Absolute quantification of protein copy number using a single-molecule-sensitive microarray

Plasmids play a major role in bacterial adaptation to environmental stress and often contribute to antibiotic resistance and disease virulence. Although the complete sequence of each plasmid is essential for studying plasmid biology, most antibiotic resistance and virulence plasmids in Salmonella are present only in a low copy number, making extraction and sequencing difficult. Long read sequencing technologies require higher concentrations of DNA to provide optimal results. To resolve this problem, we assessed the sufficiency of multiple displacement amplification (MDA) for replicating Salmonella plasmid DNA to a satisfactory concentration for accurate sequencing and multiplexing. Nine Salmonella enterica isolates, representing nine different serovars carrying plasmids for which sequence data are already available at NCBI, were cultured and their plasmids isolated using an alkaline lysis extraction protocol. We then used the Phi29 polymerase to perform MDA, thereby obtaining enough plasmid DNA for long read sequencing. These amplified plasmids were multiplexed and sequenced on one single molecule, real-time (SMRT) cell with the Pacific Biosciences (Pacbio) Sequel sequencer. We were able to close all Salmonella plasmids (sizes ranged from 38 to 166 Kb) with sequencing coverage from 24 to 2,582X. This protocol, consisting of plasmid isolation, MDA, and multiplex sequencing, is an effective and fast method for closing high-molecular weight and low-copy-number plasmids. This high throughput protocol reduces the time and cost of plasmid closure.

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Prospect and Competence of Quantitative Methods via Real-time PCR in a Comparative Manner: An Experimental Review of Current Methods

The Open Bioinformatics Journal ◽

10.2174/1875036201811010001 ◽

2018 ◽

Vol 11 (1) ◽

pp. 1-11

Author(s):

Hossein Mahboudi ◽

Negin Mohammadizadeh Heidari ◽

Zahra Irani Rashidabadi ◽

Ali Houshmand Anbarestani ◽

Soroush Karimi ◽

...

Keyword(s):

Internal Control ◽

Copy Number ◽

Quantitative Methods ◽

Absolute Quantification ◽

Relative Quantification ◽

Absolute Quantitation ◽

Standard Curve ◽

Qpcr Quantification ◽

Comparative Manner

Background: There are numerous approaches dealing with relative and absolute quantitation. The methods differ in their efficiency assumption and applicability. Objective: Current methodologies and rations used in qPCR quantification were compared in an experimental study of transgenic copy number determination of a monoclonal antibody Daclizumab. Methods: With an inter and intra-methodical view, variations in relative and absolute quantification strategies were discretely extracted and compared to one another. Results: In relative quantification, six methods were studied and the ratios were computed relative to Glucagon as internal control. For Absolute quantification, the calculations were based on standard curve. Relative quantification considers the relative changes in expression levels while Absolute quantification relates the PCR signal to input copy number with a calibration curve. Conclusion: The observed unevenness of the ratios in Relative approach pointed mainly to the efficiency changes and its calculation formula. Whereas results in Absolute approach strategies showed homogeneity which indicates the consistency of the calculation method.

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BRCA Testing by Single-Molecule Molecular Inversion Probes

Clinical Chemistry ◽

10.1373/clinchem.2016.263897 ◽

2017 ◽

Vol 63 (2) ◽

pp. 503-512 ◽

Cited By ~ 28

Author(s):

Kornelia Neveling ◽

Arjen R Mensenkamp ◽

Ronny Derks ◽

Michael Kwint ◽

Hicham Ouchene ◽

...

Keyword(s):

