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Tumor Discovery Haplotype and LD of BRCA genes in GBM

groups, they were of harmonized age and sexual category neighboring sequences around the coding arrangement
with malignancy findings. Individuals who did not meet of the investigated genes. The amplification procedure
the inclusion criteria were excluded from the study. The involved a 20-µL scale using the Ion AmpliSeq Library Kit
justification criteria for the selection of GBM patients (Thermo Fisher Scientific, USA). The assay was performed
were those who had not received any treatments (adjuvant using 4 µL of 5× Ion AmpliSeq HiFi Master Mix, 10 µL of
therapy, including chemo- or radiotherapy), patients 2× Ion AmpliSeq Primer Pool (every primer in a separate
who were amenable to follow-up, and patients who had well for each sample), 10 ng of genomic DNA/sample
undergone surgery, and whose survival duration was (2 µL of 5 ng/µL from the test store), and lastly 4 µL of
above 1 month. nuclease-free water. The applied thermal settings for the
reaction were as follows: 99°C for 2 min, one round for
2.2. Medication strategy
triggering the enzyme activating by (99°C for 2 s, and 60°C
All patients underwent MRI for analysis. Then, GBM for 4 min) for 16 cycles using a thermal cycler (SureCycler
patients were cured with clinical resection, near-total 8800, Agilent, USA). After enlargement, the primers were
resection, or excisional biopsy. Thereafter, all patients were processed using the Fragmentase Universal Primer Assay
cured with regular therapy, which included radiotherapy (FuPa) Mixture and thermal conditions: 50°C for 10 min,
(total dose of 60 Gy—administered in 30 parts over 42 days) 55°C for 10 min, and 60°C for 10 min, then ligated with two
with concomitant TMZ chemotherapy (100 mg/day for adaptors (IonCode™ Barcode Adapters, Life Technologies
45 days) followed by 6 cycles of TMZ treatment at a dose of Ltd., USA) and the barcodes with the Ion Xpress Barcode
150 mg/m of body surface area for days 1 – 5. All patients Connectors-16 kit (Life Technologies Ltd., USA). Then,
2
were followed up and assessed clinically and radiologically magnification was performed using the thermal conditions:
by MRI. 22°C for 30 min, 68°C for 5 min, and 72°C for 5 min, as
The tumor was accessed based on the radiological indicated in the Ion AmpliSeq Library Kit user guide.
RANO response standards (2010) 22,23 as follows: Sanitization of the formulated libraries was carried out and
Complete response (CR), defined as the disappearance measured by the Ion Library TaqMan Measurement Kit
of all identified brain injuries; partial response (PR), (Life Technologies Ltd., USA) by quantitative PCR using
defined as a 50% or greater reduction of the assessable the real-time PCR technique (Stratagene Max3005P QPCR
brain lesion or an objective progression of the system, Agilent Biotechnology, USA). The QPCR was
assessable brain lesion; stable disease (SD), defined as performed with the following thermal conditions: 50°C
an unchanged brain lesion (<50% reduction or <25% for 2 min, 95°C for 2 min, 40 rounds at 95°C for 15 s, and
proliferation in the size of the assessable lesions); and 60°C for 1 min. The amplified libraries were additionally
progressive disease (PD), defined as ≥25% increase in subjected to template formulation.
the size of some or all brain injuries and/or the presence 2.5. Template formulation
of new brain lesions.
The cleaned and measured libraries were assembled
2.3. DNA extraction on molar comparable portions to achieve at least ×500
The QIAamp DNA mini blood kit (Cat. No. #51104, coverage for every sample. The collective collections
Qiagen, Germany) was used to extract DNA from were clonally magnified using the Ion PGM Hi-Q view
ethylenediaminetetraacetic acid blood samples, according OT2 kit (Life machineries, USA) on the Ion OneTouch
to the manufacturer’s guidelines. The kit procedure was 2 apparatus (Life Technologies Ltd., USA) according
similar to the one established for the extraction of DNA to the manufacturer’s guidelines. Then, the template
using a spin column. For the extracted DNA, both purity ion sphere particles ISP were improved using Ion PGM
and concentration were assessed using a nano-drop Enhancement Beads (Life Technologies Ltd., USA) on the
spectrophotometer (Quawell, Q-500, Scribner, USA) Ion OneTouch ES system (Life Technologies Ltd., USA)
before further examination and then stored at −80°C until based on the manufacturer’s guidelines. Positive ISP
further estimates. excellence was analyzed on the Qubit 2.0 Fluorometer
(Life Technologies Ltd., USA) and then sequencing was
2.4. DNA library formulation and sanitization performed.
Amplification of the target regions for BRCA1 and 2.6. Sequencing with Ion Torrent PGM Proposal
BRCA2 was carried out using the Ion AmpliSeq BRCA1
and BRCA2 Panel (Life Technologies Ltd., USA). This Before starting sequencing using Ion PGM, calibration
panel comprises three primer pools (167 amplicons) and pH amendments were carried out according to the
covering the entire coding region with 10 – 20 bp intronic user guide of the Ion PGM Hi-Q View Sequencing kit (Life

Volume 3 Issue 1 (2024) 3 https://doi.org/10.36922/td.1480