Back to all available

Protocol paper: bioinformatics workflow optimization for enhancing 18S amplicon sequencing analysis in human gut parasite detection (Protozoa and Helminths)

bioinformatics
computational-biology
microbiology
molecular-biology
parasitology

Abstract

Background: 18S amplicon-based sequencing is an essential technology for investigating non-human eukaryotes within the human gut microbiome community, serving as a significant approach to shed light on how eukaryotic organisms affect host health and illness.

Method: This method uses bioinformatics tools, parameter adjustments, and framework customization to improve 18S amplicon sequencing data interpretation, bioinformatics accuracy, and efficiency. DNA was extracted and amplified from stool samples of various cancer types using a modified 18S primer set (616*F/1132R) that targets 18S rDNA gene V4 regions. OTUs (operational taxonomic units) were assigned for taxonomic annotation using SILVA and PR 2 18S databases, and parasite-specific databases used were VEuPathDB and WormBase Parasite. Furthermore, microbiome statistical methods were employed accordingly.

Results: The integration of parasite-specific databases markedly improved the detection of gut parasite identification and their variety in intricate samples. The unclassified OTUs in the mainstream databases SILVA and PR 2 were reclassified as parasites using VEuPathDB and WormBase Parasite specialized databases. The conventionally identified OTUs as non-human eukaryotes were 11.5% (n= 27) of the total 230 OTUs, whereas our optimized framework increased non-human eukaryotes identification to 97% (n=224) of the OTUs, considering the overlapping data resolution among the pathogen-centric databases.

Discussion: This modification is tailored to suit the researcher’s study and can enhance the precision and reliability of microbiome analysis, facilitating the targeted exploration of specific research questions. The conventional framework can be modified for various scientific investigations, such as multi-omics. Our approach provides a reproducible framework for researchers seeking to enhance the sequencing results and analysis.

Ethical Compliance: All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards.

Ask to review this manuscript

Notes for potential reviewers

  • Volunteering is not a guarantee that you will be asked to review. There are many reasons: reviewers must be qualified, there should be no conflicts of interest, a minimum of two reviewers have already accepted an invitation, etc.
  • This is NOT OPEN peer review. The review is single-blind, and all recommendations are sent privately to the Academic Editor handling the manuscript. All reviews are published and reviewers can choose to sign their reviews.
  • What happens after volunteering? It may be a few days before you receive an invitation to review with further instructions. You will need to accept the invitation to then become an official referee for the manuscript. If you do not receive an invitation it is for one of many possible reasons as noted above.
  • PeerJ does not judge submissions based on subjective measures such as novelty, impact or degree of advance. Effectively, reviewers are asked to comment on whether or not the submission is scientifically and technically sound and therefore deserves to join the scientific literature. Our Peer Review criteria can be found on the "Editorial Criteria" page - reviewers are specifically asked to comment on 3 broad areas: "Basic Reporting", "Experimental Design" and "Validity of the Findings".
  • Reviewers are expected to comment in a timely, professional, and constructive manner.
  • Until the article is published, reviewers must regard all information relating to the submission as strictly confidential.
  • When submitting a review, reviewers are given the option to "sign" their review (i.e. to associate their name with their comments). Otherwise, all review comments remain anonymous.
  • All reviews of published articles are published. This includes manuscript files, peer review comments, author rebuttals and revised materials.
  • Each time a decision is made by the Academic Editor, each reviewer will receive a copy of the Decision Letter (which will include the comments of all reviewers).

If you have any questions about submitting your review, please email us at [email protected].