This work is supported by Geneplus&#8211;Beijing Institute.

Tumor specimens and blood samples were obtained according to a protocol approved by the Ethics Committee of Peking University People&#39;s Hospital. Written informed consent was obtained from the patients to use their samples. Participants were screened according to the following criteria: female, advanced Non-Small Cell Lung Cancer, EGFR p.L858R mutation indicated by previous Sanger Sequencing, disease progression following two session of EGFR targeted therapy with Erlotinib, and ER-Seq which was applied to ctDNA to ana...

<ol>
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V., Newman, A. M., Alizadeh, A. A., Diehn, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potential+clinical+utility+of+ultrasensitive+circulating+tumor+DNA+detection+with+CAPP-Seq.">Potential clinical utility of ultrasensitive circulating tumor DNA detection with CAPP-Seq.</a> Expert Rev. Mol. Diagn. 15, 715-719 (2015).</li><li>Diaz, L. A., Bardelli, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liquid+biopsies:+genotyping+circulating+tumor+DNA.">Liquid biopsies: genotyping circulating tumor DNA.</a> J. Clin. 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M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrated+digital+error+suppression+for+improved+detection+of+circulating+tumor+DNA.">Integrated digital error suppression for improved detection of circulating tumor DNA.</a> Nat Biotechnol. 34 (5), 547-555 (2016).</li><li>Alix-Panabie&#768;res, C., Schwarzenbach, H., Pantel, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+tumor+cells+and+circulating+tumor+DNA.">Circulating tumor cells and circulating tumor DNA.</a> Annu. Rev. Med. 63, 199-215 (2012).</li><li>Dawson, S. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+circulating+tumor+DNA+to+monitor+metastatic+breast+cancer.">Analysis of circulating tumor DNA to monitor metastatic breast cancer.</a> N. Engl. J. Med. 368, 1199-1209 (2013).</li><li>Li, H., Durbin, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+and+accurate+short-read+alignment+with+Burrows-+Wheeler+transform.">Fast and accurate short-read alignment with Burrows- Wheeler transform.</a> Bioinformatics. 25, 1754-1760 (2009).</li><li>Youn, A., Simon, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identifying+cancer+driver+genes+in+tumor+genome+sequencing+studies.">Identifying cancer driver genes in tumor genome sequencing studies.</a> Bioinformatics. 27, 175-181 (2011).</li><li>Forshew, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noninvasive+identification+and+monitoring+of+cancer+mutations+by+targeted+deep+sequencing+of+plasma+DNA.">Noninvasive identification and monitoring of cancer mutations by targeted deep sequencing of plasma DNA.</a> Sci. Transl. Med. 4, (2012).</li><li>Kobayashi, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EGFR+mutation+and+resistance+of+non-small-cell+lung+cancer+to+gefitinib.">EGFR mutation and resistance of non-small-cell lung cancer to gefitinib.</a> N. Engl. J. Med. 352, 786-792 (2005).</li><li>Kuang, Y., Rogers, A., Yeap, Y., Wang, L., Makrigiorgos, M., Vetrand, K., Thiede, S., Distel, R. J., Ja&#776;nne, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noninvasive+detection+of+EGFR+T790M+in+gefitinib+or+erlotinib+resistant+non-small+cell+lung+cancer.">Noninvasive detection of EGFR T790M in gefitinib or erlotinib resistant non-small cell lung cancer.</a> Clin. Cancer Res. 15, 2630-2636 (2009).</li><li>Taniguchi, K., Uchida, J., Nishino, K., Kumagai, T., Okuyama, T., Okami, J., Higashiyama, M., Kodama, K., Imamura, F., Kato, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+detection+of+EGFR+mutations+in+circulating+tumor+DNA+derived+from+lung+adenocarcinomas.">Quantitative detection of EGFR mutations in circulating tumor DNA derived from lung adenocarcinomas.</a> Clin. Cancer Res. 17, 7808-7815 (2011).</li><li>Leary, R. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+personalized+tumor+biomarkers+using+massively+parallel+sequencing.">Development of personalized tumor biomarkers using massively parallel sequencing.</a> Sci. Transl. Med. 2, (2010).</li><li>Forshew, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noninvasive+identification+and+monitoring+of+cancer+mutations+by+targeted+deep+sequencing+of+plasma+DNA.">Noninvasive identification and monitoring of cancer mutations by targeted deep sequencing of plasma DNA.</a> Sci. Transl. Med. 4, (2012).</li><li>Bratman, S. V., Newman, A. M., Alizadeh, A. A., Diehn, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potential+clinical+utility+of+ultrasensitive+circulating+tumor+DNA+detection+with+CAPP-Seq.">Potential clinical utility of ultrasensitive circulating tumor DNA detection with CAPP-Seq.</a> Expert Rev. Mol. Diagn. 15, 715-719 (2015).</li></ol>

The authors have nothing to disclose.

