Size Of DNA Fragments Research Articles

Abstract 3975: Tumor mutation burden and microsatellite instability in colorectal cancer

Abstract Introduction In colorectal cancer (CRC), the use of immunotherapy is currently based on the evaluation of microsatellite instability (MSI) for the identification of patients who may benefit from this treatment. Recently, tumour mutation burden (TMB) has been evaluated as a useful biomarker to predict response to immune checkpoint (IO) therapy. TMB can be measured by means of targeted next generation sequencing with panels covering approximately 1 Mb of the human genome. We performed TMB analysis on tumor cell lines and CRC samples and compared the results with MSI status to explore the relationship between these two biomarkers. Materials and methods MSI status was evaluated by means of routine immunohistochemistry (IHC), the Idylla MSI assay (Biocartis) and with fluorescence capillary electrophoresis-based DNA fragment size analysis of a T17 mononucleotide sequences within intron 8 of heat shock protein 110 kDa HSP110 (17 polythymine, T17). TMB evaluation was performed with the Oncomine Tumor Mutation Load Assay (Thermofisher) on the Ion S5XL platform. Data analysis was carried out using Ion Reporter Software v5.6. TMB was calculated as the total number of somatic single nucleotide variants (SNVs) divided by number of bases with sufficient coverage. Results We first evaluated the TMB in 13 tumor cell lines and correlated the results with massively parallel sequencing data from >1600 genes for the same cell lines available on cBioPortal, demonstrating an high correlation between the Oncomine Tumor Mutation Load Assay and the TMB value obtained by wide genetic profiling (R² = 0.976). We next analyzed a cohort of 23 MMR deficient (MMR-D) and 32 MMR-proficient (MMR-P) formalin fixed paraffin embedded (FFPE) CRC, as classified by IHC. The MMR data were compared to TMB and revealed TMB values not in line with the expected results, since some samples in the MMR-D group had low TMB values (TMB range: 5.5-134.4). We re-evaluated the 23 MMR-D samples by means of the Idylla System. The Idylla MSI assay re-classified 7 samples as microsatellite stable (MSS) and categorized the remaining 16 samples as MSI-High (MSI-H). The results were confirmed by the T17 analysis. In the MSS group (n. 39), the mean and median TMB value were 11.48 and 10.39, with TMB ranging from 4.88 to 33.29. The mean and median TMB for the MSI-H group (n. 16) were 43.89 and 38.63, and the TMB values ranged from 19.63 to 134.4. While the medians of the two groups were significantly different (Mann-Whitney test P<0.0001), these data confirm a high heterogeneity of TMB within MSI-H CRC. One MSS case displayed a TMB close to the median TMB of the MSI-H group (TMB: 33.29). The MSS status of this sample was confirmed by the Idylla System. Conclusions These data suggest that routine IHC methods for MSI evaluation can lead to a wrong classification of CRC. On the other hand, TMB assessment might better identify patients who may benefit from IOs within both the MSI-H and the MSS subgroups. Citation Format: Francesca Fenizia, Raffaella Pasquale, Cristin Roma, Francesca Bergantino, Anna Maria Rachiglio, Nicoletta Chicchinelli, Paolo Graziano, Gerardo Botti, Fabiana Tatangelo, Giosuè Scognamiglio, Christopher Allen, Matilde Lambiase, Alessia Iannaccone, Nicola Normanno. Tumor mutation burden and microsatellite instability in colorectal cancer [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 3975.

