The Experts below are selected from a list of 18528 Experts worldwide ranked by ideXlab platform
Sandrine Dudoit - One of the best experts on this subject based on the ideXlab platform.
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Biases in Illumina transcriptome sequencing caused by Random Hexamer priming
Nucleic acids research, 2010Co-Authors: Kasper D. Hansen, Steven E. Brenner, Sandrine DudoitAbstract:Generation of cDNA using Random Hexamer priming induces biases in the nucleotide composition at the beginning of transcriptome sequencing reads from the Illumina Genome Analyzer. The bias is independent of organism and laboratory and impacts the uniformity of the reads along the transcriptome. We provide a read count reweighting scheme, based on the nucleotide frequencies of the reads, that mitigates the impact of the bias.
Kasper D. Hansen - One of the best experts on this subject based on the ideXlab platform.
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Biases in illumina transcriptome sequencing caused by Random Hexamer priming. Nucleic acids research
2013Co-Authors: Kasper D. Hansen, Steven E. Brenner, Rine DudoitAbstract:by Random Hexamer primin
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Biases in Illumina transcriptome sequencing caused by Random Hexamer priming
Nucleic acids research, 2010Co-Authors: Kasper D. Hansen, Steven E. Brenner, Sandrine DudoitAbstract:Generation of cDNA using Random Hexamer priming induces biases in the nucleotide composition at the beginning of transcriptome sequencing reads from the Illumina Genome Analyzer. The bias is independent of organism and laboratory and impacts the uniformity of the reads along the transcriptome. We provide a read count reweighting scheme, based on the nucleotide frequencies of the reads, that mitigates the impact of the bias.
John Richard Nelson - One of the best experts on this subject based on the ideXlab platform.
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Current Protocols in Molecular Biology - Random‐Primed, Phi29 DNA Polymerase‐Based Whole Genome Amplification
Current protocols in molecular biology, 2014Co-Authors: John Richard NelsonAbstract:Whole-genome amplification by multiple displacement amplification (MDA) is a patented method to generate potentially unlimited genomic material when researchers are challenged with trace samples, or the amount of genomic DNA required for analysis exceeds the amount on hand. It is an isothermal reaction, using Phi29 DNA polymerase and Random Hexamer primers for unbiased amplification of linear DNA molecules, such as genomic DNA. The Random-primed MDA reaction provides extensive amplification coverage of the genome, generates extremely long DNA products, and provides high DNA yields. This unit explains the reaction, and describes use of the commercial kits available.
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Isothermal strand-displacement amplification applications for high-throughput genomics.
Genomics, 2002Co-Authors: John C. Detter, John Richard Nelson, Jamie Jett, Susan Lucas, Eileen Dalin, Andre R. Arellano, Mei Wang, Jarrod Chapman, Yunian Lou, Daniel S. RokhsarAbstract:Amplification of source DNA is a nearly universal requirement for molecular biology applications. The primary methods currently available to researchers are limited to in vivo amplification in Escherichia coli hosts and the polymerase chain reaction. Rolling-circle DNA replication is a well-known method for synthesis of phage genomes and recently has been applied as rolling circle amplification (RCA) of specific target sequences as well as circular vectors used in cloning. Here, we demonstrate that RCA using Random Hexamer primers with Φ29 DNA polymerase can be used for strand-displacement amplification of different vector constructs containing a variety of insert sizes to produce consistently uniform template for end-sequencing reactions. We show this procedure to be especially effective in a high-throughput plasmid production sequencing process. In addition, we demonstrate that whole bacterial genomes can be effectively amplified from cells or small amounts of purified genomic DNA without apparent bias for use in downstream applications, including whole genome shotgun sequencing.
Koen J. F. Verhoeven - One of the best experts on this subject based on the ideXlab platform.
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Consistent errors in first strand cDNA due to Random Hexamer mispriming.