The existence of circulating tumor DNA (ctDNA) was discovered more than 30 years ago, however the application of ctDNA analyses is still not routine in clinical practice. Interest in the practical application of ctDNA methods has increased with the development of technologies for ctDNA detection and analysis. Tumor monitoring with ctDNA offers a minimally-invasive approach for the assessment of microscopic residual disease, response to therapy, and tumor molecular profiles under the background of tumor evolution and intr...

Next-generation sequencing (NGS), a powerful tool to investigate the mysteries of the genome, can provide a large quantity of information, which may reveal genetic alterations. The application of NGS analysis in the clinic has become more common, especially for personalized medicine. One of the greatest limitations of NGS, however, is a high error rate. Although it is deemed suitable for studying inherited mutations, the analysis of rare mutations is greatly limited1,2, especially when analyzing DNA obtained from a &#34;liquid biopsy&#34;.
Circulating tumor DNA (ctDNA) is cell-free DNA (cfDNA) in the blood that is shed from tumor cells. In most cases, the quantity of ctDNA is extremely low, which make its detection and analysis very challenging. However, ctDNA has many attractive features: its isolation is minimally invasive, it can be detected in the early stages of tumor growth, the ctDNA level reflects therapeutic efficiency, and ctDNA contains DNA mutations found in both primary and metastatic lesions3,4,5. Therefore, given the rapid development of the NGS technique and analysis, the application of ctDNA detection has become more attractive.
Different massively parallel sequencing approaches have been utilized for ctDNA detection but none of these approaches have been accepted for routine use in clinics due to their limitations: low sensitivity, lack of versatility, and a relatively high cost6,7,8. For example, duplex sequencing, based on a unique identifier tag (UID), repeatedly corrects errors in the consensus bidirectionally, rectifying most sequencing errors. However, the feasibility of this method is lost due to its high cost and low data utilization9,10. Similarly, CAPP-Seq and its improved iteration, CAPP-IDES11,12, have greater practicality in cfDNA detection, though the accuracy and universality of these methods need improvement.
To meet the current need for accurate ctDNA detection and analysis, we developed a new strategy, Enrich Rare Mutation Sequencing (ER-Seq). This approach combines the following: unique sequencing adapters to efficiently recover ctDNA molecules, with bidirectional error correction and the ability to distinguish a single mutation out of &#62; 1 &#215; 107 wild-type nucleotides; 1021 kb probes which enrich measurement of target regions that cover over 95% of the tumor-related mutations from 12 tumors, including lung cancer, colorectal cancer, gastric cancer, breast cancer, kidney cancer, pancreatic cancer, liver cancer, thyroid cancer, cervical cancer, esophageal cancer, and endometrial carcinoma (Table 1); and baseline database screening making it efficient and easy to precisely detect rare mutations in ctDNA.
To build a baseline database, find all the gene mutations by ER-Seq from a number of the same type of samples (~1000 at the beginning). These real mutations must be verified by several other reliable detection methods and analysis. Next, summarize the pattern of false mutations and cluster all the false mutations to build the initial baseline database. Continue adding false mutations found from subsequent sequencing experiments to this database. Therefore, this baseline database becomes a dynamic expanded database, which significantly improves sequencing accuracy.
To promote progress in tumor diagnosis and monitoring, we present ER-Seq, a low cost and feasible method for the acquisition of universal data. We present a case study which underwent ER-Seq analysis, demonstrating its accuracy for detecting rare mutations and feasibility for use in the clinic.

