Fundamentals: The Role of Library Prep in NGS
Next-Generation Sequencing (NGS) is one of the most powerful tools in modern biology, and library preparation is central to every workflow as the starting point for turning sample into sequence. Pretty much every sequencer, no matter which platform is used, will consume two essentials types of items per run: a solid consumable like a flowcell, and liquid reagents that drive the chemistry. To learn more about sequencing platforms, say tuned for the next blog in this series. Without properly prepared material, these consumables are wasted as no sequence can be generated and the consumables have nothing to act upon. Library prep is the bridge that transforms raw biological molecules into something the sequencer can recognize and read.
In this post, we’ll explore what library prep is, how it applies to microbiome research, where the main challenges arise, and how researchers can best select methods for microbiome library preparation that best suit their needs and capabilities.
What Is Library Prep?
"That which does not engage the flowcell does not end up in the data." —Paraphrased from too many NGS experts to count.
Library preparation ensures that biological-origin nucleic acid sequences (DNA and, more rarely, RNA) can both initiate sequencing reactions and physically bind to the sequencing platform. This usually involves attaching synthetic sequences, or adapters, to the ends of biological nucleic acid fragments. These adapters not only enable the molecules to engage with the flowcell or equivalent in the sequencing platform, but also often carry additional functions, such as sample barcodes that allow multiplexing, or unique molecular identifiers that help ensure the same original biological sequence fragment is not being counted twice (known as a duplicate).
Over the years, new twists on this process have emerged. Some workflows use tagmentation, which simultaneously fragments DNA and adds adapters in one step. Others aim to minimize amplification altogether through PCR-free methods, reducing the risk of bias introduced by uneven copying of sequences. Whether simple or sophisticated, every library prep method shares the same central goal: making biological molecules compatible with sequencing chemistry.
As an instructor who has taught the basics of NGS technologies for over a decade now, I always ask my students: “What are the two essential functions that any library preparation method must fulfill at a minimum?” While many different additional functions have been added to library preparation over the years, some becoming effectively standard at this point, any library preparation must, at a minimum, provide some means of initiating the sequencing of the biological-origin fragment and having the library molecule interact with the sequencing platform.
Library Prep in Microbiome Studies
Microbiome researchers have a wide menu of library preparation strategies, each suited to different questions. One of the earliest and most widely used is targeted amplicon sequencing, particularly of the bacterial 16S rRNA gene or the fungal ITS region. These methods provide a snapshot of community membership and have long served as the entry point into microbial ecology. These methods are still used and have the distinct advantage of minimizing the amount of host-derived sequence that is included in the dataset, focusing the limited number of available reads on the targeted microbes. Additionally, the bioinformatic methods for analyzing these reads are well-developed and relatively simple.
Shotgun metagenomic sequencing (sometimes referred to as shotgun whole-genome sequencing, or shotgun WGS) takes a broader approach. Instead of targeting one region, all DNA in the sample is fragmented and sequenced, revealing not just who is present but also their potential functions as determined by the genes they carry. More specialized DNA-based methods are also emerging. Proximity ligation, for example, allows episomes or plasmids to be definitively linked to their host genomes, helping researchers understand the genetic context of mobile elements.
The RNA side of library prep has recently grown in popularity with metatranscriptomics. Rather than focusing on genetic potential, this approach reveals which genes are actively being expressed, offering a dynamic picture of microbial activity. Taken together, these DNA and RNA strategies illustrate how library prep shapes not just what data are collected, but also the kinds of biological stories those data can tell.
Deficiencies and Challenges
Each library type has its strengths, but none are without limitations. Amplicon sequencing can reveal identity but rarely function—much like reading a nametag and trying to guess the personality of the person wearing it. Shotgun metagenomic sequencing provides a much fuller picture, but in host-associated samples, microbial DNA often makes up only a fraction of the total. As a result, vast numbers of reads may map to host sequences that must later be discarded. Zymo’s HostZERO™ Microbial DNA Kit is designed to address this problem by reducing host background before sequencing, increasing the fraction of microbial reads that actually matter.
Metatranscriptomics introduces a different challenge: ribosomal RNA. In most samples, rRNA overwhelms messenger RNA, meaning the majority of sequencing output is wasted on molecules that reveal little about gene expression. Tools like Zymo’s RiboFree® rRNA Depletion Kit help tilt the balance back toward bacterial mRNA, enabling a much clearer view of functional activity. These examples show how library prep decisions and technical solutions directly influence the efficiency and interpretability of sequencing experiments.
To DIY or Not
When setting up a library prep workflow, researchers must decide whether to build their own methods or rely on standardized solutions. DIY approaches can work—homegrown 16S workflows are still common—but they often demand heavy optimization and strict controls. Small deviations can easily introduce bias, and results may not compare well across studies.
Commercial kits, in contrast, provide validated protocols and reagents that minimize variability. This standardization gives researchers greater confidence that their data will be robust and more easily compared with others. For those who prefer to focus on analysis rather than lab work, outsourcing the entire workflow can be the best option. By sending samples to a trusted service provider, researchers can ensure that preparation, sequencing, and even bioinformatics are handled consistently, freeing them to concentrate on interpretation. The Zymo Research Quick 16S kit provides an excellent method to implement a standard kit in a workflow that will generate data comparable to other users of the kit if similar methods are used throughout the pipeline. Likewise, for a laboratory more interested in data analysis than sample processing, Zymo Research services can provide end-to-end processing of the sample and even initiate the bioinformatic analysis of the generated sequences, allowing the running of sophisticated research projects with minimal hands at the bench.
Conclusion
Library preparation is the gateway to NGS success. Whether the goal is to identify microbes with amplicon sequencing, profile community function through shotgun metagenomics, or explore activity with metatranscriptomics, the quality of the data depends heavily on how the library was built. Kits and services designed to reduce host contamination or ribosomal RNA overload can make a critical difference, especially in complex microbiome samples.
As always, success lies in aligning the biological question with the right preparation method and technical support. And while library prep is an essential step, it is only one part of the journey. In the next installment of the Master the Microbiome series, we’ll explore the sequencing platforms themselves and how their unique chemistries further shape the data that researchers rely on.