Name: simple and rapid method to obtain high-quality tumor dna from clinical-pathological specimens using touch imprint cytology
Uploaded: 2018-03-21T13:00:00
Description: Watch this Scientific Journal Video about Simple and Rapid Method to Obtain High-quality Tumor DNA from Clinical-pathological Specimens Using Touch Imprint Cytology at JoVE.com

We thank all the medical and ancillary staff of the hospital and the patients for consenting to participate. We thank Gabrielle White Wolf, PhD, from Edanz Group (www.edanzediting.com/ac) for editing a draft of this report. This study was supported by a Grant-in-Aid for Genome Research Project from the Yamanashi Prefecture (Y.H. and M.O.) and a grant from The YASUDA Medical Foundation (Y.H.).

1. TIC Preparation for Quick Microscopic Assessment Using Normal Glass Slides
<ol>
	<li>Perform the TIC preparation as soon as possible after clinical pathological tissue materials are available. If TIC specimens cannot immediately be prepared, keep the tissue materials covered with saline moistened sterile-gauze and store in the refrigerator to prevent drying of tissues.</li>
	<li>Prepare 5 mm3 tissue material such as solid tumors (e.g., liver, lung, and breast tissues) clinically obtained by surge...

<ol>
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In this study, we presented an alternative method for obtaining tumor DNA from clinical pathological specimens using TIC. TIC preparation is very simple and needs less time compared with FFPE methods, without the requirement for special instruments10. All procedures from the TIC preparation to DNA extraction can be completed within two days (Figure 1). This method thus shortens the turnaround time for performing genetic testing. Notably, this provides a significant ad...

Next generation sequencing technology has provided researchers significant advancements in analyzing genome information in genetic variations, Mendelian disease, hereditary predisposition, and cancer 1,2,3. The Cancer Genome Atlas (TCGA) and International Cancer Genome Consortium (ICGC) have pursued the identification of genetic alterations in several types of common cancers4. Hundreds of essential cancer driver genes have been successfully identified, and some of these molecules are being targeted for drug development1,5,6.
In the clinical setting, FFPE specimens are commonly used for pathological diagnosis and molecular testing for various diseases, including cancer. However, during the fixation process with formalin, DNA-protein or DNA-DNA cross-linking occurs and DNA fragmentation is induced. Thus, FFPE DNA samples are not always suitable for genetic analysis because of low quality and quantity of DNA7,8,9. Additionally, it takes several days to prepare FFPE specimens, and technical skill is necessary to accurately prepare the sections. Therefore, it is desirable to develop a simple and rapid method for obtaining high-quality intact DNA.
Cytology is an alternative method for pathological diagnosis. Cytological sample preparation is a simpler, less expensive, and more rapid approach compared with FFPE preparation10. The TIC technique has been performed on sentinel lymph nodes and marginal tissues from breast cancer patients for intraoperative rapid diagnosis for some years11,12. However, there are few reports that have examined whether high-quality genomic DNA can be extracted from TIC specimens and used for subsequent genetic analysis. Cytological specimens are commonly stained with Papanicolaou (Pap) or Giemsa staining, and we previously reported that the amount and quality of DNA extracted from TIC specimens (especially Giemsa-stained samples) are superior to samples obtained from FFPE tissues13. Compared with Pap staining, Giemsa staining has an advantage in requiring less staining procedures. In Pap staining, after the samples have been fixed and stained, they must be mounted with mounting medium (e.g., Malinol) for distinguishing sample contents, such as tumor cells, normal cells, and inflammatory cells under a microscope. If the Pap specimen is prepared without the mounting step, it is almost impossible to observe the cells under a microscope because the specimen is dried. In comparison, Giemsa staining can be observed in the dried state, therefore, the mounting step is not necessary for quick cellular evaluation. For microdissection, Giemsa staining is more suitable because it requires dry specimens.
In this report, we introduce a simple and rapid method for preparing TIC specimens with Giemsa staining and demonstrate that TIC is a better source for DNA compared with FFPE specimens.

