Two-Target Quantitative PCR To Predict Library Composition for Shallow Shotgun Sequencing

ABSTRACT When determining human microbiota composition, shotgun sequencing is a powerful tool that can generate high-resolution taxonomic and functional information at once. However, the technique is limited by missing information about host-to-microbe ratios observed in different body compartments. This limitation makes it difficult to plan shotgun sequencing assays, especially in the context of high sample multiplexing and limited sequencing output and is of particular importance for studies employing the recently described shallow shotgun sequencing technique. In this study, we evaluated the use of a quantitative PCR (qPCR)-based assay to predict host-to-microbe ratio prior to sequencing. Combining a two-target assay involving the bacterial 16S rRNA gene and the human beta-actin gene, we derived a model to predict human-to-microbe ratios from two sample types, including stool samples and oropharyngeal swabs. We then validated it on two independently collected sample types, including rectal swabs and vaginal secretion samples. This assay enabled accurate prediction in the validation set in a range of sample compositions between 4% and 98% nonhuman reads and observed proportions varied between −18.8% and +19.2% from the expected values. We hope that this easy-to-use assay will help researchers to plan their shotgun sequencing experiments in a more efficient way. IMPORTANCE When determining human microbiota composition, shotgun sequencing is a powerful tool that can generate large amounts of data. However, in sample compositions with low or variable microbial density, shallowing sequencing can negatively affect microbial community metrics. Here, we show that variable sequencing depth decreases measured alpha diversity at differing rates based on community composition. We then derived a model that can determine sample composition prior to sequencing using quantitative PCR (qPCR) data and validated the model using a separate sample set. We have included a tool that uses this model to be available for researchers to use when gauging shallow sequencing viability of samples.

A large part of the contribution of the manuscript is the dataset. Please add an explicit "Data Availability" paragraph at the end of the Materials and Methods section that includes a data description, name of the repository, and the accession numbers (which I believe are currently in the response to reviewers but not in the manuscript). Please ensure that the data availability section includes access to the qPCR data as well as the sequence data and has explicit instructions on how to link the qPCR identifiers to sequence identifiers (as much as is possible given the limitation that not all of your subjects allowed sequence release). You could consider adding supplementary directions on how to download and link the qPCR and sequence data if this is more detailed than can be addressed in the "Data Availability" paragraph. These instructions would presumably be more explicit than the UNIX code fragment currently present in the supplementary materials in allowing users to reproduce the figures in the paper.

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Dear Dr. Bryan Coburn:
Thank you for submitting your manuscript to mSystems. Your manuscript was reviewed by one of the original reviewers as well as two members of the editorial board (including myself). I am pleased to inform you that, in principle, we expect to accept it for publication in mSystems. However, acceptance will not be final until you have adequately addressed the following comments: Reviewer #2 had a suggestion for the discussion. Please incorporate the text from your response into the discussion as noted below.
A large part of the contribution of the manuscript is the dataset. Please add an explicit "Data Availability" paragraph at the end of the Materials and Methods section that includes a data description, name of the repository, and the accession numbers (which I believe are currently in the response to reviewers but not in the manuscript). Please ensure that the data availability section includes access to the qPCR data as well as the sequence data and has explicit instructions on how to link the qPCR identifiers to sequence identifiers (as much as is possible given the limitation that not all of your subjects allowed sequence release). You could consider adding supplementary directions on how to download and link the qPCR and sequence data if this is more detailed than can be addressed in the "Data Availability" paragraph. These instructions would presumably be more explicit than the UNIX code fragment currently present in the supplementary materials in allowing users to reproduce the figures in the paper.
We have added an explicit "Data Availability" paragraph at L284-292, which includes the file type of the sequencing data, the name of the repository, the accession number, and a hyperlink to the sequencing data. We have also made the raw qPCR Ct values accessible in the paper in Supplementary table 3, L345-347.

Preparing Revision Guidelines
To submit your modified manuscript, log onto the eJP submission site at https://msystems.msubmit.net/cgi-bin/main.plex. Go to Author Tasks and click the appropriate manuscript title to begin the revision process. The information that you entered when you first submitted the paper will be displayed. Please update the submission to address these last concerns. Please let me know if you have questions.
For complete guidelines on revision requirements, please see the Instructions to Authors at https://msystems.asm.org/sites/default/files/additional-assets/mSys-ITA.pdf. Submissions of a paper that does not conform to mSystems guidelines will delay acceptance of your manuscript.

Dr. Bryan Coburn University Health Network Toronto, Ontario M5G 1L7 Canada
Re: mSystems00552-21R1 (Two-target quantitative PCR to predict library composition for shallow shotgun sequencing) Dear Dr. Bryan Coburn: Your manuscript has been accepted, and I am forwarding it to the ASM Journals Department for publication. For your reference, ASM Journals' address is given below. Before it can be scheduled for publication, your manuscript will be checked by the mSystems senior production editor, Ellie Ghatineh, to make sure that all elements meet the technical requirements for publication. She will contact you if anything needs to be revised before copyediting and production can begin. Otherwise, you will be notified when your proofs are ready to be viewed.
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