Abstract 438: Multicenter evaluation of circulating tumor DNA assays

Abstract Measurements of circulating tumor DNA (ctDNA) hold great potential to detect residual disease, therapeutic response monitoring and tumor evolution. However, there are many technical challenges including trace levels of circulating cell free DNA 1-10 ng/ml in plasma (300-3,000 genome equivalents/ml), DNA fragment size 170 bps found in the circulation, low variant allelic fractions ~ 0.1% and the need to detect all variant types SNVs, insertions, deletions, CNVs and structural variants. CANCER-ID partners comprise a consortium of experts, companies and institutions for blood -based biomarker validation, assay development, clinical sciences and bioinformatics. This CANCER-ID ring trial was established to help standardize complex ctDNA diagnostic assays by testing a highly complex reference material. The trial looks to improve concordance and to evaluate analytical performance by collecting metrics covering the entire workflow from sample preparation through variant reporting. The common reference material, Seraseq ctDNA Complete, used in the trial is human genomic DNA background material from the Genome-in-a-bottle consortium mixed with synthetic DNA sized to ~170 bp and formulated in artificial plasma. The material contains 25 variants per sample including 12 SNVs (8 genes), 5 deletions (3 genes), 2 insertions (2 genes), 3 CNVs (3 genes) and 3 fusion pairs (3 genes). The variant allelic fractions range from 0.1% to 5% for the SNVs, INDELS and ~ 1 -6 additional copy number for ERBB2, MET and MYC genes. Interlab and intralab testing was done on Roche AVENIO targeted Assay, Agilent SureSelect XT custom assay, QiaSeq Targeted DNA panel, Qiagen GeneRead QIAAct Lung Assay, one clinically validated digital droplet assay and one research digital droplet PCR assays and Agena Bioscience’s UltraSEEK Lung Panel. Data analysis, peer comparison and reporting of common and unique QC metrics was accomplished through the cloud based, analytical QC software iQ NGS QC Management. The results demonstrate that a common full process reference material can be used to compare distinctly different assays formats at different levels of input DNA by mutations. For the 1% AF material, the BRAF V600 variant was detected by all assay formats and the cv’s ranged from 12%-52% or on average for all variants measured by the assay ranged from 11% to 46%. The study furthermore shows that peer review analytical QC software is useful in establishing standardization of ctDNA assays. This work is supported by IMI JU & EFPIA (grand no. 115749, CANCER-ID). Citation Format: Dan Brudzewsky, Rita Lampignano, Sabrina Weber, Alexander Sartori, Sumitra Mohan, Yves Konigshofer, Lorn Davis, Dominic Rothwell, Ed Schuuring, Russell K. Garlick, Thomas Schlange, Ellen Heitzer. Multicenter evaluation of circulating tumor DNA assays [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 438.

Abstract 2563: A comparison of microsatellite instability analysis methods for detection of mismatch repair deficiency

Abstract Use of biomarkers for patient treatment stratification is an increasingly important topic with the recent FDA approval of Pembrolizumab and Nivolumab for microsatellite instability (MSI) high/mismatch repair (MMR) deficient cancers. This ground-breaking approval allows physicians to make patient treatment decisions based on molecular biomarker status. Thus, performance of detection methods for these biomarkers is of great importance. The gold standard for microsatellite instability (MSI) analysis is PCR followed by fragment analysis using capillary electrophoresis to resolve DNA fragment size. Recently, PCR followed by melt curve analysis and Next Generation Sequencing (NGS) have been presented as alternative approaches. In this set of experiments, these methods will be compared using a subset of human cancer samples. A cohort of matched cancer and adjacent normal FFPE samples were used for this comparison. From this cohort, individuals were selected that contained subtle MSI shifts, low tumor volume, or heterozygosity at microsatellite loci. Samples from these individuals were tested for MSI by either fragment or melt curve analysis or by sequencing. Samples were then classified as MSI-High, MSI-Low or microsatellite stable (MSS) according to manufacturer specifications for melt curve analysis or by an interpretive software for fragment analysis and NGS. Preliminary results indicate that there may be differences in sensitivity when using challenging samples with melt curve analysis compared to the gold standard method, fragment analysis. The standard method for determining MSI status utilizes PCR and fragment analysis. Recently, melt curve analysis and NGS have been proposed as alternative methods. We present preliminary findings of decreased sensitivity with melt curve compared to fragment analysis. Additional studies with a larger, more diverse sample set will be required to define this relationship further. Citation Format: Samantha R. Lewis, Shaun Peterson, Kathryn Oostdik, Heather Tomlinson. A comparison of microsatellite instability analysis methods for detection of mismatch repair deficiency [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 2563.

Genetic diversity analysis and molecular characterization of grain amaranth genotypes using inter simple sequence repeat (ISSR) markers

BackgroundGrain amaranth (Amaranthus spp.) has been cultivated since ancient times in some countries in the world and it is one of the oldest food crops. At present, the crop has gained more importance in the plains of India, especially in parts of Gujarat and Maharashtra. Grain amaranth exhibits an incredible extent of morphological diversity and an extensive adaptability to diverse eco-geographical conditions. Hence, the aim of the recent research was to evaluate the genetic diversity of 19 genotypes from four diverse species of Amaranthus from India using ISSR markers.ResultsThe set of 11 polymorphic ISSR primers produced a total of 114 amplicons, among which 98 amplicons were polymorphic. The mean number of polymorphic amplicons per primer was 8.91. Overall, the size of PCR-amplified DNA fragments ranged from 200 to 3702 bp. The average percent polymorphism was 87.15%, and the average PIC value was 0.853, which indicates good selection of primers in the present study for the assessment of genetic diversity. The unique amplicon (marker)-producing primers were also found which can be used for identification of genotypes. The dendrogram grouped 19 grain amaranth genotypes into two major clusters. The groups formed on the principle component analysis (PCA) plot resembles with the results of the dendrogram although some genotypes have been diverted on the PCA plot.ConclusionsThe technique may be used to obtain reasonably precise information on the genetic relationship among grain amaranth genotypes. Such information may be useful for selecting the diverse parents and monitoring the genetic diversity periodically in the breeder’s working collection of grain amaranth.

