CRE : a cost effective and rapid approach for PCR-mediated concatenation of and exons KRAS EGFR

Molecular diagnostics has changed the way lung cancer patients are treated worldwide. Of several different testing methods available, PCR followed by directed sequencing and amplification refractory mutation system (ARMS) are the two most commonly used diagnostic methods worldwide to detect mutations at KRAS exon 2 and EGFR kinase domain exons 18-21 in lung cancer. Compared to ARMS, the PCR followed by directed sequencing approach is relatively inexpensive but more cumbersome to perform. Moreover, with a limiting amount of genomic DNA from clinical formalin-fixed, paraffin-embedded (FFPE) specimens or fine biopsies of lung tumors, multiple rounds of PCR and sequencing reactions often get challenging. Here, we report a cost-effective single multiplex-PCR based method, CRE (for Co-amplification of five K RAS and E GFR exons), followed by concatenation of the PCR product as a single linear fragment for direct sequencing. CRE is a robust protocol that can be adapted for routine use in clinical diagnostics with reduced variability, cost and turnaround time requiring a minimal amount of template DNA extracted from FFPE or fresh frozen tumor samples. As a proof of principle, CRE is able to detect the activating EGFR L858R and T790M EGFR mutations in lung cancer cell line and primary tumors.

We are particularly grateful to Reviewer 1 for describing the study as "a well conducted proof of principle report", and Reviewer 2 for their comments that, "The methods are well described and the test is of clinical relevance, particularly in settings with limited resources and without access to tumor next generation sequencing". Further, incorporating the suggestions made by the reviewers have contributed to an improved version of the manuscript. Specifically, we have, in the revised version: In response to Reviewer 1 a) We have incorporated the suggestion of the reviewer by correcting the original submission in response their comments 1, 3, 4 and 7.
b) We have included the relevant references as pointed by the reviewer comment 2. c) We have detailed our response to rest of the queries.

Introduction
The growing significance of identifying EGFR and KRAS mutations in lung cancer using molecular diagnostic approaches underlines the emphasis on the use of personalized medical care by physicians to help design optimal therapeutic regimens (Lynch et al., 2004;Paez et al., 2004;Pao et al., 2004;Pao et al., 2005a;Pao et al., 2005b). While EGFR and KRAS mutations largely occur mutually exclusively in non-small cell lung cancer (NSCLC), and predict contrasting response rate to tyrosine-kinase inhibitors (TKI) (Chougule et al., 2013;Fukuoka et al., 2011;Ihle et al., 2012;Lynch et al., 2004;Mao et al., 2010;Mok et al., 2009), some recent studies, including ours, suggest co-occurrence of EGFR and KRAS mutations in the same patients, albeit at low frequency (Choughule et al., 2014;Li et al., 2014). While no direct evidence exists as yet, these studies may have implications for carrying out routine KRAS molecular testing along with EGFR mutations for precluding a patient with NSCLC from therapy with EGFR inhibitors, as approved for colorectal cancer (Lievre et al., 2006). Such information is especially important for lung cancer patients at an advanced-stage, who are not candidates for surgical intervention-wherein biopsy specimens obtained through fine-needle aspiration (FNA) may represent the only opportunity to obtain tissue material for diagnosis and molecular diagnostic analysis.
EGFR mutations in NSCLC are characterized by approximately 39 unique mutations present across exons 18-21. Of these, most common are activating mutations, which account for approximately 90% of all EGFR mutations and are closely related to the efficacy of EGFR-TKIs. These activating mutations include point mutations G719S, T790M, L858R, and L861Q in exons 18, 20 and 21 respectively and in-frame deletions/insertions in exon 19 (Kosaka et al., 2004). The most common mutations that result in an amino acid substitution at position 12 and 13 in KRAS are G12V and G13D (Choughule et al., 2014). Several screening and target based methods are currently in use for to infer the EGFR and KRAS hot spot mutations, viz; PCR-RFLP (Restriction fragment length polymorphism), Amplification Refractory Mutation System (ARMS), PCR-Invader, TaqMan PCR, allele specific qPCR, high resolution melting analysis and ultra-deep pyrosequencing, SNaPshot analysis and co-amplification at lower denaturation temperature (COLD)-PCR (Angulo et al., 2012;Borràs et al., 2011;Ellison et al., 2013;Santis et al., 2011;van Eijk et al., 2011;Zinsky et al., 2010). Of these, direct sequencing is the most commonly used method worldwide (Yatabe et al., 2015). However, a typical PCR reaction that precedes the sequencing step to amplify 4 EGFR and 1 KRAS exon(s) essentially consists of five rounds of independent PCR requiring separate aliquots of genomic DNA template for each reaction, followed by ten rounds of sequencing reactions. With a limited amount of genomic DNA from clinical FFPE specimens or fine biopsies of lung tumors, multiple rounds of PCR and sequencing reactions can often be challenging to perform.
In-frame concatenation or assembly of individually amplified exons from genomic DNA to generate a coding fragment has been described in earlier research, wherein the total number of PCR reactions corresponds to the number of exons to be concatenated (An et al., 2007;Fedchenko et al., 2013;Mitani et al., 2004;Tuohy & Groden, 1998). Here, we describe a novel methodology to co-amplify all four EGFR exons 18-21 along with KRAS exon 2 in a single multiplex PCR followed by directional or ordered concatenation of the products in the form of a single linear fragment. This concatenated product can be used to detect mutations by direct sequencing, at a much reduced cost and duration, and with a much smaller amount of template.

