NERD-seq: A novel approach of Nanopore direct RNA sequencing that expands representation of non-coding RNAs

  1. Luke Saville
  2. Yubo Cheng
  3. Babita Gollen
  4. Liam Mitchell
  5. Matthew Stuart-Edwards
  6. Travis Haight
  7. Majid Mohajerani
  8. Athanasios Zovoilis

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Abstract

The new next-generation sequencing platforms by Oxford Nanopore Technologies for direct RNA sequencing (direct RNA-seq) allow for an in-depth and comprehensive study of the epitranscriptome by enabling direct base calling of RNA modifications. Non-coding RNAs constitute the most frequently documented targets for RNA modifications. However, the current standard direct RNA-seq approach is unable to detect many of these RNAs. Here we present NERD-seq, a sequencing approach which enables the detection of multiple classes of non-coding RNAs excluded by the current standard approach. Using total RNA from a tissue with high known transcriptional and non-coding RNA activity in mouse, the brain hippocampus, we show that, in addition to detecting polyadenylated coding and non-coding transcripts as the standard approach does, NERD-seq is able to significantly expand the representation for other classes of RNAs such as snoRNAs, snRNAs, scRNAs, srpRNAs, tRNAs, rRFs and non-coding RNAs originating from LINE L1 elements. Thus, NERD-seq presents a new comprehensive direct RNA-seq approach for the study of epitranscriptomes in brain tissues and beyond.

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  1. Review Commons

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    Reply to the reviewers

    General Statements

    We are grateful to the reviewers for reviewing our manuscript. In general, the reviewers agree that our method presents a new approach of Nanopore direct RNA-seq that is not limited to the standard use of only the adenylated fraction of RNAs within a sample but they have also requested more evidence about the effectiveness and usefulness of this approach. Reviewer 1 notes that NERD-seq "extends nanopore direct RNA-Sequencing to beyond the poly(A) fraction." Reviewer 2 notes that "This manuscript has the potential of having major significance to researchers in the field of transcriptomics". At the same time Reviewer 1 remarks that, "the authors need to expand more on why this is useful, and what scenarios this would be used in" and need "to demonstrate that NERD-Seq is more than an incremental improvement to existing approaches". Reviewer 2 notes that "This technique has great potential, as ONT direct RNA sequencing can be used to detect RNA modifications... There are however some issues that need to be addressed before the manuscript is suitable to publication" and agrees with Reviewer 1 that the authors need to demonstrate "that their newly sequencing techniques could indeed improve RNA detection beyond the current techniques." We are grateful to the reviewers for these comments, and we fully agree that the manuscript in its initially submitted form falls "short from providing strong arguments supporting the fidelity, accuracy and coverage of the new technique." and that more "proof of the increased accuracy or utility of the new technique" was needed.

    We attach a substantially revised and expanded version of the manuscript that includes additional data needed to ensure the methodology is replicable and further supports the rationale why NERD-seq is a useful addition to the current direct RNA sequencing methodology repertoire. We now provide:

    • the repetition of all performed NERD-seq runs with a new enzyme (commercially available), as the one used in our initial study (Omniamp polymerase) is not anymore commercially available

    • 6 revised main figure panels based on the new sequencing runs and a new main figure (Fig.7),

    • 20 new supplementary figures, and

    • 1 new supplementary table (Suppl. Tables 1),

    that correspond to the points raised by the reviewers. We have also revised the main text accordingly.

    Please find below the reviewers 'comments and a detailed point by point response to these comments. A document with the changes from the initially submitted manuscript being highlighted is attached at the end of this response.

    Point-by-point description of the revisions

    Reviewer 1

    *------------------------------------------------------------------------------ *

    *Reviewer #1 (Evidence, reproducibility and clarity (Required)): *

    ***Summary:** *

    This reviewer generally remarks that: "Saville et al detail a new method, NERD-Seq, which extends nanopore direct RNA-Sequencing to beyond the poly(A) fraction. In principle, this allows the capture of additional RNA types in a single sequencing reaction, albeit at the expense of sequencing depth. However, the authors need to expand more on why this is useful, and what scenarios this would be used in. The rationale for many of their experimental choices are not explained/contextualized appropriately. The manuscript also suffers from excessive jargon and grammatical errors."

