Preparing a DNA fragment library for Illumina sequencing

ALERT! ALERT! ALERT! WARNING ALERT! ALERT! ALERT! THIS PROTOCOL IS DEPRECATING RAPIDLY, KAPA BIOSYSTEM KITS ARE NOW BEING USED RATHER THAN NEB KITS, SEVERAL MODIFICATIONS HAVE CHANGED. THIS PROTCOL WILL WORK, BUT SEE DAN FOR CURRENT PROTOCOL.

Overview

  1. Harvest DNA and determine concentration.
  2. Fragment DNA.
  3. End Repair DNA.
  4. dA tail 3' end of DNA fragments
  5. Ligate barcoded adapters on
  6. Size select window of library(s) accounting for addition of adapter sequences
  7. PCR amplify library
  8. Determine concentration
  9. Send for high sensitivity bioanalyzer
  10. Submit for sequencing

Before Beginning and Important Notes

  1. Low retention tubes and PCR tubes should be used at every step.
  2. Filter tips should be used for every step where DNA or reagents are being transferred. Only exception to this should be EtOH washes during cleanup.
  3. PCR step should NEVER be preformed on block A.
  4. Before starting make sure ligated adapters are available in cardboard box labeled 'Anealed adapters'.
    ALERT! Importance of this box/these tubes never reaching RT cannot be underscored enough
  5. WFI water should be used for all steps where water is needed. Aliquots of water should be taken from bottle to avoid contamination.
  6. Between 1 and 4 samples can be processed in parallel for E-Gel size selection. Up to 6 samples can be processed in parallel if doing bead size selection.
  7. Anealed Adapters are restocked 1 'batch' at a time in an effort to minimize barcode overlap. If you plan to run your samples with someone associated with the lab be sure to check with them about what barcodes they have used. Especially if the adapters need to be restocked for your samples.
  8. Sample should never be vortexed, always thoroughly mixed by pipetting. This is done to avoid sample getting near the top of the tube and increasing contamination risk during magnetic bead clean up.
  9. "Freshly prepared 80% EtOH" for cleanup reactions should be prepared daily, and be made combining 100% EtOH with WFI water.

Adapter Anealation

TIP Note, this step is only necessary if 'Anealed adapters' box and backup box are both empty. This should only happen once every 152 libraries made based on current barcodes available.
  1. Mix the following in PCR tube for a 40M adapter concentrated stock:
    1. 5l 100M Universal Barcode with Thioate
    2. 5l 100M Barcode Adapter # (# represents whatever barcode is being made, ideally all should be prepped together)
    3. 2.5l WFI water
  2. Use thermocycler with following conditions:
    1. 97C 2minutes
    2. 97C 1minute
      1. Repeat previous step for 72 total cycles at -1C per cycle
    3. 25C 5 minutes
      After this step, adapters should not EVER be warmed above 25C.
  3. Transport tube(s) back to lab on ice.
  4. Add 20.5l of CHILLED WFI water to create 15M working stock.
  5. Alliquot 4l to 8 PRECHILLED 600l low retention eppendorf tubes. Make sure tubes are labeled.
  6. Store 1 'set' of barcodes (ie 78-96) in 'anealed adpaters' box. Store rest of sets in illumina box.

Harvest DNA and determine Concentration

  1. Harvest DNA from sample using Invitrogen PureLink Genomic DNA kit using standard protocol.
  2. It is important to elute in elution buffer rather than water.
  3. 2l of sample should be used to determine concentration via Qubit BR assay.

Fragment DNA

  1. Place between 1 and 10g of DNA in a covaris microtube (typically between 1 and 5).
  2. Bring total volume up to 130l using Invitrogen Elution Buffer.
  3. Signup for an appropriate amount of time in the GSAF core, and take all samples to core.
  4. Use appropriate shearing conditions for the Covaris instrument.
    1. 150bp (80-300 distribution) Intensity: 5 ; Duty Cycle: 10% ; Cycles per burst: 200 ; Time: 600 sec (10 cycles of 60 sec)
    2. Consult Covaris manual for other size distributions, and enter them here for future reference.
  5. Determine concentration of post-sheared DNA via BR Qubit.

