Reviewer ’s report  
 
Title: Identification of novel alternative splicing isoform biomarkers and their association with  
overall survival in colorectal cancer  
 
Version: 0 Date: 15 Jul 2019  
 
Reviewer: Malgorzata Komor  
 
Reviewer's repo rt: 
 
In the manuscript "Identification of novel alternative splicing isoform biomarkers and their 
association with overall survival in color ectal cancer" by Wang et al. the authors performed 
differential splicing analysis in normal colon, colorectal cancer (CRC) and metastatic tissue 
samples. The aim of this study was not precisely defined but includes alternative splicing 
characterization in CRC and metastatic CRC and association of alternative splicing with overall 
survival. Even though the results of this study are intriguing, the way the manuscript was written 
does not allow a proper evaluation of the methods applied and the data generated.   
Major concerns:  
1. Aim of the study is not clearly defined. Especially, in the context of Zong, Li et al. 
2018, the novelty of the current study should be better defined.  
2. In the Introduction the authors claim "(…) it's still unclear what the most comm on 
alternative splicing events are in large CRC patient populations". I disagree and suggest the 
authors refer to the following:  
a. Snezhkina, Krasnov et al. 2016  
b. Climente -Gonzalez, Porta -Pardo et al. 2017  
c. Kahles, Lehmann et al. 2018  
3. Materials an d methods are not sufficiently described:  
a. Number of samples in each cohort should be described in materials and methods.  
b. How were RPM values obtained?  
c. Custom perl script should be presented in the manuscript or published. The explanation 
what the script does should be more detailed.  
d. What were the thresholds applied for DEXSeq results?  
e. What were the thresholds applied for the PSI analysis?  
f. How ma ny TCGA samples were used, in detail: how many primary tumors, metastatic 
samples or normal colon samples? E.g. "431 CRC or metastatic samples" is not precise enough.  
4. The results are not sufficiently described:  
a. It is unclear what the research questi ons for some paragraph were, e.g. why is CD44 in 
a separate paragraph than CLK1 and COL6A3?  
b. Are CLK1, COL6A3 and CD44 results of DEXSeq or PSI analysis?  
c. How many cohorts were analyzed for each research question? It is unclear why cohort 
CRC3P is incl uded or excluded in different paragraphs.  
d. What were the criteria for candidate selection? Do they differ per paragraph?  
e. How the authors handle using two different analyses: DEXSeq and PSI calculation? 
Are the results a union/overlap between the meth ods? Often it is unclear which candidate came 
from which method?  
f. The Figures do not match the tables. E.g. Supplementary Table 3 has 84 events, while 
the Venn diagram from Figure 2B has 34, was there any additional filtering performed?  
g. The DEXSeq res ults for non -terminal exons seem to be missing.  
h. Column names in the Supplementary Tables are unclear and should be explained.  
i. It is unclear how metastatic isoforms were identified; Figure 1 suggests comparison MS 
vs NC, while the Results state "We se lected a subset of splicing events with the further increase 
or decrease of PSI values in metastatic tissues compared to CRC."  
j. The number of TCGA samples mentioned in the Introduction/Results and Discussion 
do not match.  
5. The authors mention the splic ing signature of 10 genes, which is confusing as the 10 
genes were never analyzed together, in a signature manner, for any of the paragraphs. The genes 
seem to be always considered separately, therefore, the phrasing "signature" is not adequate in 
this con text. 
6. The discussion is intermingled in the Results section.  
 
Other remarks:  
1. References to the original publications from which the data was obtained are missing.  
2. The alternative splicing events are not presented consistently in the same way 
throu ghout the manuscript. It is unclear why different visualizations were chosen for different 
events and makes the reader confused about the results.  
a. The alternating usage of IGV Sashimi plots and UCSC visualization is unclear.  
b. Not all the events are pr esented in the boxplots, e.g. SERPINA1, FBLN2, CALD1 in 
the CRC18P cohort.  
c. Not all the events are presented in the heatmap.  
3. Was the overlap from Figure 2B significant?  
4. What is the distribution of PSI values? Can the authors justify using t -test?  
5. Experimental validation of the identified isoforms (e.g. RT -qPCR) would strengthen 
the manuscript, especially when a candidate was defined solely based on PSI calculation with 
thresholds arbitrarily chosen by the authors.  
6. In the validation of splicin g events using TCGA data p -values should be adjusted for 
multiple hypothesis testing.  
7. The authors mention that some of the isoforms are expressed in stroma. The authors 
could further discuss if the abundance of stroma in different samples may have an im pact on 
differential splicing analysis.  
 
