Megaruptor^®

Validation using two different DNA sources and two different methods of analysis. A: Shearing of lambda phage genomic DNA (20 ng/μl; 150 μl/sample) sheared at different speed settings and analyzed on 1% agarose gel. B: Fragment Analyzer profiles of human genomic DNA (25 ng/μl; 200 μl/sample) sheared at different software settings of 2, 3, 5 and 8 kb. (Standard Sensitivity Large Fragment Analysis Kit ; Advanced Analytical Technologies, Inc. was used for separation and fragment sizing).

The Norwegian sequencing centre (NSC) is a national core facility fully equipped to handle a diverse set of sequencing projects. We see an increased interest in long read technologies, in which our PacBio Single Molecule sequencing service is important. PacBio, (or SMRT) sequencing, like any other single molecule system, starts with a critical library preparation step to generate long fragment libraries of 10-20kb length. Even longer libraries can be made, but common to all library types is to start with good quality DNA of high molecular weight. Physical shearing of the DNA, targeting the desired library length, is the first critical step in the procedure. NSC has experience with different methods to fragment DNA like nebulization, enzymatic, tagmentation, covaris AFA, g-tubes (also covaris), custom syringe/needle protocols and hydroshearing. The best fragmentation method should produce a sharp fragmentation pattern close to the target length as defined by the protocol settings, with as little “smear like” pattern of lower molecular length as possible. The final step of PacBio library preparation is to size select only the longest fragments (> 7kb) as this increases the overall success rate of only sequencing the longest library fragments. It is not uncommon to loose a lot of material during this step. However, if the initial fragmentation yields sharp fragmentation patterns with minimal smear, more of the total DNA is retained in the final library. This is perhaps most important on samples were DNA amounts are limited. The optimal fragmentation method has to produce similar fragmentation pattern each time, with little deviation with sample type and input amount. We have found the new Megaruptor from Diagenode to be one of the best instruments for routine shearing of DNA to lengths of 2-40kb. It is easy to use, with walk away shearing, and the instrument does all the washes before, in between samples and after shearing without user intervention. Disposable hydropores is beneficial for lowering the contamination risk. The fact that it is so easy to use make it a good choice for laboratories with multiple users and/or when training new staff.

Morten Skage and Dr Ave Tooming-Klunderud, Norwegian Sequencing Centre, Oslo, Norway

We will use  the Megaruptor^® within our PacBio^® Single Molecule Sequencing workflows, especially for the construction of very big insert  (>20kb) libraries.

Andrea Patrignani, Anna Bratus and Ralph Schlapbach, Functional Genomics Center Zurich (ETHZ/UZH), Switzerland