Simple sequence repeat analysis of new potato varieties developed in Alberta, Canada.

The worldwide demand for potato production requires the constant development of new potato varieties with improved yield, quality, disease resistance, and abiotic tolerance. However, cultivar registration is preceded by a long process to morphologically and physiologically characterize the plants. Notably, this process can be expedited by DNA marker analysis. Simple sequence repeats (SSRs), also known as microsatellites, are the most common reliable DNA markers used to discriminate between genotypes. In this study, 20 potato varieties, including five new genotypes developed in Alberta, Canada, were fingerprinted using 10 SSR markers selected for their high discriminatory power. Different SSRs were amplified from potato DNA using specific primers, and the DNA fragment sizes were analyzed by denaturing polyacrylamide gel electrophoresis. The number of alleles per locus ranged from two for the SSR marker STPoAc58 to six for STM0030 and STM0037 with an average of 4.4. In addition, a total of 77 unique patterns were observed for the 10 SSR markers. The polymorphic information content ranged from 0.477 to 0.802 with an average of 0.675 per locus. In this study, STM0037, STM1016, and STM1104 were found to be the best SSR markers to detect genetic differences between potato varieties. A minimum of two markers was required to distinguish between all 20 genotypes. Most importantly, this highly informative molecular tool confirmed that the developed potato varieties were genetically different from their respective maternal lines and potentially constituted new cultivars.

Molecular Identification of Mammalian Blood Meals in Mosquito Vectors in Nile Delta, Egypt

The degree of contact of the vector and the vertebrate host is an important variable in determining the vectorial capacity of mosquito species for the arthropod-borne disease. This study conducted in Monufia Governorate, Egypt, to describe the mosquito community composition and species-specific host-feeding patterns. Mosquitoes were surveyed over a 2-years period and their host-feeding patterns were determined in relation to species relative abundance by polymerase chain reaction (PCR).This diagnostic technique was used to identify mammalian blood meals from female mosquitoes by sized DNA fragments following agarose gel electrophoresis. One universal reverse primer and five animal-specific forward primers included Human, Pig, Cow, Dog and Goat were used. Multiple blood meals from distinctive mammalian hosts were identified from single mosquito abdomens. Ninety-nine mosquito blood meals from four mosquito species were identified, 67.7% (67) were mixed blood. Both Cx. pipiens and Cx. antennatus fed on human and animals but feeding strategies differed from outdoors to indoors. Inside houses engorged female Cx. pipiens accounted for (94) 74% of collections and out of this, 53.8% fed on humans as single blood and 40% as mixed blood. However, outdoor, collected Ochlerotatus caspius constituted 7.1% of the collected females. Results suggested that, Cx. pipiens an important bridge of disease vector to humans in Egypt.

DNA Quality and Quantity Analysis of Camellia sinensis Through Processing from Fresh Leaves to a Green Tea Extract.

Background: Although there has been some success using DNA barcoding to authenticate raw natural health product (NHP) botanical ingredients, there are many gaps in our understanding of DNA degradation, which may explain low PCR and sequencing success in processed NHPs. Objective: In this study, we measured multiple DNA variables after each step in the processing of a green tea extract in order to document DNA quality and quantity. Methods: We sampled plant material after each step of green tea extract processing: five steps at a Chinese tea farm (n = 10) and five at an NHP processing facility (n = 3). We hypothesized that processing treatments degrade and remove DNA from NHPs, reflected by decreasing quantities of extractable genomic DNA (gDNA), an increasing proportion of small DNA fragments in genomic extracts, and decreasing quantitative PCR (QPCR) efficiency [higher cycle threshold (Ct) values]. DNA from end-production green tea extract was sequenced in order to try to validate material as the botanical of interest. Results: We saw a 41.1% decrease in mean extractable gDNA through farm processing (P < 0.01) and a 99.7% decrease through facility processing (P < 0.05). There was a 26.3% decrease in mean DNA fragment size through farm processing (P < 0.001) and an 82.0% decrease through facility processing (P < 0.05). QPCR efficiency was reduced through processing, marked by significant increases in Ct values with 100 base pair (bp) and 200 bp PCR targets (P < 0.05), and an inability to amplify 300 bp targets when using DNA template from end-production green tea extract. Conclusions: Although there was significant degradation and removal of DNA through processing, sufficiently intact DNA was able to be recovered from highly processed green tea extract for further sequencing and identification. Highlights: This work addresses a key gap in the understanding of DNA degradation through processing and provides useful information to consider when designing molecular diagnostic techniques for NHP identification.

