A High-Throughput Method for Illumina RNA-Seq Library Preparation.

With the introduction of cost effective, rapid, and superior quality next generation sequencing techniques, gene expression analysis has become viable for labs conducting small projects as well as large-scale gene expression analysis experiments. However, the available protocols for construction of RNA-sequencing (RNA-Seq) libraries are expensive and/or difficult to scale for high-throughput applications. Also, most protocols require isolated total RNA as a starting point. We provide a cost-effective RNA-Seq library synthesis protocol that is fast, starts with tissue, and is high-throughput from tissue to synthesized library. We have also designed and report a set of 96 unique barcodes for library adapters that are amenable to high-throughput sequencing by a large combination of multiplexing strategies. Our developed protocol has more power to detect differentially expressed genes when compared to the standard Illumina protocol, probably owing to less technical variation amongst replicates. We also address the problem of gene-length biases affecting differential gene expression calls and demonstrate that such biases can be efficiently minimized during mRNA isolation for library preparation

Evaluation of a new high-throughput method for identifying quorum quenching bacteria

Quorum sensing (QS) is a population-dependent mechanism for bacteria to synchronize social behaviors such as secretion of virulence factors. The enzymatic interruption of QS, termed quorum quenching (QQ), has been suggested as a promising alternative anti-virulence approach. In order to efficiently identify QQ bacteria, we developed a simple, sensitive and high-throughput method based on the biosensor Agrobacterium tumefaciens A136. This method effectively eliminates false positives caused by inhibition of growth of biosensor A136 and alkaline hydrolysis of N-acylhomoserine lactones (AHLs), through normalization of beta-galactosidase activities and addition of PIPES buffer, respectively. Our novel approach was successfully applied in identifying QQ bacteria among 366 strains and 25 QQ strains belonging to 14 species were obtained. Further experiments revealed that the QQ strains differed widely in terms of the type ofQQenzyme, substrate specificity and heat resistance. The QQ bacteria identified could possibly be used to control disease in aquaculture

SORTCERY—A High–Throughput Method to Affinity Rank Peptide Ligands

Uncovering the relationships between peptide and protein sequences and binding properties is critical for successfully predicting, re-designing and inhibiting protein–protein interactions. Systematically collected data that link protein sequence to binding are valuable for elucidating determinants of protein interaction but are rare in the literature because such data are experimentally difficult to generate. Here we describe SORTCERY, a high-throughput method that we have used to rank hundreds of yeast-displayed peptides according to their affinities for a target interaction partner. The procedure involves fluorescence-activated cell sorting of a library, deep sequencing of sorted pools and downstream computational analysis. We have developed theoretical models and statistical tools that assist in planning these stages. We demonstrate SORTCERY's utility by ranking 1026 BH3 (Bcl-2 homology 3) peptides with respect to their affinities for the anti-apoptotic protein Bcl-x[subscript L]. Our results are in striking agreement with measured affinities for 19 individual peptides with dissociation constants ranging from 0.1 to 60 nM. High-resolution ranking can be used to improve our understanding of sequence–function relationships and to support the development of computational models for predicting and designing novel interactions.National Institutes of Health (U.S.) (Award GM096466)German Academic Scholarship Foundation (Grant RE 3111/1-1

A high throughput method for genome-wide analysis of retroviral integration

Retroviral and lentiviral vectors integrate their DNA into the host cell genome leading to stable transgene expression. Integration preferentially occurs in the proximity of active genes, and may in some case disturb their activity, with adverse toxic consequences. To efficiently analyze high numbers of lentiviral insertion sites in the DNA of transduced cells, we developed an improved high-throughput method called vector integration tag analysis (VITA). VITA is based on the identification of Genomic Tags associated to the insertion sites, which are used as signatures of the integration events. We use the capacity of MmeI to cleave DNA at a defined distance of its recognition site, in order to generate 21 bp long tags from libraries of junction fragments between vector and cellular DNA. The length of the tags is sufficient in most cases, to identify without ambiguity an unique position in the human genome. Concatenation, cloning and sequencing of the tags allow to obtain information about 20–25 insertion sites in a single sequencing reaction. As a validation of this method, we have characterized 1349 different lentiviral vector insertion sites in transduced HeLa cells, from only 487 sequencing reactions, with a background of <2% false positive tags