End Repair

Mix the following in PCR tube and incubate at 20C for 30 minutes with 30C lid
Item Amount
1-5 g of Fragmented DNA 1-85 l
NEBNext End Repair Reaction Buffer (10x) Green lid 10 l
NEBNext End Repair Enzyme Mix Green lid 5 l
WFI Water 85-1 l
Total Volume 100 l
700ng is maximum amount for loading on an Egel at the size selection stage. Therefore if using Egel size selection, 1g is recommended amount. Alternatively, 2g can be split in half etc. If too much DNA is lost during fragmentation, concentrate sample via beads, not speed vac. (Untested concern that speed vac concentrates tris in the TE buffer.)
Reaction buffer contains DTT and can be very difficult to thaw at RT (precipitation remains as rest of solution thaws). Warming with fingers and vortexing recommended to get everything into solution.
ALERT! If buffer lacks 'the DTT smell' the buffer is probably bad based on previous experience.

Cleanup

  1. Transfer 100 l to new 1.5ml eppendorf tube.
  2. Add 160 l of Ampure XP Beads. Mix by pipetting up and down at least 10x for solution to be homogenous brown color.
  3. Incubate 5 min @ RT.
  4. Place tube in magnetic stand for 5 min @ RT.
    TIP slightly angel the tube so the wall of the tube is physically in contact with tube
  5. Remove liquid without disturbing the beads and add 200 l of freshly prepared 80% EtOH by pipetting against the tube wall farthest away from the magnet. Incubate at least 30 seconds before proceeding.
  6. Repeat previous step 1 time.
  7. Air dry 10 minutes.
    1. As long as all liquid is at the bottom of the tube and a good job is done of removing liquid at each of the washes this should be sufficient. If additional liquid is clearly visible, it should be pipetted out with a new tip. Alternatively, an additional 10minutes of air dry can be performed.
  8. Remove tube from rack, add 44 l WFI water against wall with beads. Pippette at least 10x to completely resuspend beads. Place on rack for 1 minute.
    TIP Important to be semi gentle with resuspension so as to avoid bubbles and splashing.
  9. When solution is visibly clear, transfer 42 l directly to new PCR tube.

dA Tailing

Mix the following in PCR tube and incubate at 37C for 30 minutes with 47C lid
Item Amount
End repaired, blunt ended DNA 42 l
NEBNext dA-Tailing Reaction Buffer (10x) Yellow lid 5 l
Klenow Fragment (3`-5` exo-) Yellow lid 3 l
Total Volume 50 l
ALERT! Important to prechill WFI water for elution and PCR tube for adapter ligation. Failure to do so can result in denature adapters, and unsucessful ligation.

Cleanup

  1. Transfer 50 l to new 1.5ml eppendorf tube.
  2. Add 90 l of Ampure XP Beads. Mix by pipetting up and down at least 10x for solution to be homogenous brown color.
  3. Incubate 5 min @ RT.
  4. Place tube in magnetic stand for 5 min @ RT.
    TIP slightly angel the tube so the wall of the tube is physically in contact with tube
  5. Remove liquid without disturbing the beads and add 200 l of freshly prepared 80% EtOH by pipetting against the tube wall farthest away from the magnet. Incubate at least 30 seconds before proceeding.
  6. Repeat previous step 1 time.
  7. Air dry 10 minutes.
    1. As long as all liquid is at the bottom of the tube and a good job is done of removing liquid at each of the washes this should be sufficient. If additional liquid is clearly visible, it should be pipetted out with a new tip. Alternatively, an additional 10minutes of air dry can be performed.
  8. Remove tube from rack, add 36 l PRECHILLED WFI water against wall with beads. Pippette at least 10x to completely resuspend beads. Place on rack for 1 minute.
    TIP Important to be semi gentle with resuspension so as to avoid bubbles and splashing.
    ALERT! Failure to use prechilled water will result in lower levels of successful library generation, or complete failure
  9. When solution is visibly clear, transfer 34 l directly to new PRECHILLED PCR tube.
    ALERT! Prechilled PCR tube is important for same reason as already listed.

Adapter Ligation

Mix the following in a prechilled PCR tube on ice allowing tube to cool after each addition.
Transfer to thermocycler on ice. Incubate at 20C for 15 min with lid at 30C
Item Amount
dA Tailed DNA 34 l
Quick Ligation Reaction Buffer (5x) Red lid 10 l
15uM DNA Adaptors 1 l
Quick T4 DNA Ligase Red lid 5 l
Total Volume 50 l

ALERT! Adapter should be thawed on ice, and not allowed to reach room temperature. After incubation temperature is not a concern for assay

