers for forensic DNA mixtures.

Short tandem repeat analysis is challenging when dealing with unbalanced mixtures in forensic cases due to the presence of stutter peaks and large amplicons. In this research, we propose a novel genetic marker called DIP-TriSNP, which combines deletion/insertion polymorphism (DIP) with tri-allelic single nucleotide polymorphism in less than 230bp length of human genome. Based on multiplex PCR and SNaPShot, a panel, including 14 autosomal DIP-TriSNPs and one Y chromosomal DIP-SNP, had been developed and applied to genotyping 102 unrelated Han Chinese individuals in Sichuan of China and simulated a mixture study. The panel sensitivity can reach as low as 0.1ng DNA template, and the minor contributor of DNA can be detected with the highest ratio of 19:1, as indicated by the obtained results. In the Sichuan Han population, the cumulative probability of informative genotypes reached 0.997092, with a combined power of discrimination of 0.999999998801. The panel was estimated to detect more than two alleles in at least one locus in 99.69% of mixtures of the Sichuan Han population. In conclusion, DIP-TriSNPs have shown promising as an innovative DNA marker for identifying the minor contributor in unbalanced DNA mixtures, offering advantages such as short amplifications, increased polymorphism, and heightened sensitivity.

Abstract LB095: ASPYRE-Lung addresses critical gaps in NGS-based biomarker testing: Robust variant calling from NGS QC fails

Abstract Advanced NSCLC treatment guidelines recommend testing patients for genomic biomarkers to utilize over 30 FDA-approved targeted therapies. A major challenge with current NGS-based testing methods is that ~45% of patient samples fail NGS quality control (QC), leading to inadequate patient care. The ASPYRE® technology addresses the urgent need for rapid, accessible and affordable diagnostics informing actionable genomic target variants. The ASPYRE-Lung® panel covers 114 variants in 11 genes (ALK, BRAF, EGFR, ERBB2, KRAS, RET, ROS1, MET &amp; NTRK1/2/3) to robustly inform clinical management, based on clinical practice guidelines. The assay concurrently detects single nucleotide variants, insertions, deletions, and gene fusions from NSCLC tissue-derived DNA and RNA. In this study, we investigated an NSCLC patient sample set from Precision for Medicine, including biobanked clinical specimens that had previously failed NGS QC, and for which genomic biomarker data were unavailable. 120 matched DNA and RNA biobanked NSCLC FFPE patient samples previously analyzed by Illumina TSO 500 were tested using ASPYRE-Lung in Biofidelity’s CLIA laboratory, including 94 samples which failed NGS QC parameters for DNA, RNA, or both, and 26 control samples which passed NGS QC. Samples were tested by ASPYRE-Lung using an input of 20 ng DNA and 6 ng RNA, except for 5 patient samples that were run at lower inputs, ranging from 4.25 to 14 ng DNA. Key findings:•Of the 94 patient samples that failed NGS QC, 98% of samples (92/94) passed ASPYRE-Lung QC and could inform patient care.•In the 92 patient samples that passed ASPYRE-Lung QC, 47% (43/92) had a detectable variant identified by ASPYRE-Lung.•All 26 control samples that passed NGS QC also passed ASPYRE-Lung QC.•Of the 5 samples with 4.25 to 14 ng DNA inputs to ASPYRE-Lung, data were generated on 4/5 samples and 2/4 had a detectable variant.•ASPYRE-Lung has the potential to provide actionable clinical information on patient samples deemed to be of insufficient quantity for NGS testing.ASPYRE-Lung has a high success rate, is easily adoptable and cost effective making it suitable as a first-line testing option, or for samples that are either QNS or fail NGS QC, and can provide a large fraction of NSCLC patients with actionable biomarker information, with potentially smaller tissue requirements. ASPYRE-Lung genomic testing is transformative for cancer care management, and allows more patients with NSCLC to benefit from effective and better tolerated therapies. Citation Format: Ryan T. Evans, Elyse Shapiro, Elizabeth Gillon-Zhang, Julia N. Brown, Candace King, Cory Kiser, Mary Beth Rossi, Darren Davis, Michelle L. Taylor, Jennifer Clower, Jian Yang, Cullen A. Taylor, Robert W. Snyder, Wendy J. Levin. ASPYRE-Lung addresses critical gaps in NGS-based biomarker testing: Robust variant calling from NGS QC fails [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB095.

