Coextraction of microbial metagenomic DNA and RNA from deep-sea sediment

A protocol to coextract the microbial metagenomic DNA and RNA from deep-sea sediment was developed for the microbiological study of environmental samples. The obtained pure metagenomic DNA with the size larger than 23 kb and stable RNA could be used directly for PCR and reverse transcription - PCR （ RT - PCR） respectively. The direct lysis including the treatments of SDS, proteinase and lysozyme was applied to acquiring the metagenomic DNA and RNA furthest. Prior to the lysis treatment, the glass bead and denaturing solution were added to enhance the lysis efficiency and keep the integrity of RNA respectively. Denaturing gradient gel electrophoresis （DGGE） was applied in accessing the microbial 16S rRNA diversity by PCR and RT -PCR amplification from a single extraction. The pattern obtained by this analysis revealed some differences between them, indicating the efficiency of the protocol in extracting the metagenomic DNA and total RNA from deep-sea sediment.

An evaluation of multiple annealing and looping based genome amplification using a synthetic bacterial community

The low biomass in environmental samples is a major challenge for microbial metagenomic studies. The amplification of a genomic DNA was frequently applied to meeting the minimum requirement of the DNA for a high-throughput next-generation-sequencing technology. Using a synthetic bacterial community, the amplification efficiency of the Multiple Annealing and Looping Based Amplification Cycles（MALBAC） kit that is originally developed to amplify the single-cell genomic DNA of mammalian organisms is examined. The DNA template of 10 pg in each reaction of the MALBAC amplification may generate enough DNA for Illumina sequencing. Using 10 pg and 100 pg templates for each reaction set, the MALBAC kit shows a stable and homogeneous amplification as indicated by the highly consistent coverage of the reads from the two amplified samples on the contigs assembled by the original unamplified sample. Although Genome Plex whole genome amplification kit allows one to generate enough DNA using 100 pg of template in each reaction, the minority of the mixed bacterial species is not linearly amplified. For both of the kits, the GC-rich regions of the genomic DNA are not efficiently amplified as suggested by the low coverage of the contigs with the high GC content. The high efficiency of the MALBAC kit is supported for the amplification of environmental microbial DNA samples, and the concerns on its application are also raised to bacterial species with the high GC content.