A high-throughput method for isolation of salicylic acid metabolic mutants

<p>Abstract</p> <p>Background</p> <p>Salicylic acid (SA) is a key defense signal molecule against biotrophic pathogens in plants. Quantification of SA levels in plants is critical for dissecting the SA-mediated immune response. Although HPLC and GC/MS are routinely used to determine SA concentrations, they are expensive and time-consuming. We recently described a rapid method for a bacterial biosensor <it>Acinetobacter </it>sp. ADPWH_<it>lux</it>-based SA quantification, which enables high-throughput analysis. In this study we describe an improved method for fast sample preparation, and present a high-throughput strategy for isolation of SA metabolic mutants.</p> <p>Results</p> <p>On the basis of the previously described biosensor-based method, we simplified the tissue collection and the SA extraction procedure. Leaf discs were collected and boiled in Luria-Bertani (LB), and then the released SA was measured with the biosensor. The time-consuming steps of weighing samples, grinding tissues and centrifugation were avoided. The direct boiling protocol detected similar differences in SA levels among pathogen-infected wild-type, <it>npr1 </it>(nonexpressor of pathogenesis-related genes), and <it>sid2 </it>(SA induction-deficient) plants as did the previously described biosensor-based method and an HPLC-based approach, demonstrating the efficacy of the protocol presented here. We adapted this protocol to a high-throughput format and identified six <it>npr1 </it>suppressors that accumulated lower levels of SA than <it>npr1 </it>upon pathogen infection. Two of the suppressors were found to be allelic to the previously identified <it>eds5 </it>mutant. The other four are more susceptible than <it>npr1 </it>to the bacterial pathogen <it>Pseudomonas syringae </it>pv. <it>maculicola </it>ES4326 and their identity merits further investigation.</p> <p>Conclusions</p> <p>The rapid SA extraction method by direct boiling of leaf discs further reduced the cost and time required for the biosensor <it>Acinetobacter </it>sp. ADPWH_<it>lux</it>-based SA estimation, and allowed the screening for <it>npr1 </it>suppressors that accumulated less SA than <it>npr1 </it>after pathogen infection in a high-throughput manner. The highly efficacious SA estimation protocol can be applied in genetic screen for SA metabolic mutants and characterization of enzymes involved in SA metabolism. The mutants isolated in this study may help identify new components in the SA-related signaling pathways.</p

A high-throughput method for genotyping S-RNase alleles in apple

We present a new efficient screening tool for detection of S-alleles in apple. The protocol using general and multiplexed primers for PCR reaction and fragment detection on an automatized capillary DNA sequencer exposed a higher number of alleles than any previous studies. Analysis of alleles is made on basis of three individual fragment sizes making the allele interpretation highly accurate. The method was employed to genotype 432 Malus accessions and exposed 25 different S-alleles in a selection of Malus domestica cultivars of mainly Danish origin (402 accessions) as well as a selection of other Malus species (30 accessions). The allele S3 (28 %) was the most common among the Danish cultivars followed by S1 and S7 (both 27 %). The alleles S36 and S40 not previously reported from M. domestica were found in 6 and 17 cultivars, respectively. Complete allelic composition was found in 91 % of the 369 diploid accessions and in 86 % of the 63 triploids concerned. We further identified a relatively high frequency of S33 and S34, which has not been considered by most previous studies. The protocol presented here is easy to adopt and saves both time and work effort compared to previous methods. The robustness is illustrated by the great accuracy and a high number of S-alleles presented. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11032-016-0448-0) contains supplementary material, which is available to authorized users

Fluidized-bed development at JPL

Silicon deposition on silicon seed particles by silane pyrolysis in a fluidized bed reactor (FBR) was investigated as a low cost, high throughput method to produce high purity polysilicon for solar cell applications. The emphasis of the research is fundamental understanding of fluidized bed silicon deposition. The mechanisms involved were modeled as a six-path process: heterogeneous deposition; homogeneous decomposition; coalescence; coagulation; scavenging; and chemical vapor deposition growth on fines

Kinetic study of an on-chip isocyanate derivatization reaction by on-line nano-esi ms

A high-throughput method is presented for the study of reaction kinetics by nano- electrospray ionization mass spectrometry (nano-ESI MS). The reaction of propyl isocyanate (2), benzyl isocyanate (3), and toluene-2,4-diisocyanate (4) with 4-nitro-7- piperazino-2,1,3-benzoxadiazole (NBDPZ) (1) to yield the corresponding urea derivatives (5) was carried out in a continuous flow glass microchip. Real-time monitoring of the reactions was done by nano-ESI MS. Rate constants of 1.6 ␣ 104 M-1 min-1, 5.2 ␣ 104 M-1 min-1, and 2.5 ␣ 104 M-1 min-1 were determined for isocyanate 2, 3 and 4, respectively

A robust, high-throughput method for quantitative, in-depth analisys of proteomes

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