Association of cell-free DNA (cfDNA) levels with myeloid-derived suppressor cells (MDSC) levels in blood of patients (pts) with muscle invasive (MI) and metastatic (met) bladder cancer (BC).

4528 Background: cfDNA can be detected in healthy individuals but higher concentrations are present in pts with cancer. MDSC are immature immunosuppressive cells that can be mobilized from bone marrow by tumor-related factors. Higher blood MDSC levels have been associated with worse outcomes in pts with solid tumors including BC. We assessed correlations between cfDNA and MDSC levels in pts with MIBC and met BC. Methods: Peripheral blood from pts with MIBC and met BC was collected in Streck BCT tubes and processed to obtain cf nucleic acid extracts. Total cfDNA was determined by fluorimetry. Cell-free DNA fragment size was measured by Bioanalyzer DNA analysis; 100-400 bp fragments (mono- and di-nucleosomal fragments linked to granulocytic processing of apoptotic and necrotic tumor cells) were designated low molecular weight (LMW-frags). The % of MDSC (CD33+/HLADR-) and subtypes were measured. MDSC subtypes were defined as polymorphonuclear (PMN-MDSC: CD15+/CD14-), monocytic (M-MDSC: CD15-/CD14+) and uncommitted (UNC-MDSC: CD15-/CD14-). Spearman’s correlation test was used for analysis. Results: Blood from 37 pts (19% women, 40% never smokers) with MIBC or met BC was collected: 15 (41%) with MIBC and 22 (59%) with met BC at time of collection. There was a significantly positive correlation between total MDSC and cfDNA levels (r = 0.57, P = 0.0003). Among MDSC subtypes, there was a significantly positive correlation between PMN-MDSC and cfDNA levels (r = 0.61, P &lt; 0.0001). The higher level of LMW-frags was significantly but moderately associated with higher total MDSC (r = 0.43, P 0.008) and PMN-MDSC (r = 0.41, P 0.01) levels. There was no significant correlation between cfDNA level and other MDSC subtypes. Conclusions: There was a positive correlation between total and PMN-MDSC with cfDNA levels in blood from pts with MIBC and met BC. That may suggest a putative role for MDSC in mediating cfDNA release into the circulation, consistent with prior reports of granulocyte-mediated ctDNA processing. Further studies need to identify mechanisms and implications of our findings and potential correlation with clinical outcomes.

Rasta Disease of Tomato in Ghana is Caused by the Pospiviroids Potato spindle tuber viroid and Tomato apical stunt viroid.

Rasta is a virus-like disease of unknown etiology affecting tomato (Solanum lycopersicum) plants in Ghana. Symptoms include stunting; epinasty, crumpling, and chlorosis of leaves; and necrosis of leaf veins, petioles, and stems. Leaf samples with rasta symptoms were collected from commercial tomato fields in Ghana in October 2012 and applied to FTA cards, and RNA extracts were prepared. Reverse-transcription polymerase chain reaction (RT-PCR) tests with primers for Columnea latent viroid, which causes rasta-like symptoms in tomato plants in Mali, were negative, whereas tests with degenerate viroid primer pairs were inconclusive. However, tomato seedlings (Early Pak 7) mechanically inoculated with RNA extracts of 10 of 13 samples developed rasta-like symptoms. In RT-PCR tests with RNA from leaves of the 10 symptomatic seedlings and primers for Potato spindle tuber viroid (PSTVd) or Tomato apical stunt viroid (TASVd), the expected size (approximately 360 bp) of DNA fragment was amplified from eight and two seedlings, respectively. Sequence analyses confirmed that these fragments were from PSTVd and TASVd isolates, and revealed a single PSTVd haplotype and two TASVd haplotypes. The PSTVd and TASVd isolates from Ghana had high nucleotide identities (>94%) with isolates from other geographic regions. In a host range study, PSTVd and TASVd isolates from Ghana induced rasta symptoms in the highly susceptible tomato cultivar Early Pak 7 and mild or no symptoms in Glamour, and symptomless infections in a number of other solanaceous species. PSTVd and TASVd isolates were seed associated and possibly seed transmitted.