 
Are the methods appropriate and well described?  
If not, please specify what is required in your comments to the authors.  
 
No 
 
Does the work include the necessary controls?  
If not, please  specify which controls are required in your comments to the authors.  
 
Yes 
 
Are the conclusions drawn adequately supported by the data shown?  
If not, please explain in your comments to the authors.  
 
No 
 
Are you able to assess any statistics in the manuscript o r would you recommend an  
additional statistical review?  
If an additional statistical review is recommended, please specify what aspects require further  
assessment in your comments to the editors.  
 
I am able to assess the statistics  
 
Quality of written Englis h 
Please indicate the quality of language in the manuscript:  
 
Needs some language corrections before being published  
 
Declaration of competing interests  
Please complete a declaration of competing interests, considering the following questions:  
 
1. Have you in  the past five years received reimbursements, fees, funding, or salary from an  
organisation that may in any way gain or lose financially from the publication of this  
manuscript, either now or in the future?  
 
2. Do you hold any stocks or shares in an organisation that may in any way gain or lose  
financially from the publication of this manuscript, either now or in the future?  
 
3. Do you hold or are you currently applying for any patents relating to the content of the  
manuscript?  
 
4. Have you receiv ed reimbursements, fees, funding, or salary from an organization that 
holds  or has applied for patents relating to the content of the manuscript?  
 
5. Do you have any other financial competing interests?  
 
6. Do you have any non -financial competing interests i n relation to this paper?  
 
If you can answer no to all of the above, write 'I declare that I have no competing interests' 
below.  If your reply is yes to any, please give details below.  
 
I declare that I have no competing interests  
 
I agree to the open peer re view policy of the journal. I understand that my name will be included  
on my report to the authors and, if the manuscript is accepted for publication, my named report  
including any attachments I upload will be posted on the website along with the authors'  
responses. I agree for my report to be made available under an Open Access Creative Commons  
CC-BY license ( http://creativecommons.org/licenses/by/4.0/ ). I understand that any comments  
which I do not wish to be included in my named report can be included as  confidential comments  
to the editors, which will not be published.  
 
I agree to the open peer review policy of the journal  
Reviewer ’s report  
 
Title: Identification of novel alternative splicing isoform biomarkers and their association with  
overall survival in colorectal cancer  
 
Version: 0 Date: 15 Jul 2019  
 
Reviewer: Malgorzata Komor  
 
Reviewer's repo rt: 
 
