Name: wild-type blocking pcr combined with direct sequencing as a highly sensitive method for detection of low-frequency somatic mutations
Uploaded: 2017-03-29T15:00:00.000+00:00
Duration: 10 min 41 s
Description: Watch this Scientific Journal Video about Wild-type Blocking PCR Combined with Direct Sequencing as a Highly Sensitive Method for Detection of Low-Frequency Somatic Mutations at JoVE.com

The authors have no acknowledgements.

Ethics Statement: All testing of human samples was performed after obtaining Institutional Review Board (IRB) approval.
1. DNA Extraction from FFPE Tissue, Peripheral Blood, and Bone Marrow Aspirate
<ol>
	<li>For bone marrow FFPE tissue with DNA FFPE extraction kit
	<ol>
		<li>Begin with FFPE tissue from unstained slides (5 - 10 sections at 5 - 10 &#181;m thickness). 
		NOTE: If beginning with tissue shavings, use 3 - 6 sections at 5 - 10 &#181;m thickness and skip to step 1.1.6.</li>
		<li>Place slides in a slide basket and prepare four wash reservoirs (two for Xylene and two for 100% Alcohol). Add a minimum volume of 600 mL of solution to each basket.</li>
		<li>Deparaffinize the slides by doing a 5 min xylene wash in the first tray. Transfer the slides to the second xylene wash reservoir for an additional 5 min.</li>
		<li>After deparaffinization, wash the slides with 100% alcohol for 5 min. Transfer the slides to the second alcohol wash reservoir for an additional 5 min.</li>
		<li>Allow the slides to dry completely under a hood before scraping them with a razor blade into a microcentrifuge tube. 
		NOTE: If an H&#38;E slide with tumor region indicated is available, align the slides and scrape only the tumor region.</li>
		<li>Proceed with instructions from manufacturer handout included in kit.</li>
	</ol>
	</li>
	<li>For BM aspirate and Peripheral Blood
	<ol>
		<li>Extract according to the manufacturer&#39;s instructions handout with these specifications. 
		NOTE: There are numerous commercially available kits and methods for DNA extraction from FFPE tissues and cells. Practically, all can be used. Use a method that is established in the laboratory.
		<ol>
			<li>Use 200 &#181;L peripheral blood (PB) or 100 &#181;L BM + 100 &#181;L PBS.</li>
			<li>Use 4 &#181;L RNase A stock solution.</li>
			<li>Elute with 100 &#181;L elution buffer.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>DNA Quantification
	<ol>
		<li>Measure DNA concentrations using a spectrophotometer ensuring a 260 nm/280 nm ratio of approximately 1.8 (for pure DNA). If the ratio is appreciably lower, it may indicate the presence of protein, phenol, or other contaminants that may interfere with downstream applications.</li>
		<li>Adjust DNA concentrations to approximately 50 - 100 ng/&#181;L with appropriate elution buffer.</li>
	</ol>
	</li>
</ol>
2. Wild-Type Blocking PCR
<ol>
	<li>Primer Design
	<ol>
		<li>Design and obtain primers for genes of interest according to previously described general guidelines of PCR primer design13. Include M13-forward and reverse universal sequencing primers in PCR primers. 
		NOTE: The MYD88 assay was developed to amplify exon 5 of MYD88 (G259 - N291) and 110 nucleotides located in the 5&#39; intronic region to cover the splice site and part of intron 4. The forward and reverse primers were designed with a 5&#39;-M13 sequence (M13-forward: tgt aaa acg acg gcc agt; M13-reverse: cag gaa aca gct atg acc) to allow for annealing of complementary sequencing primers (see Materials Table).</li>
	</ol>
	</li>
	<li>Locked Nucleic Acid Oligonucleotide Design
	<ol>
		<li>Design the blocking oligonucleotide to be approximately 10 - 15 bases in length and complementary to the WT template where mutant enrichment is desired. 
		NOTE: A shorter oligo will improve mismatch discrimination. To achieve high target specificity, it is important not to use too much blocking nucleotides since this can result in a very &#34;sticky&#34; oligonucleotide. The content and the length and blocking nucleotide should to be optimize according to the specific nucleotide that need to be blocked. It is important to achieve high binding specificity without compromising secondary structure and risking self-complementarity.</li>
		<li>Design the blocking oligo to have a Tm 10 - 15 &#176;C above the extension temperature during thermocycling. Adjust the Tm by adding or removing blocking bases or by substituting blocking bases for DNA while avoiding long stretches (3 - 4 bases) of blocking G or C bases.
		<ol>
			<li>Navigate to the oligo tools website (see Table of Materials) and select the &#34;blocking Oligo Tm Prediction&#34; tool.</li>
			<li>Paste the sequence of the WT template that is desired to be blocked into the &#34;Oligo Sequence&#34; box. Add &#34;+&#34; in front of bases to indicate blocker bases.</li>
			<li>Select the &#34;Calculate&#34; button to determine the approximate Tm of the DNA-Blocker hybrid.</li>
		</ol>
		</li>
		<li>Design the blocking oligo to avoid secondary structure formation or self-dimers.
		<ol>
			<li>Navigate to the oligo tools website (see Table of Materials) and select the &#34;blocking Oligo Optimizer&#34; tool.</li>
			<li>Paste the sequence of the WT template that is desired to be blocked into the box. Add &#34;+&#34; in front of bases to indicate blocking bases.</li>
			<li>Select the two boxes for &#34;Secondary Structure&#34; and &#34;Self Only&#34;. Press the Analyze button to review the oligo for potentially troublesome secondary structures or self-dimers. 
			NOTE: The scores represent very rough estimates of the Tm&#39;s of the secondary structures and self-dimers. Lower scores are therefore optimal and can be achieved by limiting blocker-blocker pairing. The blocking oligonucleotide for MYD88 [MYD88blocker (TCAGA+AG+C+G+A+C+T+G+A+T+CC/invdT/)] was designed to cover amino acids Q262-I266 and features a 3&#39;-inverted dT to inhibit both extension by DNA polymerase and degradation by 3&#39;-exonuclease. This specific blocking oligo is a 17mer with 10 blocking bases which are denoted as &#34;+N&#34;. The remaining 7 bases are ordinary DNA nucleotides.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>WTB-PCR Setup and Thermocycling
	<ol>
		<li>Prepare a WTB-PCR master mix (MMX) using 2.5 &#181;L PCR reaction buffer 10x w/ 20 mM MgCl2, 250 &#181;M dNTPs, 0.2 &#181;M forward and reverse primer, 1.2 &#181;M MYD88blocker, and DNAse, RNAse-free, ultra-pure H2O to create a final solution volume of 21.75 &#181;L per reaction. 