Concatenation of exons and sequencing analysis
For concatenation of KRAS exon 2 and EGFR exons 18-21, 2 µl of multiplex PCR product was used as template in a 50 µl PCR reaction containing 0.2 µM of each OAD176 and OAD152 primers. PCR was carried out in a Verity thermal cycler (Applied Biosystems) with an initial hot-start denaturation at 95°C for 15 min, followed by 35 cycle of denaturation at 94°C for 30 seconds, annealing at 57°C for 90 seconds, polymerization at 72°C for 60 seconds, and final incubation for 30 min at 60°C. Concatenated PCR product was analyzed by agarose gel electrophoresis. Sequencing of concatenated PCR products were performed by Sanger sequencing. Sequences were analyzed using Mutation Surveyor software V4.0.9 (Minton et al., 2011).

Results
CRE (Co-amplification of KRAS and EGFR) exons is a costeffective multiplex-PCR based method followed by concatenation of the PCR product as a single fragment for direct sequencing ( Figure 1). It is a robust methodology to determine the mutation status of KRAS and EGFR with reduced variability, cost and turnaround time, requiring a minimal amount of template DNA extracted from FFPE or fresh frozen tumor samples.
CRE-based KRAS-EGFR concatenation from fresh frozen primary tumors and tumor-derived cell lines Following CRE-based multiplex PCR of KRAS exon 2 and EGFR exons 18-21 with overlapping PCR bands ( Figure 2A, lane 6), concatenation of the PCR product was performed with OAD176 and OAD152 primers using genomic DNA extracted from NCI-H1975 cells, a non-small-cell lung adenocarcinoma cell line. Concatenation PCR resulted in the enrichment of a concatenated product of about 915 base pairs ( Figure 2B). This concatenated, gel purified PCR product of 915 base pair was used for Sanger sequencing. Sequencing analysis of the concatenated PCR product confirmed concatenation as a single fragment ( Figure 3) along with the presence of EGFR T790M and L585R mutations in NCI-H1975 cells (Supplementary Figure S1). A similar concatenation of a 915 bp single fragment was performed with genomic DNA extracted from fresh frozen tumor cells ( Figure 2C).

CRE-based KRAS-EGFR concatenation from paraffinembedded clinical cancer specimens
The amount of genomic DNA obtained from FFPE tissue is always limiting and thus there is a substantial need to develop a technique with a limited amount of starting DNA as a template for mutation detection. CRE demonstrates the ability to co-amplify all five exons (KRAS exon 2 and EGFR exon 18-21) in a single multiplex PCR reaction with a limited amount of starting template DNA followed by the enrichment of concatenated product ( Figure 2D) by concatenation PCR using first multiplex PCR product as a template.The concatenated product confirmed EGFR L858R mutation in the FFPE tissues (Supplementary Figure S2), as reported earlier (Choughule et al., 2014). Thus our CRE method can be routinely used for the mutational analysis of KRAS and EGFR genes.