In the manuscript "Identification of novel alternative splicing isoform biomarkers and their 
association with overall survival in color ectal cancer" by Wang et al. the authors performed 
differential splicing analysis in normal colon, colorectal cancer (CRC) and metastatic tissue 
samples. The aim of this study was not precisely defined but includes alternative splicing 
characterization in CRC and metastatic CRC and association of alternative splicing with overall 
survival. Even though the results of this study are intriguing, the way the manuscript was written 
does not allow a proper evaluation of the methods applied and the data generated.   
Major concerns:  
1. Aim of the study is not clearly defined. Especially, in the context of Zong, Li et al. 
2018, the novelty of the current study should be better defined.  
2. In the Introduction the authors claim "(…) it's still unclear what the most comm on 
alternative splicing events are in large CRC patient populations". I disagree and suggest the 
authors refer to the following:  
a. Snezhkina, Krasnov et al. 2016  
b. Climente -Gonzalez, Porta -Pardo et al. 2017  
c. Kahles, Lehmann et al. 2018  
3. Materials an d methods are not sufficiently described:  
a. Number of samples in each cohort should be described in materials and methods.  
b. How were RPM values obtained?  
c. Custom perl script should be presented in the manuscript or published. The explanation 
what the script does should be more detailed.  
d. What were the thresholds applied for DEXSeq results?  
e. What were the thresholds applied for the PSI analysis?  
f. How ma ny TCGA samples were used, in detail: how many primary tumors, metastatic 
samples or normal colon samples? E.g. "431 CRC or metastatic samples" is not precise enough.  
4. The results are not sufficiently described:  
a. It is unclear what the research questi ons for some paragraph were, e.g. why is CD44 in 
a separate paragraph than CLK1 and COL6A3?  
b. Are CLK1, COL6A3 and CD44 results of DEXSeq or PSI analysis?  
c. How many cohorts were analyzed for each research question? It is unclear why cohort 
CRC3P is incl uded or excluded in different paragraphs.  
d. What were the criteria for candidate selection? Do they differ per paragraph?  
e. How the authors handle using two different analyses: DEXSeq and PSI calculation? 
Are the results a union/overlap between the meth ods? Often it is unclear which candidate came 
from which method?  
f. The Figures do not match the tables. E.g. Supplementary Table 3 has 84 events, while 
the Venn diagram from Figure 2B has 34, was there any additional filtering performed?  
g. The DEXSeq res ults for non -terminal exons seem to be missing.  
h. Column names in the Supplementary Tables are unclear and should be explained.  
i. It is unclear how metastatic isoforms were identified; Figure 1 suggests comparison MS 
vs NC, while the Results state "We se lected a subset of splicing events with the further increase 
or decrease of PSI values in metastatic tissues compared to CRC."  
j. The number of TCGA samples mentioned in the Introduction/Results and Discussion 
do not match.  
5. The authors mention the splic ing signature of 10 genes, which is confusing as the 10 
genes were never analyzed together, in a signature manner, for any of the paragraphs. The genes 
seem to be always considered separately, therefore, the phrasing "signature" is not adequate in 
this con text. 
6. The discussion is intermingled in the Results section.  
 
Other remarks:  
1. References to the original publications from which the data was obtained are missing.  
2. The alternative splicing events are not presented consistently in the same way 
throu ghout the manuscript. It is unclear why different visualizations were chosen for different 
events and makes the reader confused about the results.  
a. The alternating usage of IGV Sashimi plots and UCSC visualization is unclear.  
b. Not all the events are pr esented in the boxplots, e.g. SERPINA1, FBLN2, CALD1 in 
the CRC18P cohort.  
c. Not all the events are presented in the heatmap.  
3. Was the overlap from Figure 2B significant?  
4. What is the distribution of PSI values? Can the authors justify using t -test?  
5. Experimental validation of the identified isoforms (e.g. RT -qPCR) would strengthen 
the manuscript, especially when a candidate was defined solely based on PSI calculation with 
thresholds arbitrarily chosen by the authors.  
6. In the validation of splicin g events using TCGA data p -values should be adjusted for 
multiple hypothesis testing.  
7. The authors mention that some of the isoforms are expressed in stroma. The authors 
could further discuss if the abundance of stroma in different samples may have an im pact on 
differential splicing analysis.  
 
 
Are the methods appropriate and well described?  
If not, please specify what is required in your comments to the authors.  
 
No 
 
Does the work include the necessary controls?  
If not, please  specify which controls are required in your comments to the authors.  
 
Yes 
 
Are the conclusions drawn adequately supported by the data shown?  
If not, please explain in your comments to the authors.  
 
No 
 
Are you able to assess any statistics in the manuscript o r would you recommend an  
additional statistical review?  
If an additional statistical review is recommended, please specify what aspects require further  
assessment in your comments to the editors.  
 
I am able to assess the statistics  
 
Quality of written Englis h 
Please indicate the quality of language in the manuscript:  
 
Needs some language corrections before being published  
 
Declaration of competing interests  
Please complete a declaration of competing interests, considering the following questions:  
 
1. Have you in  the past five years received reimbursements, fees, funding, or salary from an  
organisation that may in any way gain or lose financially from the publication of this  
manuscript, either now or in the future?  
 
