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Sage Science develops sample prep technologies for life science research. We focus on electrophoretic approaches that improve and automate high-value steps in Next Gen sequencing workflows.

Sage sells the Pippin™ line of DNA size selection instruments, which are widely used for DNA, RNA, and ChIP-seq library construction for short-read sequencing. Our systems are also used for preparing high molecular weight DNA for 3rd generation, long-range genomics platforms.

Our products are manufactured at our headquarters in Beverly, Massachusetts, USA.

DNA Extraction for Next Gen Sequencing

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DNA size selection for NGS libraries

The advent of Next Gen Sequencing (NGS) has been truly amazing. One of the marvels that is often overlooked is how advances in DNA extraction technology have helped streamline NGS workflows. The original standard – phenol/chloroform extraction – is not well suited to the automated nature of today’s sequencing workflows (though with the emergence of long read sequencing it is making a bit of a comeback).

Most methods use silica-based solid phase extraction that was worked out in the early 1980s. This is based on the principle that DNA will adhere to glass while proteins can be digested from cells and washed away.

Packaged in easy-to-use spin columns, these are staples of NGS workflows and can completely automated if need be. Eluted DNA is then sheared to an optimal fragment size- a critical step. For instance, if you are running a 150X150 chip on an Illumina MiSeq, its good to have a library where most DNA fragments are around 150 bp. Sometimes a DNA size-selection step can help dial in
that optimum even further.

The Challenge of Long-Read Sequencing

With new platforms that can produce sequencing reads in the 10s to 100s of kilobases, creating libraries with large inserts can be difficult. Silica based extractions will fragment DNA. Bead clean-ups will also further fragment DNA – in fact every successive pipetting step (wide-bore tip recommended!) can break the longer DNA fragments in the sample.

Luckily, silica-based spin columns have been formulated to minimize shearing. High molecular weight (HMW) versions will produce fragments to 200 kb and even beyond, but they will include a “trail” of DNA fragments down to 10kb.

There are alternatives; anion exchange gravity columns elute 50-150 kb fragments, and then there’s the old standby: phenol-chloroform extraction. If great care is taken, phenol-chloroform can produce fragments ›300kb with minimal low molecular weight DNA present.

As with short-read sequencing, HMW DNA extractions need to be sheared to a Gaussian fragment size profile in order to maximize the performance of the long-read sequencer. This typically involves pushing the DNA through a pore which can be fairly inexpensive, using specialized centrifugation tubes or simply pushing the DNA through a syringe needle.

There are also more sophisticated instruments that provide more shearing options. For these workflows, a DNA size selection step can also be very beneficial and is typically used to filter out smaller fragments below a size threshold.

Ultra-HMW DNA Extraction

There are methods to isolate ultra-HMW DNA. Though onerous, this can be the only option for very complex genomes (usually plants) and involves using agarose plugs. Cells or protoplasts (this won’t work with plant cell walls) can be embedded in agarose. The agarose can then provide a solid but porous matrix which allows for the diffusion lysis reagents for DNA purification while preventing the

DNA from being sheared.  In this way, almost intact genomic DNA can be isolated with the agarose gel. The DNA can be cut with restriction enzymes and Ultra-HMW DNA gently eluted with electrophoresis. The SageHLS platform by Sage Science uses a similar approach but with a novel twist. The agarose gel electrophoresis is semi-automated, and CRISPR/Cas9 technology (a process called CATCH) is used to target extract specific HMW genomic regions up to 500 kb. This can provide very large genetic target areas without requiring the sequencing of the whole genome.

Further Reading:

  1. Boom R, et al. (1990) Rapid and simple method for purification of nucleic acids.. J Clin Microbiol. 495–503.
  2. Schwartz  DC, Cantor CR.(1983) Separation of yeast chromosome-sized DNAs by pulsed field gradient gel electrophoresis. Cell. 31(1):67–75.
  3. Volgelstein B, Gillespie D. (1979)  Preparative and analytical purification of DNA from agarose.. Proc. Natl. Acad. Sci. 76(2):615-619.
  4. Zhang H, Zhao X, Ding X, Paterson AH, Wing RA (1995) Preparation of megabase-size DNA from plant nuclei. The Plant Journal. 7(1):175-184.
  5. Gong L, et al. (2019) Ultra-long Read Sequencing for Whole Genomic DNA Analysis. J. Vis. Exp. (145), e58954.
  6. Shin G, et al. (2019) Targeted short read sequencing and assembly of re-arrangements and candidate gene loci provide megabase diplotypes  Nucleic Acids Research.
Image Credit: Sage Science

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