搜索

Plant Sequence Capture Optimised for Illumina Sequencing
优化用于Illumina测序的植物序列捕获技术   

下载 PDF 引用 收藏 提问与回复 分享您的反馈 Cited by

本文章节

Abstract

Plant Sequence Capture is used for targeted resequencing of whole exomes (all exons of a genome) of complex genomes e.g. barley and its relatives (Mascher et al., 2013). Sequencing and computing costs are significantly reduced since only the greatly enriched and gene-coding part of the barley genome is targeted, that corresponds to only 1-2% of the entire genome. Thus, applications such as genetic diversity studies and the isolation of single genes (“cloning-by-sequencing”) are greatly facilitated. Here, a protocol is provided describing the construction of shotgun DNA libraries from genomic barley DNA for sequencing on the Illumina HiSeq/MiSeq systems. The shotgun DNA sequencing libraries are hybridized to an oligonucleotide pool (Exome Library) encompassing the whole exome of barley. The Exome Library is provided as a liquid array containing biotinylated probes (Roche/NimbleGen). Subsequently, genomic shotgun DNA fragments hybridized to the Exome Library are affinity-purified using streptavidin coated magnetic beads. The captured library is PCR-amplified and sequenced using high-throughput short read sequencing-by-synthesis.

Keywords: Sequence capture(序列捕获), Targeted resequencing(靶向基因测序), Exome(外显子组), Sequencing-by-synthesis(合成测序), Barley(大麦)

Materials and Reagents

  1. SYBR Gold (Life Technologies, catalog number: S11494 )
  2. UltraPure  Agarose (Life Technologies, InvitrogenTM, 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, InvitrogenTM, 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, InvitrogenTM, catalog number: 123-21D )
  20. DynaMag-96 Side Skirted Magnetic Particle Concentrator (MPC96) (Life Technologies, InvitrogenTM, 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

  1. Concentration of genomic DNA
    1. The genomic DNA should be isolated using standard methods for barley (e.g. Stein et al., 2001). Dissolve DNA in TE (pH 8.0). It is important to make sure that the genomic DNA is completely dissolved. Any insoluble matter should be removed by centrifugation (16,000 x g, 10 min, RT). The soluble supernatant is transferred into a fresh tube for fragmentation.
    2. Determine precisely the concentration of genomic DNA [dissolved in TE (pH 8.0)] using a system specific for dsDNA. The basic quantification protocol is described here for the Qubit 2.0 fluorometer. Any other picogreen-based assays may be used as well. Depending on the DNA starting concentration use the HS Assay (0.2 ng– 100 ng) or the BR Assay (2 ng-1,000 ng), respectively.
      For the Qubit device 1 µl genomic DNA is diluted with 199 µl Working Solution (1 µl dye/200 µl buffer, equilibrated to RT) in Qubit assay tubes.
      Dilute in Qubit assay tubes 10 µl of the two standards with 190 µl Working Solution.
      Vortex Qubit assay tubes, incubate for 2 min and measure the DNA concentration.
       Note: To ensure reproducibility of the measurements make 3 independent quantifications.
    3. Using TE (pH 8.0) adjust the DNA concentration to 1 µg in a total volume of 53 µl and store the sample on ice for fragmentation.

  2. DNA fragmentation
    1. For DNA fragmentation a Covaris S220 device is employed. The instrument generates adaptive focused acoustic energy resulting in mechanical disruption of DNA. Operate the instrument according to the manufacturer instructions. Fill the water bath of the Covaris S220 according to the water level indicator on the front of the water bath (“FILL” column) to the “12” level. Use deionized or distilled water. The water bath must be degassed and cooled (7 °C) for proper operation of the device. Preparation of the instrument will take about 40 min.
      Use the following fragmentation parameters (Mascher et al., 2013):
      175 W ultrasonic power
      10% duty factor
      200 cycles per burst and 50 sec treatment, 30 sec pause followed by additional 50 sec treatment.
    2. Pipette the genomic DNA (1 µg in 53 µl) into the Covaris microTUBE (precooled on ice). Insert the tube into the holder and fragment the DNA.  
    3. Transfer the fragmented DNA into a fresh 96-well plate. The DNA can be stored at -20 °C or used for subsequent capture library construction.
    4. Analyse the fragments (1 µl) electrophoretically with an Agilent 2100 Bioanalyzer (High Sensitivity DNA Chip). The size peak should be between 200 and 300 bp (Figure 1).


      Figure 1. Typical size profile of fragmented genomic DNA. Following fragmentation with the Covaris S220 device, the genomic DNA (1 µl) was size-fractionated using the Agilent 2100 Bioanalyzer. The lower (LM) and upper marker peaks (UM) are indicated. FU: Fluorescence unit.

  3. Options for Capture library construction
    The capture libraries can be made using:
    1. Illumina TruSeq DNA [Low-Throughput (LT) Protocol; gel method]
    2. TruSeq DNA PCR-free sample preparation kits following the manufacturer instructions (Illumina, Inc., San Diego, CA). The final library is eluted in 30 µl Resuspension Buffer.
      Notes:
      1. For sequencing pooled captured Illumina libraries the appropriate index adapters must be selected according to the “Pooling Guidelines” (e.g. see Reference 2).
      2. If option a. or b. is chosen, prepare the libraries according to the manufacturer`s instructions, skip steps D to G and continue with step H. “Gel-purification of the library”.
    3. Alternatively, the library can be prepared based on a protocol for DNA Illumina multiplex (IM) libraries (Meyer and Kircher, 2010). This approach is transparent and avoids expensive components such as sequencing library construction kits and commercial reagents for Solid-Phase Reversible Immobilization (SPRI). Flexibility in experimental design is given by a large number of possible indices. As an example 33 indices for the generation of sequencing libraries are provided (Table 1 in Supplementary Material). Rules for pooling indices are described elsewhere (Meyer and Kircher, 2010). If deeper multiplexing is required, additional index sets with the corresponding blocking oligos can be created (Meyer and Kircher, 2010). Libraries can be prepared in single tubes as well as in 96-well plates using multichannel pipettes.
      Steps D to G describe the construction of DNA Illumina multiplex (IM) libraries using 96-well plates.

  4. Blunt-end repair
    Note: Prepare the adapter mix P57 required for adapter ligation. A purified PCR product [Test fragment for DNA Illumina multiplex (IM) libraries; see Recipies] should be included as a positive control. EBT buffer serves as a negative control. The controls are added to empty wells and are subjected to all manipulations during library preparation, thereby permitting to monitor the success of the enzymatic reactions.
    1. Prepare a master mix from the following reagents (volume per sample). Add the components in the order indicated:
      Water
      7.12 µl
      10x Buffer Tango
      7.00 µl
      dNTPs (25 nM each)
      0.28 µl
      ATP (100 mM)
      0.70 µl
      T4 polynucleotide kinase (10 U/µl)
      3.50 µl
      T4 DNA polymerase (5 U/µl)
      1.40 µl
      Mix the components by flicking the tube gently.
      Caution: Vortexing may inactivate the enzymes.
    2. Add 20 µl of the master mix to the DNA (50 µl). Mix gently by pipetting.
    3. Seal the plate and incubate the samples (70 µl) in a PCR-cycler 15 min at 25 °C, 5 min at 12 °C and keep at 4 °C.
    4. Immediately proceed to the sample clean-up by adding 126 µl MagNA beads (equilibrated to RT and vortexed). The suspension is mixed to homogeneity by pipetting (5x) and incubated for 5 min at RT.
    5. The 96-well plate is placed on a DynaMag-96 Side Skirted Magnetic Particle Concentrator (MPC96) for 5 min to separate the beads carrying the DNA from the solution.
    6. The supernatant is removed completely using a pipette while the 96-well plate remains in the MPC96.
    7. Leave the plate in the MPC96 and wash the beads with 150 µl freshly prepared 70% ethanol. Wait for 1 min and remove the supernatant completely using a pipette.
    8. Repeat step D7 once.
    9. Air-dry the beads completely for ~ 30 min while the open plate is located in the MPC96.
    10. Remove 96-well plate from MPC96 and elute the DNA in 22 µl EBT. Pipette up and down (10x) to resuspend the beads. Incubate for 1 min (RT). Take care that the liquid is located in the bottom of the well. If this is not the case, close the plate with adhesive tape and spin down all droplets using a centrifuge with a swing-out rotor (1,000 x g, 2 min, RT).
    11. The 96-well plate is placed on a DynaMag-96 Side Skirted Magnetic Particle Concentrator (MPC96) for 5 min to separate the eluted DNA from the beads.
    12. 20 µl DNA are transferred into a new well. Subsequent reactions are not impeded by small carryover of beads.
    13. Sealed 96-well plates can be stored at -20 °C.

  5. Adapter ligation
    1. Set up a master mix from the following reagents (volume per sample). Add the components in the order indicated:
      Water
      10.0 µl
      10x T4 DNA ligase buffer
      4.0 µl
      PEG 4000 (50%)
      4.0 µl
      Adapter mix P57 (100 µM each)
      1.0 µl
      T4 DNA ligase (5 U/µl)
      1.0 µl
      Note: If a white precipitate is observed, incubate the 10x T4 DNA ligase buffer 5 min at 37 °C. Vortex until the buffer is clear. The master mix should be vortexed after the addition of the viscous PEG 4000 (50%) solution. After the final addition of the T4 DNA ligase the master mix should be mixed gently by flicking the tube.
      Caution: Vortexing may inactivate the enzyme.
    2. Add 20 µl of the master mix to the DNA (20 µl). Mix gently by pipetting.
    3. Seal the plate and incubate the samples (40 µl) in a PCR-cycler 30 min at 22 °C and keep at 8 °C.
    4. Immediately proceed to the sample clean-up by adding 72 µl MagNA beads (equilibrated to RT and vortexed). The suspension is mixed to homogeneity by pipetting (5x) and incubated for 5 min at RT.
    5. The 96-well plate is placed on a DynaMag-96 Side Skirted Magnetic Particle Concentrator (MPC96) for 5 min to separate the beads carrying the DNA from the solution.
    6. The supernatant is removed completely using a pipette while the 96-well plate remains in the MPC96.
    7. Leave the plate in the MPC96 and wash the beads with 150 µl freshly prepared 70% ethanol. Wait for 1 min and remove the supernatant completely using a pipette.
    8. Repeat step E7 once.
    9. Air-dry the beads completely for ~ 30 min while the open plate is located in the MPC96.
    10. Remove 96-well plate from MPC96 and elute the DNA in 22 µl EBT. Pipette up and down (10x) to resuspend the beads. Incubate for 1 min (RT). Take care that the liquid is located in the bottom of the well. If this is not the case, close the plate with adhesive tape and spin down all droplets using a centrifuge with a swing-out rotor (1,000 x g, 2 min, RT).
    11. The 96-well plate is placed on a DynaMag-96 Side Skirted Magnetic Particle Concentrator (MPC96) for 5 min to separate the eluted DNA from the beads.
    12. 20 µl DNA are transferred into a new well. Subsequent reactions are not impeded by small carryover of beads.
    13. Sealed 96-well plates can be stored at -20 °C.