Discussion
CRE is a novel, simple and effective strategy to concatenate multiple amplicons obtained from a multiplex PCR, using primers with overlapping complementary overhangs. Compared to ARMS, and other genotyping technologies, CRE is relatively inexpensive with faster turnaround time involving lesser amount of template genomic DNA.
Using CRE, in vitro tandem reconstitution of KRAS exon 2 with EGFR exons 18-21 can be effectively performed to generate a concatenated single PCR product of 915 bp, as a template for sequencing. Most commercially-available allele-specific and genotyping technologies are restricted by their ability to probe only for eight out of the approximately 39 known commonly occurring EGFR and KRAS activating mutations. However, growing clinical data on the less common mutations are now emerging to fully inform their predictable outcomes on EGFR TKIs (Lohinai et al., 2015;Yang et al., 2012). Currently available methodologies, if extended to genotype all known 39 mutations would not only be cost-prohibitive but challenging to perform due to a limiting amount of template genomic DNA available from clinical cancer specimens that are mostly available in the form of formalin-fixed, paraffin-embedded (FFPE) tissue. While a directed sequencing approach -classical or next-generation sequencing (NGS) -based-can determine a whole spectrum of rare and co-occurring mutations in an individual, the question of template genomic DNA availability still remains. CRE circumvents the issue of a limiting amount of template genomic DNA with increased affordability by multiplexing PCR for all exons to a single reaction and concatenating the PCR product as a single fragment, thereby further reducing the cost of multiple sequencing reactions.
In this era of genome sequencing, applicability of the CRE strategy could be of immense significance to effectively reduce the cost and turnaround time taken to determine the mutational status across the whole KRAS exon 2 and EGFR kinase domain exons. As the limitation of the CRE strategy is defined by the sensitivity and resolution of the sequencing methodology adopted, concatenated EGFR and KRAS PCR products from multiple individuals-each tagged with unique bar code sequence-can be pooled and deep-sequenced using a NGS platform. The CRE strategy described here can reduce the labor and cost of performing individual PCR for all exons for each patient and effectively circumvent the noise due to variation in normalization for equimolar pooling of exons within the same sample at a resolution of single base. Additionally, the current version of CRE is limited by exclusion of fewer number of exons of EGFR and KRAS. Inclusion of known extracellular EGFR and KRAS exon 3 codon 61 mutation may help to immediately expand the scope of its application to other cancers, such as glioblastoma.

Competing interests
The authors declared no competing interests.     The authors have provided satisfactory responses to all the concerns. Within the limits of sensitivity of Sanger sequencing, and currently limited to the the exons examined, the relatively simple, cost effective assay system presented here is a practical solution to an important clinical question in resource limited environment.

I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.
No competing interests were disclosed. In the age of precision medicine with an expanding number of oncogenic drivers in lung cancers that may be treated with targeted agents, multiplexed genomic testing is increasingly important in clinical practice. The study by Ramteke et al. describes a rapid and relatively inexpensive multiplexed test for EGFR and KRAS mutations. The methods are well described and the test is of clinical relevance, particularly in settings with limited resources and without access to tumor next generation sequencing. I recommend making the following minor revisions: In the introduction, it is incorrect to suggest that the reason for KRAS testing in lung cancers is to preclude patients from EGFR inhibitors. The rationale for EGFR inhibitors in lung cancers is very different to that of colorectal cancers, as activating EGFR mutations in lung cancers predict response to EGFR TKIs. However, it is still important to test all lung cancers for KRAS mutations as it is a common oncogenic driver occuring in over 25% of lung adenocarcinomas. Being a driver KRAS is highly unlikely to co-exist with other actionable drivers, therefore once KRAS is found one could justify that further genomic testing for other drivers is not necessary, especially in a resource 2.
could justify that further genomic testing for other drivers is not necessary, especially in a resource limited setting.
It should be acknowledged that the authors' CRE method will not capture all KRAS mutations, especially KRAS mutations in exon 3 codon 61. However, the ability to capture the majority of KRAS and EGFR mutations in one single inexpensive test is still of value for patients with lung cancers.