    We appreciate these comments made by the reviewer. We now expand more on why and how NERD-seq is useful, and what scenarios this could be used in. In particular:

    In the introduction, we now denote that a significant number of the most well described RNA modification positions are located in classes of RNAs such as tRNAs, snoRNAs and other ncRNAs like 7SK RNA. In the results section, we build on the second reviewer's suggestion that "This technique has great potential, as ONT direct RNA sequencing can be used to detect RNA modifications...", and we first show in revised Figures 3-5 that the standard approach misses the above classes of RNAs. Subsequently, in two new figures (Fig. 7 and Suppl.Fig. S23) we present an example of the ability of NERD-seq to decipher known RNA modifications in a well-studied ncRNA, 7SK, compared with the inability of the standard approach to do so. Widely studied RNAs such as 7SK have the potential and thus often serve as controls for known positions of certain RNA modifications when validating them for novel positions in other RNAs. This however means that the application of the standard approach, which is not able to detect RNAs and thus their modifications for RNAs such as 7SK, makes it difficult to use them as controls. In other words, the ability to perform genome wide studies of RNA modifications using the direct RNA-seq approach relies on the ability to be able to confirm at the same time these findings in already known control positions located in the above classes of RNAs. If these known controls such as short ncRNAs are missing, as we show that it is the case with standard RNA-seq, it is difficult to perform such genome wide epitranscriptome studies. Thus, the usefulness of our approach is not only that it can sequence certain classes of shorter RNAs beyond mRNAs, but that it can do this without affecting the ability to study mRNAs simultaneously or use targeted sequencing adapters. This is now discussed in the text. We have also tried to avoid the excessive use of jargon language and correct any grammatical errors.

    *------------------------------------------------------------------------------ *

    ***Major comments:** *

    Point 1. The reviewer mentions:* "To my understanding, the primary purpose of NERD-Seq is to allow the sequencing of the non-adenylated fraction of RNAs within sample with a size filter step designed to exclude ncRNAs larger than ~200 nt. While this allow for subsequent polyadenylation of the small fraction, it remains quite likely that larger non-adenylated non-rRNAs are being missed by the protocol. It is also not clear why NERD-Seq should be considered the optimal strategy. The authors should show that they have considered/evaluated other strategies such as: *

    *- Depletion/Seperation of the poly(A) fraction prior to size selection performed on non-adenylated fraction. *

    - Targeted degradation of the rRNA fraction using rRNA depletion kits."

    We thank the reviewer for this comment. The primary purpose of NERD-seq is not only to allow the sequencing of the non-adenylated fraction of shorter RNAs but to do this while maintaining the ability to sequence longer polyadenylated RNAs. In particular, we are not sequencing only the short RNA fraction but also the longer RNA fraction of naturally polyadenylated RNAs. This includes both mRNAs and ncRNAs that are polyadenylated. We apologize that this may have not be presented clearly in our initial text, and we have now revised the respective results part (page 8, lines 13-14 and 30-31). As mentioned below, we now also provide evidence that our approach does not affect the ability to efficiently sequence mRNAs and decipher effectively their isoforms (new Suppl. Fig. S19). Only large non poly-A ncRNAs still evade our detection. Now, we discuss this limitation in the last paragraph of the discussion section (page 19, lines 12-20). We have now also evaluated other strategies such as those mentioned by the reviewer (depletion of rRNA, size selection using magnetic beads, different enzymes) and in the new Suppl. Figure S5 we show that they were suboptimal in providing sufficient reads that pass the quality thresholds in base calling compared to our NERD-seq approach.

    To sum up, we certainly don't claim to be able to capture longer ncRNAs, due to their lack of polyadenylated tails. We show though that while the ability to capture simultaneously all reads under the current Nanopore sequencing protocols may be unattainable, the NERD-seq methodology demonstrates a useful addition to the Nanopore sequencing repertoire due to its ability, in addition to polyadenylated transcripts, to simultaneously capture multiple classes of short ncRNAs, hitherto until now only achievable one transcript at a time with custom adaptor ligation.

    Point 2. The reviewer mentions that "Related to this, the authors note that NERD-Seq is designed to allow the sequencing of both adenylated and (short) non-adenylated fractions of RNAs within a study. What is the actual value of this versus, say, simply targeting the short non-adenylated fraction directly? "

    Please see our response to the general comment by this reviewer and the new figures Fig. 7 and Suppl. Fig. S23 and the completely new results section at pages 15 and 16 about examples on how NERD-seq expands the study of epitranscriptomic signatures to additional RNA classes while maintaining the ability to efficiently assess the protein coding transcriptome (new Suppl. Fig. S19).