  6. Adapter fill-in
    1.  Prepare a master mix from the following reagents (volume per sample). Add the components in the order indicated:
      Water
      14.1 µl
      10x ThermoPol reaction buffer
      4.0 µl
      dNTPs (25 mM each)
      0.4 µl
      Bst polymerase, large fragment (8 U/µl)
      1.5 µl
      Note: After the final addition of the enzyme, the master mix should be mixed by flicking the tube gently.
      Caution: Vortexing may inactivate the enzyme.
    2. Add 20 µl of the master mix to the DNA (20 µl). Mix gently by pipetting.
    3. Seal the plate and incubate the samples (40 µl) in a PCR-cycler 20 min at 37 °C and keep at 8 °C.
    4. Immediately proceed to the sample clean-up by adding 72 µl MagNA beads (equilibrated to RT and vortexed). The suspension is mixed to homogeneity by pipetting (5x) and incubated for 5 min at RT.
    5. The 96-well plate is placed on a DynaMag-96 Side Skirted Magnetic Particle Concentrator (MPC96) for 5 min to separate the beads carrying the DNA from the solution.
    6. The supernatant is removed completely using a pipette while the 96-well plate remains in the MPC96.
    7. Leave the plate in the MPC96 and wash the beads with 150 µl freshly prepared 70% ethanol. Wait for 1 min and remove the supernatant completely using a pipette.
    8. Repeat step F7 once.
    9. Air-dry the beads completely for ~ 30 min while the open plate is located in the MPC96.
    10. Remove 96-well plate from MPC96 and elute the DNA in 22 µl EBT. Pipette up and down (10x) to resuspend the beads. Incubate for 1 min (RT). Take care that the liquid is located in the bottom of the well. If this is not the case, close the plate with adhesive tape and spin down all droplets using a centrifuge with a swing-out rotor (1,000 x g, 2 min, RT).
    11. The 96-well plate is placed on a DynaMag-96 Side Skirted Magnetic Particle Concentrator (MPC96) for 5 min to separate the eluted DNA from the beads.
    12. 20 µl adapter ligated DNA are transferred into a new well. Subsequent reactions are not impeded by small carryover of beads.
    13. Sealed 96-well plates can be stored at -20 °C.
    14. The performance of the enzymatic reactions should be monitored by separating 20 µl of the positive and negative controls next to the initial purified PCR-product with A-overhanging ends and a DNA ladder using a 2% agarose gel (Figure 2). The size of the ethidium bromide stained positive control (adapter ligated test fragment) should be 67 bp larger than the initial test fragment.
    15. Determine the concentration of the adapter ligated DNA using the Qubit HS assay.


      Figure 2. Controls for the adapter ligation. Test fragment (T; 750 ng) with A-overhanging ends was size-fractionated along with the test fragment provided with adapters (positive control; P, 20 µl) and a negative control (N, 20 µl). In the negative control the test fragment was replaced by EBT buffer. The samples were separated using standard agarose (2%) electrophoresis and stained with ethidium bromide. L: DNA ladder. The size of the standards is indicated (bp).

  7. Indexing PCR and reaction clean-up of the library
    1.  Add 8 µl containing 20 - 100 ng adapter ligated DNA (template) to a new well in the PCR plate.
      Note: It is sufficient to use part of the adapter ligated DNA for the PCR. The remaining material can serve as a back-up or can be labelled later with a different index (if required). Index selection rules for pooling and additional index sets are described elsewhere (Meyer and Kircher, 2010).
    2. Add 1.4 µl of the appropriate (index) primer (10 µM) from the Index primer 7001 to 7033 series (Table 1 in Supplementary Material).
    3. Prepare a master mix from the following reagents (volume per sample). Add the components in the order indicated and mix:
      Water
      28.7 µl
      5x Phusion HF buffer
      10.0 µl
      dNTPs (25 mM each)
      0.4 µl
      Primer IS4 (10 µM; Table 1 in Supplementary Material)
      1.0 µl
      Phusion Hot Start Flex DNA polymerase (2 U/µl)
      0.5 µl
    4. Distribute 40.6 µl of the master mix into the wells containing the template and Index primer (50 µl reaction), mix by pipetting gently and cover the plate with an adhesive tape for PCR.
    5. Perform PCR using the following conditions:
      -Denaturation        
      98 °C      
      30 sec
      12 cycles of:
      -Denaturation    
      98 °C 10 sec
      -Annealing
      60 °C 
      20 sec
      -Elongation
      72 °C   
      20 sec
      And a final extension:
      -Elongation        
      72 °C   
      10 min
      The reactions are kept at 8 °C.
    6. Proceed to the sample clean-up by adding 90 µl MagNA beads (equilibrated to RT and vortexed). The suspension is mixed to homogeneity by pipetting (5x up and down) and incubated for 5 min at RT.
    7. The 96-well plate is placed on a DynaMag-96 Side Skirted Magnetic Particle Concentrator (MPC96) for 5 min to separate the beads carrying the DNA from the solution.
    8. The supernatant is removed completely using a pipette while the 96-well plate remains in the MPC96.
    9. Leave the plate in the MPC96 and wash the beads with 150 µl freshly prepared 70% ethanol. Wait for 1 min and remove the supernatant completely using a pipette.
    10. Repeat step G9 once.
    11. Air-dry the beads completely for ~ 30 min while the open plate is located in the MPC96.
    12. Remove 96-well plate from MPC96 and elute the DNA in 27 µl EBT. Pipette up and down (10 x) to resuspend the beads. Incubate for 1 min (RT). Take care that the liquid is located in the bottom of the well. If this is not the case, close the plate with adhesive tape and spin down all droplets using a centrifuge with a swing-out rotor (1,000 x g, 2 min, RT).
    13. The 96-well plate is placed on a DynaMag-96 Side Skirted Magnetic Particle Concentrator (MPC96) for 5 min to separate the eluted DNA from the beads.
    14. 25 µl indexed library DNA are transferred into a new well, and sealed 96-well plates can be stored at -20 °C.
    15. Quality and quantity of randomly selected PCR products is controlled by standard agarose gel electrophoresis (Figure 3).


      Figure 3. Products of the indexing PCR reactions. PCR products from randomly selected samples (3 µl, step G15) were size-separated using standard agarose (2 %) gel electrophoresis and stained with ethidium bromide. L: GeneRuler 50 bp DNA ladder. The size of the ladder is indicated (bp).

  8. Gel-purification of the library
    Note: The gel-purification is required for all types of libraries in order to make sure that appropriately sized DNA fragments are used for the pre-capture library amplification. DNA is revealed in agarose gels using SYBR-Gold dye and visible blue light emitted from a “Dark reader” transilluminator as the excitation source.
    Caution: Do not use ethidium bromide stained gels. Ultra-violett radiation used for excitation of ethidium bromide will damage the DNA.
    1. Prepare a 2% agarose gel: Melt 3 g UltraPure Agarose in 150 ml 1x TAE using an Erlenmeyer flask (500 ml) and a microwave. Make sure that the agarose is completely dissolved and cool down the solution to 60 °C. Add 15 µl SYBR-Gold, swirl gently without forming bubbles and cast the gel using standard equipment (15 cm x 15 cm tray, comb etc.).
      Protect the gel from light by covering with aluminium foil. Let the gel solidify.
    2. Dilute the GeneRuler 50 bp DNA ladder: Take 250 ng ladder, 4 µl 6x loading dye and fill up the volume to 24 µl using EBT (ladder for one lane).
    3. Add 5 µl 6x loading dye to the indexed library DNA (25 µl), mix gently.
    4. Remove the comb and place the gel into the electrophoresis chamber.
    5. Fill the tank with 1x TAE until the gel is covered with buffer.
    6. Load each library in two adjacent slots of the agarose gel (2x 15 µl). Load the diluted ladder between two different libraries to prevent contaminations by spill-over.
    7. Perform electrophoresis for 2 h with 4 V/cm (distance between anode and cathode). Protect the gel from light during electrophoresis.
    8. Reveal the DNA using a “Dark reader transilluminator” and use a clean scalpel to excise the region of interest (320 to 430 bp). A typical result is shown in Figure 4. Place the agarose block into a clean 2 ml (or 5 ml) tube.
    9. Determine the volume of the block using a balance (e.g. 240 mg corresponds to 240 µl).
    10. Purify the DNA using the Qiagen MinElute kit (spin column) essentially as described by the manufacturer.
    11. Add 6 volumes QG buffer to the block (e.g. 240 µl block: 1,440 µl QC buffer).
    12. Dissolve the gel (RT) completely under gentle agitation.
    13. Add one volume isopropanol (e.g. 240 µl block: 240 µl isopropanol) and mix by inverting (5x).
    14. Apply 700 µl of the mixture to a MinElute column placed in a collection tube and spin (16,000 x g, 1 min).
    15. Discard the flow-through.
    16. Stepwise apply the remainder to the column by repeating steps H14-15.
    17. Add 740 µl PE to wash the column, incubate for 3 min at RT and spin the column (16,000 x g, 1 min).
      Note: Place the columns in the rotor consistently, since the columns will be turned 180° in step H19.
    18. Discard the flow-through.
    19. Turn the column (180°) in the rotor and spin (16,000 x g, 1 min) to remove traces of the PE buffer.
    20. Discard the collection tube, place the MinElute column into a clean 1.5 ml tube and add 21 µl EB to the center of the resin to elute the DNA.
    21. Incubate for 1 min and spin the column (16,000 x g, 1 min).
    22. The eluted DNA can be stored at -20 °C.
    23. Determine the DNA concentration using the Qubit BR assay.
      Expected concentrations:
      TruSeq libraries (15-50 ng/µl) and DNA Illumina multiplex (IM) libraries (0.5-10 ng/µl).


      Figure 4. Gel-purification of the libraries. Libraries (1, 2) were size-separated using standard agarose gel electrophoresis (2% agarose, 1xTAE buffer) and stained with SYBR Gold. The DNA was revealed using a visible blue light emitting “Dark reader transiIlluminator”. A gel image is shown after electrophoresis (A.) and after the isolation of the area between 320 and 430 bp (B.). R: Ruler (cm), L: GeneRuler 50 bp DNA ladder. The size of the ladder is indicated (bp).

  9. Pre-capture library amplification using LM-PCR
    1. To enrich for correctly ligated DNA fragments the library [TruSeq DNA, TruSeq DNA PCR-free or DNA Illumina multiplex (IM) libraries] is used as a template in the pre-capture LM-PCR (ligation-mediated PCR). Prepare a master mix (80 µl per reaction) by adding the reagents in the order indicated and mix well by pipetting:
      2x Phusion High-Fidelity PCR Master Mix           
      50 µl
      PCR-grade water                      
       26 µl
      TS-PCR Oligo 1 (100 µM; Table 1 in Supplementary Material)   
      2 µl
      TS-PCR Oligo 2 (100 µM; Table 1 in Supplementary Material)     
      2 µl
    2. Distribute 80 µl of the master mix to PCR tubes and add 20 µl of the library. Mix by pipetting (5x up and down) the reagents. LM-PCR cycling, clean-up and elution of the sample were essentially as described (Haun et al., 2011).
    3. Perform PCR using the following conditions:
      -Denaturation
      98 °C   
      30 sec
      8 cycles of:
      -Denaturation 
      98 °C  
      10 sec
      -Annealing
      60 °C    
      30 sec
      -Elongation
      72 °C   
      30 sec
      And a final extension:
      -Elongation   
      72 °C     
      5 min
      The reactions are kept at 8 °C.
    4. Transfer the amplified library into a 1.5 ml tube and add 5 volumes (500 µl) Qiagen buffer PBI. Mix by pipetting (5x up and down).
    5. Transfer the mixture to a QIAquick column placed in a collection tube and centrifuge 16,000 x g (1 min). Discard flow-through.
    6. Add 740 µl PE wash buffer and centrifuge 16,000 x g (1 min).
      Note: Place the columns in the rotor consistently, since the columns will be turned 180° in step I8.
    7. Discard the flow-through.
    8. Turn the column (180°) and centrifuge the column 16,000 x g (1 min) to remove residual PE wash buffer.
    9. Discard the collection tube, place the MinElute column in a new 1.5 ml tube and elute the DNA by adding 50 µl of PCR-grade water to the column matrix. (Caution: Do not use TE or EB for elution.) Incubate 1 min at RT.
    10. Centrifuge the column 16,000 x g (1 min) to collect the pre-capture LM-PCR amplified library.
    11. Perform quality checks:
      1. Quantify the amount of library using a Qubit device or a picogreen based assay. The yield should be > 1 µg.
      2. Determine the size profile of the sample library (1 µl) electrophoretically with an Agilent 2100 Bioanalyzer and a DNA 7500 chip. The fragments of the sample library should be between 250 and 500 bp (Figure 5).
    12. If the pre-capture LM-PCR amplified library meets the quality requirements, proceed to the hybridization of the sample library to exome library.
      Optional: Store the sample at -20 °C.