<table>
	<tbody>
		<tr>
			<td>QIAamp Circulating Nucleic Acid Kit</td>
			<td>Qiagen</td>
			<td>55114</td>
			<td>DNA Extraction from Peripheral Blood for cfDNA</td>
		</tr>
		<tr>
			<td>QIAamp DNA Blood Mini Kit</td>
			<td>Qiagen</td>
			<td>51105</td>
			<td>DNA Extraction from Peripheral Blood for gDNA</td>
		</tr>
		<tr>
			<td>Quant-iT dsDNA HS Assay Kit</td>
			<td>Invitrogen</td>
			<td>Q32854</td>
			<td>Measure cfDNA concentration</td>
		</tr>
		<tr>
			<td>Quant-iT dsDNA BR Assay Kit</td>
			<td>Invitrogen</td>
			<td>Q32853</td>
			<td>Measure library concentration</td>
		</tr>
		<tr>
			<td>Agilent DNA 1000 Reagents</td>
			<td>Agilent</td>
			<td>5067-1504</td>
			<td>Measure cfDNA and library fragments</td>
		</tr>
		<tr>
			<td>The NEBNext UltraII DNA Library Prep Kit for Illumina</td>
			<td>NEB</td>
			<td>E7645L</td>
			<td>Library Preparation</td>
		</tr>
		<tr>
			<td>Agencourt SPRIselect Reagent</td>
			<td>Beckman</td>
			<td>B23317</td>
			<td>DNA fragment screening and purification</td>
		</tr>
		<tr>
			<td>Tris-HCl (10 mM, pH 8.0)-100ML</td>
			<td>Sigma</td>
			<td>93283</td>
			<td>Dissolution</td>
		</tr>
		<tr>
			<td>xGen Lockdown Probes</td>
			<td>IDT</td>
			<td>&#8212;&#8212;</td>
			<td>xGen Custom Probe</td>
		</tr>
		<tr>
			<td>Human Cot-1 DNA</td>
			<td>Life</td>
			<td>15279-011</td>
			<td>Targeted DNA capture</td>
		</tr>
		<tr>
			<td>Dynabeads M-270 Streptavidin</td>
			<td>Life</td>
			<td>65305</td>
			<td>Targeted DNA capture</td>
		</tr>
		<tr>
			<td>xGen Lockdown Reagents</td>
			<td>IDT</td>
			<td>1072281</td>
			<td>Targeted DNA capture</td>
		</tr>
		<tr>
			<td>KAPA HiFi HotStart ReadyMix</td>
			<td>KAPA</td>
			<td>KK2602</td>
			<td>post-capture PCR enrichment libraries</td>
		</tr>
		<tr>
			<td>KAPA Library Quantification Kit</td>
			<td>KAPA</td>
			<td>KK4602</td>
			<td>Measure library concentration</td>
		</tr>
		<tr>
			<td>NextSeq 500 High Output Kit v2((150 cycles)</td>
			<td>illumina</td>
			<td>FC-404-2002</td>
			<td>Sequence</td>
		</tr>
		<tr>
			<td>Centrifuge5810</td>
			<td>eppendorf</td>
			<td>5810</td>
			<td></td>
		</tr>
		<tr>
			<td>Nanodrop8000</td>
			<td>Thermo Scientific</td>
			<td>8000</td>
			<td>Measure gDNA concentration</td>
		</tr>
		<tr>
			<td>Qubit 2.0</td>
			<td>Invitrogen</td>
			<td></td>
			<td>Quantify</td>
		</tr>
		<tr>
			<td>Agilent 2100 Bioanalyzer</td>
			<td>Agilent</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ThermoMixer C</td>
			<td>eppendorf</td>
			<td></td>
			<td>Incubation</td>
		</tr>
		<tr>
			<td>16-tube DynaMagTM-2 Magnet</td>
			<td>Life</td>
			<td>12321D</td>
			<td></td>
		</tr>
		<tr>
			<td>Concentrator plus</td>
			<td>eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PCR</td>
			<td>AB</td>
			<td>simplyamp</td>
			<td></td>
		</tr>
		<tr>
			<td>QPCR</td>
			<td>AB</td>
			<td>7500Dx</td>
			<td></td>
		</tr>
		<tr>
			<td>NextSeq 500</td>
			<td>illumina</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

detection of rare mutations in ctdna using next generation sequencing

The analysis of circulating tumor DNA (ctDNA) using next-generation sequencing (NGS) has become a valuable tool for the development of clinical oncology. However, the application of this method is challenging due to its low sensitivity in analyzing the trace amount of ctDNA in the blood. Furthermore, the method may generate false positive and negative results from this sequencing and subsequent analysis. To improve the feasibility and reliability of ctDNA detection in the clinic, here we present a technique which enriches rare mutations for sequencing, Enrich Rare Mutation Sequencing (ER-Seq). ER-Seq can distinguish a single mutation out of 1 x 107 wild-type nucleotides, which makes it a promising tool to detect extremely low frequency genetic alterations and thus will be very useful in studying disease heterogenicity. By virtue of the unique sequencing adapter&#39;s ligation, this method enables an efficient recovery of ctDNA molecules, while at the same time correcting for errors bidirectionally (sense and antisense). Our selection of 1021 kb probes enriches the measurement of target regions that cover over 95% of the tumor-related driver mutations in 12 tumors. This cost-effective and universal method enables a uniquely successful accumulation of genetic data. After efficiently filtering out background error, ER-seq can precisely detect rare mutations. Using a case study, we present a detailed protocol demonstrating probe design, library construction, and target DNA capture methodologies, while also including the data analysis workflow. The process to carry out this method typically takes 1-2 days.