Abstract 949: A single stranded DNA library preparation workflow used for hybridization capture based WES assay showed superior uniformity

Abstract Introduction: In many instances in Precision Oncology Medicine fragmented DNA from formalin-fixed paraffin-embedded (FFPE) samples is the only option. The majority of such degradedsamples need to be sequenced using next generation sequencing (NGS) [1]. Standardizedguidelines highlight that a minimum of 500 × coverage depth is required for tissue analyses [2].However, coverage isn’t the only metric required for a successful NGS analysis. Highly uniformDNA libraries allow clinical labs to generate more hits from their screens, saving both time andmoney across the course of diagnosis therefore ultimately benefits patient care. The commerciallyavailable single stranded DNA library prep kit tested in this study renders all input moleculessingle-stranded before ligation therefore it recovers template molecules containing DNA nicks andlabile lesions thus maximizing library complexity. Method: 10 solid tumor FFPE derived DNA samples went through three different NGS workflows:100ng of DNA input in an amplicon-based assay 325 gene panel (Assay A), 60ng input for a 500gene mechanical sheared-based double strand DNA library preparation with hybridization captureassay (~500 gene panel, Assay B), 50ng and 10ng input for a enzymatic shear-based singlestranded DNA library preparation with hybridization capture assay (WES with 1000+ enhancedgene content, Assay C). Data from assays A and C was processed with in-house bioinformaticpipeline, while Assay B’s data was processed with a commercially available bioinformatic pipelinethat was bundled with the assay. NGS QC metrics were compared across the three datasets. Results: This novel single stranded DNA library preparation with hybridization capture workflowshowed superior QC metrics compared to the other two workflows, especially on coverageuniformity and percent target above 100X, with as low as 10ng input. Conclusion: Employing a novel commercially available DNA library preparation workflow in anNGS assay, NeoGenomics was able to rescue poor quality samples that had previously failed tomeet our internal uniformity metric on other NGS assays and generate robust and reportableresults with as low as 10ng DNA input.[1] Nagahashi et al., J Surg Res. 2017 Dec; 220: 125-132.[2] Cappello et al., J Pers Med. 2022 May; 12(5): 750. Citation Format: Jieying Chu, Rachel Schell, Faqiang Wu, Michal Krawczyk, Daniel Whang, Segun Jung, Hyunjun Nam, Kenneth B. Thomas, Fernando J. Lopez-Diaz, Vincent Funari, Julie A. Mayer, Shashi Kulkarni, Jiannan Guo, Steven P. Lau-Rivera. A single stranded DNA library preparation workflow used for hybridization capture based WES assay showed superior uniformity [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 949.

