The paradigm shift from 16S rRNA Sanger sequencing to high-throughput whole genome sequencing (HT-WGS) to calculate the relative abundance of microorganisms present in complex samples has increased since the first microbiome paper was published in 2006.1 As the leading mechanical shearing technology in the NGS space, Covaris products deliver superior NGS sample preparation quality compatible with a number of different commercial library prep kits and sequencing platforms including: Illumina, Ion Torrent from Thermo Fisher Scientific, Pacific Biosciences, and Oxford Nanopore Technologies.
The Covaris Adaptive Focused Acoustics® (AFA®) technology provides users with the flexibility to fragment DNA to various sizes ranging from 150 bp to 5 Kb 2 (Figure 1).
Figure 1.Fragment size distribution profiles. Electropherogram trace files demonstrate the feasibility of fragmenting DNA to multiple insert sizes using the Covaris AFA technology. Product sizes range from 150-5,000 bp.
AFA delivers unbiased fragmentation across a variety of microbial genomes irrespective of the purine (AG) and pyrimidine (TC) content (Figure 2).
Figure 2. Reproducible shearing profiles across multiple genera. Electropherogram trace files of DNA sheared to 150 bp for multiple bacterial samples with different GC-content genetic makeup.
Variable Input Mass
The desired insert sizes can be achieved at varying input masses (pg to µg) without alteration of processing conditions because the technology’s performance is not influenced by the concentration of the sample (Figure 3).
Figure 3. Sample fragmentation is independent of DNA concentration. Electropherogram trace files of DNA sheared to 200 bp with varying input masses ranging from as low as 50 ng and up to 2 µg.
Accurate and Precise
In order to ensure adequate coverage is achieved, the fragmentation needs to be precisely controlled in order to successfully sequence the samples and make meaningful biological interpretations from the data (Figure 4).
Figure 4. Read depth at varying positions in the genome. Uniform sequencing coverage observed across the E. coli genome. The read depth (y-axis) is plotted against the position along the genome (x-axis).
Mechanical Shearing for LONG read PLATFORMS
Complex metagenomic studies that require larger contiguous fragments for accurate taxonomic classification can be prepared using the Covaris g-TUBE. The g-TUBE is specifically designed for researchers who need longer read lengths (6-20 Kb) sequenced using the single-molecule real-time circular consensus sequencing (SMRT CSS) technology from Pacific Biosciences and the MinION sequencing platform from Oxford Nanopore Technologies 3, 4.
Extraction for Difficult to Lyse Mycobacteria
The patented Adaptive Focused Acoustics (AFA) technology can be utilized for other applications beyond nucleic acid shearing for NGS. For microbiologists looking to extract both stabile and labile molecules from difficult samples such as mycobacteria, Covaris is pleased to offer molecular extraction solutions. If you are looking for custom extraction solutions, please contact us for further information.