Next Generation Sequencing

An evaluation of a sequencing method was performed on samples using the Ion Proton and a targeted gene panel. The workflow of the entire process is summarized in Figure 2.5. The first step requires the creation of a library from the genomic DNA by am-plification of targeted regions and subsequent purification using magnetic beads. Next, emulsion PCR is performed to obtain clonal amplification of specific templates on in-dividual Ion Sphere Particles (ISPs). The template-positive ISPs are enriched with the Ion One Touch and magnetic beads. Finally, the templates are sequenced on the Ion Proton, which calls bases by detecting the pH change upon each base addition. Kits used for this procedure are listed in section2.1.5.

Figure 2.4: Next Generation Sequencing workflow [108].

Table 2.9: Cancer Hotspot Panel v2 Gene Coverage. 50 target genes with a total of 207 amplified regions [109]. Listed as: GENE (number of amplicons)

ABL1 (4) EZH2 (1) JAK2 (1) PTEN (8) AKT1 (2) FBXW7 (5) JAK3 (3) PTPN11 (2) ALK (2) FGFR1 (2) KDR (9) RB1 (10)

APC (7) FGFR2 (4) KIT (9) RET (5)

ATM (17) FGFR3 (5) KRAS (3) SMAD4 (9) BRAF (2) FLT3 (4) MET (6) SMARCB1 (4) CDH1 (3) GNA11 (1) MLH1 (1) SMO (5) CDKN2A(2) GNAQ (1) MPL (1) SRC (1) CSF1R (2) GNAS (2) NOTCH1 (3) STK11 (5) CTNNB1 (1) HNF1A(2) NDM1 (1) TP53 (8) EGFR (8) HRAS (2) NRAS (3) VHL(3) ERBB2 (3) IDH1 (1) PDGFRA (4)

ERBB4 (8) IDH2 (1) PIK3CA (11)

2.2.9.1 | Library Construction

The construction of the template library was performed following the Ampliseq Library Preparation User Guide. The Cancer Hotspot Panel was used to create the library, which amplifies gene regions commonly mutated in various cancers. The panel covers 50 genes with a total of 207 primer pairs (Table. 2.9)

Chapter 2. Materials and Methods 39

Target Amplification A cell line sample and two spiked normal blood samples were evaluated in this experiment. A 1:10 dilution of cell line cDNA was made and 10 ng added to the target mix. 12 µl (maximum volume) of each the spiked samples was added. To prepare the libraries for amplification the following was added to each DNA template: 5X Ion AmpliSeq HiFi Mix (4 µl), 5X Ion Ampliseq Primer Pool (4 µl), and nuclease-free water to a total of 20 µl. The samples were mixed by vortexing and subsequently spun down and were amplified in the thermocycler.

Digest Primer Sequences The primer sequences were partially digested by adding 2µl of FuPa Reagent to a total reaction colume of 22µl. The samples were mixed, spun down, and again placed in the thermocycler.

Ligate Adapters & Amplify Since multiple libraries were prepared and would be run on a single chip, a unique barcoded adapter was used for each library. 2 µl of the 1:4 barcode adapter mix and 4µl Switch Solution was added to each digested amplicon library. DNA Ligase (2µl) was then added to each sample and the samples were placed in the thermocycler for ligation.

Purify Libraries For purification of the libraries, 45 µl of Agencourt AMPure XP Reagent was added to each, incubated for 5 minutes at room temperature, and sub-sequently placed in a magnetic rack. To wash the beads, 70% ethanol was added to each tube, removed and the tube incubated for 5 minutes to dry. Immediately following drying, the libraries were amplified for quantification.

Library Amplification, Purification and Quantification Libraries were amplified by first adding Platinum PCR Supermix High Fidelity (50µl) and Library Amplification Primer Mix (2 µl) to each bead pellet and replaced in the magnet for 2 minutes. The sample libraries were placed in the thermocycler and following amplification, the libraries were purified by AmpPure beads with Agencourt AMPure XP Reagent. The first round was at a 0.5X bead-to-sample ratio for the removal of any residual high molecular-weight DNA. The second round was at a 1.2X bead-to-original-sample-volume ratio. Here the amplicons bind to the beads and primers are left in solution. The bead pellet was saved and the amplicons were eluted from the beads.

Library concentrations were measured on the Qubit 2.0 Fluorometer (Life Technologies).

A fresh 1:200 working dilution of the Qubit dsDNA HS reagent was prepared. Each amplified library aliquot was combined with 190 µl of dye reagent and incubated for 2 minutes.

Combine Libraries After quantification, the libraries were diluted accordingly (to 15 ng/ml) for an even mix of 3.5µl each for template preparation. Nuclease-free water was added to a final volume of 100 µl for template-positive ISP preparation.

2.2.9.2 | Template Preparation

Template-positive ISPs were prepared by emulsion PCR for clonally amplified DNA, and subsequent enrichment of the template-positive particles using the Ion One Touch system. The Ion PI Template OT2 200 guide was followed for this technique. After emulsion PCR and before enrichment of template-positive ISPs, the percent of templated ISPs were measured in the Qubit fluorometer. The ISP sample was measured by inserting the sample into the Qubit and under the Ion option, AF 488 was selected. The value was recorded and then the AF 647 fluorescence was measured and recorded. These values were entered into a spreadsheet containing the factor calculator to determine the percent of templated ISPs in the unenriched sample. The sample was then enriched for template-positive ISPs and stored at 4℃ for the sequencing run.

2.2.9.3 | Run Sequence

For the sequencing chip preparation and run, the Ion PI Sequencing 200 guide was followed. First, the instrument and was initialized to obtain the proper pH in the sequencing. The chip was also prepared with multiple washes and calibrated by the instrument to ensure the correct pH was present in the chip. The chip was then loaded;

55 µl of the ISP solution was foamed and injected into the chip. Further dispersion of the ISP foam into the chip wells was performed by centrifugation. The chip was placed in the instrument and the sequencing run was started.