      Figure 5. Typical size profile of the purified pre-capture PCR amplified library. Following pre-capture PCR and reaction clean-up of the amplified library, the DNA (1 µl) was size-fractionated using the Agilent 2100 Bioanalyzer. The lower (LM) and upper marker peaks (UM) are indicated. FU: Fluorescence unit.

  10. Hybridization of sample library to exome library
    1.  Add 1 µg of the pre-capture LM-PCR amplified library and 10 µl of Roche NimbleGen`s proprietary Sequence Capture Developer Reagent to a 1.5 ml tube.
      Note: Several pre-capture LM-PCR amplified libraries provided with different indexes can be pooled (total amount 1 µg). Sequence Capture Developer Reagent was previously known as Plant Capture Enhancer (PCE) from Roche NimbleGen.
    2. Add 1 µl TruSeq HE Universal Oligo 1 (1 mM).
      Note: The TruSeq HE Universal Oligo 1 was designed to block the universal segment of TruSeq DNA and DNA Illumina multiplex (IM) library adapters during sequence capture hybridization.
      1. For TruSeq DNA or TruSeq DNA PCR-free libraries (pre-capture LM-PCR amplified)
        Add 1 µl of the appropriate 1 mM TruSeq INV-HE Index Oligo.
        Note: The TruSeq INV-HE Index Oligos were designed, to block the corresponding indexed segment of the TruSeq DNA library adapters AD002, AD004, AD005, AD006, AD007 and AD012 during sequence capture hybridization, respectively.
      2. For DNA Illumina multiplex (IM) libraries (pre-capture LM-PCR amplified)
        Add 1 µl of the appropriate 1 mM IM INV-HE Index Oligo.
        Note: The IM INV-HE Index Oligos were designed, to block the corresponding indexed segment of the IM DNA library adapters (Indices 7001 to 7033) during sequence capture hybridization, respectively.
        Important: If pooled pre-capture LM-PCR amplified libraries are used, the amount of the corresponding blocking oligos is proportional to the concentration of the sample in the pool. Example: If four pre-capture LM-PCR amplified libraries provided with TruSeq adapter (AD002, AD004, AD005 and AD007) are pooled (0.25 µg/library) for hybridization, use 1 µl TruSeq HE Universal Oligo 1 (1 mM) together with 0.25 µl TruSeq INV-HE Index Oligo 2 (1 mM), 0.25 µl TruSeq INV-HE Index Oligo 4 (1 mM), 0.25 µl TruSeq INV-HE Index Oligo 5 (1 mM) and 0.25 µl TruSeq INV-HE Index Oligo 7 (1 mM), respectively.
    3. Lock the tube, puncture the lid with a clean needle and dry down the mixture in a SpeedVac at 60 °C.
    4. Pre-heat a heating block (95 °C).
    5. The tube contains now the following dried-down components (example for one TruSeq DNA library):
      Sequence Capture Developer Reagent             
      10 µl (dried-down)
      Pre-capture LM-PCR amplified library (TruSeq DNA)           
      1 µg (dried-down)
      1 mM TruSeq HE Universal Oligo 1                 
      1 µl (dried-down)
      1 mM Appropriate IM INV-HE Index Oligo            
      1 µl (dried-down)
    6. Replace the punctured lid by an intact lid.
    7. Add to the dried-down components:
      2x Sequence Capture (SC) Hybridization Buffer (tube 5)      
       7.5 µl
      Hybridization Component A (tube 6)               
      3.0 µl
    8. The cocktail (total volume 10.5 µl) is vortexed for 10 sec and collected by centrifugation (RT, 10 sec, 16,000 rpm).
    9. The cocktail is denatured in a heating block (95 °C, 10 min).
    10. Collect the sample by centrifugation (RT, 10 sec, 16,000 rpm).
    11. Transfer the entire sample to a 0.2 ml PCR tube containing 4.5 µl Exome Library (liquid array) per capture. Mix by pipetting up and down gently (10x). Make sure that the entire sample is located at the bottom of the tube.
    12. Hybridize the sample (15 µl) in a thermocycler at 47 °C for 64-72 h (lid heated to 57 °C).

  11. Affinity purification of the captured library
    1. Dilute 10x Wash Buffers (tubes 1, 2 and 3), 10x Stringent Wash Buffer (tube 4) and 2.5x Bead Wash Buffer (tube 7) to 1x working solutions. Use PCR-grade water as diluent.
      Volumes per reaction:
      Stock                   
      PCR-grade water   
      1x working solution
      30 µl 10 x Wash Buffer I (tube 1)       
      270 µl                  
      300 µl
      20 µl 10 x Wash Buffer II (tube 2)       
      180 µl          
       200 µl
      20 µl 10 x Wash Buffer III (tube 3)       
      180 µl          
      200 µl
      40 µl 10 x Stringent Wash Buffer (tube 4)   
      360 µl           
      400 µl
      200 µl 2.5 x Bead Wash Buffer (tube 7)       
      300 µl           
      500 µl
      Note: Set a water bath to 47 °C and verify the temperature with an external calibrated thermometer. Pre-heat the 1x working solutions (Wash Buffer I and Stringent Wash Buffer) for 2 h prior to use.            
    2. Equilibrate Streptavidin Dynabeads to RT, vortex 10 sec and aliquot 50 µl (per hybridization) into a new 1.5 ml tube.
    3. Place the tube into a DynaMag-2 magnet for 2 min, discard the clear liquid, and add 100 µl of Bead Wash Buffer.
    4. Remove the tube from the magnet, vortex (10 sec) and collect the suspension to the bottom of the tube.
    5. Place the tube into a DynaMag-2 magnet for 2 min, discard the clear liquid, and add 100 µl of Bead Wash Buffer.
    6. Remove the tube from the magnet, vortex (10 sec) and collect the beads by centrifugation (5 sec, 16,000 x g).
    7. Place the tube into a DynaMag-2 magnet for 2 min, discard the clear liquid, and add 50 µl of Bead Wash Buffer.
    8. Remove the tube from the magnet, vortex (10 sec) and reclaim the beads by centrifugation (5 sec, 16,000 x g).
    9. Transfer the resuspended Dynabeads into a PCR plate. Collect the Dynabeads in a DynaMag-96 Side Skirted Magnetic Particle Concentrator (MPC96) for 2 min and discard the clear liquid.
      Note: Keep the temperature of the PCR-plate containing the wet Dynabeads as close as possible to 47 °C by using a pre-warmed heating block. Dynabeads should not dry down.
    10. Add the hybridization sample (15 µl) to the wet Dynabeads (pre-warmed 47 °C) in the PCR plate. Mix gently by pipetting up and down (5x). Proceed swiftly.
      Note: The volume of the hybridization sample may now be < 15 µl. However, this does not interfere with subsequent steps.
    11. Using a pre-heated thermocycler (57 °C lid temperature) the (biotinylated) captured sample is bound to the (streptavidin- coated) Dynabeads at 47 °C for 45 min. Vortex the plate for 3 sec in 15-min intervals to make sure that the Dynabeads remain in suspension and collect droplets to the bottom of the plate by a quick centrifugation (swing-out rotor) without pelleting the beads.
    12. Add 100 µl 1x Wash Buffer I (pre-heated in a water bath to 47 °C) to the Dynabeads plus bound DNA and vortex for 10 sec. Collect the suspension to the bottom of the plate by a quick centrifugation (swing-out rotor) without pelleting the beads.
    13. Transfer the suspension to a new 1.5 ml tube. Place the tube into a DynaMag-2 device to reclaim the Dynabeads plus bound DNA for 1 min. Discard the supernatant. Remove the tube from the magnet.
    14. Add 200 µl 1x Stringent Wash Buffer (pre-heated in a water bath to 47 °C) to the Dynabeads plus bound DNA and mix by pipetting thereby avoiding a major temperature drop and incubate for 5 min (47 °C, 300 rpm).
    15. Place the tube into a DynaMag-2 device to collect the Dynabeads plus bound DNA for 1 min. Discard the supernatant. Remove the tube from the magnet.
    16. Repeat washing with 1x Stringent Wash Buffer once (steps K14-15).
    17. Add 200 µl Wash Buffer I (room temperature) to the Dynabeads plus bound DNA and vortex for 2 min. Collect the suspension to the bottom of the tube by a quick centrifugation without pelleting the beads.
    18. Place the tube into a DynaMag-2 device to collect the Dynabeads plus bound DNA for 1 min. Discard the supernatant. Remove the tube from the magnet.
    19. Add 200 µl Wash Buffer II (room temperature) to the Dynabeads plus bound DNA and vortex for 1 min. Collect the suspension to the bottom of the tube by a quick centrifugation without pelleting the beads.
    20. Place the tube into a DynaMag-2 device to collect the Dynabeads plus bound DNA for 1 min. Discard the supernatant. Remove the tube from the magnet.
    21. Add 200 µl Wash Buffer III (room temperature) to the Dynabeads plus bound DNA and vortex for 30 sec. Collect the suspension to the bottom of the tube by a quick centrifugation without pelleting the beads.
    22. Place the tube into a DynaMag-2 device to collect the Dynabeads plus bound DNA for 1 min. Discard the supernatant. Remove the tube from the magnet.
    23. Resuspend the washed Dynabead-bound captured library in 50 µl PCR-grade water and determine precisely the volume using a pipette (storage at -20 °C possible).