Genetic Testing ◽

Copy Number Variation ◽

Single Molecule ◽

Copy Number ◽

Single Gene ◽

Work Flow ◽

Analytical Sensitivity ◽

Brca1 And Brca2 ◽

Number Variation ◽

Molecular Inversion Probes

Abstract BACKGROUND Despite advances in next generation DNA sequencing (NGS), NGS-based single gene tests for diagnostic purposes require improvements in terms of completeness, quality, speed, and cost. Single-molecule molecular inversion probes (smMIPs) are a technology with unrealized potential in the area of clinical genetic testing. In this proof-of-concept study, we selected 2 frequently requested gene tests, those for the breast cancer genes BRCA1 and BRCA2, and developed an automated work flow based on smMIPs. METHODS The BRCA1 and BRCA2 smMIPs were validated using 166 human genomic DNA samples with known variant status. A generic automated work flow was built to perform smMIP-based enrichment and sequencing for BRCA1, BRCA2, and the checkpoint kinase 2 (CHEK2) c.1100del variant. RESULTS Pathogenic and benign variants were analyzed in a subset of 152 previously BRCA-genotyped samples, yielding an analytical sensitivity and specificity of 100%. Following automation, blind analysis of 65 in-house samples and 267 Norwegian samples correctly identified all true-positive variants (>3000), with no false positives. Consequent to process optimization, turnaround times were reduced by 60% to currently 10–15 days. Copy number variants were detected with an analytical sensitivity of 100% and an analytical specificity of 88%. CONCLUSIONS smMIP-based genetic testing enables automated and reliable analysis of the coding sequences of BRCA1 and BRCA2. The use of single-molecule tags, double-tiled targeted enrichment, and capturing and sequencing in duplo, in combination with automated library preparation and data analysis, results in a robust process and reduces routine turnaround times. Furthermore, smMIP-based copy number variation analysis could make independent copy number variation tools like multiplex ligation-dependent probes amplification dispensable.

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Telomere Shortening linked to Disability and Mitochondrial DNA Copy Number in Patients with Relapsing-Remitting Multiple Sclerosis

10.20944/preprints202112.0026.v1 ◽

2021 ◽

Author(s):

José Alfonso Cruz-Ramos ◽

Gabriela del Carmen López-Armas ◽

Eduardo Ignacio Díaz-Barba ◽

Mónica Navarro-Meza ◽

Miguel Ángel Macías-Islas ◽

...

Keyword(s):

Multiple Sclerosis ◽

Mitochondrial Dna ◽

Copy Number ◽

Binary Logistic Regression ◽

Peripheral Blood Leukocyte ◽

Absolute Quantification ◽

Severe Disability ◽

Dna Copy Number ◽

Binary Logistic Regression Model ◽

Mitochondrial Dna Copy Number

Multiple sclerosis (MS) is a chronic autoimmune inflammatory disease that affects the nervous system. Peripheral blood leukocyte telomere length (LTL) and mitochondrial DNA copy number (mtDNA-CN) are potential biomarkers of disability and neurological damage. The present work evaluated LTL and mtDNA-CN in 75 relapsing-remittent MS (RRMS) patients 50 of whom had an Expanded Disability Status Scale (EDSS) 0 to 3 (mild-moderate disability), and 25 had an EDSS of 3.5 to 7 (severe disability). Absolute LTL and absolute mtDNA-CN were measured via real-time polymerase chain reaction (qPCR). The LTL and mtDNA-CN were significantly lower in RRMS severe disability than in RRMS mild-moderate disability (3.924 ± 0.124 vs 2.854 ± 0.092, p<00001; 75.14 ± 1.77 vs 68.06 ± 1.608, p<0.00001, respectively). The LTL and mtDNA-CN showed a linear correlation in RRMS with mild-moderate disability (r=0.2986, p=0.0351). In addition, in a binary logistic regression model the LTL can predict severe disability (AUC=0.697, p=0.0031, cutoff ≤ 3.0875 Kb, sensitivity= 73.1%, specificity=62.5%), the prediction is improved by including age to the model (AUC=0.765, <0.0001, sensitivity=78.26%, specificity=81.25%). Aging is closely linked to the development of disability in RRMS and can be evaluated through LTL and mtDNA-CN absolute quantification.

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High Blood BAALC Copy Numbers Determined By Digital Droplet PCR at Timepoint of Allogeneic Transplantation in Complete Remission Predicts Relapse in Patients with Acute Myeloid Leukemia

Blood ◽

10.1182/blood.v128.22.517.517 ◽

2016 ◽

Vol 128 (22) ◽

pp. 517-517

Author(s):

Madlen Jentzsch ◽

Marius Bill ◽

Julia Schulz ◽

Juliane Grimm ◽

Stefanie Beinicke ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Copy Number ◽

