Plant Sequence Capture Optimised for Illumina Sequencing

Materials and Reagents

1. SYBR Gold (Life Technologies, catalog number: S11494 )
   
2. UltraPure  Agarose (Life Technologies, Invitrogen^TM, catalog number: 16500-500 )
   
3. PCR-grade water
   
4. Tris (hydroxymethyl) aminomethane (Tris base) (multiple vendors)
   
5. Tween 20 (Bio-Rad Laboratories, catalog number: 170-6531 )
   
6. Ethylenediaminetetraacetic acid (EDTA) (multiple vendors)
   
7. Ethanol (absolute) (analytical grade) (multiple vendors)
   
8. 70 % (v/v) ethanol (analytical grade)
   
9. Isopropanol (2-Propanol) (>99.5%) (multiple vendors)
   
10. Acetic acid (glacial) (multiple vendors)
    
11. 2x Phusion High-Fidelity PCR Master Mix (New England BioLabs, catalog number: F-531L )
    
12. GeneRuler  50 bp DNA ladder (Thermo Fisher Scientific, catalog number: SM 0371 )
    
13. QIAquick PCR Purification Kit (QIAGEN, catalog number: 28106 )
    
14. Minelute PCR Purification Kit (QIAGEN, catalog number: 28006 )
    
15. Agilent DNA 7500 Kit (Agilent Technologies, catalog number: 5067-1506 )
    
16. Agilent High Sensitivity DNA Kit (Agilent, catalog number: 5067-4626 )
    
17. SeqCap EZ Hybridization Kit [containing NimbleGen SC Wash Buffers (tubes 1, 2 and 3), Stringent Wash Buffer (tube 4), 2x SC Hybridisation Buffer (tube 5), Hybridisation Component A (tube 6) and the Bead Wash Buffer (tube 7)] (Roche Diagnostics, catalog number: 05634261001 )
    
18. Sequence Capture Developer Reagent (Roche Diagnostics, catalog number: 06684335001 )
    Note: This reagent was previously known as Plant Capture Enhancer (PCE) from Roche NimbleGen.
    
19. 6x loading dye (Thermo Fisher Scientific, Fermentas, catalog number: R 0611 )
    
20. DNA away (Thermo Fisher Scientific, catalog number: 21-236-28 )
    
21. Oligonucleotides (Sigma-Aldrich)
    Note: All oligonucleotides were reverse phase cartridge purified and dissolved in PCR-grade water. To avoid cross-contaminations each oligonucleotide was purified using a fresh column. Sequences are listed in Table 1 in Supplementary Material.
    
22. Reagents for the generation of capture libraries:
    1.  Illumina TruSeq DNA kit (Illumina, catalog number: FC-121-2001 , Box A) or
       
    2. TruSeq DNA PCR-free sample preparation kit (Illumina, catalog number: FC-121-3001 , Set A)
       
    3. If the DNA Illumina multiplex (IM) protocol is used, the following additional reagents (items 27- 41) are required.
23. T4 polynucleotide kinase (10 U/µl) (Thermo Scientific Fermentas, catalog number: EK0032 )
    
24. T4 DNA polymerase (5 U/µl) (Thermo Scientific Fermentas, catalog number: EP006B )
    
25. 10x Buffer Tango (Thermo Scientific Fermentas, catalog number: BY5 )        
    
26. dNTPs (25 nM each) (Thermo Scientific Fermentas, catalog number: R1121 )
    
27. ATP (100 mM) (Thermo Fisher Scientific, catalog number: R0441 )            
    
28. T4 DNA ligase (5 U/µl) provided with 10x T4 DNA ligase buffer and 50% PEG 4000 (Thermo Scientific Fermentas, catalog number: EL0011 )                  
    
29. 10x ThermoPol reaction buffer (New England Biolabs, catalog number: B9004S )    
    