    Point 3. The reviewer notes that "The manuscript in general is written in a somewhat subversive style, seemingly focused on highlighting the 'failings' of the standard ONT protocol. This is somewhat disingenuous as these are not 'failings' per se given the design objective of the standard DRS protocol (i.e. to capture and sequence the poly(A) fraction of RNAs) works well. The authors would be better off highlighting the situations (i.e. study questions) where NERD-Seq would provide a measurable benefit over the standard DRS strategy. "

    We apologize if this was indicated in our initial submission, as this was not our intention. We now focus on highlighting what NERD-seq can do, and this is reflected in the changes we have made in the introduction and discussion section.

    Point 4. The reviewer asks* "The authors state that 1.5ug total RNA is used as input for NERD-Seq but how much input (poly-A RNA) actually goes into the short- and long-fraction parts of the DRS protocol? This is important to know.* "

    We have now included data in the manuscript for polyadenylation signals in the reads themselves (new Suppl. Fig S5F) and view it as a useful addition to the manuscript as it seems to show there is little difference in the relationship of length of transcript and polyadenylation tail length between the two methods.

    RNA RIN values differ across samples so within different samples the portions of poly-A RNA, non poly-A RNA and former poly-A RNA that has lost the poly-A due to degradation may also vary. This however has not been shown to affect the reproducibility of the standard direct RNA-seq methodology, so it should not affect also that of NERD-seq. Nevertheless, we searched the literature for a well characterized methodology for quantifying this further and found little on poly(A) specific quantification approaches that we could use. In fact, poly(A) selection has also been shown to have variable efficiency and could thus produce inaccurate measurements that would make it difficult for us to assess explicitly the exact portions of poly-A vs non poly-A portions. Thus, we feel that in the absence of a well characterized methodology to make these quantifications, developing a new one may have been beyond the scope of this manuscript.

    Point 5. The reviewer recommends:* "The authors show that NERD-Seq performs well on a tissue that is generally enriched in non-coding RNA activity but without the inclusion of biology replicates or tissue samples from other sources, it is impossible to assess (1) the reproducibility/robustness of the methodology and (2) whether NERD-Seq would produce useable data from other tissues with lower non-coding RNA activity. These experiments are required. "*

    We thank the reviewer for this suggestion and we have now included an additional biological replicate for the mouse tissue and data from another tissue in a separate organism (5-person pool source human cerebral cortex RNA).

    Point 6. The reviewer notes:* "Several figures are poorly explained. For instance, Does Figure 2A show data only from the short RNA fraction? If not then this suggests incredibly high levels of mRNA degradation in the 'standard' ONT fraction - far beyond what is seen in other studies. The legends for all figures should be far more specific and detailed."*

    We have now replaced figure 2A with a different mapping strategy and a whole sample assessment of mapped reads lengths. We have also edited the figure legends to include more information.

    The reviewer made also the following comments:

    Minor comments:

    ": Please refrain from using 'next-generation' in a sequencing context (abstract, introduction). Having a 'new' next-generation sequencing is confusing in regard to the old 'next generation' sequencing. "

    The instances mentioned have been removed as per the reviewer's suggestion.

    "Introduction should acknowledge that targeted sequencing of (individual) non-adenylated RNAs is possible (i.e. there is an ONT protocol for this)."

    We now include mention of this in the introduction, results and discussion.

    "Several figure elements not referenced in the text appropriately (e.g. red bars in Figure 2A). "

    We have now addressed this and thank the reviewer for making us aware of this.

    "The authors mention profiling of the epitranscriptome several times in the introduction and discussion but do not include any work geared toward looking for RNA modifications. Indeed it is entirely unclear whether NERD-Seq produces the depth of sequencing (on non-adenylated RNAs) required for current RNA modification detection tools. "

    We have now included a new main figure (Fig. 7) and a new supplementary figure (Suppl. Fig. S23) which profile elements of the epitranscriptome and show that indeed NERD-seq produces the depth of sequencing necessary for RNA modification detection.

