Chapter 8 Library pooling

Subsequently, each individual indexed library is analysed for fragment size distribution using capillary electrophoresis. This technique enables measuring the concentration and molarity of desired fragments (usually between 300 and 800 bp) and undesired molecules such as adaptor dimers and primer remains (usually between 30 and 150 bp). Using this information, one can calculate how much volume from each library will be needed to generate a desired amount of data.

8.1 Instruments, plasticware and reagents

Instruments

  • Magnet compatible with Microcentrifuge Tubes 1.5/2 mL such as DynaMag™-2 Magnet (Invitrogen, 12321D)
  • Fragment analysis system such as 2100 Bioanalyzer (Agilent)
  • SAFE® Screw Cap De-/Capper (LVL technogies)
  • SAFE® 2D/1D Code Reader (LVL technogies)

Plasticware

Item Brand Catalogue number
Snap cap, DNA LoBind®, 1.5/2 tube Eppendorf 30108051/0030108078

Stock reagents

Reagent Brand Catalogue number Storage
Deionized ultrapure water (ddH2O) Bionordika BN-51100 20 ºC
Absolute ethanol, EtOH 20 ºC
Elution Buffer (EB) Qiagen 19086 20 ºC
HighPrep™ PCR Clean-up System MAGBIO AC-60050 4 ºC

8.2 Protocol

1. Sample pooling

  1. Calculate how much volume is required from each indexed library to achieve the desired proportion of sequencing, and doublecheck no repeated indices are pooled together.
  2. If the required volume is lower than 1 µl (meaning library is very concentrated), dilute the indexed library to ensure accurate pipetting.
  3. Transfer the required volume of original or diluted indexed library to the library pool tube (usually 1.5 ml Eppendorf tube).

2. Final magnetic bead-based purification

Once all indexed libraries have been pooled into a single pool, it is convenient to perform one last bead-purification, to 1) get rid of short fragment remains that will likely create sequencing problems, while 2) concentrating the final library pool into a lower volume.

  1. Equilibrate the beads to room temperature for 30 min.
  2. Ensure the beads are fully resuspended by vortexing.
  3. Transfer the volume of beads that is equivalent to ~1.2 times the volume of the library pool and mix thoroughly by pipetting.
  4. Incubate the mixture for 5 minutes at room temperature.
  5. Place PCR strips/PCR plate on a magnetic rack and wait until the supernatant is clear.
  6. Discard the supernatant.
  7. Add 200 µl of 80% EtOH to each well. Discard the supernatant.
  8. Repeat step 7 and ensure that all residual ethanol is removed.
  9. Dry the beads for a maximum of 5 minutes.
  10. Add 35 µl of EBT buffer to each well and quick-spin the PCR strips/PCR plate.
  11. Incubate the mixture 10 minutes at 37°C (outside the magnet).
  12. Quick-spin the PCR strips/PCR plate.
  13. Place the PCR strips/PCR plate on a magnetic rack and wait until the supernatant is clear.
  14. Aspirate (slowly to avoid bead transfer) and dispense the supernatant (DNA libraries) to a new PCR strips/PCR plate.
  15. Transfer the purified DNA to a 200 µl LVL tube.