30. Bst polymerase (8 U/µl) (large fragment) (New England Biolabs, catalog number: M0275L )
    
31. 5x Phusion HF buffer (New England Biolabs, catalog number: B0518S )              
    
32. Phusion Hot Start Flex DNA polymerase (2 U/µl) (New England Biolabs, catalog number: M0535L )
    
33. carboxyl-modified Sera-Mag Magnetic Speed-beads (Thermo Fisher Scientific, catalog number: 1182-9912 )
    
34. PEG-8000 (Sigma-Aldrich, catalog number: 89510 )
    
35. NaCl (Sigma-Aldrich, catalog number: S3014 )
    
36. Agilent High Sensitivity DNA Kit (Agilent, catalog number: 5067-4626)
    
37. Agilent DNA 7500 Kit (Agilent, catalog number: 5067-1506)
    
38. TE (pH 8.0) (see Recipes)
    
39. Resuspension Buffer (RSB) (see Recipes)
    
40. 50x TAE (see Recipes)
    
41. EBT (see Recipes)
    
42. Test fragment for DNA Illumina multiplex (IM) libraries (see Recipes)
    
43. Adapter mix P57 for IM libraries (see Recipes)
    
44. MagNA beads for DNA clean-up (see Recipes)
    

Equipment

1. Qubit 2.0 fluorometer (Starter Kit) [including instrument, assay tubes, dsDNA HS Assay and dsDNA BR Assay] (Thermo Fisher Scientific, catalog number: Q32871 ) or other picogreen-based dsDNA quantification devices
   
2. AMPure XP Beads (Beckman Coulter, catalog number: A63882 )
   
3. Streptavidin Dynabeads (M-270) (Life Technologies, Invitrogen^TM, catalog number: 65306 )
   
4. 1.5 ml tubes (multiple vendors)
   
5. 0.2 ml PCR-tubes (multiple vendors)
   
6. 96-well plates (Greiner Bio-One GmbH, catalog number: 652250 )
   
7. Plastic seals for 96 well plate (Thermo Fisher Scientific, catalog number: AB-0558 )
   
8. Covaris S220 AFA Ultrasonicator (LGC, catalog number: KBS-500217 ) and associated equipment such as microTUBE holder, chiller, software, computer etc.
   
9. Snap-Cap microTUBES with AFA-fiber and pre-split septum (Covaris, catalog number: 520045 )
   
10. Agilent 2100 Electrophoresis Bioanalyzer (Agilent, catalog number: G2939AA ) and associated material (e.g. computer)
    
11. NanoDrop 2000 Spectrophotometer (VWR International, PeQlab, catalog number: 91-ND-2000 )
    
12. HiSeq or MiSeq Illumina systems and associated materials (such as Sequencing-by-Synthesis reagents for 2x 100 cycles)
    
13. Heating block (multiple vendors)
    
14. Water Bath with external calibrated thermometer (multiple vendors)
    
15. Microcentrifuge (16,000 x g) (multiple vendors)
    
16. SpeedVac (multiple vendors)
    
17. Thermocycler (multiple vendors)
    Note: Throughout the protocol a “Bio Rad DNA Engine Tetrad 2 Peltier Thermal Cycler” was used.
    
18. Vortex Mixer (multiple vendors)
    
19. DynaMag-2 Magnet (Life Technologies, Invitrogen^TM, catalog number: 123-21D )
    
20. DynaMag-96 Side Skirted Magnetic Particle Concentrator (MPC₉₆) (Life Technologies, Invitrogen^TM, catalog number: 120.27 )
    
21. Agarose gel electrophoresis equipment and accessories [microwave, tray (15 x 15 cm), combs, power supply, UV-transilluminator, etc.] (multiple vendors)
    
22. Disposable scalpels (multiple vendors)
    
23. Dark Reader blue light transilluminator (Clare Chemical Research, catalog number: DR46B )
    
24. Pipettes (2, 100, 200 and 1,000 µl, multiple vendors)
    
25. Filter tips (multiple vendors)
    

Procedure