    Reviewer 1's summary:

    "NERD-Seq presents a modified method for DRS of both short non-adenylated and the standard adenylated fraction of RNAs within a sample. This utility of this approach appears rather niche and it is hard to determine situations in which this approach would be generally useful versus many of the existing (Illumina-based) approaches. The burden to demonstrate that NERD-Seq is more than an incremental improvement to existing approaches lies with the authors. "

    We appreciate the reviewer's comments. While we agree with them that for various applications (such as differential gene expression, quantification of lowly expressed genes) "the existing (Illumina-based) approaches" Illumina RNA-seq or short-RNA-seq would be more appropriate due to higher sequence yields, the main advantage of nanopore direct RNA-seq is the ability to identify RNA modifications directly at the same time (see new Figs 7 and S23). While we also agree that the field of high throughput sequencing epitranscriptomics is quite nascent, it is quickly gaining interest. Based on the reviewer's suggestions we believe that we have now modified the manuscript substantially to provide justification for NERD-seq's feasibility as a methodology to capture multiple classes of ncRNAs for the study of their sequence, quantity and their epitranscriptomic signatures.

    We would also like to note that even as a BioRxiv preprint, our manuscript has received already a number of citations, including from an article published in Nature Biotechnology and feel this demonstrates interest in NERD-seq from the scientific community.

    Reviewer 2

    Reviewer #2 (Evidence, reproducibility and clarity (Required))

    This reviewer generally remarks that* "In this manuscript Saville et al. present NERD-seq, a novel method to enrich and directly sequence non-coding RNA using the Oxford Nanopore Technology (ONT). The technique is based on the addition of a poly-A tail to small non-coding RNAs which enables their sequencing by a conventional ONT direct RNA-seq protocol using a poly(T)-tethering adaptor. This technique has great potential, as ONT direct RNA sequencing can be used to detect RNA modifications. Furthermore, non-coding RNAs are known to harbor a lot of these modifications and that they are important to their function. In addition, non-coding RNAs aren't generally poly-adenylated and therefore are underrepresented when using standard ONT direct RNA-seq. The authors have shown that using NERD-seq, they are able to significantly improve the number of reads on several subsets of non-coding RNAs : tRNAs, snoRNAs, snRNAs, scRNAs and rRFs. There are however some issues that need to be addressed before the manuscript is suitable to publication."*

    We thank the reviewer for their encouraging comments.

    Major comments:

    Point 1. The reviewer recommends that "Data on RNA modifications present in the sequenced non-coding RNA would greatly improve the impact of the manuscript, as the method is presented as read tool to study RNA modifications. If the authors can't include these data, they should at least explain why in the discussion section."

    We have now included a new main figure (Fig.7) and a supplemental figure (Suppl. Fig. S23) which show the ability of NERD-seq to identify previously described RNA modifications and its superiority in the case of non-coding RNAs such as 7SK. We thank the reviewer for this recommendation that significantly improved our manuscript better revealing the rationale and impact of our approach.

    Point 2. The reviewer remarks that "The authors did not discuss possible biases of their method. For example, is the poly-A tail ligation efficiency affected by sequence and or structural differences between RNA? Is there detectable differences in the efficiency of sequencing small RNA with structured 3'end when compared to mRNA? Are the proportions of reads obtained per non-coding RNA sub-category and RNA rank supported by other biochemical data (e.g. Northern blots, primer extension, RT-qPCR? "

    We have included commentary in the discussion on these biases (page 19, lines 7-11). We also include a new supplementary figure (Suppl. Fig. S5F) regarding polyadenylation in which we see similar transcript length to polyadenylation tail length dynamics between the two methodologies.

    Point 3. The reviewer notes that "It is essential to include external size and sequence markers spike-in to directly evaluate the quality and fidelity of the newly develop sequencing technique. "

    We have now repeated the sequencing, including sequencing with the well described RNA sequins mix B. We include a new supplementary figure with this data (Suppl. Fig. S10) that shows that NERD-seq does not differ from the standard approach regarding its ability to effectively evaluate the quantity and complexity of the transcripts present.

    Point 4. The reviewer mentions that "The author compared NERD-seq to ONT standard direct RNA-seq but did not compare it with other methods that are currently used for non-coding RNA sequencing (e.g. Illumina based sequencing, TGIRTseq). There is a need for side by side comparison at least by using data available in the literature if not experimentally. "

    We now include a comparison with Illumina sequencing data for both long and short fractions from each biological replicate (Suppl.Figs S12-15,17,19 and 21). This data shows that NERD-seq combines the advantages of both standard and short RNA-seq with Illumina, enriching both long and short RNAs, while simultaneously as mentioned above, offering the benefits of direct RNA-seq. We feel this also demonstrates the utility of the unique chemistry of Nanopore sequencing where fragmenting of long RNAs is not required for sequencing, allowing the combination of long RNAs and short RNAs in the same library to reproduce what is essentially enriched in short RNA libraries as well as poly(A) sequencing libraries. Since the enzyme included in our initial submission is not anymore widely accessible, we also tested a series of other enzymes, including the commercial replacement for omniamp (the originally used enzyme), lavalamp, and other small tweaks to the library methodology, until finding the GSP SSD2.0 enzyme to be the most suitable for our research goals. The summary of our findings is in Figure S5. Initially, we considered TGIRT as a potential enzyme for the sequencing reaction because of its use for sequencing snoRNAs and other highly structured RNAs but because of its strand switching properties, we felt it would be poorly suited for a nanopore direct RNA sequencing approach.