<table border="0" cellpadding="0" cellspacing="0" width="796">
	<tbody>
		<tr height="41">
			<td height="41" style="height:41px;width:283px;">FINE FROST white20 micro slide glass</td>
			<td style="width:191px;">Matsunami Glass ind, Ltd</td>
			<td style="width:143px;">SFF-011</td>
			<td style="width:180px;"></td>
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			<td height="41" style="height:41px;">Arcturus PEN Membrane Glass Slides&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>LCM0522</td>
			<td></td>
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			<td height="41" style="height:41px;">Cyto Quick A solution</td>
			<td>Muto Pure Chemicals</td>
			<td>20571</td>
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			<td>Muto Pure Chemicals</td>
			<td>20581</td>
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			<td height="41" style="height:41px;">May-Grunwald Solution</td>
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			<td>15053</td>
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			<td height="41" style="height:41px;">Giemsa solution</td>
			<td>Muto Pure Chemicals</td>
			<td>15002</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;">QIAamp DNA FFPE tissue kit</td>
			<td>Qiagen</td>
			<td>56404</td>
			<td></td>
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			<td height="41" style="height:41px;">TaqMan Fast Advanced Master Mix</td>
			<td>Thermo Fisher Scientific</td>
			<td>4444557</td>
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			<td height="41" style="height:41px;">TaqMan RNase P Detection Reagents Kit&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>4316831</td>
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			<td height="41" style="height:41px;">TaqMan Assay from FFPE DNA QC Assay v2</td>
			<td>Thermo Fisher Scientific</td>
			<td>4324034</td>
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			<td height="41" style="height:41px;">MicroAmp Fast Optical 96-Well Reaction Plate&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>4346907</td>
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			<td height="41" style="height:41px;width:283px;">MicroAmp optical Adhesive Film</td>
			<td style="width:191px;">Thermo Fisher Scientific</td>
			<td style="width:143px;">4311971</td>
			<td style="width:180px;"></td>
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			<td height="41" style="height:41px;width:283px;">MicroMixer E36</td>
			<td style="width:191px;">TITEC</td>
			<td style="width:143px;">0027765-000</td>
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			<td height="41" style="height:41px;width:283px;">ViiA 7 Real-Time PCR System</td>
			<td style="width:191px;">Thermo Fisher Scientific</td>
			<td style="width:143px;">VIIA7-03</td>
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			<td height="41" style="height:41px;width:283px;">Himac CF16RXII</td>
			<td style="width:191px;">Hitachi-koki</td>
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			<td height="41" style="height:41px;width:283px;">Ion Library TaqMan Quantitation Kit</td>
			<td style="width:191px;">Thermo Fisher Scientific</td>
			<td style="width:143px;">4468802</td>
			<td style="width:180px;"></td>
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			<td height="41" style="height:41px;width:283px;">Ion AmpliSeq Cancer Hotspot Panel v2</td>
			<td style="width:191px;">Thermo Fisher Scientific</td>
			<td style="width:143px;">4475346</td>
			<td style="width:180px;"></td>
		</tr>
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			<td height="41" style="height:41px;width:283px;">Ion AmpliSeq Library Kit 2.0</td>
			<td style="width:191px;">Thermo Fisher Scientific</td>
			<td style="width:143px;">4480442</td>
			<td style="width:180px;"></td>
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			<td height="41" style="height:41px;width:283px;">Ion Xpress Barcode Adapters 1-16 Kit</td>
			<td style="width:191px;">Thermo Fisher Scientific</td>
			<td>4471250</td>
			<td style="width:180px;"></td>
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			<td height="41" style="height:41px;width:283px;">Ion PGM Hi-Q View Sequencing Kit (200 base)</td>
			<td style="width:191px;">Thermo Fisher Scientific</td>
			<td style="width:143px;">A30044</td>
			<td style="width:180px;"></td>
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			<td height="41" style="height:41px;width:283px;">Ion Chef System</td>
			<td style="width:191px;">Thermo Fisher Scientific</td>
			<td>4484177</td>
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			<td height="41" style="height:41px;width:283px;">Veriti 96-well Thermal Cycler</td>
			<td style="width:191px;">Thermo Fisher Scientific</td>
			<td>Veriti200</td>
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			<td height="41" style="height:41px;width:283px;">Ion 318 Chip Kit v2 BC</td>
			<td style="width:191px;">Thermo Fisher Scientific</td>
			<td>4488150</td>
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			<td height="41" style="height:41px;width:283px;">Ion PGM System</td>
			<td style="width:191px;">Thermo Fisher Scientific</td>
			<td>PGM11-001</td>
			<td style="width:180px;"></td>
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			<td height="41" style="height:41px;width:283px;">Ion PGM Wash 2 Bottle kit</td>
			<td style="width:191px;">Thermo Fisher Scientific</td>
