DNA requirements

For genomic samples, NSC offers three different library prep protocols depending on input amounts available: Standard HiFi libraries, Low input libraries and Ultra-low input libraries.

Image: Courtesy of Pacific Biosciences of California, Inc., Menlo Park, CA, USA
Image: Courtesy of Pacific Biosciences of California, Inc., Menlo Park, CA, USA

Please note that, in order to improve processing options, NSC recommends delivering more DNA than indicated in the table above. Input amount depends on genome size and number of SMRT cells needed. For 3 Gb genomes, at least 15 µg DNA is needed, for smaller genomes (< 1Gb), 7 µg DNA should be enough. For genomes <100 Mb, several samples can be sequenced on same SMRT cell and DNA amount required is lower. Each project is unique, please contact us to discuss yours!

For microbial samples, 3.4 µg DNA is requested. Samples should be normalized to 45 ng/µl and delivered on plate or strips, in 75 µl. If you do not have required amount, please send us all DNA (at least 1.5 µg) in 75 µl. 

For PacBio sequencing technology, integrity and purity of the DNA sample is of utmost importance. The DNA samples should meet these criteria:

  • The majority of the DNA must be equal or greater than 40-50 kbp.
  • Minimal DNA purity: OD 260/280 should be 1.8-2.0; OD 260/230 should be >2.0
  • Sample has undergone a minimum of freeze-thaw cycles
  • Sample has not been exposed samples to temperatures above 65°C
  • Sample has not been exposed to pH extremes (<6 or >9)
  • No insoluble material in the sample
  • Sample is RNA-free
  • Sample has not been exposed to UV or intercalating fluorescent dyes
  • Sample should not contain any chelating agents (e.g. EDTA), divalent metal ions (e.g. Mg2+), denaturants, or detergents (note: EDTA concentration in 0.1x TE buffer is OK).
  • No carry-over contamination that originates from the source organism or DNA-prep (phenols, salts, polysaccharides, lipids).

DNA extraction

For reference genome sequencing, we strongly recommend to use a single individual. If isolation of DNA from fresh tissue is not possible, sample should be flash-frozen with liquid nitrogen and stored at -80°C. 

DNA isolation protocols suited for PacBio sequencing can be found here: 

PacBio guidelines for preparing DNA for PacBio HiFi sequencing

Extractdanforpacbio.com

HMW DNA extraction from all kingdoms

Please be aware that some samples might require testing of several HMW DNA extraction protocols. 

Guidance for sample collection, preparation and storage: PacBio guidelines for preparing samples for PacBio sequencing

 

 

Published Nov. 15, 2021 12:20 PM - Last modified May 13, 2022 9:28 AM