A novel single tube amplification and barcoding approach for preimplantation genetic testing (PGT-A) for aneuploidy detectionusing Next Generation Sequencing

Introduction There are several methods to prepare embryo biopsy samples for Next Generation Sequencing (NGS) for Preimplantation Genetic Testing for Aneuploidy (PGT-A). DOPlify® provides a flexible technology to not only amplify whole genomes and target sequences using RHS’ target sequence enrichment (TSE) protocol, but also provides a mechanism to incorporate platform specific sequences for NGS. The use of PCR barcoding during whole genome amplification provides several laboratory efficiencies compared to sequential whole genome amplification followed by library preparation methods, including the reduction of hands on time, total protocol time and reagent requirements for sample preparation, all of which are equally relevant across small clinics through to high throughput service laboratories. Here we describe the development of a novel approach which allows PCR barcoding of PGT-A samples in a single tube using PCR that has been developed on multi- cell samples of known ploidy as a model of trophectoderm biopsy. Material and Methods Five-cell samples manually sorted from aneuploid cell lines (Coriell Institute) were prepared for sequencing using a two-stage, single tube protocol. DNA was amplified using standard DOPlify® kit reagents (RHS Ltd) then Ion Torrent (ThermoFisher) NGS adapter sequences and barcodes were subsequently incorporated utilising a second PCR step within the same tube. Incorporation of the adapter sequences at both the 5’ and 3’ ends of the amplified DNA was quantified using qPCR (Kapa Biosystems) with adapter sequence-specific primers (RHS Ltd). The barcoded samples were pooled and sequenced. The sequencing data was bioinformatically aligned to hg19, sequencing metrics collated and the data analysed to determine sample ploidy status. Results More than 8 different barcoding methods were trialled and 3 were successful. Measurable differences were observed for WGA DNA yield, DNA fragment size range and the 5’ and 3’ adapter sequence incorporation ratio (1:1-1:5). For the successful protocols, concordance of the sample karyotype was achieved for all cell lines sequenced (n=5 for each method). The most time efficient protocol produced amplified, sequencing ready samples within a total time of 3 hrs 30 min with a hands on time of about 30 mins, with further reduction of total protocol time to less than 3 hrs currently being evaluated. Additional time improvements may come from automation. Conclusions Leveraging the unique DOPlify® technology, this novel method provides a single tube amplification and barcoding protocol to allow rapid, scalable and economical sequencing of embryo biopsy samples for PGT-A using next generation sequencing. With the possibility of incorporating RHS’ target sequence enrichment (TSE) protocol, this novel PCR barcoding approach will then allow combined PGT-M and PGT-A.

Investigation of direct counting and sizing of DNA fragments in flow applying an improved data analysis and correction method

Direct detection of single stained DNA fragments in flow is a very sensitive method for nucleic acid detection which does not need any amplification process. We have developed an instrument for direct counting and sizing of single DNA fragments (single or double stranded DNA) in flow with integrated sample volume measurement for concentration determination. As the method is a potential reference method for DNA quantification, processes affecting the measurement uncertainty are of major interest. Additionally, comparison of this method to the orthogonal method of digital PCR is useful with the restriction of low specificity of the direct detection method. In this study, we analysed raw detector signals and the sizing performance for target identification and the effect of coincidence detection concerning concentration measurements. We present data of purified artificial DNA samples measured with the home-built setup. Main emphasis was to develop an improved data analysis method to gain insight into and carefully correct for coincident detection of DNA fragments and for estimation of the amount of fragment dimers.

Drainage of Tumor-Derived DNA into Sentinel Lymph Nodes in Breast Cancer Patients.

Circulating tumor DNA (ctDNA) is released from cancer cells by apoptosis or other mechanisms, and as tumor tissue contains both blood and lymphatic vessels, ctDNA can spread to local lymph nodes (LNs). We aimed to detect the tumor-derived free DNA in metastasis-free LNs in patients with breast cancers harboring the PIK3CA-H1047R mutation. One hundred twenty-three patients were evaluated and the PIK3CA-H1047R mutation was assayed in sentinel LNs (SLNs), non-SLNs without metastasis, and serum by digital PCR. The mutant DNA was more frequent in metastasis-free SLNs (21.6%) than in metastasis-free non-SLNs (8.6%; P = 0.038), and patients with mutation-positive SLNs were more likely to be positive for serum mutant DNA. Apoptosis in primary breast tumors was determined by TUNEL assay. The apoptotic index was significantly higher (P = 0.003) in patients with mutation-positive SLNs without metastasis (mean, 1.17%) than those with mutation-negative SLNs without metastasis (mean, 0.79%). It was also significantly higher (P = 0.006) in those with mutation-positive serum (mean, 1.41%) than in those with mutation-negative serum (mean, 0.86%). Furthermore, fragment size of PIK3CA-H1047R mutant DNA in metastatic-free SLN lysate used for the one-step nucleic acid amplification (OSNA) assay was short (<500bp). These results support the theory that DNA is released from the primary tumor via apoptotic fragmentation. In conclusion, ctDNA is detectable in metastasis-free LNs and significantly more frequent in SLNs from patients with breast tumors harboring a high apoptotic index, consistent with drainage of ctDNA from apoptotic primary tumor cells into SLNs.

