Single cell transcriptome analysis using next generation sequencing.

The heterogeneity of tissues, especially in cancer research, is a central issue in transcriptome analysis. In recent years, research has primarily focused on the development of methods for single cell analysis. Single cell analysis aims at gaining (novel) insights into biological processes of healthy and diseased cells. Some of the challenges in transcriptome analysis concern low abundance of sample starting material, necessary sample amplification steps and subsequent analysis. In this study, two fundamentally different approaches to amplification were compared using next-generation sequencing analysis: I. exponential amplification using polymerase-chain-reaction (PCR) and II. linear amplification. For both approaches, protocols for single cell extraction, cell lysis, cDNA synthesis, cDNA amplification and preparation of next-generation sequencing libraries were developed. We could successfully show that transcriptome analysis of low numbers of cells is feasible with both exponential and linear amplification. Using exponential amplification, the highest amplification rates up to 106 were possible. The reproducibility of results is a strength of the linear amplification method. The analysis of next generation sequencing data in single cell samples showed detectable expression in at least 16.000 genes. The variance between samples results in a need to work with a greater amount of biological replicates. In summary it can be said that single cell transcriptome analysis with next generation sequencing is possible but improvements leading to a higher yield of transcriptome reads is required. In the near future by comparing single cancer cells with healthy ones for example, a basis for improved prognosis and diagnosis can be realised

Amplification of Angular Rotations Using Weak Measurements

We present a weak measurement protocol that permits a sensitive estimation of angular rotations based on the concept of weak-value amplification. The shift in the state of a pointer, in both angular position and the conjugate orbital angular momentum bases, is used to estimate angular rotations. This is done by an amplification of both the real and imaginary parts of the weak-value of a polarization operator that has been coupled to the pointer, which is a spatial mode, via a spin-orbit coupling. Our experiment demonstrates the first realization of weak-value amplification in the azimuthal degree of freedom. We have achieved effective amplification factors as large as 100, providing a sensitivity that is on par with more complicated methods that employ quantum states of light or extremely large values of orbital angular momentum.Comment: 5 pages, 3 figures, contains supplementary informatio

Clinical and molecular characterization of HER2 amplified-pancreatic cancer

<p>Background: Pancreatic cancer is one of the most lethal and molecularly diverse malignancies. Repurposing of therapeutics that target specific molecular mechanisms in different disease types offers potential for rapid improvements in outcome. Although HER2 amplification occurs in pancreatic cancer, it is inadequately characterized to exploit the potential of anti-HER2 therapies.</p> <p>Methods: HER2 amplification was detected and further analyzed using multiple genomic sequencing approaches. Standardized reference laboratory assays defined HER2 amplification in a large cohort of patients (n = 469) with pancreatic ductal adenocarcinoma (PDAC).</p> <p>Results: An amplified inversion event (1 MB) was identified at the HER2 locus in a patient with PDAC. Using standardized laboratory assays, we established diagnostic criteria for HER2 amplification in PDAC, and observed a prevalence of 2%. Clinically, HER2- amplified PDAC was characterized by a lack of liver metastases, and a preponderance of lung and brain metastases. Excluding breast and gastric cancer, the incidence of HER2-amplified cancers in the USA is >22,000 per annum.</p> <p>Conclusions: HER2 amplification occurs in 2% of PDAC, and has distinct features with implications for clinical practice. The molecular heterogeneity of PDAC implies that even an incidence of 2% represents an attractive target for anti-HER2 therapies, as options for PDAC are limited. Recruiting patients based on HER2 amplification, rather than organ of origin, could make trials of anti-HER2 therapies feasible in less common cancer types.</p&gt

Comment on "A proposed method for measuring the electric dipole moment of the neutron using acceleration in an electric field gradient and ultracold neutron interferometry"

We discuss the proposal of Freedman, Ringo, and Dombeck to search for the neutron electric dipole moment use of the acceleration of neutrons in an inhomogeneous electric field followed by amplification of the resulting displacement by several methods involving spin independent interactions (gravity) or reflection from curved (spin independent) mirrors. We show that the proposed technique is inferior to the usual methods based on magnetic resonance.Comment: Prevous vesion on xxx is to be published in Nucl. Instr. and Methods. Sec. IIb of previous version has been reworked to address the reply of Dombeck and Ring

Examining peak height ratios in low template DNA samples with and without sampling using a single-tube extraction protocol