  12. Post-capture library amplification using LM-PCR
    1. Set up a master mix for the PCR reaction (volume for one reaction):
      2x Phusion High-Fidelity PCR Master Mix              
      100 µl
      PCR-grade water                         
      42 µl
      TS-PCR Oligo 1 (100 µM; Table 1 in Supplementary Material)       
      4 µl
      TS-PCR Oligo 2 (100 µM; Table 1 in Supplementary Material)           
      4 µl
      Mix the mastermix carefully by pipetting up and down (8x).
      Add the Dynabead-bound captured library (50 µl). The final volume is 200 µl.
      Mix carefully by pipetting up and down (8x). Avoid the formation of foam.
    2. Distribute 100 µl of the mixture into two 0.2 ml-PCR tubes.
    3. Perform PCR using the following conditions:
      -Denaturation              
      98 °C   
      30 sec
      18 cycles of:
      -Denaturation               
      98 °C   
      10 sec
      -Annealing       
      60 °C    
      30 sec
      -Elongation      
      72 °C   
      30 sec
      And a final extension:
      -Elongation               
      72 °C   
      5 min
      The reactions are kept at 8 °C.
    4. Combine the two samples (total 200 µl) into a 1.5 ml tube and add 1 ml Qiagen buffer PBI. Mix by pipetting (5x up and down).
    5. Transfer 600 µl of the mixture to a QIAquick column placed in a collection tube and centrifuge 16,000 x g (1 min). Discard flow-through.
    6. Repeat step L5 with the remaining mixture.
    7. Add 740 µl PE wash buffer and centrifuge 16,000 x g (1 min). Discard flow-through.
    8. Centrifuge the column 16,000 x g (1 min) to remove residual PE wash buffer.
    9. Turn the column 180° and centrifuge the column 16,000 x g (30 sec) to remove traces of PE wash buffer. Place the column into a new 1.5 ml tube.
    10. For elution add 50 µl EB (pre-heated to 50 °C) to the column matrix, incubate for 1 min.
    11. Centrifuge the column 16,000 x g (1 min) to reclaim the post-capture LM-PCR amplified library (exome capture library). Re-eluate the column by adding 50 µl of the first eluate to the column again. Incubate 1 min. Turn the column (180°) and reclaim the DNA by centrifugation (1 min, 16,000 x g). Store the library at -20 °C.
    12. Characterization of the library:
      1. Use e.g. a Nanodrop Spectrophotometer device to determine the A260/A280 ratio from 1 µl of the exome capture library (A260/A280 should be 1.7-2.0).
      2. Determine the size profile of the exome capture library (1 µl) electrophoretically with an Agilent 2100 Bioanalyzer and a DNA 7500 chip. The fragments should be between 250 and 500 bp (Figure 6).
      3. Measure the DNA concentration using a picogreen-based assay (e.g. Qubit BR). The expected yield is > 500 ng.
      4. Quantify the exome capture library using Real-Time PCR as described previously (Mascher et al., 2013).


        Figure 6. Typical size profiles of final capture libraries. Following post-capture PCR and reaction clean-up, libraries (1 µl) were size-fractionated using the Agilent 2100 Bioanalyzer. As examples, three libraries (a, b and c) derived from three independent preparations are shown. The lower (LM) and upper marker peaks (UM) are indicated. FU: fluorescence unit

  13. Sequencing-by-synthesis and data evaluation
    1. Sequencing-by-synthesis (paired-end, 2x 100 cycles) using e.g. a HiSeq Illumina system is performed as described previously (Mascher et al., 2013).
    2. Analyse data as described previously (Mascher et al., 2013).

Notes

  1. In general, filter tips and gloves are used to avoid contaminations. Labs and equipment for processing samples pre- and post-PCR are physically separated. The workspace surface (bench, instruments etc.) is treated daily with commercial solutions (e.g. DNA away) to remove traces of DNA. Good laboratory practice is used throughout the experiment.
  2. The “Bio Rad DNA Engine Tetrad 2 Peltier Thermal Cycler” was used for incubations and PCR. For incubations, the heated lid was set to “Lid Control Mode: Tracking at 5 °C above”, unless indicated otherwise. During PCR and annealing adapters P57 the lid was heated to 100 °C to prevent condensation. Thermal cyclers should be programmed before starting the experiment.
  3. All components listed (Materials and Reagents; Equipment) were validated in independent experiments for plant sequence capture. However, this list does not imply that alternative products from other suppliers cannot perform equally well.
  4. Samples can be stored at the steps indicated for at least 3 weeks at -20 °C.
  5. If not stated otherwise, manipulations such as incubations, drying of pellets and centrifugations are performed at RT.
  6. The use of the published equipment and chemistry is not indicating any competing interest.
  7. Although the protocol performed well in our laboratorium (>50 independent experiments), the authors take no liability for the success of the experiments conducted by the reader.
  8. Estimated hands-on time based on processing one array are given below (Mascher et al., 2013). Long time incubations and operating periods of instruments are indicated (brackets):
    Library preparation
    2 days
    Pre-capture library amplification using LM-PCR
    3 h
    Hybridization of sample library to exome library
    2 h (3 days)
    Affinity purification of the captured library
    4 h
    Post-capture library amplification using LM-PCR
    3 h
    Quality and quantity controls of the libraries
    6 h
    Sequencing-by-synthesis (e.g. cBot, HiSeq, 2x 100 cycles)
    (10 days)

Recipies

  1. TE (pH 8.0)
    10 mM Tris-Cl (pH 8.0)
    1 mM EDTA
  2. Resuspension Buffer (RSB)
    10 mM Tris-Cl (pH 8.5)
  3. 50x TAE
    Dissolve 242 g Tris base in deionized water, add 57.1 ml glacial acetic acid and 100 ml 0.5 M EDTA (pH 8.0).
    Adjust the volume to 1 liter and mix thoroughly
    The 50x TAE stock is diluted to 1x TAE working solution using deionized water (mix thoroughly).
  4. EBT
    10 mM Tris-Cl (pH 8.0)
    0.05 % (v/v) Tween 20
  5. Test fragment for DNA Illumina multiplex (IM) libraries (Meyer and Kircher, 2010)
    Design unmodified primers to create an amplicon with a single size (length 150-200 bp)
    Use Taq polymerase for amplification in order to generate products with A-overhangs at the 3’ ends
    Purify the PCR products using standard protocols and adjust the concentration to 250 ng/ 50 µl.
  6. Adapter mix P57 for IM libraries (Meyer and Kircher, 2010)
    Prepare 10x Oligo hybridization buffer (500 mM NaCl, 10 mM Tris-Cl, 1 mM ETDA, pH 8.0)
    Add to PCR tube 1:
    40 µl IS1_adapter_P5.F (500 µM; Table 1 in Supplementary Material)
    40 µl IS3_adapter_P5+P7.R (500 µM; Table 1 in Supplementary Material)
    10 µl 10x Oligo hybridization buffer
    10 µl PCR-grade water
    Add to PCR tube 2:
    40 µl IS2_adapter_P7.F (500 µM; Table 1 in Supplementary Material)
    40 µl IS3_adapter_P5+P7.R (500 µM; Table 1 in Supplementary Material)
    10 µl 10x Oligo hybridization buffer
    10 µl PCR-grade water
    Place PCR tubes 1 and 2 into a PCR-machine.
    Denature: 95 °C 10 sec
    Annealing: cool to 12 °C (ramp: 0.1 °C/sec)
    Combine annealed oligonucleotides from tube 1 and 2. The final concentration is 100 µM (each adapter).
    Store aliquots at -20 °C
  7. MagNA beads for DNA clean-up
    MagNA beads were essentially used as described (Rohland and Reich, 2012), thereby replacing the commercial product (Agencourt AMPure XP) in DNA clean-up steps.
    Composition of MagNA beads:
    2 % carboxyl-modified Sera-Mag Magnetic Speed-beads (v/v)
    18 % PEG-8000 (w/v)
    1 M NaCl
    10 mM Tris-HCl (pH 8.0)
    1 mM EDTA (pH 8.0)
    0.05 % Tween 20 (v/v)
    For the removal of sodium azide, new Speed-beads were washed 3 times with TE before addition. The MagNA beads were stored in the dark (4 °C) and used within 3 months.

Supplementary Materials

Table 1. List of oligonucleotides

Acknowledgments

The generation of DNA Illumina multiplex (IM) libraries was essentially as described (Meyer and Kircher, 2010). The operations leading to the captured library were based on the manufacturer’s instructions (Roche/NimbleGen, NimbleGen Arrays User’s Guide, Plant Sequence Capture Illumina Optimized, 2010) and as described previously (Mascher et al., 2013). The financial support provided by the grants GABI-BARLEX (FKZ 0314000 to N.S.) and TRITEX (FKZ 0315954 to N.S.) by the German Ministry of Education and Research (BMBF) is gratefully acknowledged.

References

  1. Haun, W. J., Hyten, D. L., Xu, W. W., Gerhardt, D. J., Albert, T. J., Richmond, T., Jeddeloh, J. A., Jia, G., Springer, N. M. and Vance, C. P. (2011). The composition and origins of genomic variation among individuals of the soybean reference cultivar Williams 82. Plant Physiol 155(2): 645-655.
  2. Illumina TruSeq Sample Preparation Pooling Guide, part # 15042173. http://support.illumina.com/downloads/truseq_sample_preparation_pooling_guide_15042173.ilmn.
  3. Mascher, M., Richmond, T. A., Gerhardt, D. J., Himmelbach, A., Clissold, L., Sampath, D., Ayling, S., Steuernagel, B., Pfeifer, M., D'Ascenzo, M., Akhunov, E. D., Hedley, P. E., Gonzales, A. M., Morrell, P. L., Kilian, B., Blattner, F. R., Scholz, U., Mayer, K. F., Flavell, A. J., Muehlbauer, G. J., Waugh, R., Jeddeloh, J. A. and Stein, N. (2013). Barley whole exome capture: a tool for genomic research in the genus Hordeum and beyond. Plant J 76(3): 494-505.
  4. Meyer, M. and Kircher, M. (2010). Illumina sequencing library preparation for highly multiplexed target capture and sequencing. Cold Spring Harb Protoc 2010(6): pdb prot5448.
  5. Rohland, N. and Reich, D. (2012). Cost-effective, high-throughput DNA sequencing libraries for multiplexed target capture. Genome Res 22(5): 939-946.
  6. Stein, N., Herren, G. and Keller, B. (2001). A new DNA extraction method for high‐throughput marker analysis in a large‐genome species such as Triticum aestivum. Plant Breeding 120(4): 354-356.

简介

植物序列捕获用于复杂基因组(例如大麦及其亲属)的整个外显子(基因组的所有外显子)的靶向重测序(Mascher等人,2013)。测序和计算成本显着降低,因为只有大麦基因组的大量富集和基因编码部分被靶向,其仅对应于整个基因组的1-2%。因此,大大促进了诸如遗传多样性研究和单个基因的分离("通过测序克隆")的应用。这里,提供了描述来自基因组大麦DNA的Shotgun DNA文库的构建以在Illumina HiSeq/MiSeq系统上测序的方案。鸟枪DNA测序文库与包含大麦整个外显子组的寡核苷酸池(Exome Library)杂交。外显子组库作为包含生物素化探针(Roche/NimbleGen)的液体阵列提供。随后,使用链霉亲和素包被的磁珠对与Exome文库杂交的基因组鸟枪DNA片段进行亲和纯化。捕获的文库被PCR扩增和测序,使用高通量短读序列合成

