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We, the students of MICI5029/5049, a Graduate Level Molecular Pathogenesis Journal Club at Dalhousie University in Halifax, NS, Canada, hereby submit a review of the following BioRxiv preprint:

Stop codon recoding is widespread in diverse phage lineages and has the potential to regulate translation of late stage and lytic genes 

Adair L. Borges, Yue Clare Lou, Rohan Sachdeva, Basem Al-Shayeb, Alexander L. Jaffe, Shufei Lei, Joanne M. Santini, Jillian F. Banfield. 2021.08.26.457843; doi: https://doi.org/10.1101/2021.08.26.457843

We will adhere to the Universal Principled (UP) Review guidelines proposed in:

Universal Principled Review: A Community-Driven Method to Improve Peer Review. Krummel M, Blish C, Kuhns M, Cadwell K, Oberst A, Goldrath A, Ansel KM, Chi H, O'Connell R, Wherry EJ, Pepper M; Future Immunology Consortium. Cell. 2019 Dec 12;179(7):1441-1445. doi: 10.1016/j.cell.2019.11.029.

SUMMARY: The authors investigated expanded genetic codes in bacteriophage genomes. Bacteriophages from humans, pigs, baboons, cattle and horses were evaluated for TAG or TGA stop codon recoding. Alternatively coded genomes were more likely to contain TAG recoding than TGA and were found in low abundance in a human “western diet” cohort. To understand the phylogenetic relationship between families of alternatively coded phages, the authors constructed a tree of large terminase subunits from AC phages and standard code relatives. They found that TGA was often reassigned to tryptophan and TAG was often reassigned to glycine. Suppressor tRNAs allow for alternate coded organisms to access the translational machinery of hosts that use a standard genetic code. Almost half of the alternate code TGA- or TAG-using phages used suppressor tRNAs, suggesting this could be a mechanism used to overcome limitations of host translational machinery. Divergent stop-codon recoding strategies were observed amongst phage with very similar genomes suggesting rapid evolution of recoding potential. The frequency of alternative code usage in phage genomes supports a link between lysis regulation and code change. This link was termed the code change model for Jade phages and consists of a suppressor tRNA, a tRNA synthetase, and a release factor directly upstream of the lysis cassette. Overall, the investigation of alternative codon placement suggests that phages use alternate codes to control gene expression and timing. This is supported by placement of alternate stop codons in Garnet and Topaz genomes; these phages have alternate codons in lytic genes, as standard code does not provide stop codons for lytic genes such as integrase. This finding suggests that these phages alter the codon use of the host to regulate the latent/lytic switch. Overall, this pre-print provides evidence that stop-codon recoding in phages and prophages is used to expand the viral proteome.

OVERALL ASSESSMENT: 

STRENGTHS: 

- The manuscript is well-written, and figures and data analysis are explained in the body of the paper, making the text easier to digest for the reader.

- Overall conclusions are well supported by the data. 

- The dataset supports the hypothesis that phage use alternate genetic codes to control gene expression. 

WEAKNESSES: 

- The manuscript would benefit from additional information to explain the genetic code numbering system referenced in the paper (code 25 etc.) to non-expert readers.

- We would like to see more discussion about what qualifies as a Western vs. Non-Western diet, and the limitations of the groups analyzed in this study. How well do they represent Western vs. Non-Western diets? 

- Differences in data presented from Western vs. Non-Western diets are initially discussed, but not mentioned in the paper's conclusion. To make the paper more cohesive, it would be beneficial to circle back to link new mechanistic understanding of phage stop codon recoding to the cohort studies. 

- Figure 2 is well done, but nevertheless it is challenging to interpret in its current form.  We suggest providing a supplementary figure that explains the main findings from figure 2. 

- In Figure 1a, how do you deal with gene density when it is similar between standard and recoded phages?

DETAILED U.P. ASSESSMENT:

OBJECTIVE CRITERIA (QUALITY)

1.   Quality: Experiments (1–3 scale; note: 1 is best on this scale) SCORE = 1.5

● Figure by figure, do experiments, as performed, have the proper controls? [note: we use this ‘figure-by-figure' section for broader detailed critiques, rather than only focusing on controls.]

Figure 1. We think that additional information about the number of participants in each group should be included, and additional information about the diets they consume. We recognize that a Western diet is typically characterized by high fat intake, and the inverse for a Non-Western diet. We also recognize that the hunter-gather group has been previously published and is an accepted representative group for Non-Western diets that are commonly used in these types of larger bioinformatic studies comparing diet choice. However, more information on sample numbers, read depth and sequencing techniques would be quite valuable here.

Figure 2. We think that the legend that accompanies the figure should be written in order of how data is presented on the figure. Right now, the legend does not follow the same organization as the figure it is describing. 

Many of the large phage clades are predicted to infect Firmicutes, which sometimes use alternative genetic codes. If alternatively coded phage infect hosts with alternative genetic codes, could it be argued that phages have adapted their codon usage to their hosts? If a host already uses an alternative genetic code, could this explain why some phages do not encode their own suppressor tRNAs?

The results section for figure 2c has an extensive discussion about weaknesses/limitations of the dataset. This is important but overlong. Could some of this be moved to the Discussion, or paraphrased? 

Figure 3. We recommend providing an explanation regarding what the colors in 3b indicate (red vs. green sequences). We recommend indicating that partial genomes are shown in this figure. This information could be provided in the figure caption or results section.