Attempts to Identify Cassava Brown Streak Virus in Western Democratic Republic of Congo

Root necrosis similar to those of the cassava brown streak disease (CBSD) were observed on cassava in western provinces of the Democratic Republic of Congo (DR.Congo) in the early 2000&amp;rsquo;s. However molecular laboratory diagnosis were not able to detect any causative agent responsible for the attacks, hence, the disease related to these symptoms was named CBSD-like disease.&#x0D; &#x0D; In order to assess the distribution and the incidence of the CBSD-like disease, surveys were carried out in four western provinces, comprising, Kwango and Kwilu, Sud Ubangi, Kinshasa and Kongo Central. CBSD-like disease was observed in all surveyed provinces on the basis of root symptoms because foliar symptoms were different to those of the documented cases of CBSD in other parts of east Africa. CBSD-like disease incidence was high in Kongo Central and Sud Ubangi, exceeding an average of 50 %, but low in Kwango and Kwilu (32.8%) and in Kinshasa (19.1%). &#x0D; &#x0D; During the surveys, cassava leaf samples were collected for lab identification of the causal agent. PCR diagnosis was done on these samples using primers specific for the two known CBSVs. All samples tested negative with no amplification of DNA fragments of the correct size.&amp;nbsp; Thus, further analysis on the causative organism is needed using Next Generation Sequencing (NGS) approaches. NGS approaches will help also to identify the causative organism in other Central Africa countries (Angola, Congo-Brazzaville and Gabon) where such cassava root necrosis have been reported or are suspected.

Enhanced detection of circulating tumor DNA by fragment size analysis.

Existing methods to improve detection of circulating tumor DNA (ctDNA) have focused on genomic alterations but have rarely considered the biological properties of plasma cell-free DNA (cfDNA). We hypothesized that differences in fragment lengths of circulating DNA could be exploited to enhance sensitivity for detecting the presence of ctDNA and for noninvasive genomic analysis of cancer. We surveyed ctDNA fragment sizes in 344 plasma samples from 200 patients with cancer using low-pass whole-genome sequencing (0.4×). To establish the size distribution of mutant ctDNA, tumor-guided personalized deep sequencing was performed in 19 patients. We detected enrichment of ctDNA in fragment sizes between 90 and 150 bp and developed methods for in vitro and in silico size selection of these fragments. Selecting fragments between 90 and 150 bp improved detection of tumor DNA, with more than twofold median enrichment in >95% of cases and more than fourfold enrichment in >10% of cases. Analysis of size-selected cfDNA identified clinically actionable mutations and copy number alterations that were otherwise not detected. Identification of plasma samples from patients with advanced cancer was improved by predictive models integrating fragment length and copy number analysis of cfDNA, with area under the curve (AUC) >0.99 compared to AUC <0.80 without fragmentation features. Increased identification of cfDNA from patients with glioma, renal, and pancreatic cancer was achieved with AUC > 0.91 compared to AUC < 0.5 without fragmentation features. Fragment size analysis and selective sequencing of specific fragment sizes can boost ctDNA detection and could complement or provide an alternative to deeper sequencing of cfDNA.

Fragment size and level of cell-free DNA provide prognostic information in patients with advanced pancreatic cancer

BackgroundIt was recently demonstrated that the size of cell-free DNA (cfDNA) fragments that originates from tumor cells are shorter than cfDNA fragments that originates from non-malignant cells. We investigated whether cfDNA fragment size and cfDNA levels might have prognostic value in patients with advanced pancreatic cancer.MethodsBlood samples were obtained from patients with advanced pancreatic cancer, before (n = 61) initiation of chemotherapy and after the first cycle of chemotherapy (n = 39). Samples were separated with density centrifugation and plasma DNA was isolated. Mode cfDNA fragment size and cfDNA levels were then determined using a 2100 Bioanalyzer. A cohort of partially age-matched healthy volunteers (n = 28) constituted the control group.ResultsBoth a pre-treatment cfDNA fragment size of ≤ 167 bp (mode) and high pre-treatment cfDNA levels were associated with shorter progression-free survival (PFS) (p = 0.002 and p < 0.001, respectively) and overall survival (OS) (p = 0.001 and p = 0.001, respectively). Furthermore, multivariable Cox regression analyses demonstrated that pre-treatment cfDNA levels could independently predict prognosis for both PFS (HR = 3.049, p = 0.005) and OS (HR = 2.236, p = 0.028).ConclusionThis study demonstrates that cfDNA fragment size and cfDNA levels can be used to predict disease outcome in patients with advanced pancreatic cancer. The described approach, using a rapid, economic and simple test to reveal prognostic information, has potential for future treatment stratification and monitoring.

