Long-reads require long DNA molecules which require high molecular weight (and undamaged) DNA. Extraction with your standard column is unlikely to be optimal. I’m starting to look at the best methods for HMW DNA extraction for long-read applications as my lab is supporting a couple of pilot projects on the 10X Genomics phased genomes/exome technology, and we’re bringing the ONT MinION into my core lab as a new service offering this year.
The last time I was thinking about HMW DNA prep was almost 15-20 years ago. A group at JIC was part of the massive Wheat BAC cloning project and I was in the lab sharing the robotics for colony screening (I was making macro-arrays for “high-throughput” gene expression analysis – 1152 PCR amplicon spots).
Two recent blog posts caught my attention; the first from Nick Loman’s group in Birmingham describing the protocol used to generate 950kb reads on MinION, the second from the 10X Genomics blog “Meet Jill Herschleb” where Jill describes their protocol for fresh-frozen tissue that yields ~300 kb fragments. Neither of these preps use gel plugs!
There are many variables that will affect gDNA quality in sample types, including sample age, transportation methods, type of tissue, additives, freezing method, etc.
The Loman protocol:
The Quick Protocol, sounds faster than the now obsolete,
Loman Protocol (sounds like a Joy Division album) is capable of generating incredibly large HMW DNA molecules with the record standing somewhere north of 900kb. It uses a modified Sambrook phenol-chloroform extraction/purification (read Cell: “Culinary Biology” a review of Maniatis from 1990 which says “the appendix is an unexpected joy…loaded with such gems as…the electronic mail address of the Japanese nucleotide sequence data bank”), with minimal pipetting steps. I used this same method for some huge DNA extraction protocols in 1997 when a research assistant at the JIC – it’s a real blast from the past.
- 5 x 10^7 human cells in a 50 ml Falcon tube (I’m unsure how much tumour tissue this equates too – please make suggestions in the comments)
- Mix in 10ml of TLB buffer (Tris, EDTA, a little SDS and RNaseA)
- Incubate at 50C for 3 hours (Nick suggests rotating the tube end over end, a tube roller might be gentler)
- Add the lot to a 50ml Phase-lock Gel tube and 10ml Phenol:Chloroform
- After phase separation the DNA is ammonium acetate + ethanol precipitated
- HMW DNA can then be “spooled” on a glass rod
Take care not to be harsh when handling the HMW DNA and the results are remarkable as attested to by Nick and Josh’s success.
— Mark Akeson (@AkesonUCSC) March 8, 2017
10X Genomics protocol:
A note of caution when reading Nick’s post (see above): the focus on MinION is on the number of molecules extracted. For 10X Genomics we need to ensure there are not too many molecules present as this messes with the expectations of what’s likely to be in a gel bead for molecular tagging.
Jill manages the 10x Genomics sample prep group (all the preps for Genome and Single-cell applications – she’s a good lady to know, follow her on Twitter @ScienceJill ). Her group have developed a HMW DNA extraction protocol for fresh-frozen tissue that is capable of generating ~300 kb fragments using magnetic bead extraction.
The 10X protocol describes extraction from fresh frozen breast tumour tissue so is immediately relevant to much of the work we’re doing in the CRUK Cambridge Institute. It does include a disclaimer that it’s not been tested with other tissues which may give sub-optimal DNA extraction e.g. liver, due to high enzyme activity.
The protocol says to handle the extraction”carefully” and suggests using a wide-bore pipette tips.
It was tested on two replicate fresh frozen breast tumour tissue samples, with two elution’s from each sample. PFGE analysis showed >150kb length HMW DNA and Chromium genome analysis returned mean DNA size of >80 kb, the highest sample quality defined in their Chromium Genome tech note.
- Start with around 25mg of tissue (0.5 cm3 so small biopsies are out)
- The protocol uses the Sigma PURE extraction kits (about £30 per sample)
- Nuclei are first isolated then digested with Proteinase K
- HMW DNA is then purified with SPRI Select (read my 2012 “How SPRI works” post – over 150,000 other people have!)
- Expect an average size of >150-200 kb by pulsed-field gel electrophoresis.
I’ll be looking at other protocols in the next few months so if you have recommendations please do suggest them in the comments.
PS: compare and contrast the differences in DNA quality with the differences in production quality – Nick’s Google Doc versus 10X professionally designed PDF.