The developments of the polymerase chain reaction (PCR) and the short tandem repeat multiplex kits increased the ease and lowered the time and sample quantity required for deoxyribonucleic acid (DNA) typing compared to previous methods. However the amplification of low mass of DNA can lead to increased stochastic effects, such as allele drop-out (ADO) and heterozygous peak height (PH) imbalance, which make it difficult to determine the true donor profile. These stochastic effects are believed to be due to: 1) pre-PCR sampling from pipetting and sample transferal of dilute samples prior to amplification resulting in unbalanced heterozygous allele templates in the amplification reaction, and 2) the kinetics of the PCR process where, when few target templates are available, there is uneven amplification of heterozygous alleles during early PCR cycles. This study looks to examine the contribution of PCR chemistry and pre-PCR sampling errors on stochastic effects by utilizing a single-tube DNA extraction and direct amplification method. Cells were collected into tubes using the McCrone and Associates, Inc. cell transfer method, which allowed for approximation of DNA mass without quantification. The forensicGEM® Saliva Kit was used to lyse the cells and inactivate nucleases without inhibiting downstream amplification. The samples were then directly amplified with the AmpFLSTR® Identifiler® Plus PCR Amplification Kit. These samples should only show the effects of PCR chemistry since pipetting and tube transferal steps prior to amplification were removed with the expectation that equal numbers of heterozygous alleles are present in the sample pre-amplification. Comparisons of PH imbalance were made to samples extracted with forensicGEM® but had one or more pipetting and tube transferal steps prior to amplification. These samples were either created through the dilution of stock DNA or from the cell transfer method where aliquots were then taken for amplification; thus these samples would exhibit the effects of both pre-PCR sampling and PCR chemistry errors and inefficiencies. The use of carrier ribonucleic acid (cRNA) was also added to cell transfer samples prior to the amplification of samples to see if it assisted with amplification and increased signal. Results show that the samples with only PCR chemistry generally have significantly higher mean peak height ratios (PHRs) than samples with both pre-PCR sampling and PCR chemistry except in cases where there were large numbers of ADOs. When compared to the diluted samples, the cell transfer samples had significantly higher mean PHR at 0.0625 ng and 0.125 ng, and higher mean PHR at 0.0375 ng when PHs from ADOs are included. Average peak heights (APHs) in the cell transfer samples were also significantly higher in these comparisons. When compared to aliquots taken from cell transfer samples, mean PHR was significantly higher at 0.0625 ng in cell transfer samples with only PCR chemistry than cell transfer samples with both pre-PCR sampling and PCR chemistry; however APH for the samples with only PCR chemistry was also significantly higher in one experiment and not significantly different in another. In a third experiment, the difference in mean PHR was not significant while APH was significantly higher in the samples with pre-PCR sampling and PCR chemistry; however there were also a large numbers of ADOs. Our results also found quantification of dilute samples unreliable but cell counting through the cell transfer method is an appropriate alternative for DNA mass approximation. Also there were no significant changes in PHR or APH in the presence or absence of cRNA

Microfluidic method for rapid turbidimetric detection of the DNA of Mycobacterium tuberculosis using loop-mediated isothermal amplification in capillary tubes

We describe a microfluidic method for rapid isothermal turbidimetric detection of the DNA of Mycobacterium tuberculosis. Loop-mediated isothermal amplification is accomplished in capillary tubes for amplifying DNA in less than 15 min, and sensitivity and specificity were compared to conventional loop-mediated isothermal amplification (LAMP). The method can detect as little as 1 pg mL−1 DNA in a sample. Results obtained with clinical specimens indicated 90 % sensitivity and 95 % specificity for microfluidic LAMP in comparison to culture methods. No interference occurred due to the presence of nonspecific DNAs. The findings demonstrate the power of the new microfluidic LAMP test for rapid molecular detection of microorganisms even when using bare eyes. © 2014, Springer-Verlag Wien

Performance of four modern whole genome amplification methods for copy number variant detection in single cells

Whole genome amplification (WGA) has become an invaluable tool to perform copy number variation (CNV) detection in single, or a limited number of cells. Unfortunately, current WGA methods introduce representation bias that limits the detection of small CNVs. New WGA methods have been introduced that might have the potential to reduce this bias. We compared the performance of PicoPLEX DNA-Seq (Picoseq), DOPlify, REPLI-g and Ampli-1 WGA for aneuploidy screening and copy number analysis using shallow whole genome massively parallel sequencing (MPS), starting from single or a limited number of cells. Although the four WGA methods perform differently, they are all suited for this application