Variable IGF1R mRNA Expression in Individual GV and In Vitro Matured M2 Human Oocytes

Objective Characterization of human insulin-like growth factor 1 receptor (IGF1R) mRNA expression in individual human oocytes to better understand the role of biologically significant mRNAs in establishing oocyte quality following ovarian hyperstimulation for in vitro fertilization (IVF) treatment. Design Germinal vesicle oocytes (GV) were identified after removal of all cumulus cells prior to ICSI and collected from consenting patients with IRB-approval. Materials and Methods Single GVs were placed directly into lysis buffer (5μl). Oocytes in vitro matured (IVM) to the M2 stage were placed into individual tubes containing lysis buffer after 24 to 48 hours (h) in culture. Total RNA isolated from cumulus granulosa cells (GC) using the TRIzol reagent (Invitrogen) as stated in the manufacturer’s protocol served as a positive control. Reverse Transcription (RT) was performed using anchored oligo-dT primers (IDT, Coralville, IA) and Moloney Murine Leukemia Virus reverse transcriptase (Invitrogen) on the entire 5μl oocyte lysate at 37°C for 1.5h or 1mg total GC RNA 42°C for 1h. Quantitative polymerase chain reaction (qPCR) was performed combining standards or 1μl cDNA with PCR buffer containing SYBR Green I (Sigma Chemical Co, St. Louis, Missouri), Taq polymerase (Genscript, Piscataway, NJ), and primers for IGF1R. Melting curves were routinely determined to ascertain generation of only the expected product. Results qPCR performed on GC cDNA demonstrated detection of the expected product under the reaction conditions in use. Gel electrophoresis of the PCR product confirmed the size of the DNA fragment generated. qPCR analysis detected a signal for IGF1R mRNA in 9 of the 15 GVs analyzed from 9 patients. Moreover, for 3 patients with multiple GVs, differences in IGF1R rnRNA expression were seen between individual GV oocytes, as well as between oocytes from different patients. Similarly, 8 of 12 IVM M2 oocytes from 11 patients gave a signal for the presence of IGF1R mRNA. Conclusions These results report the use of a direct-lysis method for the extraction and analysis of mRNA from single human oocytes. These data suggest that GV oocytes failing to respond to the hCG trigger during ovarian stimulation for IVF vary in their biological integrity. Furthermore, we observed that GV oocytes advanced to the M2 stage in vitro in the absence of any demonstrable IGF1R mRNA expression. Molecular assessment of single human oocytes can be used to better understand the low per-oocyte success rate in human IVF. Disclosures Nothing to disclose. Funding None.

Plasmid Profile Analysis of Multi-drug Resistant Proteus spp isolated from Patients with Wound Infection in Northeastern Nigeria

Aims: We analysed the prevalence of plasmid mediated multidrug resistance among Proteus spp isolated from wound infection patients attending healthcare centers in Maiduguri.&#x0D; Methodology: 320 wound swab samples were collected from August 2016 to June 2017, and investigated via microscopy, cultured on Blood agar and MacConkey agar. Suspected Proteus spp isolates were further confirmed using biochemical tests. Kirby bauer disc diffusion test was used to determine the antimicrobial susceptibility pattern. Isolates confirmed to be multidrug resistant (MDR) were subjected to gel electrophoresis for the determination of plasmid profile.&#x0D; Results: Twenty eight (28) samples yielded Proteus spp, giving a prevalence rate of 8.75%. Proteus mirabilis was the most significant specie isolated (32.14%). Isolates were most sensitive/least resistant to Ciprofloxacin (85.71%/14.29%) and most resistant/least sensitive to Augmentin (10.71%/89.29%). 64.28% of isolates observed were MDR strains and were quite significant among patients between the ages of 21-30years (21.43%). MDR Proteus mirabilis was most the significant and highly prevalent among patients suffering from wound sepsis and burns (10.71% respectively). The association between MDR Proteus spp and wound types was statistically not significant (X2 =7.342, p&gt;0.01). Plasmid profile analysis revealed that 72.22% of the MDR isolates harbour plasmids with a DNA fragment size of 100 bp and a molecular weight of 31ng/10µl. An average of 7.22% of MDR isolates were cured of their plasmids while an average of 56.67% of MDR isolates resisted curing.&#x0D; Conclusion: Here, we report a high prevalence of multidrug resistance and a high rate of plasmid carrying strains of Proteus spp in wounds of hospitalised patients. We suggest that there is a role played by plasmid in the mediation of multidrug resistance among the MDR Proteus spp isolated, where the majority of the MDR isolates observed carry plasmids.

