The perfect genome is a treasure, DNA is the key

Transcription

1 SMRT-Leiden Jun 12-14, Leiden, NL The perfect genome is a treasure, DNA is the key Olga Vinnere Pettersson, PhD Project Coordinator National Genomics Infrastructure Uppsala University

2 Outline National Genomics Infrastructure (NGI): who we are. Long-read Sequencing at NGI: what we do. HMW-DNA R&D: Lessons learned from 4 years with PacBio HMW-DNA vs normal DNA HMW-DNA extraction lab & our experience

3 NGI: Mission statement From Jan 1, 2013: NGI is a national resource for massively parallell sequencing National resource Guidelines and support State-of-theart infrastructure Our mission is to make a state-of-the-art infrastructure for massively parallel DNA sequencing and SNP genotyping available to researchers all over Sweden enabling internationally competitive research in genomics. To provide guidelines and support for study design, sample collection, protocol selection and bioinformatic analysis.

4 Platform organisation NGI-Uppsala SNP&SEQ Technology platform Ann-Christine Syvänen, UU Professor in Molecular Medicine NGI-Uppsala Uppsala Genome Center Ulf Gyllensten, UU Professor in Medical Molecular Genetics Lars Feuk, UU Senior lecturer (Deputy) NGI Uppsala NGI NGI Stockholm NGI-Stockholm Joakim Lundeberg, KTH Professor in Gene Technology

5 Project handling at NGI

6 This research infrastructure is world class and a jewel in the crown of Swedish bioscience. (Swedish Research Council)

7 Long-Read technologies in NGI service Number of PacBio projects, Short amplicons Long amplicons De novo large genomes De novo small genomes Sequence capture Base Modifications Iso-seq EVERY PROJECT IS UNIQUE

8 SMRT sequencing, lessons learned: DNA quality requirements Same yeast, different DNA FOCUS: chemical purity For Long Reads one needs to have long and pure DNA

9 Focus on chemical purity: sorting out the culprits 1. Carry-over from host cells - you name it 2. Carry-over of extraction chemicals - Phenol - Guanidinium - Ethanol - EDTA "DNA chemical structure," by Madeleine Price Ball Most have dual action: - Enzyme inhibition - DNA-binding

10 Focus on chemical purity: why so important? Lower chemical purity = worse loading and shorter reads, Ergo higher sequencing and analysis costs. Organism type, de novo application, 60x 2 Gb / SMRT 4 Gb / SMRT 7 Gb / SMRT Bacterium (3.2 Mb) 2-4 strains 6-8 strains strains Insect (300 Mb) N cells: 9 Price: 10 keur Bird (1.2 Gb) N cells: 43 Price: 47 keur Mammal (3.2 Gb) N cells: 96 Price: 105 keur N cells: 5 Price: 6 keur N cells: 22 Price: 25 keur N cells: 48 Price: 53 keur N cells: 3 Price: 4 keur N cells: 12 Price: 14 keur N cells: 27 Price: 30 keur

11 Focus on structural integrity: learning how to work with HMW-DNA Working with 25+ kb libraries is becoming a standard Do not have a Fragment Analyser of FemtoPulse yet? Consider it. Hydroshear speed code 3 Size by gel: 6 Kb Hydroshear speed code 15 Size by gel: 20 kb

12 25+ kb library an example of a good DNA sample Input QC Shearing QC Library QC Before size-selection Library QC After size-selection

13 A journey to HMW-DNA world HMW-DNA behaves very differently from LMW-DNA High viscosity: 1. Bad hydration -> topological issues -> getting HMW-DNA in solution without any concentration gradient is an issue 2. Presence of contaminants

14 Getting correct readings of HMW-DNA DNA-spa: let the molecules relax days at RT or +4 C - Gentle agitation - Playing with ionic strength HMW-DNA Read N50, kb E.coli in situ - fresh 13.3 E.coli in situ - relaxed 22.3 LMW-DNA HMW-DNA FemtoPulse measurements: several replicates (and dilution series)

15 Real life examples Bird 1 CONTIGS Primary Associated Unzipped Primary Unzipped haplotigs # contigs # >50Kb Largest 52,5Mb 0,18Mb 52,6Mb 3,11Mb N50 11,4Mb 0,05Mb 12,1Mb 0,42Mb Total 1,12Gb 97,8Mb 1,07Gb 1,01Gb 20 kb library perfect DNA 60x Bird 2 SCAFFOLDS Unphased 10 kb library short, dirty & little DNA 36X # scaffolds # >50Kb 201 Largest 2,01 Mb N50 0,21 Mb Total 1,21Gb User insisted to proceed despite warning

16 HMW-R&D at NGI: DNA lab In situ DNA extraction is a dying art (developed in late 70s early 80s). DNA extraction is carried out in agarose plugs -> melting -> dialysis (-> clean-up) Allows recovery of intact Mb-size molecules, suitable for all any Long-Read technology Objectives: Nuclei extraction worth it! Testing protocols HMW-DNA storage parameters Preservation of HMW-fraction Recording contaminant information Compiling experimental data Other types of kits & protocols we use: MagAttract, GenomicTip & Phenol-ChISAM.

17 Note on commercial HMW-DNA kits Issue: focus on length, not chemical purity One kit might not suit them all! Keep an eye for: 0.5 mm EDTA -> 0.05 mm EDTA Guanidinium 260/280 & 260/230 Carry-over of host cell contaminants Kits that we know work for sure (in most cases), as on May 2018: MagAttract, Genomic Tip, Zymogene genomic DNA clean-up and concentrator

18 CONCLUSIONS - Both molecule length and chemical purity of input DNA are equally important to successful sequencing. Spending time on optimizing DNA extractions save money for sequencing (and analysis). - Working with HMW-DNA is a craft. It is worth to learn. - The HMW-DNA field is becoming extremely popular. There are still many unknowns, however, the basic biochemistry is still the same.

19 Acknowledgements Mai-Britt Mossbech, PhD R&D engineer, DNA extractions Tomas Klingström, PhD Faithful young padawan Inger Jonasson Boss who allows me to play with DNA UGC-NGI team Who tolerates all my requests for additional DNA-QC Sponsors and vendors