			<td>A25591</td>
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			<td height="41" style="height:41px;width:283px;">Agencourt&#8482; AMPure&#8482; XP Kit</td>
			<td style="width:191px;">Beckman Coulter</td>
			<td style="width:143px;">A63881</td>
			<td style="width:180px;"></td>
		</tr>
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			<td height="41" style="height:41px;width:283px;">16-position Magnetic Stand</td>
			<td style="width:191px;">Thermo Fisher Scientific</td>
			<td style="width:143px;">4457858</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:283px;">Nonstick, RNase-free Microfuge Tubes, 1.5 mL (Low binding tube)</td>
			<td style="width:191px;">Thermo Fisher Scientific</td>
			<td style="width:143px;">AM12450</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:283px;">Nuclease-free water</td>
			<td style="width:191px;">Thermo Fisher Scientific</td>
			<td style="width:143px;">AM9938</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:283px;">MicroAmp&#8482; Optical 96-well Reaction Plates</td>
			<td style="width:191px;">Thermo Fisher Scientific</td>
			<td style="width:143px;">4306737</td>
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			<td height="41" style="height:41px;width:283px;">MicroAmp&#8482; Clear Adhesive Film</td>
			<td style="width:191px;">Thermo Fisher Scientific</td>
			<td style="width:143px;">4306311</td>
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			<td height="41" style="height:41px;width:283px;">Agencourt&#8482; AMPure&#8482; XP Kit</td>
			<td style="width:191px;">Beckman Coulter</td>
			<td style="width:143px;">A63881</td>
			<td style="width:180px;"></td>
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			<td height="41" style="height:41px;width:283px;">Ethanol(99.5)</td>
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			<td height="41" style="height:41px;width:283px;">Sodium hydroxide (10M)</td>
			<td style="width:191px;">Sigma</td>
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			<td height="41" style="height:41px;width:283px;">DTU-Neo</td>
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			<td height="41" style="height:41px;width:283px;">E-36&#160;</td>
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			<td>0027765-000</td>
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			<td height="41" style="height:41px;width:283px;">ECLIPSE Ci-L</td>
			<td style="width:191px;">Nikon</td>
			<td style="width:143px;">704354</td>
			<td style="width:180px;"></td>
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			<td height="41" style="height:41px;">Pipet-Lite LTS Pipette L-2XLS+</td>
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			<td height="41" style="height:41px;">Pipet-Lite LTS Pipette L-10XLS+</td>
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		</tr>
		<tr height="41">
			<td height="41" style="height:41px;">Pipet-Lite LTS Pipette L-20XLS+</td>
			<td>METTLER TOLEDO</td>
			<td>17014392</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;">Pipet-Lite LTS Pipette L-100XLS+</td>
			<td>METTLER TOLEDO</td>
			<td>17014384</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;">Pipet-Lite LTS Pipette L-200XLS+</td>
			<td>METTLER TOLEDO</td>
			<td>17014391</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;">Pipet-Lite LTS Pipette L-1000XLS+</td>
			<td>METTLER TOLEDO</td>
			<td>17014382</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;">petit-change</td>
			<td>WAKEN</td>
			<td>MODEL8864</td>
			<td style="width:180px;">Mini centrifuge</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;">petit-incubator</td>
			<td>WAKEN</td>
			<td>WKN-2290</td>
			<td style="width:180px;">Air incubator</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:283px;">SensiCare Powder-free Nitrile Exam Gloves</td>
			<td style="width:191px;">MEDLINE</td>
			<td>SEM486802</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:283px;">Sterile gauze</td>
			<td style="width:191px;">Osaki</td>
			<td style="width:143px;">11138</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:283px;">Refrigerator MediCool</td>
			<td style="width:191px;">SANYO</td>
			<td style="width:143px;">MPR-312DCN-PJ</td>
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		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:283px;">FEATHER TRIMMING BLAD</td>
			<td style="width:191px;">FEATHER</td>
			<td style="width:143px;">No.130</td>
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		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:283px;">FEATHER TRIMMING BLAD</td>
			<td style="width:191px;">FEATHER</td>
			<td style="width:143px;">No.260</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:283px;">FEATHER&#160; S</td>
			<td style="width:191px;">FEATHER</td>
			<td style="width:143px;">FA-10</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:283px;">Vortex Genius 3</td>
			<td style="width:191px;">IKA</td>
			<td>41-0458&#160;</td>
			<td style="width:180px;">Vortex mixer</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:283px;">Pincette</td>
			<td style="width:191px;">NATSUME</td>
			<td style="width:143px;">A-5</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:283px;">1.5 mL microtube</td>
			<td style="width:191px;">BIOBIK</td>
			<td style="width:143px;">RC-0150</td>
			<td style="width:180px;"></td>
		</tr>
	</tbody>
</table>