I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.
No competing interests were disclosed. We sincerely thank the reviewer for the elaborate and detailed constructive review. In particular, we are grateful to the reviewer for describing the study as "a well conducted proof of principle report". We hope the reviewer will find the improved version of the manuscript acceptable, without reservation. Our response to specific concerns are as follows- (Lievre et al., 2006)." Can the authors cite any reference wherein NSCLC patients are precluded from EGFR inhibitor therapy if they harbor KRAS mutations. The reference cited here is specific to colorectal cancer. and mutations occur mutually exclusive in NSCLC, which Author's response: EGFR KRAS suggests functional redundancy, however they predict contrasting response rates to tyrosine-kinase inhibitors (TKI). While mutation predicts longer progression-free survival EGFR rate, adverse prognosis is associated with patients harboring mutations. Thus, the recently KRAS reported co-occurrence of and activating mutations in 30 of 5125 patients, along with KRAS EGFR our study of co-occurrence of and activating mutations in 3 of 86 patients, raises KRAS EGFR questions about the relative value of and mutation status as predictors of outcome in EGFR KRAS NSCLC. As the reviewer may agree these studies may have obvious implications for routine KRAS testing in this disease, potentially precluding EGFR TKI therapy from some patients, similar to current practice in colorectal cancer. However, their direct mention in NSCLC is speculative.

Referee's comment 1: Introduction, "These studies have direct implications for carrying out routine KRAS molecular testing along with EGFR mutations for precluding a patient with NSCLC from therapy with EGFR inhibitors, as approved for colorectal cancer
Thus, in principle, we fully agree with the reviewer that no direct evidence exists to preclude EGFR inhibitor therapy among patients co-harboring and mutation. In accordance with the EGFR KRAS reviewer's suggestion we have revised the text to reflect the speculative implication of our methodology in NSCLC. Our modified text reads as follows: "….While no evidence exists as yet, these studies may have implications for carrying out routine KRAS molecular testing along with EGFR mutations for precluding a patient with NSCLC from therapy with EGFR inhibitors, as approved for colorectal cancer (Lievre et al., 2006)…." Referee's comment 2: Introduction, "Of these direct sequencing is the most commonly used worldwide". Is this a personal opinion or there is a reference to support this. Should be method cited.
We thank the reviewer for pointing the omission. A relevant citation has been Author's response: added. However, as our citation in manuscript is likely to be incomplete to summarize the field, some additional studies are mentioned below: We agree and thank the reviewer for bringing it our attention. Our modified Author's response: text reads as follows: "….concatenation or assembly of individually amplified exons from genomic DNA to generate a coding fragment has been described in earlier research…".
Referee's comment 4: Introduction, "Here, we describe to co-amplify all a novel methodology four EGFR exons 18-21 along with KRAS exon 2 in a single multiplex PCR". It's more like a novel application of a well described methodology supported by several previous references. The novelty, albeit rather incremental, is in combining exons from two different genes, using previously described approach. Should be stated as such.
As suggested by the reviewer, we have dropped the term "novel". Our Author's response: modified text reads as follows: "….Here, we describe a methodology to co-amplify all four exons 18-21 along with EGFR KRAS exon 2 in a single multiplex PCR…."

Referee's comment 5:
The big appeal of the study is that it affords use of a minimal amount of FFPE sample. Please specify the amount of FFPE material used and yield of DNA to convey an idea of how little/much sample is needed to carry out this analysis.
As mentioned in the methodology section subtitled, "Multiplex PCR of Author's response: KRAS exon 2 and exons 18-21", multiplex PCR (50µl per reaction) was carried out in a single tube EGFR by using multiplex PCR kit (Qiagen) containing either 10 ng of genomic DNA from the NSCLC cell line or fresh frozen primary tumor, or 50 ng of genomic DNA from FFPE blocks. Furthermore, as mentioned under the methodology section subtitled, "Concatenation of exons and sequencing analysis", 2 µl of multiplex PCR product was used as template in a 50 µl PCR reaction for analysis", 2 µl of multiplex PCR product was used as template in a 50 µl PCR reaction for concatenation.

Referee's comment 6:
The important consideration of sensitivity has not been addressed. This could be easily tested by assaying a serial dilution of known mutated cell line/FFPE DNA spiked in a wild-type background sample. This will add value to the study.
As mentioned under the last paragraph of the discussion section, "….As the Author's response: limitation of the CRE strategy is defined by the sensitivity and resolution of the sequencing methodology adopted…" -which in this study has been Sanger Sequencing, but could significantly vary if advanced contemporary sequencing methodologies are adopted. However, as the sensitivity of PCR followed by directed Sanger Sequencing is well established from FFPE samples and mutated cell line, we humbly differ from the reviewer that admixture experiment would add additional information.

