Esophageal Adenocarcinoma Methods and Protocols

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  1. The run plan specified sequencing settings, number of flows,
    kit type used in sample preparation, barcodes used and corre-
    sponding samples, sample type, i.e., DNA, and the reference
    file to be used.

  2. This allows the server to automatically assemble sequence frag-
    ments against this reference sequence. All torrent softwares
    should be updated regularly.

  3. For quality assessment of each sequencing run, 5 μL of control
    ISPs were added to half of the 100 μL volume of ISPs previ-
    ously stored in the fridge.

  4. The other 50 μL are kept as reserve stores. The “A” and “D”
    test fragments found in this control mixture provided insight
    about the quality of sequencing runs.

  5. After adding control fragments, 100 μL of annealing buffer
    was added and mixed in by pipetting up and down.

  6. The tube was centrifuged for 2 min at 15,500 × g and all but
    3 μL of supernatant was removed.

  7. The sequencing primer was then annealed to each target frag-
    ment by adding 3 μL of thawed sequencing primer followed by
    a 95 °C for 2 min and 37 °C for 2 min thermal cycling step.

  8. The chip (Fig. 4c) is placed on the PGM system via the ground-
    ing plate (Fig. 4d) and “experiment” was selected on the main
    menu to initiate a chip check.

  9. Following a successful chip check, the chip is washed once with
    50 μL of 100% isopropanol and twice with 50 μL of annealing
    buffer.

  10. Sequencing polymerase is then added to the ISPS, to bring the
    total volume to 7 μL.

  11. After an incubation period of 5 min, ISPs were loaded onto the
    chip and equally distributed through a series of centrifugations
    using Chip Minifuge (Fig. 5 ).

  12. Start the run. Each run took approximately 2½ h (see Note 5).

  13. Sequencing results of the Ion Torrent PGM run are assessed
    via the Torrent Browser, which is a web-based user interface on
    the Torrent Server.

  14. The Torrent Browser runs report comprises the statistics and
    quality metrics of the run, which include the Ion Sphere
    Particle (ISP) density, the percentage of polyclonal ISPs (ISPs
    carrying clones from two or more templates), and low quality
    percentage (percentage of ISPs with a low signal).

  15. Together, these factors make up the percentage of usable reads,
    which is the percentage of library ISPs that are not eliminated


3.3.1 Chip Loading


3.4 Data Analysis


Suja Pillai et al.
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