关键字:序列捕获, 靶向基因测序, 外显子组, 合成测序, 大麦

材料和试剂

  1. SYBR Gold(Life Technologies,目录号:S11494)
  2. 超纯 琼脂糖(Life Technologies,Invitrogen TM,目录号:16500-500)
  3. PCR级水
  4. 三(羟甲基)氨基甲烷(Tris碱)(多个供应商)
  5. 吐温20(Bio-Rad Laboratories,目录号:170-6531)
  6. 乙二胺四乙酸(EDTA)(多个供应商)
  7. 乙醇(绝对)(分析纯)(多个供应商)
  8. 70%(v/v)乙醇(分析纯)
  9. 异丙醇(2-丙醇)(> 99.5%)(多个供应商)
  10. 醋酸(冰川)(多个供应商)
  11. 2x Phusion High-Fidelity PCR Master Mix(New England BioLabs,目录号:F-531L)
  12. GeneRuler  50bp DNA梯(Thermo Fisher Scientific,目录号:SM 0371)
  13. QIAquick PCR纯化试剂盒(QIAGEN,目录号:28106)
  14. Minelute PCR纯化试剂盒(QIAGEN,目录号:28006)
  15. Agilent DNA 7500试剂盒(Agilent Technologies,目录号:5067-1506)
  16. Agilent高灵敏度DNA试剂盒(Agilent,目录号:5067-4626)
  17. SeqCap EZ杂交试剂盒[含有NimbleGen SC洗涤缓冲液(管1,2和3),严格洗涤缓冲液(试管4),2x SC杂交缓冲液(试管5),杂交组分A(试管6)和珠洗涤缓冲液 7)](Roche Diagnostics,目录号:05634261001)
  18. Sequence Capture Developer Reagent(Roche Diagnostics,目录号:06684335001)
    注意:此试剂以前称为来自Roche NimbleGen的植物捕获增强剂(PCE)。
  19. 6x负载染料(Thermo Fisher Scientific,Fermentas,目录号:R 0611)
  20. DNA离开(Thermo Fisher Scientific,目录号:21-236-28)
  21. 寡核苷酸(Sigma-Aldrich)
    注意:所有寡核苷酸 是反相盒纯化的并溶解在PCR级水中。 为了避免交叉污染,使用新鲜的柱纯化每个寡核苷酸。 序列在补充材料的表1中列出。
  22. 用于生成捕获库的试剂:
    1.   Illumina TruSeq DNA试剂盒(Illumina,目录号:FC-121-2001,Box A)或
    2. TruSeq DNA无PCR样品制备试剂盒(Illumina,目录号:FC-121-3001,Set A)
    3. 如果使用DNA Illumina多重(IM)方案,则需要以下另外的试剂(项目27-41)。
  23. T4多核苷酸激酶(10U /μl)(Thermo Scientific Fermentas,目录号:EK0032)
  24. T4 DNA聚合酶(5U /μl)(Thermo Scientific Fermentas,目录号:EP006B)
  25. 10x Buffer Tango(Thermo Scientific Fermentas,目录号:BY5)       
  26. dNTP(各25nM)(Thermo Scientific Fermentas,目录号:R1121)
  27. ATP(100mM)(Thermo Fisher Scientific,目录号:R0441)           
  28. 提供有10×T4DNA连接酶缓冲液和50%PEG 4000(Thermo Scientific Fermentas,目录号:EL0011)的T4 DNA连接酶(5U /μl)                
  29. 10x ThermoPol反应缓冲液(New England Biolabs,目录号:B9004S)   
  30. Bst聚合酶(8U /μl)(大片段)(New England Biolabs,目录号:M0275L)
  31. 5x Phusion HF缓冲液(New England Biolabs,目录号:B0518S)             
  32. Phusion Hot Start Flex DNA聚合酶(2U /μl)(New England Biolabs,目录号:M0535L)
  33. 羧基修饰的Sera-Mag磁速珠(Thermo Fisher Scientific,目录号:1182-9912)
  34. PEG-8000(Sigma-Aldrich,目录号:89510)
  35. NaCl(Sigma-Aldrich,目录号:S3014)
  36. Agilent高灵敏度DNA试剂盒(Agilent,目录号:5067-4626)
  37. Agilent DNA 7500试剂盒(Agilent,目录号:5067-1506)
  38. TE(pH 8.0)(参见配方)
  39. 重悬缓冲液(RSB)(参见配方)
  40. 50x TAE(请参阅配方)
  41. EBT(见配方)
  42. DNA Illumina多重(IM)文库的测试片段(参见配方)
  43. IM库的适配器组合P57(参见配方)
  44. 用于DNA清理的MagNA珠(参见配方)

设备

  1. Qubit 2.0荧光计(起始试剂盒)[包括仪器,测定试管,dsDNA HS测定和dsDNA BR测定](Thermo Fisher Scientific,目录号:Q32871)或其他基于picogreen的dsDNA定量装置
  2. AMPure XP Beads(Beckman Coulter,目录号:A63882)
  3. 链霉亲和素Dynabeads(M-270)(Life Technologies,Invitrogen TM ,目录号:65306)
  4. 1.5 ml管(多个供应商)
  5. 0.2 ml PCR管(多个供应商)
  6. 96孔板(Greiner Bio-One GmbH,目录号:652250)
  7. 用于96孔板(Thermo Fisher Scientific,目录号:AB-0558)的塑料密封件
  8. Covaris S220 AFA超声波发生器(LGC,目录号:KBS-500217)和相关设备如微型TUBE夹具,冷却器,软件,计算机等。
  9. 具有AFA光纤和预分裂隔膜的Snap-Cap microTUBES(Covaris,目录号:520045)
  10. Agilent 2100电泳生物分析仪(Agilent,目录号:G2939AA)和相关材料(例如计算机)
  11. NanoDrop 2000分光光度计(VWR International,PeQlab,目录号:91-ND-2000)
  12. HiSeq或MiSeq Illumina系统和相关材料(如2x 900次循环测序合成试剂)
  13. 加热块(多个供应商)
  14. 带有外部校准温度计的水浴(多个供应商)
  15. 微型离心机(16,000 x g )(多个供应商)
  16. SpeedVac(多个供应商)
  17. 热循环仪(多个供应商)
    注意:在整个协议中,使用了"Bio Rad DNA Engine Tetrad 2 Peltier Thermal Cycler"。
  18. 涡流混合器(多个供应商)
  19. DynaMag-2磁体(Life Technologies,Invitrogen TM ,目录号:123-21D)
  20. DynaMag-96侧缘磁性粒子浓集器(MPC 96)(Life Technologies,Invitrogen TM ,目录号:120.27)
  21. 琼脂糖凝胶电泳设备及配件(微波,托盘(15 x 15厘米),梳子,电源,紫外线透射仪等)。
  22. 一次性手术刀(多个供应商)
  23. Dark Reader blue light transilluminator(Clare Chemical Research,目录号:DR46B)
  24. 移液器(2,100,200和1,000μl,多个供应商)
  25. 过滤提示(多个供应商)

程序

  1. 基因组DNA的浓度
    1. 应使用大麦的标准方法分离基因组DNA (例如, Stein等人,2001)。 将DNA溶解于TE(pH8.0)中。 这很重要 以确保基因组DNA完全溶解。 任何不溶   应通过离心(16,000×g/min,10分钟,RT)除去物质。 将可溶性上清液转移到新鲜管中 碎片。
    2. 准确地确定基因组的浓度  DNA [溶解于TE(pH8.0)]中,使用对dsDNA特异性的系统。的 基本量化协议在这里描述的Qubit 2.0 荧光计。也可以使用任何其他基于picogreen的测定。 根据DNA起始浓度,使用HS测定(0.2ng- 100ng)或BR测定(2ng-1,000ng)。
      为了  Qubit装置1μl基因组DNA用199μl工作溶液稀释 (1μl染料/200μl缓冲液,平衡至室温)在Qubit测定管中 稀释在Qubit测定管10微升的两个标准与190微升工作解决方案 涡旋Qubit测定管,孵育2分钟,并测量DNA浓度   注意:为确保测量的可重复性,请进行3次独立量化。
    3. 使用TE(pH8.0)将DNA浓度总计调整为1μg 体积53微升,并将样品储存在冰上碎裂。

  2. DNA断裂
    1. 对于DNA片段化,使用Covaris S220装置。 仪器 产生导致机械的自适应聚焦声能 DNA的破坏。 根据制造商操作仪器 说明。 填写Covaris S220的水浴根据 水浴前面的水位指示器("FILL"栏)至 "12"级。 使用去离子水或蒸馏水。 水浴必须   脱气和冷却(7℃)以适当地操作该装置。 仪器的准备需要约40分钟。
      使用以下碎片参数(Mascher 等,2013):
      175 W超声波功率
      10%占空因数
      每次爆发200个循环,50秒处理,30秒暂停,然后再额外处理50秒
    2. 吸取基因组DNA(1微克在53微升)到Covaris microTUBE (在冰上预冷却)。 将管插入支架,碎片 脱氧核糖核酸。  
    3. 将片段化的DNA转移到新鲜的96孔板中。 所述DNA可以储存在-20℃或用于随后的捕获文库 施工
    4. 通过电泳分析片段(1μl) 使用Agilent 2100生物分析仪(高灵敏度DNA芯片)。 尺寸 峰值应在200至300 bp之间(图1)

      图1.典型 片段基因组DNA的大小分布。 Covaris S220装置,使用基因组DNA(1μl)进行大小分级   Agilent 2100生物分析仪。 下(LM)和上标记峰 (UM)。 FU:荧光单位。

  3. Capture库构造的选项
    捕获库可以使用:
    1. Illumina TruSeq DNA [低通量(LT)协议; 凝胶法]
    2. TruSeq DNA无制造商提供的无PCR样品制备试剂盒 说明书(Illumina,Inc.,San Diego,CA)。 最后的库是 在30μl重悬缓冲液中洗脱。
      注意:
      1. 用于测序   合并捕获的Illumina库中适当的索引适配器必须 根据"池化指南"选择(例如,参见参考文献 2)。
      2. 如果选项a。 或b。 被选择,准备库 根据制造商的说明,跳过步骤D到G和 继续进行步骤H."凝胶纯化文库"。
    3. 或者,可以基于DNA方案制备文库 Illumina多重(IM)图书馆(Meyer和Kircher,2010)。 这个 方法是透明的,并避免昂贵的组件,例如 测序文库构建试剂盒和商业试剂 固相可逆固定(SPRI)。灵活性 实验设计由大量可能的指数给出。如 用于产生测序文库的实例33指数是 (补充材料表1)。汇集指数规则 (Meyer和Kircher,2010)。如果更深 需要多路复用,附加索引集与对应 可以产生阻断寡核苷酸(Meyer和Kircher,2010)。图书馆可以 在单管中以及在96孔板中使用制备 多通道移液器。
      步骤D至G描述了使用96孔板的DNA Illumina多重(IM)文库的构建。

  4. 平头端修复
    注意:准备适配器连接所需的适配器组合P57。纯化的PCR产物[DNA Illumina Multiplex(IM)文库的测试片段;见Recipies]应作为阳性对照。 EBT缓冲液用作阴性对照。将对照加入空孔中,并在文库制备期间进行所有操作,从而允许监测酶反应的成功。
      固相可逆固定(SPRI)。灵活性 实验设计由大量可能的指数给出。如 用于产生测序文库的实例33指数是 (补充材料表1)。汇集指数规则 (Meyer和Kircher,2010)。如果更深 需要多路复用,附加索引集与对应 可以产生阻断寡核苷酸(Meyer和Kircher,2010)。图书馆可以 在单管中以及在96孔板中使用制备 多通道移液器。
      步骤D至G描述了使用96孔板的DNA Illumina多重(IM)文库的构建。

