Tuesday, September 27, 2011

Preparing high-quality DNA and RNA for Illumina sequencing

The MasterPure™ line of DNA and RNA purification products has become one of the tools of choice for researchers wishing to obtain pure, high-quality nucleic acids for deep sequencing and microarray-based analysis. The MasterPure procedure uses gentle detergent/salt and proteolysis-based extraction technology that does not require the use of toxic organics or spin-columns. The resulting high-quality nucleic acids--RNA (RIN > 9.5) and DNA (A260/280 >1.8)--are of high molecular weight. Recent publications have highlighted the use of MasterPure kits to generate DNA for standard Illumina® library construction and GAII sequencing for developing reference genomes and de novo deep sequencing of various genomes. Other successful applications using MasterPure-purified DNA and RNA include SNP detection, methylation analysis, and expression profiling.

Platform MasterPure™ Kit Sample Type Reference
GAII MasterPure Gram-Positive DNA Kit Bacteria (Riemerella anatipestifer type) Mavromatis, K et al. (2011)
doi:10.4056/sigs.1553862
MasterPure Gram-Positive DNA Kit Bacteria (Bacteroides salanitronis) Gronow S et al. (2011)
doi:10.4056/sigs.1704212

MasterPure Complete Kit

Bacteria (Geobacter sulfurreducens)

Nagarajan H et al. (2010)
doi:10.1371/journal.pone.0010922

MasterPure Gram-Positive Kit

Bacteria (Aminomonas paucivorans)

Pitluck S et al. (2010)
doi:10.4056/sigs.1253298

MasterPure Gram-Positive DNA Kit

Bacteria (Marivirga tractuosa)

Pagani, I et al. (2011)
doi:10.4056/sigs.1623941

GoldenGate™ MasterPure DNA Purification Kit Colorectal carcinoma lines Irizarry, RA et al. (2008)
doi: 10.1101/gr.7301508
BeadArray™ MasterPure DNA Purification Kit Blood lymphocytes; cystic fibrosis patients Darrah R et al. (2010)
doi: 10.​1152/​physiolgenomics.​00185.​2009

Wednesday, September 14, 2011

Nextera libraries aid in study of honey bee pathogens

Recently, honey bee (Apis mellifera) populations in North America and in Europe have been experiencing increased annual losses due to a phenomenon known as Colony Collapse Disorder (CCD). Population loss of honey bee colonies poses grave risks to agriculture, due to the importance of these insects in pollination of food crops. Since the effect of environmental pathogens on bees has been poorly studied, the causes of CCD have not beeen well characterized. Thus, it is necessary to build a database of bee pathogens to learn more about pathogen transmission and to determine their role in CCD.

To this end, Runckel et al. wanted to identify what constitutes an abnormal pathophysiological condition in a honey bee colony. Using the Nextera™ DNA Sample Prep Kit (Illumina®-compatible), the researchers developed pools of sequence data from 20 different monitor hives in an ultra-deep sequencing experiment, and these data were compared to sequence data from known bee pathogen types, including known viruses, Nosema sp., Crithidia mellificae, and bacteria. The authors state that theirs is the first U.S. honey bee pathogen monitoring study to report both comprehensive pathogen incidence and relative abundance of specific pathogens over time. Results from their molecular analysis pipeline (microarray, PCR, qPCR, ultra-deep sequencing) identified four novel RNA viruses, and provide a basis for future epidemiologic studies aimed at determining the causes of CCD.

ResearchBlogging.orgRunckel, C. et al. (2011). Temporal Analysis of the Honey Bee Microbiome Reveals Four Novel Viruses and Seasonal Prevalence of Known Viruses, Nosema, and Crithidia PLoS ONE, 6 (6) DOI: 10.1371/journal.pone.0020656

Friday, September 2, 2011

Nextera library prep used to characterize novel virus variants

A significant advantage of Nextera™ library preparation technology is its low input requirements. A recent publication exploits this advantage to characterize the emergence and spread of a strain of new hemorrhagic fever viruses in red colobus monkeys in Uganda. Lauck et al. report that simian hemorrhagic fever virus (SHFV) has caused lethal outbreaks of hemorrhagic disease in captive primates, but has not been studied in wild primates. They describe the discovery and genetic characterization of two novel, divergent SHFV variants coinfecting a single male red colobus monkey found in the wild.

The genomic sequencing libraries were created using the Nextera DNA Sample Prep Kit (Roche Titanium-compatible) to locate and characterize any viral entities that may be residing in the wild. The Nextera libraries did not require isolation of viroids from the individual and only a simple total genomic DNA preparation was needed to isolate sufficient DNA (50 ng) for library construction and sequencing. Analysis of the sequence data revealed that the two viral genotypes seen show greater evolutionary sequence divergence than has been observed in other coinfecting virions that infect individuals within a population. The authors state that the results of this investigation will increase the knowledge base of the history and biology of these pathogens, and demonstrate that wild primates are often reservoirs for novel pathogens.

ResearchBlogging.orgLauck, M. et al. (2011). Novel, Divergent Simian Hemorrhagic Fever Viruses in a Wild Ugandan Red Colobus Monkey Discovered Using Direct Pyrosequencing PLoS ONE, 6 (4) DOI: 10.1371/journal.pone.0019056