Cleanup

  1. Transfer 50 l to new 1.5ml eppendorf tube.
  2. Add 90 l of Ampure XP Beads. Mix by pipetting up and down at least 10x for solution to be homogenous brown color.
  3. Incubate 5 min @ RT.
  4. Place tube in magnetic stand for 5 min @ RT.
    TIP slightly angel the tube so the wall of the tube is physically in contact with tube
  5. Remove liquid without disturbing the beads and add 200 l of freshly prepared 80% EtOH by pipetting against the tube wall farthest away from the magnet. Incubate at least 30 seconds before proceeding.
  6. Repeat previous step 1 time.
  7. Air dry 10 minutes.
    1. As long as all liquid is at the bottom of the tube and a good job is done of removing liquid at each of the washes this should be sufficient. If additional liquid is clearly visible, it should be pipetted out with a new tip. Alternatively, an additional 10minutes of air dry can be performed.
  8. Remove tube from rack, add X l WFI water against wall with beads. Pippette at least 10x to completely resuspend beads. Place on rack for 1 minute.
    • X l of WFI water to add based on size selection method to be used:
      • Egel = 52
      • Beads = 102
        TIP Important to be semi gentle with resuspension so as to avoid bubbles and splashing.
  9. When solution is visibly clear, transfer X-2 l directly to new PCR tube.

Egel Size Selection

ALERT! Skip this section if using bead based size selection

Removal of Adapter dimers prior to Egel Selection.

  1. Transfer 50 l to new 1.5ml eppendorf tube.
  2. Add 42.5 l of Ampure XP Beads. Mix by pipetting up and down at least 10x for solution to be homogenous brown color.
    ALERT! This ratio determined to be optimal to remove ~150bp without removing 200bp using 50bp ladder for current batch of Ampure beads. This needs to be validated each time new beads are purchased due to documented lot to lot variability.
  3. Incubate 5 min @ RT.
  4. Place tube in magnetic stand for 5 min @ RT.
    TIP slightly angel the tube so the wall of the tube is physically in contact with tube
  5. Remove liquid without disturbing the beads and add 200 l of freshly prepared 80% EtOH by pipetting against the tube wall farthest away from the magnet. Incubate at least 30 seconds before proceeding.
  6. Repeat previous step 1 time.
  7. Air dry 10 minutes.
    1. As long as all liquid is at the bottom of the tube and a good job is done of removing liquid at each of the washes this should be sufficient. If additional liquid is clearly visible, it should be pipetted out with a new tip. Alternatively, an additional 10minutes of air dry can be performed.
  8. Remove tube from rack, add 27 l WFI water against wall with beads. Pippette at least 10x to completely resuspend beads. Place on rack for 1 minute.
    TIP Important to be semi gentle with resuspension so as to avoid bubbles and splashing.
  9. When solution is visibly clear, transfer 25 l directly to appropriate Egel well.
    TIP While Egel needs to be run within 15 minutes of opening package, it is highly reccomended to have all water pre-loaded on gel, and marker to sample is the last thing being added to avoid diffusion.

Egel Size Selection.

  1. Place 2% size select egel in the base.
    1. Important that gel be run be started within 15 minutes of opening package.
  2. Place 25ul of WFI water in all bottom wells (10 ul for the marker lane).
  3. Place 25ul of Sample or water in each top well.
    TIP If using multiple samples you want to skip every other lane and the 2 lanes next to the marker (ie load 1, 3, 6, 8 with sample if 4 samples, 3, 6 if 2 samples)
    ALERT! If started with more than 1 g of DNA, necessary to split DNA over multiple wells, and pool to a single tube. DNA should then be concentrated via beads with 1.8X ampure beads.
  4. Run sample for ~12-15 minutes to reach ~200 bp. After 8 minutes, add ~10 ul of water to each collection well.
  5. When size wanted is in the well, stop current, and carefully remove water transferring it to new tube.
    ALERT! Important to remember that the adapters add 123 bases to each product, so if you want a 100bp insert library, need to collect DNA of 223bp
  6. Add 30ul additional water, pipette up and down 2x, run 10 seconds, transfer to same tube.
  7. Add ~10ul, run in reverse 5 seconds, transfer to same tube.
  8. Check that you have ~50ul total volume.
    TIP If able/desired, add 25 ul water to each collection well, and continue running until appropriate size is in the collection window and collect larger size fractions by repeating the above collection steps. This should be based on downstream applications for this particular library.