The 1021 kb probes enriched target regions used in ER-Seq are shown in Table 1, which covers over 95% of the gene mutations in 12 common tumors. The wide range of these probes makes this process applicable to a majority of cancer patients. Additionally, our unique sequencing adapters and baseline database screening make it possible to detect rare mutations precisely.
Due to the different propert...

Watch this Scientific Journal Video about Detection of Rare Mutations in CtDNA Using Next Generation Sequencing at JoVE.com

Detection of Rare Mutations in CtDNA Using Next Generation Sequencing

This manuscript describes a technique for detecting mutations of low frequency in ctDNA, ER-Seq. This method is differentiated by its unique use of two-directional error correction, a special background filter, and efficient molecular acquirement.

The analysis of circulating tumor DNA (ctDNA) using next-generation sequencing (NGS) has become a valuable tool for the development of clinical oncology. ...

The overall goal of this technique is to detect mutations of low frequency in circulating tumor or ctDNA to provide clinical physicians a powerful tool to diagnose tumors and monitor tumor dynamics in response to therapy. The method of testing ctDNA can help answer key questions about what kinds of mutations a patient carries such as single nuclear-type variation, insertion deletion, copy number variation, and structural variation. The main advantage of this technqiue is high sensitivity and specificity which precise detection of low frequency mutations in ctDNA.Demonstrating the procedures will be Yuliang Yang, a technician from my laboratory. To begin, draw 10 milliliters of peripheral blood in a collection tube and gently invert the tube up and down six to eight times to mix the contents. Store blood samples in the collection tubes at six to 37 degrees Celsius for up to 72 hours.Centrifuge the collection tube at 1, 600 times g at room temperature for 10 minutes. Then use a disposal pipette to transfer the supernatant or plasma from the tube to four clean two milliliter appropriately labeled centrifuge tubes without disturbing the pellet. Use a clean disposal pipette to transfer one milliliter of the cells to a two milliliter appropriately labeled centrifuge tube.Store the cells at minus 20 degrees Celsius or colder before genomic or gDNA isolation. Next, centrifuge the plasma at 1, 600 times g at four degrees Celsius for 10 minutes. Then transfer the plasma to clean centrifuge tubes properly labeled as plasma leaving approximately 0.1 milliliter of residual volume at the bottom to avoid contamination.The buffy coat should be avoided from the plasma during the separation process. Otherwise, large DNA fragments extracted from the cells might dilute the ctDNA and affect the sensitivity of the test. Using a commercially available kit, isolate cfDNA from three milliliters of the collected plasma following the manufacturer's instructions.Then also with a kit, isolate gDNA from 200 microliters of white blood cells following the manufacturer's instructions. To fragment the gDNA control sample by sonication, prepare one microgram of gDNA sample in 100 microliters of Tris-EDTA buffer in a clean sonication tube. Set the sonication program to 30 seconds on and 30 seconds off for 12 cycles for a total of 12 minutes.After confirming the product contains 200 to 250 base pair fragments, transfer all fragmentation products to a new 1.5 milliliter tube containing 150 microliters of magnetic beads and incubate the sample for five minutes to select the correct fragments. Next, place the tube on a magnetic rack for 30 seconds and remove the supernatant. Then while keeping the tube on the rack, add 200 microliters of freshly prepared 80%ethanol to wash the beads.Incubate the beads for 30 seconds before removing the supernatant and repeat the wash. Keep the tube on the rack with the lid open to air dry the beads. Then elute the DNA fragments by adding 32 microliters of 10 millimolar Tris-HCL to the beads.To carry out the end prep reaction following the manufacturer's instructions, to a sterile nuclease-free tube, add seven microliters of reaction buffer, three microliters of the enzyme mix, 30 nanograms of cfDNA, and doubly distilled water for a total volume of 60 microliters. In a separate tube, add the same components but with one microgram of gDNA instead of cfDNA. Incubate the mixture in a thermal cycler at 20 degrees Celsius for 30 minutes followed by 65 degrees Celsius for 30 minutes without the lid heated.Immediately following the incubation, add 30 microliters of the ligation master mix, one microliter of the ligation enhancer, and four microliters of the unique sequencing adapter to the tube. Incubate