    The reviewer made also the following comments/suggestions:

    Minor comments

    "Page 5 line 3, NERD-seq is misspelled. "

    This is now fixed.

    "Throughout this section µL and µg are written as uL and ug."

    This is now fixed.

    "Several temperatures are missing the o symbol between the number and C."

    This is now fixed.

    Final comments:

    "This manuscript has the potential of having major significance to researchers in the field of transcriptomics if the author demonstrated that their newly sequencing techniques could indeed improve RNA detection beyond the current techniques. Unfortunately, the manuscript only show the capacity of the technique to detect non-coding RNA but fall short from providing strong arguments supporting the fidelity, accuracy and coverage of the new technique. The use of Nanopore sequencing is not unique and was used in the past for sequencing non-coding RNA. What is needed now is a proof of the increased accuracy or utility of the new technique to justify yet another publication about Nanopore sequencing paper. The manuscript would potentially be of interest to researcher working on different type of non-coding RNA and transcriptomics. 

    I am in the field of non-coding RNA sequencing and functional analysis and very familiar with this approach."

    We thank the reviewer again for their encouraging comments and valid concerns. We agree that the initial manuscript fell short of providing evidence of its utility as an addition to the Nanopore sequencing repertoire. We hope the additional data we provide (rerunning with an additional enzyme, use of replicates, use of standard spike in controls, testing an additional organism and tissue, testing and comparing different enzymes and other sequencing platforms) provide the necessary assurances about reproducibility and accuracy, while the new data concerning identification of RNA modifications and detection of important RNA classes missed by the standard protocol provides the necessary assurances about the utility of our approach.

  2. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

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    Referee #2

    Evidence, reproducibility and clarity

    In this manuscript Saville et al. present NERD-seq, a novel method to enrich and directly sequence non-coding RNA using the Oxford Nanopore Technology (ONT). The technique is based on the addition of a poly-A tail to small non-coding RNAs which enables their sequencing by a conventional ONT direct RNA-seq protocol using a poly(T)-tethering adaptor. This technique has great potential, as ONT direct RNA sequencing can be used to detect RNA modifications. Furthermore, non-coding RNAs are known to harbor a lot of these modifications and that they are important to their function. In addition, non-coding RNAs aren't generally poly-adenylated and therefore are underrepresented when using standard ONT direct RNA-seq. The authors have shown that using NERD-seq, they are able to significantly improve the number of reads on several subsets of non-coding RNAs : tRNAs, snoRNAs, snRNAs, scRNAs and rRFs. There are however some issues that need to be addressed before the manuscript is suitable to publication.

    Major Comments:

    • Data on RNA modifications present in the sequenced non-coding RNA would greatly improve the impact of the manuscript, as the method is presented as read tool to study RNA modifications. If the authors can't include these data, they should at least explain why in the discussion section.

    • The authors did not discuss possible biases of their method. For example, is the poly-A tail ligation efficiency affected by sequence and or structural differences between RNA? Is there detectable differences in the efficiency of sequencing small RNA with structured 3'end when compared to mRNA? Are the proportions of reads obtained per non-coding RNA sub-category and RNA rank supported by other biochemical data (e.g. Northern blots, primer extension, RT-qPCR?

    • It is essential to include external size and sequence markers spike-in to directly evaluate the quality and fidelity of the newly develop sequencing technique.

    • The author compared NERD-seq to ONT standard direct RNA-seq but did not compare it with other methods that are currently used for non-coding RNA sequencing (e.g. Illumina based sequencing, TGIRTseq). There is a need for side by side comparison at least by using data available in the literature if not experimentally.

    Minor Comments:

    • Page 5 line 3, NERD-seq is misspelled.

    • Throughout this section µL and µg are written as uL and ug.

    • Several temperatures are missing the o symbol between the number and C.

    We recommend that all these issues should be addressed before publication.