2. Do you hold any stocks or shares in an organisation that may in any way gain or lose  
financially from the publication of this manuscript, either now or in the future?  
 
3. Do you hold or are you currently applying for any patents relating to the content of the  
manuscript?  
 
4. Have you receiv ed reimbursements, fees, funding, or salary from an organization that 
holds  or has applied for patents relating to the content of the manuscript?  
 
5. Do you have any other financial competing interests?  
 
6. Do you have any non -financial competing interests i n relation to this paper?  
 
If you can answer no to all of the above, write 'I declare that I have no competing interests' 
below.  If your reply is yes to any, please give details below.  
 
I declare that I have no competing interests  
 
I agree to the open peer re view policy of the journal. I understand that my name will be included  
on my report to the authors and, if the manuscript is accepted for publication, my named report  
including any attachments I upload will be posted on the website along with the authors'  
responses. I agree for my report to be made available under an Open Access Creative Commons  
CC-BY license ( http://creativecommons.org/licenses/by/4.0/ ). I understand that any comments  
which I do not wish to be included in my named report can be included as  confidential comments  
to the editors, which will not be published.  
 
I agree to the open peer review policy of the journal  
Please review this paper.
user
Reviewer ’s report  
 
Title: Identification of novel alternative splicing isoform biomarkers and their association with  
overall survival in colorectal cancer  
 
Version: 0 Date: 15 Jul 2019  
 
Reviewer: Malgorzata Komor  
 
Reviewer's repo rt: 
 
In the manuscript "Identification of novel alternative splicing isoform biomarkers and their 
association with overall survival in color ectal cancer" by Wang et al. the authors performed 
differential splicing analysis in normal colon, colorectal cancer (CRC) and metastatic tissue 
samples. The aim of this study was not precisely defined but includes alternative splicing 
characterization in CRC and metastatic CRC and association of alternative splicing with overall 
survival. Even though the results of this study are intriguing, the way the manuscript was written 
does not allow a proper evaluation of the methods applied and the data generated.   
Major concerns:  
1. Aim of the study is not clearly defined. Especially, in the context of Zong, Li et al. 
2018, the novelty of the current study should be better defined.  
2. In the Introduction the authors claim "(…) it's still unclear what the most comm on 
alternative splicing events are in large CRC patient populations". I disagree and suggest the 
authors refer to the following:  
a. Snezhkina, Krasnov et al. 2016  
b. Climente -Gonzalez, Porta -Pardo et al. 2017  
c. Kahles, Lehmann et al. 2018  
3. Materials an d methods are not sufficiently described:  
a. Number of samples in each cohort should be described in materials and methods.  
b. How were RPM values obtained?  
c. Custom perl script should be presented in the manuscript or published. The explanation 
what the script does should be more detailed.  
d. What were the thresholds applied for DEXSeq results?  
e. What were the thresholds applied for the PSI analysis?  
f. How ma ny TCGA samples were used, in detail: how many primary tumors, metastatic 
samples or normal colon samples? E.g. "431 CRC or metastatic samples" is not precise enough.  
4. The results are not sufficiently described:  
a. It is unclear what the research questi ons for some paragraph were, e.g. why is CD44 in 
a separate paragraph than CLK1 and COL6A3?  
b. Are CLK1, COL6A3 and CD44 results of DEXSeq or PSI analysis?  
c. How many cohorts were analyzed for each research question? It is unclear why cohort 
CRC3P is incl uded or excluded in different paragraphs.  
d. What were the criteria for candidate selection? Do they differ per paragraph?  
e. How the authors handle using two different analyses: DEXSeq and PSI calculation? 
Are the results a union/overlap between the meth ods? Often it is unclear which candidate came 
from which method?  
f. The Figures do not match the tables. E.g. Supplementary Table 3 has 84 events, while 
the Venn diagram from Figure 2B has 34, was there any additional filtering performed?  
g. The DEXSeq res ults for non -terminal exons seem to be missing.  
h. Column names in the Supplementary Tables are unclear and should be explained.  
i. It is unclear how metastatic isoforms were identified; Figure 1 suggests comparison MS 
vs NC, while the Results state "We se lected a subset of splicing events with the further increase 
or decrease of PSI values in metastatic tissues compared to CRC."  
j. The number of TCGA samples mentioned in the Introduction/Results and Discussion 
do not match.  
5. The authors mention the splic ing signature of 10 genes, which is confusing as the 10 
genes were never analyzed together, in a signature manner, for any of the paragraphs. The genes 
seem to be always considered separately, therefore, the phrasing "signature" is not adequate in 
this con text. 
6. The discussion is intermingled in the Results section.  
 
