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.
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.
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