Bead Size Selection

ALERT! Skip this section if using Egel size selection
  1. Transfer 100 l to new 1.5ml eppendorf tube.
  2. Add 80 l of Ampure XP Beads. Mix by pipetting up and down at least 10x for solution to be homogenous brown color.
  3. Incubate 5 min @ RT.
  4. Place tube in magnetic stand for 5 min @ RT.
    TIP slightly angel the tube so the wall of the tube is physically in contact with tube
  5. Transfer 100% of supernatant (~180l) to new eppendorf without disturbing beads HELP Fragments of ~400 and above are bound to the beads leaving <400 in solution
  6. Remove from stand and add 20 l of fresh Ampure beads. Mix by pipetting at least 10x for solution to be homogenous brown color. HELP Fragments of ~300 to 400 are now on beads.
  7. Incubate 5 min @ RT.
  8. Place tube in magnetic stand for 5 min @ RT.
  9. Remove liquid without disturbing the beads and add 200 l of freshly prepared 80% EtOH by pipetting against the tube wall farthest away from the magnet. Incubate at least 30 seconds before proceeding.
  10. Repeat previous step 1 time.
  11. Air dry 10 minutes.
    1. As long as all liquid is at the bottom of the tube and a good job is done of removing liquid at each of the washes this should be sufficient. If additional liquid is clearly visible, it should be pipetted out with a new tip. Alternatively, an additional 10minutes of air dry can be performed.
  12. Remove tube from rack, add 27 l WFI water against wall with beads. Pippette at least 10x to completely resuspend beads. Place on rack for 1 minute.
    TIP Important to be semi gentle with resuspension so as to avoid bubbles and splashing.
  13. When solution is visibly clear, transfer 25 l directly to appropriate Egel well.
    TIP While Egel needs to be run within 15 minutes of opening package, it is highly reccomended to have all water pre-loaded on gel, and marker to sample is the last thing being added to avoid diffusion.

PCR Amplification

Mix the following in a PCR tube
Item Amount
Size Selected DNA 20 l
P5/P7 primer mix (12.5M eac) 5 l
Phusion High-Fidelity PCR Master Mix with HF Buffer -- blue lid∆ 25 l
Total Volume 50 l
Store remaining DNA at -20C. This will be used if the PCR is unsuccessful, or if further optimization is required. 12.5 l of each 100M primer in 100l total volume ∆ Alternatively use Phusion High-Fidelity master mix with HF buffer that has been purchased separately.

PCR Conditions
Step Temp Time
1 98 30 sec
2 98 10 sec
3 65 30 sec
4 72 30 sec
5 go to Step 2
6 72 5 min
Should be repeated as few times as possible to maximize diversity of DNA molecules. For Egel selection 15+ cycles may be necessary. For standard bead library, 5-8 cycles reccomended.

Cleanup (and remove unused primers)

  1. Transfer 50 l to new 1.5ml eppendorf tube.
  2. Add 50 l of Ampure XP Beads. Mix by pipetting up and down at least 10x for solution to be homogenous brown color.
  3. Incubate 5 min @ RT.
  4. Place tube in magnetic stand for 5 min @ RT.
    TIP slightly angel the tube so the wall of the tube is physically in contact with tube
  5. Remove liquid without disturbing the beads and add 200 l of freshly prepared 80% EtOH by pipetting against the tube wall farthest away from the magnet. Incubate at least 30 seconds before proceeding.
  6. Repeat previous step 1 time.
  7. Air dry 10 minutes.
    1. As long as all liquid is at the bottom of the tube and a good job is done of removing liquid at each of the washes this should be sufficient. If additional liquid is clearly visible, it should be pipetted out with a new tip. Alternatively, an additional 10minutes of air dry can be performed.
  8. Remove tube from rack, add 27 l WFI water against wall with beads. Pippette at least 10x to completely resuspend beads. Place on rack for 1 minute.
    TIP Important to be semi gentle with resuspension so as to avoid bubbles and splashing.
  9. When solution is visibly clear, transfer 25 l directly to new 1.5 ml eppendorf tube.

DNA Concentration & Bioanalyzer

  1. DNA concentration should be determined using at least 2l of cleaned product.
    TIP If concerned that sample may have low concentration, using an increased amount of product is recommended (ie it is better to use 4 or 6 l of product 1 time rather than 2l not have enough, and need to use an additional 4-6 l)
  2. Assuming DNA concentration is greater than Undetermined, submit 1-2l for HS bioanalyzer in the GSAF core.

Sequence

ALERT! assume sample 'looks good' Need to add image files showing what a good sample looks like
  1. Create job at the GSAF website
  2. When job approved, take sample to GSAF core.
  3. For more information about data retrieval and analysis visit internal site InternalNgsData (login required).
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Topic revision: r12 - 24 Sep 2015 - 22:27:50 - Main.JordanMonk
 
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