Plasmid Profile of Multi-drug Resistant Phenotypes of Pseudomonas aeruginosa Isolated among Patients with Indwelling Catheter in Northeastern Nigeria

Aims: This study was carried out to analyze the plasmid profile of multidrug resistant Pseudomonas aeruginosa isolated among catheterized patients attending the University of Maiduguri Teaching Hospital.&#x0D; Place of Study: Department of Medical Microbiology (Laboratory Section), University of Maiduguri Teaching Hospital and Department of Biological Sciences, Abubakar Tafawa Balewa University, Nigeria.&#x0D; Methodology: 244 samples (catheter tip, urethral swab, urine) were collected from catheterised patients and investigated via microscopy and culture on Blood agar and MacConkey agar. Suspect Pseudomonas aeruginosa isolates were further confirmed using biochemical tests. Kirby bauer disc diffusion test was used to determine the antimicrobial susceptibility pattern. Isolates confirmed to be multidrug resistant (MDR) were subjected to plasmid profile analysis using agarose gel electrophoresis.&#x0D; Results: 21 yielded Pseudomonas aeruginosa which gives a recovery rate of 8.6%. A significant proportion was isolated from catheter tip samples collected from male patients (33.33%). The association between sex of patient and sample type in the isolation of Pseudomonas aeruginosa was statistically significant (X2 = 10.76, df = 2, P &lt;.01). Isolates were most-sensitive/least-resistant to Ofloxacin and Ampiclox, and least-sensitive/most-resistant to Penicillin. All isolates identified were multi-drug resistant (MDR) with an average resistance rate of 3.28 antimicrobials per isolate. Plasmid analysis revealed that 57.14% of isolates possessed similar plasmid with a DNA fragment size of 300bp and a molecular weight of 31 ng/10 µl.&#x0D; Conclusion: We establish a very high rate of multidrug resistance among Pseudomonas aeruginosa isolates. Plasmid profile analysis of MDR Pseudomonas aeruginosa observed revealed a high plasmid prevalence rate and since most isolates cannot express the resistance marker after plasmid curing, we suggest that this is indicative of the plasmidial origin of such a marker.

Differentiation and quantification of the ochratoxin A producers Aspergillus ochraceus and Aspergillus westerdijkiae using PCR‐DGGE

Ochratoxin A (OTA) is a nephrotoxic, teratogenic, immunotoxic, and carcinogenic mycotoxin which is produced in tropical zones mainly by Aspergillus carbonarius, A. niger, A. ochraceus, and A. westerdijkiae. A. ochraceus and A. westerdijkiae species are phenotypically and genomically very close but A. westerdijkiae produce OTA at a very higher level than A. ochraceus. These species have been differentiated recently. The DNA primer pairs which were drawn so far are not specific and a genomic region of the same size is amplified for both species or they are too specific, and in this case, the DNA of a single species is amplified. To help preventing OTA contamination of foodstuffs, the PCR-DGGE (Denaturing Gradient Gel Electrophoresis) method was used to discriminate between A. ochraceus and A. westerdijkiae DNA fragments of the same size but with different sequences and thus faster access to a diagnosis of the toxigenic potential of the fungal microflora. The proposed methodology was able to differentiate A. westerdijkiae from A. ochraceus with only one primer pairs in a single run. A calibration based on initial DNA content was obtained from image analysis of the DGGE gels and a method of quantification of the two strains was proposed.

Morphological and Molecular Analysis of Isolated Cultures of Tobacco Adventitious Roots Obtained by the Methods of Biolistic Bombardment and Agrobacterium-Mediated Transformation

Plant infection with Agrobacterium rhizogenes leads to the development of a hairy root disease notable for the rapid agravitropic growth of roots on hormone-free nutrient media. In order to look into the interaction of A. rhizogenes with plants and assess opportunities of practical application of hairy root culture, new approaches to their production are elaborated. A method of bacterium-free and plasmid-free production of genetically modified roots (hairy roots) by means of biolistic transformation of leaf explants with a DNA fragment (size of 5461 bp) consisting of genes rolA, rolB, rolC, and rolD are proposed. In most cases, such transformation resulted in the emergence of only adventitious roots with transient expression of rol-genes, and the growth of such roots on hormone-free media ceased in 2–3 months in contrast to genuine hairy roots capable of unrestricted growth. Molecular analysis of different systems of target genes’ expression showed an important role of transgene rolC and host gene of cyclin-dependent protein kinase CDKB1-1 in the maintenance of rapid growth of hairy roots in vitro (in isolated cultures).