  5. 平头端修复
    注意:准备适配器连接所需的适配器组合P57。纯化的PCR产物[DNA Illumina Multiplex(IM)文库的测试片段;见Recipies]应作为阳性对照。 EBT缓冲液用作阴性对照。将对照加入空孔中,并在文库制备期间进行所有操作,从而允许监测酶反应的成功。
      ... dNTPs (25 nM each)
      0.28 µl
      ATP (100 mM)
      0.70 µl
      T4 polynucleotide kinase (10 U/µl)
      3.50 µl
      T4 DNA polymerase (5 U/µl)
      1.40 µl
      Mix the components by flicking the tube gently.
      Caution: Vortexing may inactivate the enzymes.
    1. 加入20微升的主混合物的DNA(50微升)。 用移液器轻轻混匀。
    2. 密封板,并在PCR-cycler中在25℃下孵育样品(70μl)15分钟,在12℃下5分钟,并保持在4℃。
    3. 立即进行样品清理通过添加126微升MagNA 珠(平衡至室温并涡旋)。 将悬浮液混合 通过移液(5x)均质,并在室温下孵育5分钟
    4. 将96孔板置于DynaMag-96侧边带磁性仪上 粒子浓缩器(MPC 96)处理5分钟以分离携带的珠子 来自溶液的DNA。
    5. 使用移液管完全除去上清液,而96孔板保留在MPC <96>中。
    6. 将板放在MPC <96>中,并用150μl新鲜洗涤珠子 制备70%乙醇。 等待1分钟,去除上清液 完全使用移液器
    7. 重复步骤D7一次。
    8. 空气干燥珠完全〜30分钟,而打开的板位于MPC <96> 。
    9. 从MPC 96移除96孔板,并洗脱在22微升EBT中的DNA。 移液器上下(10x)重悬珠。 孵育1分钟 (RT)。 注意液体位于井底。 如果   这不是这种情况,用胶带关闭板并旋转 所有液滴使用具有摆出转子的离心机(1,000×g/g, min,RT)
    10. 将96孔板置于DynaMag-96侧 甩干磁性颗粒聚集器(MPC <96> )5分钟分离   从珠子洗脱DNA。
    11. 将20μlDNA转移到新孔中。 随后的反应不会受到小珠的小量携带的阻碍。
    12. 密封的96孔板可以储存在-20°C。

  6. 适配器连接
    1. 从以下试剂(每个样品的体积)设置主混合物。 按所示顺序添加组件:

      10.0微升
      10×T4DNA连接酶缓冲液
      4.0微升
      PEG 4000(50%)
      4.0微升
      适配器混合物P57(每种100μM)
      1.0μl
      T4 DNA连接酶(5U /μl) 1.0μl
      注意:   如果观察到白色沉淀,孵育10×T4DNA连接酶 缓冲液在37℃下5分钟。 涡旋直到缓冲液清澈。 主混合 应该在加入粘性PEG 4000(50%)后涡旋 解。 最后加入T4 DNA连接酶后,将主混合物 应轻轻混合通过轻轻管。
      注意:涡旋可能会使酶失活。
    2. 加入20微升的主混合物的DNA(20微升)。 用移液器轻轻混匀。
    3. 密封板,并在PCR-cycler中在22℃下孵育样品(40μl)30分钟并保持在8℃。
    4. 立即进行样品清理加入72微升MagNA珠   (平衡至室温并涡旋)。 将悬浮液混合 通过移液(5x)均质,并在室温下孵育5分钟
    5. 将96孔板置于DynaMag-96侧边带磁性仪上 粒子浓缩器(MPC 96)处理5分钟以分离携带的珠子 来自溶液的DNA。
    6. 使用移液管完全除去上清液,而96孔板保留在MPC <96>中。
    7. 将板放在MPC <96>中,并用150μl新鲜洗涤珠子 制备70%乙醇。 等待1分钟,去除上清液 完全使用移液器
    8. 重复步骤E7一次。
    9. 空气干燥珠完全〜30分钟,而打开的板位于MPC <96> 。
    10. 从MPC 96移除96孔板,并洗脱在22微升EBT中的DNA。 移液器上下(10x)重悬珠。 孵育1分钟 (RT)。 注意液体位于井底。 如果   这不是这种情况,用胶带关闭板并旋转 所有液滴使用具有摆出转子的离心机(1,000×g/g, min,RT)
    11. 将96孔板置于DynaMag-96侧 甩干磁性颗粒聚集器(MPC <96> )5分钟分离   从珠子洗脱DNA。
    12. 将20μlDNA转移到新孔中。 随后的反应不会受到小珠的小量携带的阻碍。
    13. 密封的96孔板可以储存在-20°C。

  7. 适配器填充
    1.  从以下试剂制备主混合物(每个样品的体积)。 按所示顺序添加组件:

      14.1微升
      10x ThermoPol反应缓冲液
      4.0微升
      dNTP(各25mM) 0.4μl
      Bst聚合酶,大片段(8U /μl)
      1.5μl
      注意:在最后添加酶之后,通过轻轻地轻轻摇动管混合主混合物。
      注意:涡旋可能会使酶失活。
    2. 加入20微升的主混合物的DNA(20微升)。 用移液器轻轻混匀。
    3. 密封板,将样品(40μl)在PCR-cycler中在37°C孵育20分钟,并保持在8°C。
    4. 立即进行样品清理加入72微升MagNA珠   (平衡至室温并涡旋)。 将悬浮液混合 通过移液(5x)均质,并在室温下孵育5分钟
    5. 将96孔板置于DynaMag-96侧边带磁性仪上 粒子浓缩器(MPC 96)处理5分钟以分离携带的珠子 来自溶液的DNA。
    6. 使用移液管完全除去上清液,而96孔板保留在MPC <96>中。
    7. 将板放在MPC <96>中,并用150μl新鲜洗涤珠子 制备70%乙醇。 等待1分钟,去除上清液 完全使用移液器
    8. 重复步骤F7一次。
    9. 空气干燥珠完全〜30分钟,而打开的板位于MPC <96> 。
    10. 从MPC 96移除96孔板,并洗脱在22微升EBT中的DNA。 移液器上下(10x)重悬珠。 孵育1分钟 (RT)。 注意液体位于井底。 如果   这不是这种情况,用胶带关闭板并旋转 所有液滴使用具有摆出转子的离心机(1,000×g/g, min,RT)
    11. 将96孔板置于DynaMag-96侧 甩干磁性颗粒聚集器(MPC <96> )5分钟分离   从珠子洗脱DNA。
    12. 20μl接头连接的DNA 转移到新井中。 随后的反应不受阻碍 小残留小珠。
    13. 密封的96孔板可以储存在-20°C
    14. 酶反应的性能应通过监测 分离20μl的阳性和阴性对照旁边 具有A突出末端的初始纯化的PCR产物和DNA梯 使用2%琼脂糖凝胶(图2)。 溴化乙锭的大小 染色阳性对照(接头连接的测试片段)应为67bp   大于初始测试片段。
    15. 使用Qubit HS测定确定接头连接的DNA的浓度。


      图   2.适配子连接的对照。测试片段(T; 750ng) 将A突出末端与测试片段一起进行大小分级 提供适配器(阳性对照; P,20μl)和阴性 对照(N,20μl)。 在阴性对照中,测试片段 替换为EBT缓冲区。 使用标准品分离样品 琼脂糖(2%)电泳并用溴化乙锭染色。 L:DNA 阶梯。 指示标准品的大小(bp)。

  8. 库的索引PCR和反应清除
    1.  向PCR板的新孔中加入含有20 - 100 ng接头连接的DNA(模板)的8μl。
      注意:   使用适配器连接的DNA的一部分用于PCR就足够了。 剩余的材料可以用作备份或者可以稍后标记 具有不同的索引(如果需要)。 合并的索引选择规则 和其他指数集描述在别处(Meyer和Kircher, 2010)。
    2. 加入1.4μl适当的(指数)引物(10μM) 从索引引物7001到7033系列(表1在补充 材料)。
    3. 从以下试剂制备主混合物(每个样品的体积)。 按所示顺序添加组件并混合:

      28.7微升
      5x Phusion HF缓冲液
      10.0微升
      dNTP(各25mM) 0.4μl
      底漆IS4(10μM;补充材料中的表1)
      1.0μl
      Phusion Hot Start Flex DNA聚合酶(2U /μl) 0.5μl
    4. 分配40.6微升的主混合物到含有的孔中 模板和引物引物(50μl反应),轻轻吹打混匀 用PCR胶带覆盖板。
    5. 使用以下条件进行PCR:
      - 饱和度         
      98°C      
      30秒
      12个循环:
      - 饱和度     
      98℃ 10秒
      - 退火
      60°C
      20秒
      - 延长
      72℃   
      20秒
      最后一个扩展:
      - 延长        
      72℃   
      10 min
      反应保持在8℃
    6. 通过添加90微升MagNA珠子进行样品清理 (平衡至室温并涡旋)。 将悬浮液混合 通过移液(5x上下)均质,并在室温下孵育5分钟
    7. 将96孔板置于DynaMag-96侧边带磁性仪上 粒子浓缩器(MPC 96)处理5分钟以分离携带的珠子 来自溶液的DNA。
    8. 使用移液管完全除去上清液,而96孔板保留在MPC <96>中。
    9. 将板放在MPC <96>中,并用150μl新鲜洗涤珠子 制备70%乙醇。 等待1分钟,去除上清液 完全使用移液器
    10. 重复步骤G9一次。
    11. 空气干燥珠完全〜30分钟,而打开的板位于MPC <96> 。
    12. 从MPC 96移除96孔板并洗脱在27微升EBT中的DNA。 移液器上下(10 x)重悬珠。 孵育1分钟 (RT)。 注意液体位于井底。 如果   这不是这种情况,用胶带关闭板并旋转 所有液滴使用具有摆出转子的离心机(1,000×g/g, min,RT)
    13. 将96孔板置于DynaMag-96侧 甩干磁性颗粒聚集器(MPC <96> )5分钟分离   从珠子洗脱DNA。
    14. 将25μl指定的文库DNA转移到新孔中,并将密封的96孔板保存在-20℃。
    15. 随机选择的PCR产物的质量和数量通过标准琼脂糖凝胶电泳控制(图3)。


      图   3.指数PCR反应的产物。 PCR产物 使用标准物将所选样品(3μl,步骤G15)进行大小分离 琼脂糖(2%)凝胶电泳并用溴化乙锭染色。 L: GeneRuler 50 bp DNA ladder。 指示梯子的尺寸(bp)。

  9. 图书馆的凝胶净化
    注意:凝胶纯化对于所有类型的文库都是必需的,以确保适当大小的DNA片段用于预捕获文库扩增。使用SYBR-Gold染料和从"Dark reader"透射仪作为激发源发射的可见蓝光在琼脂糖凝胶中揭示DNA。
    注意:不要使用溴化乙锭染色的凝胶。用于激发溴化乙锭的紫外线辐射将损害DNA。
    1. 准备2%琼脂糖凝胶:融化3克UltraPure琼脂糖在150毫升1x TAE 使用锥形烧瓶(500ml)和微波。确保 琼脂糖完全溶解并将溶液冷却至60℃。加  15微升SYBR金,漩涡轻轻没有形成气泡和铸造凝胶 使用标准设备(15cm×15cm托盘,梳子等)。
      通过用铝箔覆盖保护凝胶不受光。让凝胶凝固。
    2. 稀释GeneRuler 50 bp DNA梯:取250 ng梯,4μl6x 加载染料,并填充体积至24μl,使用EBT(梯子一 车道)。
    3. 加入5微升6x加载染料的索引库(25微升),轻轻混合。
    4. 取出梳子,将凝胶放入电泳室。
    5. 向容器中加入1倍TAE,直到凝胶被缓冲液覆盖。
    6. 加载每个库在琼脂糖凝胶的两个相邻的槽(2x 15微升)。   将稀释的梯子装载在两个不同的库之间以防止 污染。溢出
    7. 进行电泳2小时 具有4V/cm(阳极和阴极之间的距离)。 保护凝胶 电泳期间的光。
    8. 揭示DNA使用"黑暗 阅读器透照仪",并使用干净的手术刀切除该区域   兴趣(320至430 bp)。 典型的结果如图4所示 将琼脂糖块倒入干净的2ml(或5ml)管中。
    9. 使用天平确定块的体积(例如 240 mg对应于240μl)。
    10. 基本上如制造商所述,使用Qiagen MinElute试剂盒(旋转柱)纯化DNA
    11. 向块中加入6体积QG缓冲液(例如240μl块:1,440μlQC缓冲液)。
    12. 在温和搅拌下完全溶解凝胶(RT)
    13. 加入一份异丙醇(例如 240μl块:240μl异丙醇),倒置(5x)混合。
    14. 将700μl混合物施加到放置在收集管中的MinElute柱中并旋转(16,000×g,1分钟)。
    15. 丢弃流出物。
    16. 通过重复步骤H14-15,将剩余部分逐步应用到列。
    17. 加入740μlPE洗涤柱,在室温下孵育3分钟并旋转柱(16,000×g,1分钟)。
      注意:将列一直放在转子中,因为在步骤H19中列将转动180°。
    18. 丢弃流出物。
    19. 在转子中转动柱(180°)并旋转(16,000×g ,1分钟)以除去痕量的PE缓冲液。
    20. 丢弃收集管,将MinElute柱放入干净的 1.5 ml管,并加入21μlEB至树脂中心以洗脱 DNA。
    21. 孵育1分钟,旋转柱(16,000×g,1分钟)。
    22. 洗脱的DNA可以保存在-20℃
    23. 使用Qubit BR测定法确定DNA浓度。
      预期浓度:
      TruSeq文库(15-50ng /μl)和DNA Illumina多重(IM)文库(0.5-10ng /μl)。


