Tuesday, March 30, 2010

Nextera™ tagmentation of soil metagenomic DNA

High-throughput sequencing has greatly facilitated metagenomic analysis of environmental samples. Epicentre recently introduced the Meta-G-Nome™ DNA Isolation Kit for use with water, soil, and compost samples. In this analysis, we isolated metagenomic  DNA from garden soil and performed the tagmentation reaction according to the Nextera™ protocol. The isolated DNA yielded tagmentation products in the expected size range, compared to a control that was also tagmented according to the Nextera protocol (Fig. 1).

Figure 1. Nextera tagmentation of soil metagenomic DNA. Garden soil that had been stored for 6 months at 4°C was used as starting material. DNA was isolated according to the protocol for the Meta-G-Nome DNA Isolation Kit from 1 g of soil, using a final elution volume of 20 µl. The metagenomic DNA and a control (lambda DNA) were tagmented according to the Nextera protocol, and the tagmentation products were analyzed on a 1% agarose gel. Lane 1, 5 µl of metagenomic DNA before tagmentation; lane 2, tagmented lambda DNA (50 ng); lane 3, tagmented metagenomic DNA (10 µl used for tagmentation); lane 4, tagmented metagenomic DNA (17 µl used for tagmentation); lanes M, 1-kb DNA ladder.

The tagmented metagenomic DNA and lambda DNA were then used as templates in PCR with either Nextera primers (Roche FLX-compatible), or primers for the 16S rRNA gene (Fig. 2). PCR with Nextera primers yielded amplification products in the expected size range (Fig. 2, lanes 2 and 3), and metagenomic DNA yielded the expected 16S rRNA amplification product (Fig. 2, lanes 5 and 6).

Figure 2. PCR of tagmented metagenomic DNA. PCR was performed using tagmented metagenomic DNA and lambda DNA as templates, with Nextera FLX-compatible primers (lanes 1-3). Metagenomic DNA was also amplified using 16S rRNA gene primers as a control (lanes 4-6). PCR products were cleaned up using Zymo columns prior to electrophoresis in a 1% agarose gel. Lanes 1 and 4, no-DNA control; lane 2, PCR of tagmented lambda DNA; lane 3, PCR of tagmented metagenomic DNA; lanes 5 and 6, PCR of metagenomic DNA (undiluted and 1:10 diluted template, respectively).

If you’re interested in purifying metagenomic DNA from soil, compost, or water samples, and would like to evaluate the Nextera DNA Sample Prep Kits for next-generation sequencing, please fill out a short survey.

No comments: