NGI Sweden » Guidelines for sample preparation (extractions done by users)

Guidelines for sample preparation (extractions done by users)

Sources of DNA and DNA extraction

We strongly recommend that reference specimens are snap-frozen in the absence of a buffer, and stored at -80°C prior to DNA extraction. For a selection of DNA extraction protocols suited for long-read sequencing, please refer to https://www.protocols.io/groups/awesome-DNA-from-all-kingdoms-of-life and the BioNano homepage.

DNA extraction from the following sources:

Vertebrates: Fresh-frozen EDTA-blood, spleen, muscle. Avoid organs with a high-fat content, and connective tissue.
Recommended kits:
Mammalian samples: MagAttract, Zymogen for HMW DNA. For ultraHMW DNA, NanoBind discs from Circulomics or BioNano prep kits.
Nucleated blood: EDTA-blood and phenol-chloroform extractions, MagAttract, Circulomics NanoBind . For the latter, difficult-to-dissolve uHMW DNA can be subjected to gentle shearing by pipetting.
Insects, arachnids and arthropods: dissected muscles of adult individuals or dissected insect pupae. Avoid using the gut unless you are interested in studying insect microbiota!
Recommended kits:
The salting-out modification of the QiaGen MagAttract kit (https://support.10xgenomics.com/de-novo-assembly/sample-prep/doc/demonstrated-protocol-dna-extraction-from-single-insects) and Circulomics Nanobind kits.
Plants: young shoots and leaves, if possible – dark-treated for 24 hours prior to collection/extraction.
Recommended kits:
Qiagen DNeasy PowerClean Pro Cleanup and the Zymogen DNA Clean-up and Concentrator kits are designed to facilitate post-extraction purification of DNA contaminated with high levels of polysaccharides, phenolics, and secondary metabolites.
QIAGEN Genomic-tip 500/G, MagAttract, Circulomics NanoBind, Macherey-Nagel NucleoSpin, and organism-appropriate Zymogen kits.
If the DNA pellet is cleaned with a buffer containing 1% CTAB, remember to dilute it out since it can inhibit the sequencing process.
Please also ensure that submitted samples do not contain phenol, which can interfere with estimating purity and concentration of the sample.
Bacteria and yeast: Culture cells to mid-log phase in sufficient volumes to compensate for the lower A₆₀₀. Avoid culturing cells to stationary phase.
Recommended kits:
Use the QIAGEN Genomic-tip 500/G kit to extract DNA.
If a clear threadlike precipitate is observed during isopropanol precipitation, thenspool the DNA and dip-wash it in 70% EtOH.

For traditional HiFi protocol, 15 μg of chemically pure HMW DNA is required. We can process samples using our low-input or express library protocols. The low-input protocol allows construction of PacBio libraries from as little as 5 ng (ultra-low input) or 150 ng (low input) of DNA (recommended for genomes under 300 Mb).

For ONT sequencing, we require a minimum DNA amount of 3 μg for each PromethION flow cell for 20-30 kb libraries, 5 μg for 75+ kb libraries, and 40 μg for ultra-long libaries.

All samples are assessed for quality and concentration with the Qubit upon receipt by NGI.

Please contact olga.pettersson@scilifelab.uu.se for advice on sample collection, tissue preservation and DNA extraction for your specific organism type.