    Significance

    This manuscript has the potential of having major significance to researchers in the field of transcriptomics if the author demonstrated that their newly sequencing techniques could indeed improve RNA detection beyond the current techniques. Unfortunately, the manuscript only show the capacity of the technique to detect non-coding RNA but fall short from providing strong arguments supporting the fidelity, accuracy and coverage of the new technique. The use of Nanopore sequencing is not unique and was used in the past for sequencing non-coding RNA. What is needed now is a proof of the increased accuracy or utility of the new technique to justify yet another publication about Nanopore sequencing paper. The manuscript would potentially be of interest to researcher working on different type of non-coding RNA and transcriptomics.

    I am in the field of non-coding RNA sequencing and functional analysis and very familiar with this approach.

  3. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #1

    Evidence, reproducibility and clarity

    Summary:

    Saville et al detail a new method, NERD-Seq, which extends nanopore direct RNA-Sequencing to beyond the poly(A) fraction. In principle, this allows the capture of additional RNA types in a single sequencing reaction, albeit at the expense of sequencing depth. However, the authors need to expand more on why this is useful, and what scenarios this would be used in. The rationale for many of their experimental choices are not explained/contextualized appropriately. The manuscript also suffers from excessive jargon and grammatical errors.

    Major comments:

    1: To my understanding, the primary purpose of NERD-Seq is to allow the sequencing of the non-adenylated fraction of RNAs within sample with a size filter step designed to exclude ncRNAs larger than ~200 nt. While this allow for subsequent polyadenylation of the small fraction, it remains quite likely that larger non-adenylated non-rRNAs are being missed by the protocol. It is also not clear why NERD-Seq should be considered the optimal strategy. The authors should show that they have considered/evaluated other strategies such as:

    • Depletion/Seperation of the poly(A) fraction prior to size selection performed on non-adenylated fraction.
    • Targeted degradation of the rRNA fraction using rRNA depletion kits.

    2: Related to this, the authors note that NERD-Seq is designed to allow the sequencing of both adenylated and (short) non-adenylated fractions of RNAs within a study. What is the actual value of this versus, say, simply targeting the short non-adenylated fraction directly?

    3: The manuscript in general is written in a somewhat subversive style, seemingly focused on highlighting the 'failings' of the standard ONT protocol. This is somewhat disingenuous as these are not 'failings' per se given the design objective of the standard DRS protocol (i.e. to capture and sequence the poly(A) fraction of RNAs) works well. The authors would be better off highlighting the situations (i.e. study questions) where NERD-Seq would provide a measurable benefit over the standard DRS strategy.

    4: The authors state that 1.5ug total RNA is used as input for NERD-Seq but how much input (polyA RNA) actually goes into the short- and long-fraction parts of the DRS protocol? This is important to know.

    5: The authors show that NERD-Seq performs well on a tissue that is generally enriched in non-coding RNA activity but without the inclusion of biology replicates or tissue samples from other sources, it is impossible to assess (1) the reproducibility/robustness of the methodology and (2) whether NERD-Seq would produce useable data from other tissues with lower non-coding RNA activity. These experiments are required.

    6: Several figures are poorly explained. For instance, Does Figure 2A show data only from the short RNA fraction? If not then this suggests incredibly high levels of mRNA degradation in the 'standard' ONT fraction - far beyond what is seen in other studies. The legends for all figures should be far more specific and detailed.

    Minor quibbles:

    1: Please refrain from using 'next-generation' in a sequencing context (abstract, introduction). Having a 'new' next-generation sequencing is confusing in regard to the old 'next generation' sequencing.

    2: Introduction should acknowledge that targeted sequencing of (individual) non-adenylated RNAs is possible (i.e. there is an ONT protocol for this).

    3: Several figure elements not referenced in the text appropriately (e.g. red bars in Figure 2A).

    4: The authors mention profiling of the epitranscriptome several times in the introduction and discussion but do not include any work geared toward looking for RNA modifications. Indeed it is entirely unclear whether NERD-Seq produces the depth of sequencing (on non-adenylated RNAs) required for current RNA modification detection tools.

    Significance

    NERD-Seq presents a modified method for DRS of both short non-adenylated and the standard adenylated fraction of RNAs within a sample. This utility of this approach appears rather niche and it is hard to determine situations in which this approach would be generally useful versus many of the existing (Illumina-based) approaches. The burden to demonstrate that NERD-Seq is more than an incremental improvement to existing approaches lies with the authors.

  4. Version published to 10.1101/2021.05.06.442990v1 on bioRxiv