		NOTE: Working concentrations are for the final reaction volume of 25 &#181;L (after addition of DNA template and polymerase). Conventional PCR MMX is prepared by simply omitting the addition of blocker. All protocol steps remain identical for both WTB and conventional PCR. This can be used in validation and determining enrichment achieved by addition of blocker.
		<ol>
			<li>When calculating the amount of MMX to prepare make sure to account for 3 additional reactions (positive and negative controls and a non-template control to check for contamination) and at least 1 additional reaction for pipetting error.</li>
		</ol>
		</li>
		<li>Vortex MMX thoroughly. The MMX can be stored at -80 &#176;C for up to a year.</li>
		<li>Add 0.25 &#181;L Taq DNA polymerase per reaction to the MMX and invert to mix. Once the polymerase has been added to the MMX, keep it on ice.</li>
		<li>Put a new 96 well PCR plate on a cold plate and pipette 22 &#181;L of MMX with polymerase to each reaction well.</li>
		<li>Add 3 &#181;L genomic DNA (50 - 100 ng/&#181;L to each of the wells containing the MMX with polymerase.</li>
		<li>Seal the plate and centrifuge briefly to ensure the solution reaches the bottom of each well.</li>
		<li>Load the PCR plate on a thermocycler.
		<ol>
			<li>Set the thermocycling conditions for the WTB-PCR reaction as follows: initial denaturation at 95 &#176;C for 6 min; 40 cycles of denaturation at 95 &#176;C for 30 s, primer annealing at 56 &#176;C for 30 s, and extension at 72 &#176;C for 1 min 20 s; final extension at 72 &#176;C for 10 min. 
			NOTE: Best practice involves completing the remainder of the protocol in a physically separate area to avoid amplicon contamination in future setups.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Perform gel electrophoresis according to previously described protocols14 in order to determine if WTB-PCR was successful and to confirm a lack of amplification in the non-template control.</li>
	<li>Purification of PCR Product by magnetic beads
	<ol>
		<li>Remove magnetic beads from 4 &#176;C and bring to room temperature.</li>
		<li>Transfer 10 &#181;L PCR product to a new PCR plate.</li>
		<li>Vortex the magnetic beads vigorously to fully resuspend the magnetic particles.</li>
		<li>Add 18 &#181;L magnetic beads to each well on the new plate. Pipette mix 10 times.</li>
		<li>Incubate at room temperature for 5 min.</li>
		<li>Place the PCR plate onto the side-skirted magnet plate for 2 min to separate beads from solution.</li>
		<li>Aspirate the supernatant with a multichannel pipette, avoiding the bead pellet.</li>
		<li>Dispense 150 &#181;L 70% ethanol into each well and incubate at room temperature for at least 30 s. Aspirate the ethanol with a multichannel pipette and discard tips. Repeat this wash procedure once more.</li>
		<li>Using a 20 &#181;L multichannel pipette aspirate the remaining ethanol from each well and discard tips.</li>
		<li>Once wells are dry (~ 10 min), remove from magnet plate and add 40 &#181;L nuclease free water to each well and pipette mix 15 times or vortex gently.</li>
		<li>Incubate at room temperature for 2 min.</li>
		<li>Place the PCR plate onto magnet plate for 1 min to separate beads from solution.</li>
		<li>Transfer 35 &#181;L of purified product to a new PCR plate. This is purified PCR product is now ready to proceed with bi-directional sequencing or can be stored at -20 &#176;C until needed. 
		NOTE: When developing a new assay, quantification of purified PCR product may be required. 1 - 3 ng/&#181;L amplicon DNA is optimal for bi-directional sequencing. If concentration is consistently low, increase PCR product and magnetic beads proportionally (1:1.8). If concentration is consistently high, elute with a greater volume of water.</li>
	</ol>
	</li>
</ol>
3. Sequencing of WTB-PCR Product
<ol>
	<li>Bi-Directional Sequencing 
	NOTE: The following protocol is a modified form of the manufacturer&#39;s instructions that has been optimized to use fewer reagents.
	<ol>
		<li>Prepare forward and reverse sequencing reaction mixes with 0.25 &#181;L Ready Reaction mix, 1.88 &#181;L Sequencing Buffer, 1.78 &#181;M M13-F or M13-R sequencing primers and add and DNAse, RNAse-free, ultra-pure H2O to create a final solution volume of 9 &#181;L per reaction. This sequencing reaction mix can be stored at -20 &#176;C for up to a year.</li>
		<li>Pipette 9 &#181;L of the forward sequencing reaction mix into each well of a new 96 well PCR plate for the forward sequencing reaction. Repeat on a separate plate for the reverse sequencing reaction.</li>
		<li>Add 1 &#181;L of the purified WTB-PCR product to each well on both the forward and reverse plates.</li>
		<li>Seal the plate and centrifuge briefly to ensure the solution reaches the bottom of each well.</li>
		<li>Load the PCR plate on a thermocycler.
		<ol>
			<li>Set the thermocycling conditions for the sequencing reaction as follows: 96 &#176;C for 1 min; 30 cycles of 96 &#176;C for 10 s, 50 &#176;C for 5 s, 60 &#176;C for 4 min; hold at 4 &#176;C until ready for purification.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Purify Sequencing Products
	<ol>
		<li>Prepare a fresh 1:25 solution of 3 M sodium acetate (pH 5.2) and 100% ethanol.</li>
		<li>Prepare a fresh 70% ethanol solution.</li>
		<li>Add 30 &#181;L of sodium acetate/100% ethanol solution to each well of both the forward and reverse sequencing plates and pipette mix 5 times.</li>
		<li>Reseal the plate and incubate in the dark at room temperature for 20 min.</li>
		<li>Centrifuge the plate at 2,250 x g for 15 min.</li>
		<li>Remove the plate sealer and invert over a waste container. 
		NOTE: Invert only once or the pellet may loosen from the well bottoms.</li>
		<li>Place the inverted plate on a clean paper towel and centrifuge at 150 x g for 1 min.</li>
		<li>Add 150 &#181;L 70% ethanol to each well.</li>
		<li>Reseal the plate and spin at 2,250 x g for 5 min.</li>
		<li>Repeat steps 3.2.6 and 3.2.7.</li>
		<li>If the wells are not completely dry, allow them to air dry at room temperature. Make sure the samples are protected from light while drying.</li>
		<li>Add 10 &#181;L Formamide to each well and pipette mix 10 times. Reseal the plate.</li>
		<li>Denature on thermocycler at 95 &#176;C for 3 min followed by 4 &#176;C for 5 min.</li>
		<li>After denaturing, replace the plate sealer with a septa and sequence on sequencing platform according to manufacturer&#39;s instructions15.</li>
	</ol>
	</li>
</ol>
4. Analysis of Sequencing Results
<ol>
	<li>Visualize sequencing traces using a sequencing data analysis software according to manufacturer&#39;s instructions16 and align to the respective reference sequences. Align MYD88 to NCBI reference sequence &#34;NM_002468&#34;.</li>
</ol>

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The authors have nothing to disclose.

The WTB-PCR assay described here uses a generic set of primers with a blocking oligo designed to block amplification of WT DNA during extension (Figure 1). The WTB-PCR product is then sequenced for mutational analysis. The utility of WTB-PCR/Sanger lies in its simplicity, high-sensitivity, and high-throughput. Using the guidelines described here, most existing Sanger based assays can be simply modified via the addition of a blocking oligonucleotide to greatly increase sensitivity. In the example assay pr...

Sanger sequencing has traditionally been the gold standard in testing for both known and unknown somatic mutations. One of the limitations of Sanger sequencing is its limit of detection (~ 10 - 20% mutant allele in a background of WT)1. This level of sensitivity is inappropriate for detecting low level somatic mutations that may be present in samples from premalignant tissues or patients with few circulating tumor cells, or when bone marrow (BM) is patchy. This also makes assessing residual disease after therapy or detecting emerging resistance mutations during therapy difficult by conventional sequencing alone2. By replacing conventional PCR with locked nucleic acid (LNA)-mediated wild-type blocking PCR (WTB-PCR) in Sanger sequencing, sensitivities of up to 0.1% mutant allele in a background of WT can be achieved2,3,4. In WTB-PCR, enrichment for mutant alleles is achieved via the addition of a short (~ 10 - 14 NT) blocking (LNA) oligonucleotide that binds preferentially to WT DNA thereby preventing amplification of WT DNA. The mutant enriched WTB-PCR product can then be sequenced. By blocking WT DNA rather than selecting for specific mutations WTB-PCR allows for enrichment of both known and unknown mutations present in minute cell fractions.
Multiple methods are currently used for detecting mutations in small cells fractions. This includes allele-specific PCR, amplification-refractory mutation system (ARMS), denaturing high performance liquid chromatography (DHPLC), beads, emulsions, amplification, and magnetics (BEAMing), electric field-induced release and measurement (EFIRM), high resolution melting point and others. However, most of these methods are limited by false-positives and the ability to only detect one mutation that the assay was designed for4. WTB-PCR, however, allows the user to visualize sequencing traces which enables the detection of multiple mutation types and can aid in ruling out false positives due to artifacts or deamination events. Next-generation sequencing (NGS) may offer a suitable alternative to conventional sequencing, however, substantially greater costs, complexity, and longer assay time render it an unnecessary option for many disease types with few distinct molecular markers or for monitoring patients on therapy for emerging resistance mutations. Furthermore, high false positive rates when detecting variants with mutant allele frequencies of less than 5% can pose a problem for amplicon-based NGS5,6.
Here we demonstrate the increase in sensitivity achieved by WTB-PCR in screening for mutations in the myeloid differentiation factor 88 gene as described by Albitar et al.3 MYD88 mutations are important diagnostic and prognostic factors in Waldenstr&#246;m&#39;s Macroglobulinemia (WM), IgM monoclonal gammopathy of unknown significance (IgM-MGUS), splenic marginal zone lymphoma (SMZL), and diffuse large B-cell lymphoma (DLBCL). MYD88 mutations are found in almost all cases of WM and approximately 50% of patients with Immunoglobulin M (IgM)-secreting MGUS. Contrastingly, MYD88 mutations are found in only 0-6% of patients with SMZL and are absent in multiple myeloma7,8. Because overlapping morphologic, immunophenotypic, cytogenetic, and clinical characteristics between WM and SMZL or IgM-multiple myeloma can often complicate differential diagnoses, the presence of a MYD88 mutation may serve as a useful identifying factor9. MYD88 mutations have also been associated with greater disease burden in patients with DLBCL and poor overall survival following therapy7,10. Additionally, because MYD88 mutations are more frequently found in activated B-cell-like (ABC) DLBCL than germinal center B-cell-like (GCB) DLBCL or primary mediastinal B-cell lymphoma (PMBL), MYD88 mutation status may serve as a surrogate marker for the ABC subtype11,12.
The detailed protocol provided here serves as a template from which new assays can be developed or most existing sequencing assays can be easily adapted to accurately detect low frequency mutations in various sample types. The approach can also be used for monitoring and detecting resistant mutations that may develop in tumors or even bacteria that may develop while patients are being treated with targeted therapy or antibiotics. Furthermore, it addresses and remedies many of the issues associated with mutation enrichment, particularly in formalin-fixed paraffin-embedded (FFPE) tissue.

<table><tbody><tr><td>1.5 or 2 mL Safe-Lock microcentrifuge tubes</td><td>Eppendorf</td><td>05-402-25</td><td></td></tr><tr><td>100% alcohol</td><td>VWR</td><td>89370-084</td><td>Histology grade; 91.5% Ethanol, 5% Isopropyl alcohol, 4.5% Methyl alcohol</td></tr><tr><td>3730XL sequencer</td><td>ABI</td><td></td><td>or equivalent</td></tr><tr><td>Agencourt AMPure XP</td><td>Beckman Coulter</td><td>A63881</td><td>For magnetic bead PCR purification</td></tr><tr><td>Aluminum sealing foils</td><td>GeneMate</td><td>T-2451-1</td><td>For PCR and cold storage</td></tr><tr><td>BigDye Terminator v3.1 Cycle sequencing kit</td><td>Life Technologies</td><td>4337455</td><td>For bi-directional sequencing. With 5x Sequencing Buffer</td></tr><tr><td>Centrifuge 5804 Series</td><td>Eppendorf</td><td></td><td>A-2-DWP rotor (for PCR plate)</td></tr><tr><td>Cold plate for 96 well plates</td><td>Eppendorf</td><td>Z606634</td><td></td></tr><tr><td>DNAse, RNAse-free, ultra-pure water</td><td></td><td></td><td></td></tr><tr><td>dNTPs (100 mM)</td><td>Invitrogen</td><td>10297-117</td><td></td></tr><tr><td>DynaMag-96 Side-Skirted Magnet</td><td>Thermo Fisher Scientific</td><td>12027</td><td>For use in PCR Purification.</td></tr><tr><td>Ethanol Absolute&amp;nbsp;</td><td>Sigma</td><td>E7023</td><td>200 proof, for molecular biology</td></tr><tr><td>Exiqon website Oligo Tools</td><td></td><td></td><td>&lt;a href=&quot;http://www.exiqon.com/oligo-tools&quot;&gt;www.exiqon.com/oligo-tools&lt;/a&gt;</td></tr><tr><td>FastStart Taq DNA polymerase (5 U/&amp;micro;L)</td><td>Roche</td><td>12032937001</td><td>With10x concentrated PCR reaction buffer, with 20 mM MgCl&lt;sub&gt;2&lt;/sub&gt;</td></tr><tr><td>Gel electrophoresis apparatus</td><td></td><td></td><td>2% agarose gel</td></tr><tr><td>GeneRead DNA FFPE extraction Kit&amp;nbsp;</td><td>Qiagen</td><td>180134</td><td>Contains uracil DNA glycosylase necessary for reducing sequencing artifacts</td></tr><tr><td>Hi-Di Formamide</td><td>ABI</td><td>4311320</td><td>For sequencing.</td></tr><tr><td>LNA oligonucleotide</td><td>Exiqon</td><td>500100</td><td>5&#039;-TCAGA+AG+C+G+A+C+T+G+A+T+CC/invdT/ (+N = LNA bases)</td></tr><tr><td>M13-F Sequencing Primer</td><td>ABI</td><td></td><td>5&#039;-tgt aaa acg acg gcc agt</td></tr><tr><td>M13-R Sequencing Primer</td><td>ABI</td><td></td><td>5&#039;-cag gaa aca gct atg acc</td></tr><tr><td>Mastercycler Pro S Thermocycler</td><td>Eppendorf</td><td>E950030020</td><td></td></tr><tr><td>Microcentrifuge Model 5430</td><td>Eppendorf</td><td></td><td>FA-45-30-11 rotor (for 1.5/2 mL microcentrifuge tubes)</td></tr><tr><td>NanoDrop 2000 Spectrophotometer</td><td>Thermo Fisher Scientific</td><td></td><td></td></tr><tr><td>PCR forward primer</td><td>IDT</td><td></td><td>5&#039;-tgt aaa acg acg gcc agt TGC CAG GGG TAC TTA GAT GG</td></tr><tr><td>PCR reverse primer</td><td>IDT</td><td></td><td>5&#039;-cag gaa aca gct atg acc GGT TGG TGT AGT CGC AGA CA</td></tr><tr><td>PCR plates</td><td>GeneMate</td><td>T-3107-1</td><td></td></tr><tr><td>Pipettors</td><td></td><td></td><td>20, 200, 1,000 &amp;micro;L</td></tr><tr><td>Plate septa, 96 well</td><td>ABI</td><td>4315933</td><td></td></tr><tr><td>QIAamp DNA Mini Kit</td><td>Qiagen</td><td>51304</td><td>For BM aspirate and peripheral blood</td></tr><tr><td>SeqScape Sortware v3.0</td><td>ABI</td><td>4474978</td><td>For sequencing analysis</td></tr><tr><td>Slide basket</td><td></td><td></td><td></td></tr><tr><td>Sodium Acetate (3 M, pH 5.2)&amp;nbsp;</td><td>Sigma</td><td>S7899</td><td></td></tr><tr><td>Sterile filtered pipette tips</td><td></td><td></td><td>20, 200, 1,000 &amp;micro;L</td></tr><tr><td>Thermomixer C&amp;nbsp;</td><td>Eppendorf</td><td>5382000023</td><td></td></tr><tr><td>Vortex genie</td><td>Scientific Industries</td><td>SI-0236</td><td></td></tr><tr><td>Wash reservoir</td><td></td><td></td><td>~ 1,000 mL</td></tr><tr><td>Xylene</td><td>VWR</td><td>89370-088</td><td>Histology grade</td></tr></tbody></table>

wild-type blocking pcr combined with direct sequencing as a highly sensitive method for detection of low-frequency somatic mutations

Accurate detection and identification of low frequency mutations can be problematic when assessing residual disease after therapy, screening for emerging resistance mutations during therapy, or when patients have few circulating tumor cells. Wild-type blocking PCR followed by sequencing analysis offers high sensitivity, flexibility, and simplicity as a methodology for detecting these low frequency mutations. By adding a custom designed locked nucleic acid oligonucleotide to a new or previously established conventional PCR based sequencing assay, sensitivities of approximately 1 mutant allele in a background of 1,000 WT alleles can be achieved (1:1,000). Sequencing artifacts associated with deamination events commonly found in formalin fixed paraffin embedded tissues can be partially remedied by the use of uracil DNA glycosylase during extraction steps. The optimized protocol here is specific for detecting MYD88 mutation, but can serve as a template to design any WTB-PCR assay. Advantages of the WTB-PCR assay over other commonly utilized assays for the detection of low frequency mutations including allele specific PCR and real-time quantitative PCR include fewer occurrences of false positives, greater flexibility and ease of implementation, and the ability to detect both known and unknown mutations.

A conceptual overview of WTB-PCR during extension is presented in Figure 1. Because a single nucleotide mismatch in the blocker-DNA hybrid greatly decreases its melting temperature (&#916;Tm=20 - 30 &#176;C), amplification of the WT allele is blocked while mutant template DNA is free to complete extension17. In this manner, mutant DNA is amplified exponentially while WT DNA is amplified linearly.
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Watch this Scientific Journal Video about Wild-type Blocking PCR Combined with Direct Sequencing as a Highly Sensitive Method for Detection of Low-Frequency Somatic Mutations at JoVE.com

Wild-type Blocking PCR Combined with Direct Sequencing as a Highly Sensitive Method for Detection of Low-Frequency Somatic Mutations

Wild-type blocking PCR followed by direct sequencing offers a highly sensitive method of detection for low frequency somatic mutations in a variety of sample types.

Accurate detection and identification of low frequency mutations can be problematic when assessing residual disease after therapy, screening for emerging ...

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JoVE Journal

Genetics

11.6K Views.  NeoGenomics Laboratories. Wild-type blocking PCR followed by direct sequencing offers a highly sensitive method of detection for low frequency somatic mutations in a variety of sample types.

Video: Wild-type Blocking PCR Combined with Direct Sequencing as a Highly Sensitive Method for Detection of Low-Frequency Somatic Mutations

Detection of Copy Number Alterations Using Single Cell Sequencing

Detection of genomic changes at single cell resolution is important for characterizing genetic heterogeneity and evolution in normal tissues, cancers, and microbial populations. Traditional methods for assessing genetic heterogeneity have been limited by low resolution, low sensitivity, and/or low specificity. Single cell sequencing has emerged as a powerful tool for detecting genetic heterogeneity with high resolution, high sensitivity and, when appropriately analyzed, high specificity. Here we provide a protocol for the isolation, whole genome amplification, sequencing, and analysis of single cells. Our approach allows for the reliable identification of megabase-scale copy number variants in single cells. However, aspects of this protocol can also be applied to investigate other types of genetic alterations in single cells.

Single cell sequencing is an increasingly popular and accessible tool for addressing genomic changes at high resolution. We provide a protocol that uses single cell sequencing to identify copy number alterations in single cells.

Detection of genomic changes at single cell resolution is important for characterizing genetic heterogeneity and evolution in normal tissues, cancers, and ...

Robust DNA Isolation and High-throughput Sequencing Library Construction for Herbarium Specimens

Herbaria are an invaluable source of plant material that can be used in a variety of biological studies. The use of herbarium specimens is associated with a number of challenges including sample preservation quality, degraded DNA, and destructive sampling of rare specimens. In order to more effectively use herbarium material in large sequencing projects, a dependable and scalable method of DNA isolation and library preparation is needed. This paper demonstrates a robust, beginning-to-end protocol for DNA isolation and high-throughput library construction from herbarium specimens that does not require modification for individual samples. This protocol is tailored for low quality dried plant material and takes advantage of existing methods by optimizing tissue grinding, modifying library size selection, and introducing an optional reamplification step for low yield libraries. Reamplification of low yield DNA libraries can rescue samples derived from irreplaceable and potentially valuable herbarium specimens, negating the need for additional destructive sampling and without introducing discernible sequencing bias for common phylogenetic applications. The protocol has been tested on hundreds of grass species, but is expected to be adaptable for use in other plant lineages after verification. This protocol can be limited by extremely degraded DNA, where fragments do not exist in the desired size range, and by secondary metabolites present in some plant material that inhibit clean DNA isolation. Overall, this protocol introduces a fast and comprehensive method that allows for DNA isolation and library preparation of 24 samples in less than 13 h, with only 8 h of active hands-on time with minimal modifications.

This article demonstrates a detailed protocol for DNA isolation and high-throughput sequencing library construction from herbarium material including rescue of exceptionally poor-quality DNA.

Herbaria are an invaluable source of plant material that can be used in a variety of biological studies. The use of herbarium specimens is associated with ...

Rare Event Detection Using Error-corrected DNA and RNA Sequencing

Conventional next-generation sequencing techniques (NGS) have allowed for immense genomic characterization for over a decade. Specifically, NGS has been used to analyze the spectrum of clonal mutations in malignancy. Though far more efficient than traditional Sanger methods, NGS struggles with identifying rare clonal and subclonal mutations due to its high error rate of ~0.5&#8211;2.0%. Thus, standard NGS has a limit of detection for mutations that are &#62;0.02 variant allele fraction (VAF). While the clinical significance for mutations this rare in patients without known disease remains unclear, patients treated for leukemia have significantly improved outcomes when residual disease is &#60;0.0001 by flow cytometry. In order to mitigate this artefactual background of NGS, numerous methods have been developed. Here we describe a method for Error-corrected DNA and RNA Sequencing (ECS), which involves tagging individual molecules with both a 16 bp random index for error-correction and an 8 bp patient-specific index for multiplexing. Our method can detect and track clonal mutations at variant allele fractions (VAFs) two orders of magnitude lower than the detection limit of NGS and as rare as 0.0001 VAF.

Next-generation sequencing (NGS) is a powerful tool for genomic characterization that is limited by the high error rate of the platform (~0.5&#8211;2.0%). We describe our methods of error-corrected sequencing that allow us to obviate the NGS error rate and detect mutations at variant allele fractions as rare as 0.0001.

Conventional next-generation sequencing techniques (NGS) have allowed for immense genomic characterization for over a decade. Specifically, NGS has been ...

Lentiviral Mediated Production of Transgenic Mice: A Simple and Highly Efficient Method for Direct Study of Founders

For almost 40 years, pronuclear DNA injection represents the standard method to generate transgenic mice with random integration of transgenes. Such a routine procedure is widely utilized throughout the world and its main limitation resides in the poor efficacy of transgene integration, resulting in a low yield of founder animals. Only few percent of animals born after implantation of injected fertilized oocytes have integrated the transgene. In contrast, lentiviral vectors are powerful tools for integrative gene transfer and their use to transduce fertilized oocytes allows highly efficient production of founder transgenic mice with an average yield above 70%. Furthermore, any mouse strain can be used to produce transgenic animal and the penetrance of transgene expression is extremely high, above 80% with lentiviral mediated transgenesis compared to DNA microinjection. The size of the DNA fragment that can be cargo by the lentiviral vector is restricted to 10 kb and represents the major limitation of this method. Using a simple and easy to perform injection procedure beneath the zona pellucida of fertilized oocytes, more than 50 founder animals can be produced in a single session of microinjection. Such a method is highly adapted to perform, directly in founder animals, rapid gain and loss of function studies or to screen genomic DNA regions for their ability to control and regulate gene expression in vivo.

Here, we present a protocol to promote transgene integration and production of founder transgenic mice with high efficacy by a simple injection of a lentiviral vector in the perivitelline space of a fertilized oocyte.

For almost 40 years, pronuclear DNA injection represents the standard method to generate transgenic mice with random integration of transgenes. Such a ...

Amplification of Near Full-length HIV-1 Proviruses for Next-Generation Sequencing

The Full-Length Individual Proviral Sequencing (FLIPS) assay is an efficient and high-throughput method designed to amplify and sequence single, near full-length (intact and defective), HIV-1 proviruses. FLIPS allows determination of the genetic composition of integrated HIV-1 within a cell population. Through identifying defects within HIV-1 proviral sequences that arise during reverse transcription, such as large internal deletions, deleterious stop codons/hypermutation, frameshift mutations, and mutations/deletions in cis acting elements required for virion maturation, FLIPS can identify integrated proviruses incapable of replication. The FLIPS assay can be utilized to identify HIV-1 proviruses that lack these defects and are&#160;therefore potentially replication-competent. The FLIPS protocol involves: lysis of HIV-1 infected cells, nested PCR of near full-length HIV-1 proviruses (using primers targeted to the HIV-1 5&#39; and 3&#39; LTR), DNA purification and quantification, library preparation for Next-generation Sequencing (NGS), NGS, de novo assembly of proviral contigs, and a simple process of elimination for identifying replication-competent proviruses. FLIPS provides advantages over traditional methods designed to sequence integrated HIV-1 proviruses, such as single-proviral sequencing. FLIPS amplifies and sequences near full-length proviruses enabling replication competency to be determined, and also uses fewer amplification primers, preventing the consequences of primer mismatches. FLIPS is a useful tool for understanding the genetic landscape of integrated HIV-1 proviruses, especially within the latent reservoir, however, its utilization can extend to any application in which the genetic composition of integrated HIV-1 is required.

Full-length individual proviral sequencing (FLIPS) provides an efficient and high-throughput method for the amplification and sequencing of single, near full-length (intact and defective) HIV-1 proviruses and allows for determination of their potential replication-competency. FLIPS overcomes limitations of previous assays designed to sequence the latent HIV-1 reservoir.

The Full-Length Individual Proviral Sequencing (FLIPS) assay is an efficient and high-throughput method designed to amplify and sequence single, near ...

Single Droplet Digital Polymerase Chain Reaction for Comprehensive and Simultaneous Detection of Mutations in Hotspot Regions

Droplet digital polymerase chain reaction (ddPCR) is a highly sensitive quantitative polymerase chain reaction (PCR) method based on sample fractionation into thousands of nano-sized water-in-oil individual reactions. Recently, ddPCR has become one of the most accurate and sensitive tools for circulating tumor DNA (ctDNA) detection. One of the major limitations of the standard ddPCR technique is the restricted number of mutations that can be screened per reaction, as specific hydrolysis probes recognizing each possible allelic version are required. An alternative methodology, the drop-off ddPCR, increases throughput, since it requires only a single pair of probes to detect and quantify potentially all genetic alterations in the targeted region. Drop-off ddPCR displays comparable sensitivity to conventional ddPCR assays with the advantage of detecting a greater number of mutations in a single reaction. It is cost-effective, conserves precious sample material, and can also be used as a discovery tool when mutations are not known a priori.

Here, we present a protocol to accurately quantify multiple genetic alterations of a target region in a single reaction using drop-off ddPCR and a unique pair of hydrolysis probes.

Droplet digital polymerase chain reaction (ddPCR) is a highly sensitive quantitative polymerase chain reaction (PCR) method based on sample fractionation ...

Comparative Lesions Analysis Through a Targeted Sequencing Approach

Assessing intra-tumoral heterogeneity (ITH) is of paramount importance to anticipate failure of targeted therapies and design accordingly effective anti-tumor strategies. Although concerns are frequently raised due to differences in sample processing and depth of coverage, next-generation sequencing of solid tumors have unraveled a highly variable degree of ITH across tumor types. Capturing the genetic relatedness between primary and metastatic lesions through the identification of clonal and subclonal populations is critical to the design of therapies for advance-stage diseases. Here, we report a method for comparative lesions analysis that allows for the identification of clonal and subclonal populations among different specimens from the same patient. The experimental approach described here integrates three well-established approaches: histological analysis, high-coverage multi-lesion sequencing, and immunophenotypic analyses. In order to minimize the effects on the detection of subclonal events by inappropriate sample processing, we subjected tissues to careful pathological examination and neoplastic cell enrichment. Quality controlled DNA from neoplastic lesions and normal tissues was then subjected to high coverage sequencing, targeting the coding regions of 409 relevant cancer genes. While only looking at a limited genomic space, our approach enables evaluating the extent of heterogeneity among somatic alterations (single-nucleotide mutations and copy-number variations) in distinct lesions from a given patient. Through comparative analysis of sequencing data, we were able to distinguish clonal vs. subclonal alterations. The majority of ITH is often ascribed to passenger mutations; therefore, we also used immunohistochemistry to predict functional consequences of mutations. While this protocol has been applied to a specific tumor type, we anticipate that the methodology described here is broadly applicable to other solid tumor types.

This article describes a method to identify clonal and subclonal alterations among different specimens from a given patient. Although the experiments described here focus on a specific tumor type, the approach is broadly applicable to other solid tumors.

Assessing intra-tumoral heterogeneity (ITH) is of paramount importance to anticipate failure of targeted therapies and design accordingly effective ...

Behavior

Optimizing Low-Frequency Mutation Detection with Wild-Type Blocking PCR

Wild-type Blocking PCR Combined with Sanger Sequencing for Detection of Low-frequency Somatic Mutation

When monitoring minimal residual disease (MRD) after tumor treatment, there are higher requirements of the lower limit of detection than when detecting for drug resistance mutations and circulating tumor cell mutations during therapy. Traditional Sanger sequencing has 5%-20% wild-type mutation detection, so its limit of detection cannot meet the corresponding requirements. The wild-type blocking technologies that have been reported to overcome this include blocker displacement amplification (BDA), non-extendable locked nucleic acid (LNA), hot-spot-specific probes (HSSP), etc. These technologies use specific oligonucleotide sequences to block wild-type or recognize wild-type and then combine this with other methods to prevent wild-type amplification and amplify mutant amplification, leading to characteristics like high sensitivity, flexibility, and convenience. This protocol uses BDA, a wild-type blocking PCR combined with Sanger sequencing, to optimize the detection of RHOA G17V low-frequency somatic mutations, and the detection sensitivity can reach 0.5%, which can provide a basis for MRD monitoring of angioimmunoblastic T-cell lymphoma.