the reaction in the thermal cycler at 20 degrees Celsius for 15 minutes without the lid heated. Vortex to resuspend the magnetic beads and leave the beads at room temperature for at least 30 minutes.Then add 87 microliters of the resuspended magnetic beads to the ligation reaction. Mix well by pipetting up and down and then incubate the sample at room temperature for five minutes. After washing and air drying the beads, elute the DNA target from the beads by adding 20 microliters of 10 millimolar Tris-HCL.Add 20 microliters of adapter ligated DNA fragments, five microliters of index primer i7, 25 microliters of library amplification master mix, and doubly distilled water up to a total volume of 50 microliters. PCR amplify the adapter ligated cfDNA or gDNA. Then add 45 microliters of suspended magnetic beads to the PCR-enriched DNA.Mix the sample by pipetting up and down and incubate it for five minutes. After removing the supernatant and washing the beads, use 30 microliters of 10 millimolar Tris-HCL to elute the DNA fragments with adapters. To carry out targeted DNA capture, in a sterile tube, block the sample by adding 1.5 micrograms of pooling libraries, eight microliters each of P5 block 100P and P7 block 100P, and five microliters of one microgram per microliter cock one DNA.Dry the contents of the tube using a vacuum concentrator set at 60 degrees Celsius. Hybridize the DNA capture probes with the library by adding 8.5 microliters of 2X hybridization buffer, 2.7 microliters of hybridization enhancer, and 1.8 microliters of nuclease-free doubly distilled water. Mix by pipetting up and down and incubate in the thermal mixer at 95 degrees Celsius for 10 minutes.Then immediately add four microliters of the custom probe. Incubate the samples in a thermal mixer at 65 degrees Celsius with the lid heated to 75 degrees Celsius for four hours. Incubate the hybridized target DNA with streptavidin beads then wash the beads to remove unbound DNA.Use the commercial kit according to the manufacturer's instructions to get a final 20 microliters of resuspended beads with captured DNA fragments. The washing step of unbound DNA from streptavidin beads should be quick. Otherwise, the capture efficiency of the targeted DNA might be affected.Amplify the captured DNA fragments by performing PCR using a commercial kit with two micromolar backbone oligonucleotides according to the manufacturer's instructions. Finally, add 45 microliters of suspended beads directly to the PCR product and enrich the amplified target DNA fragments bound to the beads by elution with 30 microliters of 10 millimolar Tris-HCL. Quantify the fragments and carry out sequencing and data analysis according to the text protocol.This table outlines how ER-seq improves coverage depth by 23%compared with the traditional method. This is due to its efficient recovery of cfDNA molecules, thus greatly enhancing the analysis of rare mutations. It is also clear that the unique sequencing adapters used in ER-seq enable easy differentiation of natural and PCR-induced duplications.As detailed in this table on the calling results, analysis based on ER-seq was 100%consistent for EGFR pL858R detection and the frequency of detection was a bit higher when compared with a traditional analysis. Importantly, ER-seq analysis enabled the detection of other relatively low frequency mutations including EGFR pT790M which was not recognized by traditional analysis due to high background noise. Once mastered, this technique can be done in 48 hours if it is performed properly.While attempting this procedure, it's important to remember to avoid loss of a very low quantity of cfDNA during extraction, library prep, and bead wash after hybridization. After watching this video, you should have a good understanding of how ctDNA from patient was collected, prepared, and targeted based on unique identified for sequencing. After its development, this technique paved the way for the researchers in the field of liquid biopsy to explore better practices in making clinic decisions.

detection-of-rare-mutations-in-ctdna-using-next-generation-sequencing

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Cancer Research

16.6K Görüntüleme.  Geneplus-Beijing Institute. The overall goal of this technique is to detect mutations of low frequency in circulating tumor or ctDNA to provide clinical physicians a powerful tool to diagnose tumors and monitor tumor dynamics in response to therapy. The method of testing ctDNA can help answer key questions about what kinds of mutations a patient carries such as single nuclear-type variation, insertion deletion, copy number variation, and structural variation. The main advantage of this technqiue is high sensitivity and ...