Myeloid Leukemia ◽

Research Funding ◽

Residual Disease ◽

Absolute Quantification ◽

Prognostic Impact ◽

Healthy Controls ◽

Copy Numbers ◽

Acute Myeloid

Abstract Allogeneic hematopoietic cell transplantation (HSCT) is a powerful consolidation option for acute myeloid leukemia (AML) patients (pts) in hematologic complete remission (CR). Disease recurrence after HSCT remains a major clinical problem & early identification of AML pts at risk of relapse is crucial to improve outcomes. High expression of the AML associated gene BAALC (Brain and acute leukemia, cytoplasmic) at diagnosis adversely impacts on outcomes in AML pts. Little is known about its prognostic capacity during disease course & as a marker of residual disease. Here we adopted digital droplet polymerase chain reaction (ddPCR) for absolute quantification of BAALC copy numbers in peripheral blood (PB) prior to HSCT in AML pts in hematologic CR. We identified 82 AML pts with PB in first (60%) or second CR (23%) or CRi (17%) up to 28 days prior to HSCT available. Median age at HSCT was 63.9 (range 50.8-76.2) years (y). All pts received non-myeloablative (NMA) conditioning (fludarabine 3x30 mg & 2 Gy total body irradiation). At diagnosis, mutation status (mut) of the NPM1, CEBPA, IDH1, IDH2,& DNMT3A gene & presence of FLT3-ITD or FLT3-TKD were assessed. In pre-HSCT PB, absolute quantification of BAALC copy numbers was performed by ddPCR & results were normalized to ABL1 copy numbers.Additionally, absolute BAALC copy numbers wereassessedin PB of healthy controls (n=7) with a median age of 62.7 (range 39.6-82.0) y. Pts were grouped according to the European LeukemiaNet (ELN) classification in 21% favorable, 23% intermediate-I, 24% intermediate-II, 23% adverse & 9% unknown. Pts & healthy control were evenly matched in age (P=1) & sex (P=1). BAALC/ABL1 copy numbers did not differ between AML pts at HSCT (median 0.03 [range 0.01-2.48]) & the healthy controls (median 0.04 [range 0.03-0.10], P=.34, Figure 1). A cut-off point of 0.14absolute BAALC/ABL1 copies was determined using the R package 'OptimalCutpoints' & used to define pts with high (26%) & low (74%) pre-HSCT BAALC/ABL1 copy numbers. The copy number at this cut-off point was higher than the two-fold standard deviation over the median of the healthy controls (0.10 BAALC/ABL1). Pts with high & low pre-HSCT BAALC/ABL1 copy numbers did not differ significantly in pre-treatment characteristics (i.e. hemoglobin, white blood count, platelets, blasts in bone marrow or PB, ELN genetic group, FLT3-ITD, FLT3-TKD, NPM1, CEBPA, DNMT3A, IDH1 or IDH2 mut) or remission status at HSCT (CR1 vs. CR2 vs. CRi). However, pts with high pre-HSCT BAALC/ABL1 copy numbers had a significantly higher cumulative incidence of relapse (CIR, P=.02, Figure 2a) & shorter overall survival (OS, P=.02, Figure 2b). High pre-HSCT BAALC/ABL1 copy numbers especially impacted on CIR when we restricted our analysis to pts with normal cytogenetics (P=.003). In multivariate analysis for the entire cohort, high pre-HSCT BAALC/ABL1 copy numbers retained the prognostic impact on CIR (Hazard Ratio [HR] 3.6, Confidence Interval [CI] 1.6-8.2, P=.002) after adjustment for disease status at HSCT (P=.006) & the prognostic impact on OS (HR 2.2, CI 1.1-4.3, P=.02). In conclusion, ddPCR is a feasible method for absolute quantification of BAALC copy numbers in PB, which may indicate residual disease burden in AML pts. High PB BAALC/ABL1 copy numbers (>0.14) in AML pts in hematologic CR at HSCT associated with higher CIR & shorter OS in univariate & multivariate models. AML pts with high PB BAALC/ABL1 copy numbers at HSCT should be closely monitored for relapse in the post-transplant period. In the future prospective studies will be required to validate the absolute PB BAALC/ABL1 copy number cut-off point & to evaluate whether AML pts with high BAALC/ABL1 copy numbersmight benefit from additional treatment before HSCT. Figure 1 Figure 1. Figure 2 Figure 2. Disclosures Poenisch: Mundipharma: Research Funding. Niederwieser:Amgen: Speakers Bureau; Novartis Oncology Europe: Research Funding, Speakers Bureau.

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