DNA Size Selection

Demonstrated Protocol, Last Modified on July 7, 2017, Permalink

CG00018_SamplePrepDemonstratedProtocol_-_DNASizeSelection_RevD.pdf

The Chromium Genome Protocols generate long-range information across the length of individual DNA molecules. Starting the process with High Molecular Weight (HMW) genomic DNA (gDNA) will typically result in better application performance, such as increased haplotype phase block length and ability to call structural variants. Optimal performance has been characterized on input gDNA with a mean length >50 kb.

However, gDNA samples may exist that do not meet this specification, for example:

• gDNA samples may not have been extracted using an optimized HMW extraction protocol
• gDNA samples may be old and the DNA has degraded
• HMW gDNA isolation is generally difficult for certain sample types (e.g. solid tissue)

This Demonstrated Protocol outlines the use of both the BluePippin and the PippinHT instruments for removing gDNA molecules <20 kb and DNA molecules <40 kb. Briefly, gDNA was extracted from live cultured cells (cell lines NA12878 and NCI-H228) following the HMW gDNA extraction protocol outlined in the Chromium Genome User Guides. The extracted gDNA was then mechanically sheared to degrade the DNA size to ~30-40 kb and then subject to size selection on the BluePippin or PippinHT instruments. Sizing results were verified by pulsed-field gel electrophoresis (results not shown) and DNA sequencing.