simple and rapid method to obtain high-quality tumor dna from clinical-pathological specimens using touch imprint cytology

It is critical to determine the mutational status in cancer before administration and treatment of specific molecular targeted drugs for cancer patients. In the clinical setting, formalin-fixed paraffin-embedded (FFPE) tissues are widely used for genetic testing. However, FFPE DNA is generally damaged and fragmented during the fixation process with formalin. Therefore, FFPE DNA is sometimes not adequate for genetic testing because of low quality and quantity of DNA. Here we present a method of touch imprint cytology (TIC) to obtain genomic DNA from cancer cells, which can be observed under a microscope. Cell morphology and cancer cell numbers can be evaluated using TIC specimens. Furthermore, the extraction of genomic DNA from TIC samples can be completed within two days. The total amount and quality of TIC DNA obtained using this method was higher than that of FFPE DNA. This rapid and simple method allows researchers to obtain high-quality DNA for genetic testing (e.g., next generation sequencing analysis, digital PCR, and quantitative real time PCR) and to shorten the turnaround time for reporting results.

Figure 1 shows the entire process from preparing TIC specimens to DNA extraction. Notably, the procedure takes only two days to obtain genomic DNA from TIC samples. We evaluated any effects of tumor storage before the slide processing. We found that tumor cells were attached onto the glass slide when tissue specimens were immediately touched onto the slide, and when tissues were kept in saline moistened sterile-gauze for 1 h (Figure 2</st...

Watch this Scientific Journal Video about Simple and Rapid Method to Obtain High-quality Tumor DNA from Clinical-pathological Specimens Using Touch Imprint Cytology at JoVE.com

Simple and Rapid Method to Obtain High-quality Tumor DNA from Clinical-pathological Specimens Using Touch Imprint Cytology

Obtaining high-quality genomic DNA from tumor tissues is an essential first step for analyzing genetic alterations using next generation sequencing. In this article, we present a simple and rapid method to enrich tumor cells and obtain intact DNA from touch imprint cytology specimens.

It is critical to determine the mutational status in cancer before administration and treatment of specific molecular targeted drugs for cancer patients. ...

