Best practice DNA prep for SMRT. Olga Vinnere Pettersson, PhD Project coordinator NGI-Sweden / SciLifeLab (UU)

Transcription

1 Best practice DNA prep for SMRT Olga Vinnere Pettersson, PhD Project coordinator NGI-Sweden / SciLifeLab (UU)

2 National Genomics Infrastructure - Sweden NGI Stockholm NGI Uppsala NGI staff: FTE, including head of facility, lab research engineers, bioinformaticians, IT-experts, project coordinators. Operates since 1998 (separate nodes); from 2006 as an infrastructure. Hosted by

3 NGI-SciLifeLab is one of the most well-equipped NGS sites in Europe 10 Illumina HiSeq Xten 17 Illumina HiSeq 2000/ Illumina MiSeq 1 Illumina NextSeq 2 Life Technologies Ion Torrent 6 Life Technologies Ion Proton 2 Pacific Biosciences RSII 2 Sanger ABI Argus Whole Genome Map. Syst. 2 Oxford Nanopore MinIon

4 Our 2 machines have seen most of it Applications: WGS De novo Iso-seq Short amplicons Long amplicons Metagenomics Sequence capture Organisms: BACs, YACs, fosmids, plasmids, synthetics Gram positive bacteria Gram negative bacteria Archaea Parasitic protists Fungi (yeasts, moulds and mushrooms) Algae Mosses Higher plants Worms Butterflies Insects Birds Lizards Fish Mammals Human WGS and De novo are the main applications => everything must go perfect We do not make the extractions; our users do

5 Sample looked good, but WGS project of a yeast

6 And at the same time, another yeast WGS 260/ /230 1,9 2,07 1,91 2,15 1,9 2,14

7 WHY?! There is something in the DNA prep The entire workflow of PacBio De novo projects have been re-vised Collection of successful protocols, tips & tricks started Wrote a document R&D on DNA quality assessment

8 Before the order is placed: Meeting with the user Sending a document with sample quality requirements Going through the DNA extraction protocol, suggesting alterations and pointing out the most important steps. Saving time and money in the sequencing step.

9 What makes PacBio so sensitive? Library prep is PCR-free It is a Single-molecule sequencing DNA-binders: Proteins Polyphenols Secondary metabolites (e.g. toxins) Pigments Polysaccharides Polymerase inhibitors: Salts Phenol Alcohols Hamilton & Arya, Nat. Prod. Rep., 2012, 29, Physical inhibiting factors debris

10 The DNA extraction process 1 2

11 What are the main contaminants? Polysaccharides Lypopolysaccharides Growth media residuals Chitin Protein Secondary metabolites Pigments Growth media residuals Chitin Fats Proteins Pigments Polyphenols Polysaccharides Secondary metabolites Pigments

12 General DNA extraction Purpose Disruption of bi-lipid membrane Inactivation of nucleases, removal of protein Removal of phenol Preferential precipitation of DNA Chemistry Surfactants: SDS, CTAB, SLS, etc. Phenol : Chloroform : Isoamylalcohol 25:24:1 (0.6 vol) Chloroform : Isoamylalcohol Ethanol (absolute) Isopropanol (Na-acetate) Salt removal Ethanol (70%) Drying DNA precipitate Re-suspending DNA precipitate Speed-vac RT, +4 C TE (1x), TE (1:10), water Go back to pre-pcr molecular biology search for protocols from 70s-90s

13 Recommended kit: Genomic Tip G QiaGen Gravity-flow, anion-exchange Average recovered molecule size: kb Other kits: MagAttract HMW DNA kit, QiaGen Disclamer: Suggestions and advice are purely based on empirical evidence collected at Uppsala and that there is no conflict of interest.

14 Post-extraction clean-up: Use for samples contaminated with humic acids, phenol, lipids, secondary metabolites, etc. Recovers high-molecular weight DNA. PowerClean DNA clean-up kit MoBio Be prepared to lose up to 80% of the sample

15 For every new DNA sample we ask for: Gel picture 260/280 ratio 260/230 ratio Concentration estimated by TapeStation, Qubit, PicoGreen (be careful with NanoDrop), double of what is needed in the protocol. All users sign a consent form. Samples that do not conform are rejected.

16 What does gel tell us? Protein contamination - Apply phenol-chloroform RNA contamination - Apply RNase, followed by phenol-chloroform extraction Phenol carry-over or overloaded sample? If unsure, make dilution series. If problem persists try MoBio clean-up kit, or re-extract DNA

17 What do absorption ratios tell us? Pure DNA 260/280: < 1.8: Too little DNA compared to other components of the solution; presence of organic contaminants: proteins and phenol; glycogen - absorb at 280 nm. > 2.0: High share of RNA. Pure DNA 260/230: <2.0: Salt contamination, humic acids, peptides, aromatic compounds, polyphenols, urea, guanidine, thiocyanates (latter three are common kit components) absorb at 230 nm. >2.2: High share of RNA, very high share of phenol, high turbidity, dirty instrument, wrong blank. Photometrically active contaminants: phenol, polyphenols, EDTA, thiocyanate, protein, RNA, nucleotides (fragments below 5 bp)

18 Troubleshooting, Yeast Before: BEFORE Alternations 260/280 = /230 = 2.2

19 AFTER alternations Troubleshooting, Yeast After:

20 Troubleshooting: Mould BEFORE AFTER Before: Visible insoluble material in the sample, pigments. After: DNA collected with a glass rod directly after precipitation.

21 For every new de novo project with 8+ SMRT cells We make a titration series on 4 SMRT cells to determine optimal loading of the library Shearing test Making a library (as long as possible) Library QC and evaluating sizing information Load 1x, 2x, 3x and 4x per SMRT cell Determine the optimal concentration and % of reads above 10kbp Adjusting calculations and quotes

22 DropSense R&D

23 Take home: PCR-quality DNA PacBio quality DNA Time spent on DNA extraction pays off Go back to ancient protocols Wish List: New database over successful DNA extraction protocols Continued R&D Inter-lab collaboration

24 Special THANKS NGI / SciLifeLab, Uppsala Ida Höijer Susana Häggqvist Christian Tellgren-Roth Inger Jonasson PacBio Jenny Ekholm Swati Ranade Jamshid Fatehi