      图   4.文库的凝胶纯化。文库(1,2) 使用标准琼脂糖凝胶电泳(2%琼脂糖, 1xTAE缓冲液)并用SYBR Gold染色。 使用a显示DNA 可见蓝光发射"黑暗读卡器透射照明器"。 凝胶图像   在电泳(A.)后和分离区域后显示   在320和430bp之间(B.)。 R:标尺(cm),L:GeneRuler 50bp DNA 阶梯。 指示梯度的大小(bp)。

  10. 使用LM-PCR进行预捕获文库扩增
    1. 为了富集正确连接的DNA片段,文库[TruSeq DNA, TruSeq DNA无PCR或DNA Illumina多重(IM)文库]   在预捕获LM-PCR(连接介导的PCR)中的模板。 准备a 主混合物(每个反应80μl) 并通过吸移混合:
      2x Phusion高保真PCR Master Mix           
      50微升
      PCR级水                     
        26μl
      TS-PCR Oligo 1(100μM;补充材料中的表1)   
      2微升
      TS-PCR Oligo 2(100μM;补充材料中的表1)    
      2微升
    2. 分配80μl的主混合物到PCR管,加入20μl的 图书馆。 通过吸移(5x上下)试剂混合。 LM-PCR循环,   样品的清洗和洗脱基本上如(Haun等人,2011)所述。
    3. 使用以下条件进行PCR:
      - 差异
      98°C   
      30秒
      8个循环:
      - 饱和度
      98°C 
      10秒
      - 退火
      60°C   
      30秒
      - 延长
      72℃   
      30秒
      最后一个扩展:
      -Elongation   
      72°C    
      5分钟
      反应保持在8℃
    4. 将扩增的文库转移到1.5毫升管,加入5卷 (500μl)Qiagen缓冲液PBI。 用移液器混合(上下5次)。
    5. 将混合物转移至置于收集管中的QIAquick柱 并离心16,000×g(1分钟)。 丢弃流通。
    6. 加入740μlPE洗涤缓冲液,并离心16,000×g(1分钟)。
      注意:将列一直放在转子中,因为在步骤I8中列将转动180°。
    7. 丢弃流出物。
    8. 转动色谱柱(180°),离心色谱柱16,000 x g(1分钟)以除去残留的PE洗涤缓冲液。
    9. 丢弃收集管,将MinElute柱放入新的1.5 ml   通过向柱中加入50μlPCR级水洗脱DNA   矩阵。 (注意:不要使用TE或EB洗脱。)在1℃孵育1分钟 RT。
    10. 将柱子离心16,000 x g(1分钟)以收集捕获前的LM-PCR扩增文库。
    11. 执行质量检查:
      1. 使用Qubit设备或基于picogreen的测定法定量文库的量。 产率应该> 1μg。
      2. 确定样品库的大小配置文件(1μl) 使用Agilent 2100 Bioanalyzer和DNA 7500进行电泳 芯片。 样本库的片段应在250到500之间 bp(图5)。
    12. 如果预捕获LM-PCR扩增文库 满足质量要求,进行杂交 样品库到exome库。
      可选:将样品储存在-20°C。


      图   5.纯化的预捕获PCR扩增的典型大小图谱 图书馆。 进行预捕获PCR和反应净化 扩增文库,将DNA(1μl)用大小分级分离 Agilent 2100生物分析仪。 下(LM)和上标记峰(UM)为   表示。 FU:荧光单元。

  11. 样品文库与外显子组文库的杂交
    1.  加入1μg预捕获LM-PCR扩增文库和10μlRoche   NimbleGen的专有序列捕获显影剂试剂到1.5毫升 管。
      注意:提供了几个预捕获LM-PCR扩增文库 可以汇总不同指标(总量1μg)。 序列 Capture Developer Reagent以前称为植物捕获增强剂   (PCE)来自Roche NimbleGen。
    2. 加入1μlTruSeq HE Universal Oligo 1(1 mM)。
      注意: TruSeq HE通用Oligo 1旨在阻止通用 TruSeq DNA片段和DNA Illumina多重(IM)文库适配子 在序列捕获杂交期间。
      1. 对于TruSeq DNA或TruSeq DNA无PCR的文库(预捕获LM-PCR扩增)
        加入1μl适当的1 mM TruSeq INV-HE Index Oligo。
        注意:   TruSeq INV-HE Index Oligos设计,以阻止 TruSeq DNA文库适配子AD002的相应索引区段, AD004,AD005,AD006,AD007和AD012 杂交。
      2. 对于DNA Illumina多重(IM)文库(预捕获LM-PCR扩增)
        加入1μl适当的1 mM IM INV-HE Index Oligo。
        注意:   设计了IM INV-HE Index Oligos,以阻止相应的 IM DNA文库适配子的索引片段(指数7001至7033) 在序列捕获杂交期间。
        重要:如果 使用合并的预捕获LM-PCR扩增文库, 相应的封闭寡核苷酸与浓度成比例 的样品在池中。 例如:如果四次预捕获LM-PCR扩增   与TruSeq适配器(AD002,AD004,AD005和AD007)一起提供的库 (0.25μg/文库)杂交,使用1μlTruSeq HE 通用寡核苷酸1(1mM)与0.25μlTruSeq INV-HE Index Oligo   2(1mM),0.25μlTruSeq INV-HE Index Oligo 4(1mM),0.25μlTruSeq INV-HE Index Oligo 5(1mM)和0.25μlTruSeq INV-HE Index Oligo 7(1 mM)。
    3. 锁定试管,用干净的针头刺穿盖子,并在SpeedVac中在60℃下干燥混合物。
    4. 预热加热块(95°C)。
    5. 管中现在包含以下干燥组分(一个TruSeq DNA文库的实例):
      序列捕获开发人员试剂            
      10μl(干燥)
      预捕获LM-PCR扩增文库(TruSeq DNA)          
      1μg(干燥)
      1 mM TruSeq HE Universal Oligo 1                 
      1μl(干燥)
      1 mM适当的IM INV-HE Index Oligo            
      1μl(干燥)
    6. 用完好的盖子替换穿孔的盖子。
    7. 加入干燥的组分:
      2x Sequence Capture(SC)Hybridization Buffer(tube 5)      
        7.5μl
      杂交组分A(试管6)               
      3.0μl
    8. 将混合物(总体积10.5μl)涡旋10秒,并通过离心(RT,10秒,16,000rpm)收集。
    9. 混合物在加热块中变性(95℃,10分钟)
    10. 通过离心(RT,10秒,16,000rpm)收集样品
    11. 将整个样品转移到含有4.5μl的0.2 ml PCR管中 每次捕获的Exome Library(液体阵列)。 通过上下吹吸混合 轻轻(10x)。 确保整个样品位于底部 的管。
    12. 将样品(15μl)在热循环仪中在47°C杂交64-72小时(盖加热至57°C)。

  12. 捕获的文库的亲和纯化
    1. 稀释10倍洗涤缓冲液(试管1,2和3),10x严紧洗涤缓冲液 (管4)和2.5x珠洗涤缓冲液(管7)至1x工作溶液。 使用   PCR级水作为稀释剂。
      每次反应体积:
      股票                  
      PCR级水  
      1x工作溶液
      30μl10×洗涤缓冲液I(试管1)      
      270μl                  
      300微升
      20μl10×洗涤缓冲液II(试管2)      
      180μl          
        200μl
      20μl10×洗涤缓冲液III(试管3)      
      180μl          
      200μl
      40μl10×连接洗涤缓冲液(试管4)   
      360μl           
      400μl
      200μl2.5×珠洗涤缓冲液(试管7)      
      300μl           
      500微升
      注意:   将水浴设置为47°C,并用外部温度验证温度 校准温度计。 预热1x工作溶液(洗涤缓冲液I   和严格洗涤缓冲液),使用前2小时。           
    2. 平衡链霉亲和素Dynabeads到RT,涡旋10秒,等分50μl(每次杂交)到一个新的1.5毫升管。
    3. 将管放入DynaMag-2磁体中2分钟,弃去澄清的液体,并加入100μl珠洗涤缓冲液。
    4. 从磁铁上取下试管,涡旋(10秒),收集悬浮液到试管的底部
    5. 将管放入DynaMag-2磁体中2分钟,弃去澄清的液体,并加入100μl珠洗涤缓冲液。
    6. 从磁铁中取出试管,涡旋(10秒),并通过离心(5秒,16,000×g)收集珠。
    7. 将管放入DynaMag-2磁体中2分钟,弃去澄清的液体,并加入50μl珠洗涤缓冲液。
    8. 从磁铁中取出试管,涡旋(10秒)并通过离心(5秒,16,000×g)回收珠子。
    9. 将重悬的Dynabeads转移到PCR板。 收集 DynaMag-96侧边磁性粒子集中器中的Dynabeads (MPC <96>)2分钟,并丢弃透明液体 注意:保留 包含湿Dynabeads的PCR板的温度尽可能接近 通过使用预热的加热块可以达到47℃。 Dynabeads应该 不干。
    10. 加入杂交样品(15μl)到湿 在PCR板中的Dynabeads(预热的47℃)。 通过吸移轻轻混合 上下(5x)。 继续迅速。
      注意:杂交样品的体积现在可以< 15μl。 然而,这不干扰后续步骤。
    11. 使用预热的热循环仪(57℃盖温度) (生物素化的)捕获的样品结合(链霉亲和素包被的) Dynabeads在47℃下45分钟。 在15分钟内涡旋板3秒 间隔,以确保Dynabeads保持悬浮和 通过快速离心收集液滴到板的底部 (摆出转子),而不会造粒
    12. 加入100μl1x 洗涤缓冲液I(在水浴中预热至47℃)至Dynabeads 加上结合的DNA并涡旋10秒。 收集悬浮液 底部通过快速离心(摆出转子)无 造粒。
    13. 转移悬浮液到新的1.5毫升 管。 将管放入DynaMag-2设备回收Dynabeads 加上结合的DNA 1分钟。 弃去上清液。 从中取出管 磁铁。
    14. Dynabeads在47℃下45分钟。 在15分钟内涡旋板3秒 间隔,以确保Dynabeads保持悬浮和 通过快速离心收集液滴到板的底部 (摆出转子),而不会造粒
    15. 加入100μl1x 洗涤缓冲液I(在水浴中预热至47℃)至Dynabeads 加上结合的DNA并涡旋10秒。 收集悬浮液 底部通过快速离心(摆出转子)无 造粒。
    16. 转移悬浮液到新的1.5毫升 管。 将管放入DynaMag-2设备回收Dynabeads 加上结合的DNA 1分钟。 弃去上清液。 从中取出管 磁铁。
    17. ... Place the tube into a DynaMag-2 device to collect the Dynabeads plus bound DNA for 1 min. Discard the supernatant. Remove the tube from the magnet.
    18. Add 200 µl Wash Buffer II (room temperature) to the Dynabeads plus bound DNA and vortex for 1 min. Collect the suspension to the bottom of the tube by a quick centrifugation without pelleting the beads.
    19. Place the tube into a DynaMag-2 device to collect the Dynabeads plus bound DNA for 1 min. Discard the supernatant. Remove the tube from the magnet.
    20. Add 200 µl Wash Buffer III (room temperature) to the Dynabeads plus bound DNA and vortex for 30 sec. Collect the suspension to the bottom of the tube by a quick centrifugation without pelleting the beads.
    21. Place the tube into a DynaMag-2 device to collect the Dynabeads plus bound DNA for 1 min. Discard the supernatant. Remove the tube from the magnet.
    22. Resuspend the washed Dynabead-bound captured library in 50 µl PCR-grade water and determine precisely the volume using a pipette (storage at -20 °C possible).