Other remarks:  
1. References to the original publications from which the data was obtained are missing.  
2. The alternative splicing events are not presented consistently in the same way 
throu ghout the manuscript. It is unclear why different visualizations were chosen for different 
events and makes the reader confused about the results.  
a. The alternating usage of IGV Sashimi plots and UCSC visualization is unclear.  
b. Not all the events are pr esented in the boxplots, e.g. SERPINA1, FBLN2, CALD1 in 
the CRC18P cohort.  
c. Not all the events are presented in the heatmap.  
3. Was the overlap from Figure 2B significant?  
4. What is the distribution of PSI values? Can the authors justify using t -test?  
5. Experimental validation of the identified isoforms (e.g. RT -qPCR) would strengthen 
the manuscript, especially when a candidate was defined solely based on PSI calculation with 
thresholds arbitrarily chosen by the authors.  
6. In the validation of splicin g events using TCGA data p -values should be adjusted for 
multiple hypothesis testing.  
7. The authors mention that some of the isoforms are expressed in stroma. The authors 
could further discuss if the abundance of stroma in different samples may have an im pact on 
differential splicing analysis.  
 
 
Are the methods appropriate and well described?  
If not, please specify what is required in your comments to the authors.  
 
No 
 
Does the work include the necessary controls?  
If not, please  specify which controls are required in your comments to the authors.  
 
Yes 
 
Are the conclusions drawn adequately supported by the data shown?  
If not, please explain in your comments to the authors.  
 
No 
 
Are you able to assess any statistics in the manuscript o r would you recommend an  
additional statistical review?  
If an additional statistical review is recommended, please specify what aspects require further  
assessment in your comments to the editors.  
 
I am able to assess the statistics  
 
Quality of written Englis h 
Please indicate the quality of language in the manuscript:  
 
Needs some language corrections before being published  
 
Declaration of competing interests  
Please complete a declaration of competing interests, considering the following questions:  
 
1. Have you in  the past five years received reimbursements, fees, funding, or salary from an  
organisation that may in any way gain or lose financially from the publication of this  
manuscript, either now or in the future?  
 
2. Do you hold any stocks or shares in an organisation that may in any way gain or lose  
financially from the publication of this manuscript, either now or in the future?  
 
3. Do you hold or are you currently applying for any patents relating to the content of the  
manuscript?  
 
4. Have you receiv ed reimbursements, fees, funding, or salary from an organization that 
holds  or has applied for patents relating to the content of the manuscript?  
 
5. Do you have any other financial competing interests?  
 
6. Do you have any non -financial competing interests i n relation to this paper?  
 
If you can answer no to all of the above, write 'I declare that I have no competing interests' 
below.  If your reply is yes to any, please give details below.  
 
I declare that I have no competing interests  
 
I agree to the open peer re view policy of the journal. I understand that my name will be included  
on my report to the authors and, if the manuscript is accepted for publication, my named report  
including any attachments I upload will be posted on the website along with the authors'  
responses. I agree for my report to be made available under an Open Access Creative Commons  
CC-BY license ( http://creativecommons.org/licenses/by/4.0/ ). I understand that any comments  
which I do not wish to be included in my named report can be included as  confidential comments  
to the editors, which will not be published.  
 
I agree to the open peer review policy of the journal  
assistant