Figure 4. We recommend providing a statement in the results section that provides rationale about how representative data was selected and how many species from each clade were analyzed. These representative results are powerful, but we would like to see the generalized data as well so we can appreciate where these results were derived.

Figure 6. The model is compelling but not yet supported by wet lab data. Could this be indicated somewhere (unless there is in fact wet lab data to support the model. 

● Are specific analyses performed using methods that are consistent with answering the specific question?

Yes, but we recommend a few extra supplementary figures to make data presentation clearer. 

● Is there appropriate technical expertise in the collection and analysis of data presented?

Yes.

● Do analyses use the best-possible (most unambiguous) available methods quantified via appropriate statistical comparisons?

Yes, but we would like to see the generalized data from Figure 4.

● Are controls or experimental foundations consistent with established findings in the field? A review that raises concerns regarding inconsistency with widely reproduced observations should list at least two examples in the literature of such results. Addressing this question may occasionally require a supplemental figure that, for example, re-graphs multi-axis data from the primary figure using established axes or gating strategies to demonstrate how results in this paper line up with established understandings. It should not be necessary to defend exactly why these may be different from established truths, although doing so may increase the impact of the study and discussion of discrepancies is an important aspect of scholarship.

Yes.

2. Quality: Completeness (1–3 scale) SCORE = 1.8

● Does the collection of experiments and associated analysis of data support the proposed title- and abstract-level conclusions? Typically, the major (title- or abstract-level) conclusions are expected to be supported by at least two experimental systems.

The data supports the authors’ conclusions. However, we suggest discussing alternative explanations for re-coding in phages. We do not yet know how efficiently amino acids are encoded by the AC codons and how often they are decoded as a stop codon. Perhaps the efficiency/frequency of recoding regulates abundance of target proteins.

● Are there experiments or analyses that have not been performed but if ‘‘true’’ would disprove the conclusion (sometimes considered a fatal flaw in the study)? In some cases, a reviewer may propose an alternative conclusion and abstract that is clearly defensible with the experiments as presented, and one solution to ‘‘completeness’’ here should always be to temper an abstract or remove a conclusion and to discuss this alternative in the discussion section.

Unknown.

3. Quality: Reproducibility (1–3 scale) SCORE = 1.6

● Figure by figure, were experiments repeated per a standard of 3 repeats or 5 mice per cohort, etc.?’

N/A (Yes for Fig. 2).

● Is there sufficient raw data presented to assess rigor of the analysis?’

Yes.

● Are methods for experimentation and analysis adequately outlined to permit reproducibility?

We suggest that more information be provided about database versions used and how many individuals were in the                           groups analyzed. Some methods sections do not describe the parameters used to run the programs.

● If a ‘‘discovery’’ dataset is used, has a ‘‘validation’’ cohort been assessed and/or has the issue of false discovery been addressed?

Authors could verify dietary analysis in secondary cohorts.’

4. Quality: Scholarship (1–4 scale but generally not the basis for acceptance or rejection) SCORE = 1.2

● Has the author cited and discussed the merits of the relevant data that would argue against their conclusion?

            We recommend exploring alternative rationales for AC usage, as described above. 

● Has the author cited and/or discussed the important works that are consistent with their conclusion and that a reader should be especially familiar when considering the work?

We could not find another paper that examines stop codon reassignment in bacteriophages of gut microbiomes at this scale. The research presented in this manuscript is interesting and impactful, but authors could be more careful with their conclusion that stop codon re-coding can regulate lysogeny/lytic switch in phages until supportive wet lab data is obtained.

● Specific (helpful) comments on grammar, diction, paper structure, or data presentation (e.g., change a graph style or color scheme) go in this section, but scores in this area should not to be significant bases for decisions.

            See our comment regarding the legend in figure 2.

MORE SUBJECTIVE CRITERIA (IMPACT)

1.           Impact: Novelty/Fundamental and Broad Interest (1–4 scale) SCORE = 1.2 

A score here should be accompanied by a statement delineating the most interesting and/or important conceptual finding(s), as they stand right now with the current scope of the paper. A ‘‘1’’ would be expected to be understood for the importance by a layperson but would also be of top interest (have lasting impact) on the field.]

● How big of an advance would you consider the findings to be if fully supported but not extended? It would be appropriate to cite literature to provide context for evaluating the advance. However, great care must be taken to avoid exaggerating what is known comparing these findings to the current dogma (see Box 2). Citations (figure by figure) are essential here.

     This study provides key conceptual advances for the field, that could be extended by wet lab work to support Figure 6.    

2.              Impact: Extensibility (1–4 or N/A scale) SCORE = 2  

● Has an initial result (e.g., of a paradigm in a cell line) been extended to be shown (or implicated) to be important in a bigger scheme (e.g., in animals or in a human cohort)?

            Yes

● This criterion is only valuable as a scoring parameter if it is present, indicated by the N/A option if it simply doesn’t apply. The extent to which this is necessary for a result to be considered of value is important. It should be explicitly discussed by a reviewer why it would be required. What work (scope and expected time) and/or discussion would improve this score, and what would this improvement add to the conclusions of the study? Care should be taken to avoid casually suggesting experiments of great cost (e.g., ‘‘repeat a mouse-based experiment in humans’’) and difficulty that merely confirm but do not extend (see Bad Behaviors, Box 2). 

  N/A