The overall goal of this protocol is to obtain high-quality tumor DNA from clinical samples. This method can help answer key questions in basic and clinical science such as cancer research, diagnosis, and treatment decisions. The main advantage of this technique is that we can obtain tumor DNA more simply and quickly than with previous methods.To begin the protocol, touch the tumor surface of a previously prepared and resected tissue onto a normal glass slide several times with gloved hands. Visually confirm that the touched area is covering more than 80%of the glass slide. Lightly press the normal glass slide against a Polyethylene Naphthalate or PEN membrane slide and rub it gently two to three times with gloved hands.Visually confirm that the cells are transferred from the normal glass slide to the PEN membrane slide. Air dry both the glass and PEN membrane slides for five minutes at room temperature. After drying, stain the normal glass slide for direct cytological examination.Dip the glass slides into fixative solution for five seconds and then stain with Giemsa staining solution for 15 seconds. Assess and screen the tumor contents and cellularity on the normal glass slide entirely with a microscope for quick assessment. Evaluate tumor cells based on several criteria.If the sample shows tumor cellularity over 60%by quick microscopic assessment from the previous section, cut the tumor-touched film of the PEN membrane slides for DNA extraction with a knife and gloved hands. Transfer the cut film to a sterile microcentrifuge tube with a pin set and gloved hands. Store the film-containing microcentrifuge tube at four degrees Celsius until DNA extraction.Open the software and plan the run condition and set the run parameter within the software. Click the plan tab and select template, then select the appropriate run method. Select the application and technique type and click next.Select the instrument, sample preparation kit, library kit type, template kit, sequencing kit, base calibration mode, chip type, control sequence, and barcode set and click next. Select plugins and click next. Select project and click next.Then select default reference and BED files of the targeted region. Type the sample name, select the barcode, and click plan run. Next, perform template preparation and chip loading in an automated instrument according to the manufacturer's instructions.Thaw the reagent cartridge at room temperature for 45 minutes before use. Dilute the undiluted library with nuclease-free water according to the library concentration calculated in step 6.8 and make 20 picomolar libraries. Prepare a pooled library for sequencing and store it on ice.Add 25 microliters of the pooled library with a 100 microliter pipette to the bottom of the sample tube. Turn on the power and open the cover of the automated instrument. Place the sequencing chip, chip adapter, enrichment cartridge, tip cartridge, PCR plate, PCR frame seal, recovery tube, solution cartridge, and reagent cartridge to the appropriate position of the automated instrument.Touch set up run and step by step on the screen. Close the cover and touch start check on the screen. After the deck scan process, touch next.Check the display contents, kit type, chip ID, sample ID, chip type, plans. Set the time and touch OK.After finishing the chip loading, touch next and open the cover. Unload the sequencing chip from the chip adapter with gloved hands.Place the chip into the chip container, wrap it with parafilm, and store the chip at four degrees Celsius until the sequencing reaction. Then remove the enrichment cartridge, PCR plate, PCR frame seal, recovery tube, solution cartridge, and reagent cartridge from the appropriate position of the automated instrument with gloved hands. Transfer an empty tip cartridge to the waste tip position of the automated instrument with gloved hands.Touch next and close the cover. Then touch start and clean the automated instrument with ultraviolet rays for four minutes. Dissolve a sodium chlorite tablet in 1, 000 milliliters of ultra-pure water and filter the solution with a 0.22 micron filter.Once the sequencing instrument has warmed up, touch clean and next on the screen of the sequencing instrument. Clean the sequencing instrument with 250 milliliters of filter-sterilized sodium chlorite solution, then 250 milliliters of ultra-pure water. Then touch initialize and select the appropriate sequencing kit on the screen.Install a gray shipper with gloved hands. Initialize the sequencing instrument with the wash solution, the pH adjustment solution, and the pH standard solution. Next, add 20 microliters of DATP, DGTP, DCTP, and DTTP nucleotides in 50 milliliter tubes with a 100 microliter pipette.Install a gray shipper at the appropriate location with gloved hands. Load the 50 milliliter tube and screw it onto the sequencing instrument. Select next on the screen to start the initialization step.After completing the initialization step, touch run on the screen and select the appropriate library preparation instrument. Scan the two-dimensional barcode of the chip. Insert the sequencing chip in the appropriate position.Close the chip clamp and instrument door and touch chip check, next, and OK on the screen to start the sequencing run. After the sequencing reaction, transfer the data and perform data analyzing pipeline on the sequencing server. The absolute DNA quantities and the RQ value, an indicator of the degradation level of genomic DNA, show that a higher DNA yield is achieved using the TIC specimens compared with the FFPE specimens.In addition, the RQ values from the TIC DNA were significantly higher compared with that of the FFPE DNA across different tumor types. APC Q1367*was identified in both FFPE and TIC DNA extracted from site one and APC S1356*KRAS G12D, and TP53 M237i were detected in both FFPE and TIC DNA extracted from site two, three, and four suggesting that the identical somatic mutations were identified in paired FFPE and TIC DNA samples prepared from the same tumor site. Notably, the same somatic mutations were detected between primary colorectal cancer and two metastatic liver cancer samples suggesting that the tumor clones from site two metastasized to liver.After its development, this technique paved the way for researchers in the field of molecular and medical research to identify genetic cancers.

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