DNA RNA

Introduction

The demand for low cost, rapid genomic sequencing has driven the development of the present high-throughput massively parallel sequencing technology. This technology requires an efficient and reliable method to produce small DNA fragments.

DNA shearing methods

Small-insert random DNA fragments are necessary to create efficient libraries for genomic sequencing projects. The success and efficiency of sequencing a large genome is dependent on the randomness of the fragments generated by the shearing of target DNA. Physical shearing methods (i.e. sonication, nebulization, and hydrodynamic shearing; references 1, 2, 3) are preferentially chosen over enzymatic digestion due to the randomness and size of the fragments produced resulting in a suitable overlapping collection of fragments for sub-cloning. The SonicManTM offers these properties in a high-throughput sonication format allowing for a straightforward, user-friendly, customizable method to generate random DNA fragments.

Using sonication

The following is possible using sonication:

Sonication scalable to 96, 384, or 1536 formats with working volumes ranging from 10 µL to 1.5 mL.

Incorporated chiller ensures sample temperatures do not increase significantly during sonication.

Sonication procedures allow for an uncomplicated, quick process as opposed to techniques like hydrodynamic shearing.

Shear 96 sample to (50 – 200) bp fragments in 4 hours as compared to 24 hours for in focused acoustic methods.

The instrument

The work described is performed using an instrument developed by MatriCal, configurable with 96, 384, and 1536 well formats. SonicMan uses disposable pin lids to transfer sonic energy to each individual well and to prevent well-to-well cross contamination. Plates are placed on a retractable shuttle. The sonicator provides variable power outputs up to 1150 W and configurable time intervals from 0.1 to 20 seconds.

Fragment size is correlated with sonication settings (power/time) and is controllable by the user. Fragments centered around a desired length can be repeatedly generated in seconds Samples may be sonicated in volumes ranging from 25ul to 1.5ml per well. Formats are scalable to 96-well and 384-well plates Unlike enzyme based digestion, the random fragments generated are suitable for the sequencing of large genomes Sonication is an uncomplicated, quick, process compared with techniques like hydrodynamic shearing No specialized reagents are needed minimizing solution variation and providing easy carryover to downstream applications

Sonication is delivered via an acoustic horn that directs the acoustic wave to a series of pins, 1 per well of the microplate.

High-Throughput Preparation of
Fragmentor Mate-Paired Libraries Why Shear?

Randomly sheared fragments are required for shotgun based sequencing methods in order to produce the redundant overlapping library of fragments needed for sequence reconstruction. As enzymatic methods do not produce random fragments, mechanical based shearing methods are required. Sonication is arguably the most popular method for producing DNA fragments and has been used for library construction in some of the earliest shotgun sequencing experiments (4). As methodologies move to the high-throughput arena, one true high-throughput option for mechanical library construction is the high-throughput capable sonicator, the SonicMan™.

The Uniqueness of DNA shearing by acoustics

The SonicMan™ is a high-throughput plate sonicator that employs a disposable 96, 384, or 1536 probed lid to simultaneously sonicate each well of a high density plate. Like in all sonicators, a piezoelectric crystal converts electrical energy to mechanical energy producing longitudinal vibration in a titanium alloy horn. This horn is tightly coupled to the disposable pinned lid which is able to transfer the sonic energy to the samples producing the cavitation that is the basis of the biological effects of sonication.

High Throughput Shearing

Shear 96 samples to peak fragments sizes of 50 -200 bp in 4 hours as opposed to alternative methods which take over 24 hours to shear 96 samples (below).

Low Fragment Size & Tight Resolution

The SonicMan™ can shear fragments to sizes from 50 and 200 bp, the low length tight resolution desired for single end sequencing libraries. Libraries sheared to relevant size ranges for separation inserts of mate-paired reads may also be produced, e.g. bp ranges of 200 to 300 or 300 to 500 or higher.

Shearing Efficiency: Shear to 100 bp fragments

DNA fragments reaching as low as 200 bp may be achieved after 5 minutes of total sonication time. Shears producing DNA peak fragments of 150 bp may be achieved in 30 total minutes of sonication time (below). From left to right there are a standard in lanes 1 and 9, sonication times for lane 2 to lane 8 of 0 to 30 seconds in 5 second increases.

Shearing Uniformity

The tube plate is sonicated in a custom designed chiller shuttle that in conjunction with strict quality assurance procedures of the pin and PinLid dimensions as well as silicone application methods ensures a highly uniform intra plate DNA shearing procedure (left).

References Deininger PL 1983. Anal. Biochem. 129: 216-223. Bodenteich AS, Chissoe S, Wang Y-F, and Roe BA 1994. In Automated DNA sequencing and analysis techniques (ed. MD Adams, C Fields, and C Venter), pp.42-50. Academic Press, London, UK. Thorstenson YR, Hunicke-Smith SP, Oefner PJ, Davis RW 1998. Genome Res. 8:848-55. Fuhrman, S., Deininger, P., LaPorte, P., Friedmann, T. & Geiduschek, E. (1981) Analysis of transcription of the human Alu family ubiquitous repeating elementbyeukaryotic RNA polymerase III. Nucleic Acids Res., 9 (23), 6439–56. Conclusion

Small fragment DNA shearing to can be achieved reliably and accurately using an automated sonication process.  The SonicMan™ is a high-throughput sonicator capable of providing fragment libraries appropriate for today’s massively parallel sequencing technology.

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