Jonathan Shum, Natasha Paul.
Analytical Biochemistry, In press | doi:10.1016/j.ab.2009.02.033 | PMID:19258004
Multiplexed PCR, the amplification of multiple targets in a single reaction, presents a new set of challenges that further complicate more traditional PCR set-ups. These complications include a greater probability for non-specific amplicon formation and for imbalanced amplification of different targets, each of which can compromise quantification and detection of multiple targets. Despite these difficulties, multiplex PCR is frequently used in such applications as pathogen detection, RNA quantification, mutation analysis and now, next generation DNA sequencing. Herein, we investigate the utility of primers with one or two thermolabile 4-oxo-1-pentyl phosphotriester modifications in improving multiplex PCR performance. Initial endpoint and real-time analyses reveal a decrease in off-target amplification and subsequent increase in amplicon yield. Furthermore, the use of modified primers in multiplex set-ups revealed a greater limit of detection and more uniform amplification of each target as compared to unmodified primers. Overall, the thermolabile modified primers present a novel and exciting avenue in improving multiplex PCR performance.
Steven C. Slater, Barry S. Goldman, Brad Goodner, João C. Setubal*, Stephen K. Farrand, Eugene W. Nester, Thomas J. Burr, Lois Banta, Allan W. Dickerman, Ian Paulsen, Leon Otten, Garret Suen, Roy Welch, Nalvo F. Almeida, Frank Arnold, Oliver T. Burton, Zijin Du, Adam Ewing, Eric Godsy, Sara Heisel, Kathryn L. Houmiel, Jinal Jhaveri, Jing Lu, Nancy M. Miller, Stacie Norton, Qiang Chen, Waranyoo Phoolcharoen, Victoria Ohlin, Dan Ondrusek, Nicole Pride, Shawn L. Stricklin, Jian Sun, Cathy Wheeler, Lindsey Wilson, Huijun Zhu, Derek W. Wood.
J. Bacteriol. Accepts | doi:10.1128/JB.01779-08 | PMID:19251847
The family Rhizobiaceae contains plant-associated bacteria with critical roles in ecology and agriculture. Within this family, many Rhizobium and Sinorhizobium strains are nitrogen-fixing plant mutualists, while many strains designated as Agrobacterium are plant pathogens. These contrasting lifestyles are primarily dependent on the transmissible plasmids each strain harbors. Members of Rhizobiaceae also have diverse genome architectures that include single chromosomes, multiple chromosomes, and plasmids of various sizes. Agrobacterium strains have been divided into three Biovars, based on physiological and biochemical properties. The genome of a Biovar I strain, A. tumefaciens C58, has been previously sequenced. In this study the genomes of the Biovar II strain A. radiobacter K84, a commercially available biological control strain that inhibits certain pathogenic agrobacteria, and the Biovar III strain A. vitis S4, a narrow host range strain that infects grapes and invokes a hypersensitive response on non-host plants, were fully sequenced and annotated. Comparison with other sequenced members of the α-proteobacteria provides new data on evolution of multi-partite bacterial genomes. Primary chromosomes show extensive conservation of both gene content and order. In contrast, secondary chromosomes share smaller percentages of genes, and conserved gene order is restricted to short blocks. We propose that secondary chromosomes originated from an ancestral plasmid to which genes have been transferred from a progenitor primary chromosome. Similar patterns are observed in select β- and γ-proteobacteria species. Together these results define the evolution of chromosome architecture and gene content among the Rhizobiaceae and support a generalized mechanism for second chromosome formation among bacteria.
Jared T Simpson, Kim Wong, Shaun D Jackman, Jacqueline E Schein, Steven JM Jones, Inanc Birol.
Genome Res., Published in Advance | doi:10.1101/gr.089532.108 | PMID:19251739
Widespread adoption of massively parallel DNA sequencing instruments has prompted the recent development of de novo short read assembly algorithms. A common shortcoming of the available tools is their inability to efficiently assemble vast amounts of data generated from large-scale sequencing projects, such as the sequencing of individual human genomes to catalog natural genetic variation. To address this limitation, we developed ABySS (Assembly By Short Sequences), a parallelized sequence assembler. As a demonstration of the capability of our software, we assembled 3.5 billion paired-end reads from the genome of an African male publicly released by Illumina Inc. Approximately 2.76 million contigs ≥100bp in length were created with an N50 size of 1499bp, representing 68% of the reference human genome. Analysis of these contigs identified polymorphic and novel sequences not present in the human reference assembly, which were validated by alignment to alternate human assemblies and to other primate genomes.
Paolo Fontana, Alessandro Cestaro, Riccardo Velasco, Elide Formentin, Stefano Toppo.
PLoS ONE 4, e4619 (2009) | doi:10.1371/journal.pone.0004619 | PMID:19247487
Background
Large-scale sequencing projects have now become routine lab practice and this has led to the development of a new generation of tools involving function prediction methods, bringing the latter back to the fore. The advent of Gene Ontology, with its structured vocabulary and paradigm, has provided computational biologists with an appropriate means for this task.
Methodology
We present here a novel method called ARGOT (Annotation Retrieval of Gene Ontology Terms) that is able to process quickly thousands of sequences for functional inference. The tool exploits for the first time an integrated approach which combines clustering of GO terms, based on their semantic similarities, with a weighting scheme which assesses retrieved hits sharing a certain number of biological features with the sequence to be annotated. These hits may be obtained by different methods and in this work we have based ARGOT processing on BLAST results.
Conclusions
The extensive benchmark involved 10,000 protein sequences, the complete S. cerevisiae genome and a small subset of proteins for purposes of comparison with other available tools. The algorithm was proven to outperform existing methods and to be suitable for function prediction of single proteins due to its high degree of sensitivity, specificity and coverage.
Comparative analysis of H2A.Z nucleosome organization in human and yeast genome.
Michael Y Tolstorukov, Peter V Kharchenko, Joseph A Goldman, Robert E Kingston, Peter J Park.
Genome Res., Published in Advance | doi:10.1101/gr.084830.108 | PMID:19246569
Eukaryotic DNA is wrapped around a histone protein core to constitute the fundamental repeating units of chromatin, the nucleosomes. The affinity of the histone core for DNA depends on the nucleotide sequence; however, it is unclear to what extent DNA sequence determines nucleosome positioning in vivo, and if the same rules of sequence-directed positioning apply to genomes of varying complexity. Using the data generated by high-throughput DNA sequencing combined with chromatin immunoprecipitation, we have identified positions of nucleosomes containing the H2A.Z histone variant and histone H3 tri-methylated at lysine 4 in human CD4+ T cells. We find that the 10-bp periodicity observed in nucleosomal sequences in yeast and other organisms is not pronounced in human nucleosomal sequences. This result was confirmed for a broader set of mononucleosomal fragments that were not selected for any specific histone variant or modification. We also find that human H2A.Z nucleosomes protect only about 120-bp of DNA from MNase digestion and exhibit specific sequence preferences, suggesting a novel mechanism of nucleosome organization for the H2A.Z variant.
