Abstract
Sequence-based approaches to study microbiomes, such as 16S rRNA gene sequencing and metagenomics, are uncovering associations between microbial taxa and a myriad of factors. A drawback of these approaches is that the necessary sequencing library preparation and bioinformatic analyses are complicated and continuously changing, which can be a barrier for researchers new to the field. We present three essential components to conducting a microbiome experiment from start to finish: first, a simplified and step-by-step custom gene sequencing protocol that requires limited lab equipment, is cost-effective, and has been thoroughly tested and utilized on various sample types; second, a series of scripts to integrate various commonly used bioinformatic tools that is available as a standalone installation or as a single downloadable virtual image; and third, a set of bioinformatic workflows and tutorials to provide step-by-step guidance and education for those new to the microbiome field. This resource will provide the foundations for those newly entering the microbiome field and will provide much-needed guidance and best practices to ensure that quality microbiome research is undertaken. All protocols, scripts, workflows, tutorials, and virtual images are freely available through the Microbiome Helper website (https://github.com/mlangill/microbiome_helper/wiki). IMPORTANCE As the microbiome field continues to grow, a multitude of researchers are learning how to conduct proper microbiome experiments. We outline here a streamlined and custom approach to processing samples from detailed sequencing library construction to step-by-step bioinformatic standard operating procedures. This allows for rapid and reliable microbiome analysis, allowing researchers to focus more on their experiment design and results. Our sequencing protocols, bioinformatic tutorials, and bundled software are freely available through Microbiome Helper. As the microbiome research field continues to evolve, Microbiome Helper will be updated with new protocols, scripts, and training materials.
Highlights
Sequence-based approaches to study microbiomes, such as 16S rRNA gene sequencing and metagenomics, are uncovering associations between microbial taxa and a myriad of factors
Amplicon samples in our presented protocol are run on the Illumina MiSeq using 2 ϫ 300 bp paired ends (PE) v3 chemistry which allows for overlap and stitching together of paired amplicon reads into one full-length read of higher quality
As presented further below for the bioinformatics approaches, the open lab protocol will be continuously revised/refined as new molecular approaches are introduced by the community, or novel products arrive in the marketplace, and we have had the opportunity to validate them
Summary
Sequence-based approaches to study microbiomes, such as 16S rRNA gene sequencing and metagenomics, are uncovering associations between microbial taxa and a myriad of factors. We present three essential components to conducting a microbiome experiment from start to finish: first, a simplified and step-by-step custom gene sequencing protocol that requires limited lab equipment, is cost-effective, and has been thoroughly tested and utilized on various sample types; second, a series of scripts to integrate various commonly used bioinformatic tools that is available as a standalone installation or as a single downloadable virtual image; and third, a set of bioinformatic workflows and tutorials to provide step-by-step guidance and education for those new to the microbiome field. As the microbiome research field continues to evolve, Microbiome Helper will be updated with new protocols, scripts, and training materials
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