Next-Gen Sequencing has quickly become one of the most important tools in genomics research and nucleic acid fragmentation is a crucial first step in the sequencing workflow. While there are a variety of methods available to fragment nucleic acids, mechanical shearing remains the method of choice for achieving high sensitivity and unbiased results. 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.
AFA Process for DNA and RNA Shearing
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. 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 inexpensive M-Series to the single-tube full-power S-Series to higher throughput E and L-Series instruments.
g-TUBETM for Longer DNA Fragments
For sequencing applications requiring longer DNA fragments, Covaris developed the patented g-TUBE™. Generating shearing forces through centrifugation, the g-TUBE produces your selected fragment length in the 6 kb – 20 kb range. Apart from a benchtop centrifuge, no other equipment is needed.
Mechanical DNA and RNA shearing with AFA technology is the gold standard for nucleic acid fragmentation prior to NGS library preparation. All major sequencing instrument providers recommend the use of Covaris technology to obtain the highest quality data:
Unbiased results without GC or temperature bias
Consistent DNA/RNA shearing to a wide range of target sizes with low c.v. (quantify)
Concentration independent shearing
Optimization free protocols readily available
Highest quality data from amplified, non-amplified and whole genome sequencing experiments
AFA processing completely scalable from a single sample to 96 well plate
Wide range of consumables for different volumes and desired fragment size with easy selection
For customers requiring 1.5-5 Kb fragments, Covaris also offers the miniTUBE for use with the AFA™ Focused-ultrasonicators. miniTUBEs are an ideally suited for use in RainDance Technology workflows, as an example.
The Covaris AFA process is extensively cited in peer reviewed research articles for DNA fragmenting on all available sequencing platforms. Covaris technology is a mainstay for leading genome centers worldwide, including The Broad Institute, Wellcome Trust Sanger Institute, and Beijing Genome Institute.
Features & Benefits
Processing at controlled temperatures provides highest yields while preserving sample fidelity
Accurate and precise.
Highly reproducible process, day to day, user to user. Protocols can be transferred instrument to instrument without further optimization
Non-contact, closed vessel
No cross-contamination, clean-up, or sample loss
Generate tight fragment distribution centered from 100 bp to 20 kbp
For DNA fragments distribution in the 150bp – 1,500kb range: microTUBE
For DNA fragments distribution in the 2 – 5kb range: miniTUBE
For DNA fragments distribution in the 6 – 20kb range: g-TUBE
A scalable, fully automated process for construction of sequence-ready human exome targeted capture libraries. Fisher et al. Genome Biology 2011, 12:R1
In this paper, scientist from the Broad Institute describe how the use of Covaris E210 system in conjunction with AFA microTUBEs is part of the process improvements that resulted in an dramatic increase in scale and throughput of sequence ready libraries produced. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3091298/?tool=pubmed
A large genome center’s improvements to the Illumina sequencing system. Quail et al. 2008. Nature Methods Vol 5 No 12.
In this paper, scientists from the Wellcome Trust Sanger Intitute discus how the use of AFA energy improves the Illumina protocol, making it more reliable in a high throughput environment. Main benefits from Covaris AFA technology are the tight fragment size distribution, allowing to skip the size selection step in some cases, and its compatibility with high throughout workflows. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2610436/?tool=pubmed
Analyzing and minimizing PCR amplification bias in Illumina sequencing libraries. Aird et al. Genome Biology 2011, 12:R18
In this paper, scientists from the Broad Institute studied the origin of GC bias in Illumina sequencing libraries. They isolated each step of the library preparation process, and checked for GC bias. Their results show that DNA shearing with Covaris AFA technology doesn’t introduces any bias in the library preparation http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3188800/?tool=pubmed
Genome-wide copy number analysis of single cells. Baslan et al. Nature Protocols. 2012, 7:6 1024-1041
“Previously we reported sonication of WGA DNA using the Bioruptor ultrasonic disruptor24. However, we have switched to using the Covaris focus acoustics system, as it allows for higher throughput.” http://www.nature.com/nprot/journal/v7/n6/abs/nprot.2012.039.html
Solution-based targeted genomic enrichment for precious DNA samples. Shearer, AE et al. BMC Biotech 12:20 2012
“We found that after shearing with the Covaris, a tight size range is attained… negating the need for any size selection with gel electrophoresis, bead-based selection, or specialized equipment. The primary benefit of this method is that DNA loss is minimized during elution or tube transfers, as the sample is maintained in the same tube. “ http://www.biomedcentral.com/1472-6750/12/20/abstract
Impact of three Illumina library construction methods on GC bias and HLA genotype calling “Compared to standard TruSeq Nano, GC bias was more prominent in transposase-based protocols, particularly Nextera XT, likely through a combination of transposase insertion bias being coupled with a high number of PCR enrichment cycles. Importantly, our findings demonstrate non-uniform read depth can have a direct and negative impact on the robustness of HLA genotyping, which has clinical implications for users when choosing a library construction strategy that aims to balance cost and throughput with data quality.” http://www.ncbi.nlm.nih.gov/pubmed/25543015