PloS one, 2013Co-Authors: Thomas P. Van Gurp, Lauren M. Mcintyre, Koen J. F. VerhoevenAbstract:Priming of Random Hexamers in cDNA synthesis is known to show sequence bias, but in addition it has been suggested recently that mismatches in Random Hexamer priming could be a cause of mismatches between the original RNA fragment and observed sequence reads. To explore Random Hexamer mispriming as a potential source of these errors, we analyzed two independently generated RNA-seq datasets of synthetic ERCC spikes for which the reference is known. First strand cDNA synthesized by Random Hexamer priming on RNA showed consistent position and nucleotide-specific mismatch errors in the first seven nucleotides. The mismatch errors found in both datasets are consistent in distribution and thermodynamically stable mismatches are more common. This strongly indicates that RNA-DNA mispriming of specific Random Hexamers causes these errors. Due to their consistency and specificity, mispriming errors can have profound implications for downstream applications if not dealt with properly.
A Samad - One of the best experts on this subject based on the ideXlab platform.
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new record of catharanthus yellow mosaic virus and a betasatellite associated with lethal leaf yellowing of kalmegh andrographis paniculata in northern india
Plant Disease, 2015Co-Authors: Asifa Khan, S T Saeed, A SamadAbstract:Andrographis paniculata (Family Acanthaceae), also called Kalmegh, is a medicinal herb in India well-known for its various pharmaceutical properties (1). In August 2012, during a survey in the northern parts of India, several Kalmegh plants in Barabanki District of Uttar Pradesh Province showed typical virus-like symptoms along with prominent lethal leaf yellowing. The infected plants initially showed some chlorotic streaks, which later turned completely yellow, ultimately leading to premature death. Mechanical/sap inoculation failed to transmit the pathogen. Based on the symptomology, a heavy infestation of whiteflies (Bemisia tabaci) in the infected fields, and lack of mechanical transmission, the association of a begomovirus was suspected. The disease incidence was calculated to be about 15 to 20% on the basis of plant population. Twenty samples from naturally infected plants of A. paniculata were collected from various field locations. Total genomic DNA from the symptomatic and non-symptomatic samples was isolated by the modified CTAB method (4). The initial PCR-based detection was performed using begomovirus coat protein gene specific primers (forward 5'-ATGGCGAAGCGACCAG-3' and reverse 5'-TTAATTTGTGACCGAATCAT-3'), which generated an amplicon of 771 bp in most of the (17/20) symptomatic samples. No amplification was obtained in healthy or non-symptomatic plant samples. The full-length genome was amplified via rolling-circle amplification (RCA) according to the manufacturer's instructions using Random Hexamer primers and φ29 DNA polymerase. A portion of the RCA product (1 μl) was subjected to digestion with different restriction enzymes, out of which BamHI yielded DNA fragments of approximately 2.7 and 1.3 kb, corresponding to DNA-A and β satellite molecules, respectively. These fragments were eluted from the gel and cloned into the suitable restriction site of pGreen0029 vector. The positive clones were checked by restriction digestion. Twelve out of 20 clones were found to be positive and sequenced. The complete genome sequences of DNA A (2,754 bp) and β (1,366 bp) satellites were deposited in the GenBank database with the accession numbers KM359406 and KM359407, respectively. The absence of DNA-B molecule was ascertained, as no PCR amplification was detected with DNA-B-specific primers. Sequence analysis showed highest nucleotide identity (90%) with Catharanthus yellow mosaic virus (CYMV) (HE580234) and ≤85% identity with other begomoviruses of the database. Sequence analysis of the associated betasatellite showed 96% identity with Andrographis yellow vein leaf curl betasatellite (KC967282). CYMV was first reported on Catharanthus roseus with no associated betasatellite from Pakistan (2). However, this is the first report of CYMV along with a betasatellite infecting A. paniculata in India. Recently a begomovirus (Eclipta yellow vein virus) infection was reported on A. paniculata in association with Andrographis yellow vein leaf curl betasatellite from India for the first time (3); now the crop has also become a host of CYMV. Thus, this study highlights the spread of CYMV from its preliminary host to a new host plant (A. paniculata), across the South Asian countries. Therefore, it is important to take measures for the management of its transmitting vector so as to curtail the spread of the virus to new economically and commercially important crops. References: (1) S. Akbar. Altern. Med. Rev. 16:1, 2011. (2) M. Ilyas et al. Arch. Virol. 158:505, 2013. (3) A. Khan and A. Samad. Plant Dis. 98:698, 2014. (4) S. P. S. Khanuja et al. Plant Mol. Biol. Rep. 17:1, 1999.