  13. Post-capture library amplification using LM-PCR
    1. 设置PCR反应的主混合物(一个反应的体积):
      2x Phusion高保真PCR Master Mix              
      100微升
      PCR级水                        
      42微升
      TS-PCR Oligo 1(100μM;补充材料中的表1)      
      4微升
      TS-PCR Oligo 2(100μM;补充材料中的表1)          
      4微升
      通过上下吹吸(8x)小心混合mastermix。
      添加Dynabead绑定捕获的库(50微升)。 最终体积为200μl。
      通过上下吹吸混匀(8x)。 避免形成泡沫。
    2. 将100μl混合物分配到两个0.2ml-PCR管中
    3. 使用以下条件进行PCR:
      - 饱和度              
      98°C   
      30秒
      18个循环:
      - 饱和度                
      98°C   
      10秒
      - 退出       
      60°C   
      30秒
      -Elongation      
      72℃   
      30秒
      最后一个扩展:
      -Elongation                
      72℃   
      5分钟
      反应保持在8℃
    4. 将两个样品(总共200μl)合并到1.5ml管中并加入1ml   Qiagen缓冲液PBI。 用移液器混合(上下5次)。
    5. 转让   将600μl混合物置于置于收集管中的QIAquick柱中 并离心16,000×g(1分钟)。 丢弃流通。
    6. 用剩余混合物重复步骤L5
    7. 加入740μlPE洗涤缓冲液,并离心16,000×g(1分钟)。 丢弃流通。
    8. 将柱子离心16,000 x g(1分钟)以除去残留的PE洗涤缓冲液。
    9. 将色谱柱旋转180°,并将色谱柱离心16,000 x g(30秒)至 去除痕量的PE洗涤缓冲液。 将柱子放入新的1.5 ml 管
    10. 对于洗脱,向柱基质中加入50μlEB(预热至50℃),孵育1分钟。
    11. 将柱子离心16,000英寸x 1英寸(1分钟)以回收后捕获 LM-PCR扩增文库(外显子捕获文库)。 重新洗脱色谱柱 通过再次向柱中加入50μl的第一洗脱液。 孵育1分钟。   转动柱(180°)并通过离心回收DNA(1分钟, 16,000 x g )。 将库保存在-20°C。
    12. 图书馆的特征:
      1. 使用例如 Nanodrop分光光度计装置来确定 A260/A280比例从1μl的外显子组捕获库(A260/A280应该   为1.7-2.0)。
      2. 确定外显子组捕获的大小配置文件 文库(1μl)用Agilent 2100 Bioanalyzer和a   DNA 7500芯片。 片段应在250和500 bp之间(图 6)。
      3. 使用基于picogreen的测定法(例如,Qubit BR)测量DNA浓度。 预期产率为> 500 ng。
      4. 使用如前所述的实时PCR定量外显子捕获文库(Mascher等人,2013)。


        图   6.最终捕获库的典型大小配置文件 后捕获PCR和反应清除,文库(1μl) 使用Agilent 2100生物分析仪进行大小分级。 作为例子,三   来自三种独立制剂的文库(a,b和c) 显示。 指示下标(LM)和上标记峰(UM)。 FU: 荧光单元

  14. 按合成顺序排列和数据评估
    1. 使用例如HiSeq的合成测序(配对末端,2x100个循环) Illumina系统如先前所述进行(Mascher等人 2013)。
    2. 按照之前所述分析数据(Mascher等人,2013年)。

笔记

  1. 通常,使用过滤嘴和手套以避免污染。 用于在PCR之前和之后处理样品的实验室和设备是物理分离的。 每天用商业解决方案(例如,离开DNA)处理工作空间表面(台架,仪器等)以除去痕量的DNA。 在整个实验中使用良好的实验室规范。
  2. "Bio Rad DNA Engine Tetrad 2 Peltier Thermal Cycler"用于温育和PCR。 对于孵育,除非另有说明,加热盖设置为"盖控制模式:在5℃以上跟踪"。 在PCR和退火衔接头P57期间,将盖子加热至100℃以防止冷凝。 热循环仪应在开始实验之前进行编程
  3. 列出的所有组分(材料和试剂;设备)在用于植物序列捕获的独立实验中验证。然而,此列表并不意味着来自其他供应商的替代产品不能表现同样良好
  4. 样品可以在指定的步骤在-20℃下储存至少3周。
  5. 如果没有另外说明,在RT下进行操作,如温育,颗粒干燥和离心
  6. 使用已发布的设备和化学品并不表示任何竞争性的兴趣
  7. 虽然该方案在我们的实验室(> 50个独立实验)中表现良好,但作者对于读者所进行的实验的成功没有责任。
  8. 基于处理一个数组的估计的动手时间在下面给出(Mascher等人,2013)。仪器的长时间孵育和操作时间(括号):
    图书馆准备
    2天
    使用LM-PCR的预捕获文库扩增
    3小时
    样品库与exome库的杂交
    2小时(3天)
    捕获库的亲和纯化
    4小时
    使用LM-PCR的后捕获文库扩增
    3小时
    图书馆的质量和数量控制
    6小时
    按合成排序(例如 cBot,HiSeq,2x 100个周期)
    (10天)

收据

  1. TE(pH 8.0)
    10mM Tris-Cl(pH8.0) 1mM EDTA
  2. 重悬缓冲液(RSB)
    10mM Tris-Cl(pH8.5)
  3. 50x TAE
    将242g Tris碱溶解在去离子水中,加入57.1ml冰醋酸和100ml 0.5M EDTA(pH 8.0)。
    将体积调至1升,然后彻底混匀
    使用去离子水将50x TAE原液稀释至1x TAE工作溶液(彻底混合)
  4. EBT
    10mM Tris-Cl(pH8.0) 0.05%(v/v)吐温20
  5. DNA Illumina多重(IM)文库的测试片段(Meyer和Kircher,2010)
    设计未修饰的引物以产生具有单一大小(长度150-200bp)的扩增子 使用Taq聚合酶进行扩增,以产生在3'端具有A突出端的产物
    使用标准方案纯化PCR产物,并将浓度调整至250ng /50μl。
  6. IM库的适配器组合P57(Meyer和Kircher,2010)
    制备10x Oligo杂交缓冲液(500mM NaCl,10mM Tris-Cl,1mM ETDA,pH 8.0)
    加入PCR管1:
    40μlIS1_adapter_P5.F(500μM;补充材料中的表1)
    40μlIS3_adapter_P5 + P7.R(500μM;补充材料中的表1)
    10μl10x Oligo杂交缓冲液
    10μlPCR级水
    加入PCR管2:
    40μlIS2_adapter_P7.F(500μM;补充材料中的表1)
    40μlIS3_adapter_P5 + P7.R(500μM;补充材料中的表1)
    10μl10x Oligo杂交缓冲液
    10μlPCR级水
    将PCR管1和2放入PCR机。
    变性:95℃10秒
    退火:冷却至12℃(斜坡:0.1℃/秒) 组合来自管1和2的退火的寡核苷酸。最终浓度为100μM(每个衔接子)。
    将等分试样储存在-20°C
  7. 用于DNA清洗的MagNA珠子
    MagNA珠基本上如所述使用(Rohland和Reich,2012),从而在DNA清洁步骤中替换商业产品(Agencourt AMPure XP)。
    MagNA珠的组成:
    2%羧基修饰的Sera-Mag磁速珠(v/v) 18%PEG-8000(w/v)
    1 M NaCl
    10mM Tris-HCl(pH8.0) 1mM EDTA(pH8.0) 0.05%吐温20(v/v) 为了除去叠氮化钠,在加入之前用TE将新的Speed-beads洗涤3次。 将MagNA珠存储在黑暗(4℃)中并在3个月内使用。

补充材料

表1. 寡核苷酸列表

致谢

DNA Illumina多重(IM)文库的产生基本上如所述(Meyer和Kircher,2010)。导致捕获的文库的操作基于制造商的说明书(Roche/NimbleGen,NimbleGenArrays User's Guide,Plant Sequence Capture Illumina Optimized,2010)和如前所述(Mascher等人,2013) 。非常感谢德国教育和研究部(BMBF)的赠款GABI-BARLEX(FKZ 0314000到N.S.)和TRITEX(FKZ 0315954到N.S.)的资金支持。

参考文献

  1. Haun,W.J.,Hyten,D.L.,Xu,W.W.,​​Gerhardt,D.J.,Albert,T.J.,Richmond,T.,Jeddeloh,J.A.,Jia,G.,Springer,N.M.and Vance,C.P。 大豆参考品种威廉姆斯82的个体之间的基因组变异的组成和来源。 a> Plant Physiol 155(2):645-655
  2. Illumina TruSeq示例准备池化指南,零件号15042173. http://support.illumina.com/downloads/truseq_sample_preparation_pooling_guide_15042173.ilmn。
  3. Mascher,M.,D'Ascenzo,M。,M.,Richmond,TA,Gerhardt,DJ,Himmelbach,A.,Clissold,L.,Sampath,D.,Ayling,S.,Steuernagel, Akhunov,ED,Hedley,PE,Gonzales,AM,Morrell,PL,Kilian,
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用:Himmelbach, A., Knauft, M. and Stein, N. (2014). Plant Sequence Capture Optimised for Illumina Sequencing. Bio-protocol 4(13): e1166. DOI: 10.21769/BioProtoc.1166.
提问与回复

(提问前,请先登录)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片或者视频的形式来说明遇到的问题。由于本平台用Youtube储存、播放视频,作者需要google 账户来上传视频。

当遇到任务问题时,强烈推荐您提交相关数据(如截屏或视频)。由于Bio-protocol使用Youtube存储、播放视频,如需上传视频,您可能需要一个谷歌账号。