Esophageal Adenocarcinoma Methods and Protocols

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13. Carefully remove the plate seal, and then add the following
    components in the order listed to each well containing
    digested amplicons (Table 4 ). The total volume is now 30 μL
    (including ~22 μL of digested amplicon).


14. Mix by pipetting at least five times before sealing the plate.
    Strip tubes were placed in a thermal cycler and temperature
    program was performed as indicated in Table 5. This again
    could be the STOPPING POINT. Samples can be stored for
    up to 24 h at 10 °C on the thermal cycler. For longer periods,
    store at − 20 °C.


15. AMPure® XP reagent should be brought to room temperature
    and vortexed thoroughly to disperse the beads before use.


16. The solution should be pipetted slowly, then 45 μL (1.5× sam-
    ple volume) of Agencourt AMPure XP Reagent should be
    added to each library and pipetted up and down five times to
    thoroughly mix the bead suspension with the DNA.


17. The mixture should then be incubated for 5 min at room
    temperature.


18. The tube should be placed in a magnetic rack and incubated
    for 2 min until solution clears.


19. The supernatant has to be discarded without disturbing the
    pellet.

3.1.1 Library Purification

Table 4

Components to be added to the digested amplicons

Component

Volume

(μL)

Switch Solution 4

Ion Ampliseq Adapter Mix (P1 + Barcode) 2

DNA ligase 2

Total volume 15

Table 5

Thermal program for adaptor ligation

Temperature (°C) Time

22 30 min

72 10 min

10 Hold

Next Generation Sequencing and Esophageal Adenocarcinoma