Lake Vostok accretion ice metagenomics/metatranscriptomics
Quartered ice core sections were decontaminated and melted. Meltwater was collected in 50 ml aliquots, filtered and ultracentrifuged. Four sections (126 ml each) were combined into two 250 ml samples. Pellets were rehydrated and subjected to nucleic acids extraction. DNA copies of RNA were produces with cDNA kit. Then EcoRI (NotI) adapters were ligated to each end of the cDNA and DNA for subsequent PCR amplification. Each sample was size fractionated by column chromatography and reamplified. Fractions smaller than 200 bp and larger than 2 kb were excluded from the analysis. Each remaining sample was reamplified with primers containing EcoRI/NotI sequences on their 3' ends and 454-specific primer (A or B) sequences on their 5' ends. PCR products were purified and sent to Roche Life Sciences 454 Technologies for 454 sequencing. Sequences were extracted with Python software on the Ohio Super Computer (OSC) and subjected to Blast analysis optimized for automated megablast and manual recheck. Taxonomic representation was determined with MG-RAST and Galaxy - a web-based genome analysis tool. The 454 sequences were also submitted to KAAS KEGG server; the results were manually rechecked with NCBI blastx search.