DNA/RNA Shearing for NGS

Nucleic acid fragmentation is a crucial first step in the Next-Gen Sequencing workflow. While there are a variety of methods available, mechanical DNA shearing powered by AFA-energetics® remains the method of choice for achieving high sensitivity and unbiased results.

AFA Process for DNA and RNA Shearing

Adaptive Focused Acoustics® (AFA®) technology is firmly established as the fragmentation method of choice for NGS, and Covaris continues to innovate the tools required to shear DNA and RNA without GC bias or thermal damage.
The Covaris AFA process is conducted under isothermal conditions, ensuring the integrity of the nucleic acid sample is maintained and providing high recovery of double-stranded DNA during the shearing process.
Combined with the specifically engineered AFA Tubes it is possible to precisely and accurately fragment DNA and RNA to the 100 – 1500bp range (microTUBE), or 2 – 5kb range (miniTUBE). Focused-ultrasonicators range from the low through-put single-tube full-power S-Series, the mid-throughput, more affordable M-Series, to the higher throughput E and L-Series instruments.

Benefits of RNA or DNA Shearing using AFA Technology

Physical shearing of DNA or RNA using Covaris AFA is concentration independent, isothermal, and highly reproducible. Physical shearing does not require a cleanup step after shearing, thereby preserving the complexity of the sample for library preparation. This shearing method provides unmatched control and reproducibility of shearing.
FEATUREBENEFIT
Temperature ControlledProcessing at controlled temperatures provides highest yields while preserving sample fidelity
Accurate and preciseHighly reproducible process, day to day, user to user. Protocols can be transferred instrument to instrument without further optimization
Non-contact, closed vesselNo cross-contamination, clean-up, or sample loss
VersatileGenerate tight fragment distribution centered from 100 bp to 20 kbp
DNA Shearing of Clinical Samples


Unparalleled acoustic energy control enables shearing across a wide range of fragment sizes ranging from 150 bp to 5 kb and everything in between. AFA-energetics is the gold standard for DNA shearing providing you with highest sample complexity and unbiased sample yield paving the way for the most cost-effective library prep.
RNA Shearing
mRNA Fragmentation
Agilent BioAnalyzer electropherogram of 100 ng of mRNA processed in a volume of 130 µl of TE in a microTUBE™ according to the operating conditions, showing the mean fragment size at 400 bases.

Total mRNA Shearing


Agilent BioAnalyzer electropherogram of 5 µg of mRNA processed in a volume of 130 µl of TE in a microTUBE according to the operating conditions, showing the mean fragment size at 200 bases.

Comparison of Shearing Fragment Sizes

Fragment Size*Sample VolumeCovaris Products
175 to 1.5 kbp5 to 50 µlAFA-TUBE
150 to 1.5 kbp130 µlmicroTUBE-130
150 to 1.5 kbp55 µlmicroTUBE-50
150 to 1.5 kbp15 µlmicroTUBE-15
2 kbp200 µlminiTUBE-Clear
3 kbp200 µlminiTUBE-Blue
5 kbp200 µlminiTUBE-Red
6 – 20 kb150 µlg-TUBE
*For validated fragment sizes, refer to the Covaris Focused-ultrasonicator Protocol.

Resources

References

A scalable, fully automated process for construction of sequence-ready human exome targeted capture libraries. Fisher et al. Genome Biology 2011
Analyzing and minimizing PCR amplification bias in Illumina sequencing libraries. Aird et al. Genome Biology 2011
Solution-based targeted genomic enrichment for precious DNA samples. Shearer, AE et al. BMC Biotech 2012