Abstract 4416: Rapid, low-input, targeted NGS workflow for DNA methylation

Abstract DNA methylation is a fundamental epigenetic process that regulates human gene expression. In cancer, methylation changes promote proliferation networks and metastasis. Development of biomarkers for methylation will be enabled by flexible, fast, low-input next generation sequencing workflows. We describe the first Ion AmpliSeq™ Methylation targeted panel and protocol on the turn-key Genexus™ Integrated Sequencer as part an ongoing collaboration with Ontario Institute for Cancer Research (OICR) to detect and predict response in early stage breast cancer and improve diagnostics for Black and Asian women. A 2-pool Ion AmpliSeq™ Methylation Panel for Breast Cancer Research was developed as a demonstration of design, workflow, and reporting for targeted, low-input methylation assessment in a multiplex setting across a variety of samples sources including FFPE. The panel contains 327 amplicons and was designed to target both strands and 10ng DNA input into bisulfite conversion was used for controls. A complete workflow begins at bisulfite conversion and progresses through the Genexus™ Integrated Sequencer which combines library construction, template preparation, and sequencing into a single run. The bioinformatics pipeline provides DNA methylation calls on both Watson and Crick strands at single base resolution and methylated:unmethylated ratios for each CpG. The entire end-to-end workflow was completed in 2 days, including a full analysis software solution. The panel was evaluated using 2 control gDNA samples. The first had an expected average methylation state across all CpGs of &amp;gt;98% and the second &amp;lt;5%. An equal mixture of these two samples was also tested. The Methylation Panel for Cancer Research performed well on control samples. The single lane Loading, Total Bases, Final Reads, and Raw Read Accuracy were &amp;gt;92%, &amp;gt;1G, &amp;gt;14 Million, and &amp;gt;90% respectively. The workflow was also demonstrated on research FFPE cancer samples. This Methylation Panel protocol offers a 2-day, end-to-end workflow with high resolution, targeted and quantitative methylation analysis from DNA input as low as 10ng into bisulfite conversion. The option to design custom methylation panels for interrogation of targets of interest without the need for whole genome methylation is now available. &amp;lt;For Research Use Only. Not for use in diagnostic procedures. Early access materials were provided by Thermo Fisher Scientific™ to OICR in support of this abstract, but no other financial support. This study was conducted with the support of the Ontario Institute for Cancer Research through funding provided by the Government of Ontario. © 2023 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified.&amp;gt; Citation Format: Loni Pickle, Andrew Hatch, Gokhan Yavas, Melanie Spears, Louis Gasparini, Vida Talebian, Anna Ying-Wah Lee, Mathieu Lariviere, Jane Bayani, Seth Sadis, Jeffrey M. Smith. Rapid, low-input, targeted NGS workflow for DNA methylation [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 4416.

Abstract 2291: Bravo automation of Agilent Avida targeted enrichment for high-throughput detection of genomic alteration and DNA methylation

Abstract Background: Current genomic and epigenomic profiling of cancer tissue DNA or cfDNA (cell-free DNA) in liquid biopsy relies upon separate, time- and sample-consuming technologies for somatic variant detection or methylation analysis. Here we describe workflow and performance of the Agilent Bravo automated liquid handling platform with the Agilent Avida targeted enrichment solution for next generation sequencing of somatic variants and methylation profiling. This solution can effectively analyze low-input tumor DNA or cfDNA samples. The Avida Duo workflow enables highly sensitive detection of single nucleotide variant (SNV), insertions and deletions (INDEL), copy number variation (CNV) and DNA methylation profiles from a single sample, without any sample splitting. Methods and Results: Panels, reagents, and automated workflows for Avida DNA, Avida Methyl, and Avida Duo Methyl (combined DNA &amp; methylation) kits were developed to accommodate up to 96 samples on the Bravo NGS workstation. The automated solution supports independent single-day workflows for somatic variants or methylation sample preparation. The Avida Duo analysis combines both workflows without sample splitting, streamlining the process and reducing sample consumption. Leveraging a focused cancer hotspot panel, we demonstrate excellent reproducibility and low allele frequency (SNV at sub 1%) variant detection in cfDNA samples and reference standards with as little as 10ng DNA input. We exhibit similar performance with a larger ~300 kb target region tumor profiling panel. Finally, we demonstrate DNA methylation detection in the standalone or combined (“Duo”) workflows across differentially methylated regions (DMRs) with a panel of ~3400 targets. Conclusions: Automation of the Avida targeted enrichment solution with the Bravo NGS workstation enables a sensitive, high-throughput, end-to-end analysis for detection of genomic alterations and DNA methylation changes from a single low-input cfDNA or tumor DNA sample. Citation Format: Ashraf Wahba, Tony Ho, Sarah Johns, Aswati Aravind, Heng Wang, Neelima V. Mehendale, Gilbert P. Amparo, Khine Win, Manuel Gomez, Grace Zhao, Douglas N. Roberts. Bravo automation of Agilent Avida targeted enrichment for high-throughput detection of genomic alteration and DNA methylation [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 2291.