Referee's comment 7: Addition of KRAS codon 61 should be considered as well. Or the difficulty in scaling up should be discussed. How difficult is it to add additional exons.
codon 12 is mutated at a frequency of 25-50% in Caucasian Author's response: KRAS population and 5-15% among East Asians. In a recent study we reported 18.6% codon 12 KRAS among Indian population (n=86)-- (Choughule , 2014). Given that codon 61 mutation et al.
KRAS exist at frequency < 1%; and, that none were found in our study in 86 patients, we decided to not include codon 61 mutation in this study to only present the proof of principle of the CRE KRAS methodology. However, we do agree with the reviewer about the significance of codon 61 KRAS mutation, and do hope to include it along with other known activating mutations in NSCLC in an improved version of CRE.
However, to reflect the pertinent suggestion made by the reviewer we have modified our discussion to read as follows: "…Additionally, the current version of CRE is limited by exclusion of fewer number of exons of and . Inclusion of known extracellular and exon 3 codon 61 mutation EGFR KRAS EGFR KRAS may help to immediately expand the scope of its application to other cancers, such as glioblastoma ." Referee's comment 8: The concatenated PCR product may be amenable to Pyrosequencing to improve sensitivity of detection (particularly in case of low tumor content, low clonality of mutations as is expected in case of dynamically evolving tumors). This should be attempted/ discussed.
We fully agree with the reviewer's insights that CRE can be utilized at high Author's response: throughput mode to determine complete spectrum of and mutations using targeted EGFR KRAS next generation sequencing. Consistent with the reviewer's suggestion the last paragraph of the discussion section, "… the limitation of the CRE strategy is defined by the sensitivity and resolution of the sequencing methodology adopted, concatenated and PCR products from EGFR KRAS multiple individuals-each tagged with unique bar code sequence-can be pooled and deep-sequenced using a NGS platform. The CRE strategy described here can reduce the labor and cost of performing individual PCR for all exons for each patient and effectively circumvent the noise due to variation in normalization for equimolar pooling of exons within the same sample at a resolution of single base."

Referee's comment 9: A direct comparison with the standard technique(s) currently used to test
Referee's comment 9: A direct comparison with the standard technique(s) currently used to test these mutations-in terms of amount of starting material needed, sensitivity of detection, time, and cost will help the argument of the new approach as a superior option.
As detailed in the manuscript, this proof of principle study introduces CRE as Author's response: a methodology involving single multiplex-PCR followed by concatenation of the PCR product as one linear fragment for direct sequencing, as opposed to 5 rounds of PCR reaction followed by 10 rounds of sequencing reactions. A systematic comparative analysis is currently underway at our center using clinical cancer specimens for CRE compared to Sanger sequencing based methodology; SNaPShot PCR; Cobas system; Mass spec genotyping on a larger cohort sample, beyond the scope of this manuscript. Hence, we express our inability to include analysis from this ongoing study at this early on stage. We sincerely thank reviewer for approving our submission. We are particularly grateful to the reviewer for describing the study as, "The methods are well described and the test is of clinical relevance, particularly in settings with limited resources and without access to tumor next The suggestions made by the reviewers have contributed to an improved generation sequencing". version of the manuscript. Specifically, we have, in the revised version: Referee's comment 1: In the introduction, it is incorrect to suggest that the reason for KRAS testing in lung cancers is to preclude patients from EGFR inhibitors. The rationale for EGFR inhibitors in lung cancers is very different to that of colorectal cancers, as activating EGFR mutations in lung cancers predict response to EGFR TKIs. However, it is still important to test all lung cancers for KRAS mutations as it is a common oncogenic driver occuring in over 25% of lung adenocarcinomas. Being a driver KRAS is highly unlikely to co-exist with other actionable drivers, therefore once KRAS is found one could justify that further genomic testing for other drivers is not necessary, especially in a resource limited setting.
As described in or response to Reviewer 1's first comment, we agree we with Author's response: the reviewer that no direct evidence exists to preclude EGFR inhibitor therapy among patients co-harboring and mutation. In accordance with the reviewer's suggestion we have EGFR KRAS revised the text to reflect the speculative implication of our methodology in NSCLC. Our modified text reads as follows: "….While no evidence exists as yet, these studies may have implications for carrying out routine KRAS molecular testing along with EGFR mutations for precluding a patient with NSCLC from therapy with EGFR inhibitors, as approved for colorectal cancer (Lievre et al., 2006)

