The Integration of Data from Different Long-Read Sequencing Platforms Enhances Proteoform Characterization in Arabidopsis
Round 1
Reviewer 1 Report
In their manuscript, García-Campa et al. study how sequencing technologies that generate long reads, such as PacBio's Single Molecule, Real-Time (SMRT) technology, are helping to establish more complete reference transcriptomes, and the impact of such transcriptomes on the identification of different proteins in large-scale protein identification experiments using proteomics techniques. The authors demonstrate that incorporating data from Iso-seq and ONT-DRS into transcriptomes enables the identification of a greater number of proteoforms than using transcriptomes derived from short reads only. Although some of these results may seem trivial, the conclusions reached by the authors are valuable and highlight the importance of using high quality transcriptomes to accurately identify a greater number of proteins in experimental samples.
Overall, the manuscript reads very well. I came across a couple of typographical errors that the authors might want to fix:
- line 114: change "build" to "built"
- l. 146: ATONT-DRS -> AtONT-DRS
- l. 273: leave -> leaf
- l. 275: probed -> proved?
- Please spell out DRS (direct RNA sequencing)
- the authors might want to revise their use of "what" throughout the ext (e.g. on line 420)
Author Response
We are grateful for nice and useful comments that helped us to improve the manuscript. We replied to comments below:
- line 114: change "build" to "built"
The typo was corrected.
- l. 146: ATONT-DRS -> AtONT-DRS
Corrected.
- l. 273: leave -> leaf
Done.
- l. 275: probed -> proved?
Done.
- Please spell out DRS (direct RNA sequencing)
Done first time it appears in the body text (line 59). The same was done for Iso-Seq (line 58).
- the authors might want to revise their use of "what" throughout the ext (e.g. on line 420)
We did as suggested and some changes were made in different parts of the manuscript (lines 354, 433).
Reviewer 2 Report
In this manuscript, the authors reported that the combination of ONT-DRS and Iso-Seq data enhances proteoform characterization, by using Arabidopsis as an example. Because protein identification is a central step in bottom-up proteomics workflow, this study likely will be of great interests not only for plant scientists, but also for animal and microbiologist. Specific comments are as follows:
1. It would be better to add a summary sentence for Figure 1, Figure 2, Figure3, and figure 4.
2. In figure 1AB figure 2B and figure 3B, “frecuency” should be changed into “frequency”.
3. Line 222, try not to use “novel” in this scenario.
4. Some of the paragraphs are too long, for instance, the 2nd paragraph of the introduction section and the 2nd paragraph of the discussion section.
5. This study use Arabidopsis as an example to demonstrate that The combination of ONT-DRS and Iso-Seq data enhances proteoform characterization. The author should have a separated paragraph to discuss to what extent the approach/thought can also be used for other species.
Author Response
Thank you for useful and convenient comments for improving our manuscript. Our replies to specific comments follows:
- It would be better to add a summary sentence for Figure 1, Figure 2, Figure3, and figure 4.
We added a summary sentence to figure legends as suggested.
- In figure 1AB figure 2B and figure 3B, “frecuency” should be changed into “frequency”.
Typo corrected in the new version of the figures.
- Line 222, try not to use “novel” in this scenario.
Lines 224-225: we changed “novel” by “a higher number of”.
- Some of the paragraphs are too long, for instance, the 2ndparagraph of the introduction section and the 2nd paragraph of the discussion section.
We revised paragraph organization in both the intro and the discussion to avoid long paragraphs.
- This study use Arabidopsis as an example to demonstrate thatThe combination of ONT-DRS and Iso-Seq data enhances proteoform characterization. The author should have a separated paragraph to discuss to what extent the approach/thought can also be used for other species.
Following reviewer´s suggestion, we have added a short paragraph covering the potential applicability of our combined approach to other species at the end of the discussion (lines 402 – 412).