Author Spotlight: Advancing the Detection of Low-Frequency Mutations in Cancer Tissues

This article introduces the application of a low-frequency detection method based on Sanger sequencing in angioimmunoblastic lymphoma. Provide a basis for applying this method to other diseases.

When monitoring minimal residual disease (MRD) after tumor treatment, there are higher requirements of the lower limit of detection than when detecting ...

Integration of Wet and Dry Bench Processes Optimizes Targeted Next-generation Sequencing of Low-quality and Low-quantity Tumor Biopsies

All next-generation sequencing (NGS) procedures include assays performed at the laboratory bench (&#34;wet bench&#34;) and data analyses conducted using bioinformatics pipelines (&#34;dry bench&#34;). Both elements are essential to produce accurate and reliable results, which are particularly critical for clinical laboratories. Targeted NGS technologies have increasingly found favor in oncology applications to help advance precision medicine objectives, yet the methods often involve disconnected and variable wet and dry bench workflows and uncoordinated reagent sets. In this report, we describe a method for sequencing challenging cancer specimens with a 21-gene panel as an example of a comprehensive targeted NGS system. The system integrates functional DNA quantification and qualification, single-tube multiplexed PCR enrichment, and library purification and normalization using analytically-verified, single-source reagents with a standalone bioinformatics suite. As a result, accurate variant calls from low-quality and low-quantity formalin-fixed, paraffin-embedded (FFPE) and fine-needle aspiration (FNA) tumor biopsies can be achieved. The method can routinely assess cancer-associated variants from an input of 400 amplifiable DNA copies, and is modular in design to accommodate new gene content. Two different types of analytically-defined controls provide quality assurance and help safeguard call accuracy with clinically-relevant samples. A flexible &#34;tag&#34; PCR step embeds platform-specific adaptors and index codes to allow sample barcoding and compatibility with common benchtop NGS instruments. Importantly, the protocol is streamlined and can produce 24 sequence-ready libraries in a single day. Finally, the approach links wet and dry bench processes by incorporating pre-analytical sample quality control results directly into the variant calling algorithms to improve mutation detection accuracy and differentiate false-negative and indeterminate calls. This targeted NGS method uses advances in both wetware and software to achieve high-depth, multiplexed sequencing and sensitive analysis of heterogeneous cancer samples for diagnostic applications.

An integrated system for targeted next-generation sequencing of oncology specimens is described. This cross-platform system is optimized for low-quality and low-quantity tumor biopsies, accommodates low DNA inputs, includes well-characterized multi-variant controls, and features a novel variant caller that is informed by quantitative pre-analytical quality control measures.

All next-generation sequencing (NGS) procedures include assays performed at the laboratory bench (&#34;wet bench&#34;) and data analyses conducted using ...

Medicine

Wild-Type Blocking PCR to Detect Low-Frequency Somatic Mutations

The forward and reverse primers were designed with a 5&#39;-M13 sequence to allow for annealing of complementary sequencing primers. Design the blocking oligonucleotide to be approximately 10 to 15 bases in length and complementary to the wild-type template where mutant enrichment is desired.
A shorter oligo will improve mismatch discrimination. To achieve high target specificity, it&#39;s important not to use too much of the blocking nucleotides, as this will result in a very &#34;sticky&#34; oligonucleotide.
To design the blocking oligonucleotide, begin by navigating to the Oligo Tools website. Select the &#34;Oligo TM Prediction&#34; tool. A new window will open up. Paste the sequence of the wild-type template to be blocked into the &#34;Oligo Sequence&#34; box. Add a plus sign in front of the blocker bases to mark them. Click on the &#34;Calculate&#34; button to determine the approximate TM of the DNA blocker hybrid. The calculated melting temperatures will appear in the boxes below.
Design the blocking oligo to have a melting temperature 10 to 15 degrees Celsius above the extension temperature during thermocycling. Here, the extension temperature is 72 degrees Celsius. To adjust the melting temperature, add, remove, or substitute blocking bases. Avoid long stretches of three to four blocking C or G bases.
Next, to avoid secondary structure formation or self-dimerization, go back to the Oligo Tools website home screen and select the &#34;Oligo Optimizer&#34; tool. A new window will open. Paste the sequence of the wild-type template to be blocked into the box. Add a plus sign to indicate blocking bases. Select the two boxes for &#34;Secondary Structure&#34; and &#34;Self Only&#34; and press the &#34;Analyze&#34; button to see the scores for hybridization and secondary structure.
These scores represent very rough estimates of the melting temperatures of the self-dimers and secondary structures, respectively. Lower scores are optimal and can be achieved by limiting blocker-blocker pairing. Remove or reposition blocking nucleotides in order to achieve lower scores. The optimized blocking oligonucleotide for MyD88, shown here, strikes a balance between the DNA blocker hybrid melting temperature and low enough hybridization and secondary structure scores.
It was designed to cover amino acids Q262 to I266 and features a 3&#39;-inverted dT to inhibit both extension by DNA polymerase and degradation by 3&#39;-exonuclease. Once the primers have been designed, set up the wild-type blocking PCR and perform thermocycling, as described in the accompanying document.