1.
It's more like a novel application of a well described methodology supported by several previous references. The novelty, albeit rather incremental, is in combining exons from two different genes, using previously described approach. Should be stated as such.
The big appeal of the study is that it affords use of a minimal amount of FFPE sample. Please specify the amount of FFPE material used and yield of DNA to convey an idea of how little/much sample is needed to carry out this analysis.
The important consideration of sensitivity has not been addressed. This could be easily tested by assaying a serial dilution of known mutated cell line/FFPE DNA spiked in a wild-type background sample. This will add value to the study.
Addition of KRAS codon 61 should be considered as well. Or the difficulty in scaling up should be discussed. How difficult is it to add additional exons.
The concatenated PCR product may be amenable to Pyrosequencing to improve sensitivity of detection (particularly in case of low tumor content, low clonality of mutations as is expected in case of dynamically evolving tumors). This should be attempted/ discussed.
A direct comparison with the standard technique(s) currently used to test these mutations-in terms of amount of starting material needed, sensitivity of detection, time, and cost will help the argument of the new approach as a superior option.
I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.
No competing interests were disclosed.

Competing Interests:
Author Response 31 Jul 2015 , ACTREC, Tata Memorial Center,, India Amit Dutt We thank the reviewer for describing the study as "a well conducted proof of principle report", and for sharing elaborate comments and suggestions that has significantly improved the quality of the manuscript. Our response to specific concerns are as follows: Referee's comments: Introduction, "These studies have direct implications for carrying out routine KRAS molecular testing along with EGFR mutations for precluding a patient with from therapy with EGFR inhibitors, as approved for colorectal cancer (Li NSCLC èvre et al., 2006)." Can the authors cite any reference wherein NSCLC patients are precluded from EGFR inhibitor therapy if they harbor KRAS mutations. The reference cited here is specific to colorectal cancer. and mutations occur mutually exclusive in NSCLC, Author's response: EGFR KRAS which suggests functional redundancy. However, they predict contrasting response rate to tyrosine-kinase inhibitors (TKI). While mutation predicts longer progression-free EGFR survival rate, adverse prognosis is associated with patients harboring mutations. KRAS Thus, the recently reported co-occurrence of and activating mutations in 30 of KRAS EGFR 5125 patients, along with our study of co-occurrence of and activating KRAS EGFR mutations in 3 of 86 patients, raises a clinical concern about the relative value of and EGFR mutation status as predictors of outcome in NSCLC. As the reviewer may agree KRAS 8.

9.
We submit that based on literature, additional exons can be added to the current methodology, as at least up to 10 genomic spliced exons fragment of 2295 bp has been described in literature using similar methodology.

Referee's comments:
The concatenated PCR product may be amenable to Pyrosequencing to improve sensitivity of detection (particularly in case of low tumor content, low clonality of mutations as is expected in case of dynamically evolving tumors). This should be attempted/ discussed. Author's response: We fully agree with the reviewer's insights that CRE can be utilized at high throughput mode to determine complete spectrum of and mutations using EGFR KRAS targeted next generation sequencing. Consistent with the reviewer's suggestion the last paragraph of the discussion section reads, "… the limitation of the CRE strategy is defined by the sensitivity and resolution of the sequencing methodology adopted, concatenated and PCR products from multiple individuals-each tagged with unique bar EGFR KRAS code sequence-can be pooled and deep-sequenced using a NGS platform. The CRE strategy described here can reduce the labor and cost of performing individual PCR for all exons for each patient and effectively circumvent the noise due to variation in normalization for equimolar pooling of exons within the same sample at a resolution of single base."

Referee's comments: A direct comparison with the standard technique(s) currently used
to test these mutations-in terms of amount of starting material needed, sensitivity of detection, time, and cost will help the argument of the new approach as a superior option.
Author's response: As detailed in the manuscript, this proof of principle study introduces CRE as a methodology involving single multiplex-PCR followed by concatenation of the PCR product as one linear fragment for direct sequencing, as opposed to 5 rounds of PCR reaction followed by 10 rounds of sequencing reactions. A systematic comparative analysis is currently underway at our center using clinical cancer specimens for CRE compared to Sanger sequencing based methodology; SNaPShot PCR; Cobas system; Mass spec genotyping on a larger cohort sample, beyond the scope of this manuscript. Hence, we express our inability to include analysis from this ongoing study at this early on stage. We sincerely thank the reviewer for the elaborate and detailed constructive review. Hope the reviewer will find the improved version of the manuscript acceptable for indexation.
No competing interests were disclosed. Competing Interests: