Since long-read sequencing protocols (PacBio, Nanopore) analyze single DNA molecules, success depends on high-quality DNA samples. 10X Genomics linked-read sequencing has similarly high sample requirements with regards to purity and integrity, but requires far lower amounts. Thus we are now offering high-molecular-DNA (HMW-DNA) isolations as a service. The applied DNA isolation protocols will vary with the sample types. Our protocols aim to isolate HMW-DNA samples with mean fragment lengths of 50 kb or higher, if the sample allows. Longer fragments can be expected for examples for cell culture or fresh blood samples. However, we can’t promise any specific performance metrics. For larger projects we will test isolations first. The HMW-DNA isolation service includes DNA sample QC by pulsed field electrophoresis (PFGE), spectrometry and fluorometry.
The highest quality DNA samples can be isolated from cell culture and fresh blood samples. If tissue samples need to be used soft tissues are preferable. In any case submitting sterile samples should be a priority; this means that guts, gills, skin tissues should be removed if possible before flash freezing. Smaller organisms should best be not be fed for appropriate time spans before collection to avoid isolating food DNA and reduce microbiotic content.
Please note that our lab is not set up to work with samples that are potentially infectious to humans.
Please find detailed sample collection recommendations here: Sample Collection Recommendations for Long-Read Sequencing and Gene Annotations
HMW-DNA isolations from animal samples:
Cell culture samples: Please submit 10-50 million cells or more for HMW DNA extraction. Cells should be pelleted, washed with 1X PBS, supernatant removed, flash frozen in liquid nitrogen and stored at -80C. Please ship the cell pellets on dry ice. For 10X Genomics sequencing 2 million cells are sufficient.
Tissue samples:
Fresh tissue must be snap frozen in liquid nitrogen right after harvesting and stored at -80°C. Frozen sample should be shipped on dry ice. Any freeze thaw cycles should be avoided. Please submit at least 500 mg of soft tissue; not including guts.
Blood samples:
Samples should be collected fresh using EDTA as anticoagulant. We prefer to receive fresh blood samples shipped overnight on cold packs; these samples should be stored in a refrigerator temporarily. Blood samples can also be flash frozen in liquid nitrogen and stored at -80 0 C. Frozen samples should be shipped on dry ice. About 5 mls of mammalian blood sample will be required each for HWM DNA isolation (for 10X Genomics sequencing 2 ml blood will be sufficient; potentially less). Please contact us beforehand to set up an appointment if you plan to ship fresh blood samples with overnight shipping. If blood samples will arrive within two days after the blood draw it is generally better to not freeze the samples; instead, keep the samples cold at all times (refrigerator; 4°C) and transport them between a generous number of cold packs.
Plant samples:
Please submit 2-5 g of young leaf tissue. Flash freeze the tissue in liquid nitrogen and store at -80. Please transport the tissue on dry ice.
For the sequencing over a larger number of flowcells or SMRT-cells additional material might be required.
Our HMW-DNA isolation expert is Ruta Sahasrabudhe, PhD. Please contact her at rmsaha@ucdavis.edu for questions about HMW DNA extractions, Nanopore Sequencing & Hi-C projects.
