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During library preparation, DNA or RNA targets are processed and converted into the appropriate format for a sequencing run. Library preparation workflows can differ greatly between kits and the needs of the experiment. Each library prep kit contains different components necessary to prepare the target nucleic acid for sequencing. The constant development of sequencing techniques has led to a growing number of library prep options, and the process of identifying the appropriate library prep kit has become more demanding. Selection of the appropriate DNA library prep kit requires many considerations to ensure a successful sequencing experiment.
Steps of DNA library preparations
The process of converting target DNA into libraries ready for sequencing varies based on the library prep kit, application, and sequencing instrument. Below are some of the most common steps of DNA library preparations.
1. Extractions and QC
Prior to starting the library prep, the DNA must be properly isolated from the source. Although extractions are not directly part of the library prep process, successful isolation of the target DNA, the extraction method used, and the quality of the DNA can affect the library preparation process. After extractions, the quality and concentration should be checked.
2. Fragmentation or target selection
The beginning of DNA library preparation typically begins with a fragmentation step or enrichment of target regions. The fragmentation process can be done enzymatically or mechanically. For long-read systems, the DNA may not require fragmentation. Instead of fragmentation, targeted approaches often amplify the regions of interest or use probes to capture the target DNA.
3. Addition of adapters
Adapters are short sequences added onto the ends of the target DNA that contain important elements for sequencing, such as indexes, molecular barcodes, and primer and flow cell-binding sites. The addition of the adapters can occur through a variety of methods and may be done concurrently with the fragmentation step of some workflows.
4. Optional: Amplification
Depending on the needs of the study and the concentration of the DNA, the libraries are often amplified. PCR amplification of sequencing libraries assists with quantification and allows for lower amounts of starting material.
5. Cleanup/ size selection
After amplification, the sequencing libraries undergo a cleanup or size selection step to prevent extra primers, adapters, and additional contaminants from the previous steps from being loaded onto the sequencer. This process is often completed with magnetic beads, gels, or columns.
6. Quantification, normalization, and quality check
Quantification and quality checks are necessary for ensuring a successful library prep. This step can be completed with a number of options including qPCR, ddPCR, spectrophotometry, electrophoresis, or fluorometry. If samples are multiplexed, they also need to be normalized to prevent an uneven distribution of reads.
Considerations for DNA library preparations
Sequencer and applications
One of the most direct ways to narrow down the appropriate library preparation begins with the application. “Taking into consideration the application or goal of sequencing is key,” says Kaylinnette Pinet, Ph.D., Applications and Product Development Scientist II at New England Biolabs.
Pinet recommends, “knowing whether the goal is to do short- or long-read sequencing, broad or targeted, low or high input, DNA or RNA sequencing, etc. Then, factoring in what question should be answered by your sequencing, because this informs not only the type and number of samples being sequenced, but the depth of sequencing required, and the sequence analysis bioinformatic pipelines that will need to be applied once you have your sequencing data.”
Choosing library preparation kits tailored to a specific application and sequencing technology ensures that the libraries can run on the intended sequencing instrument and produce sufficient data. For example, a library prep kit for performing whole genome sequencing on an Illumina instrument would be different from the one used for investigating specific genomic regions on a sequencer from Pacific Biosciences.
In addition, by knowing the application and sequencer, users can narrow down their search with vendor-supported tools. Pinet believes, “The NEBNext Selector tool is a helpful first step for anyone new to NEBNext’s large portfolio of library prep reagents for NGS. By answering just a few questions about your needs, the tool narrows down the options, showing you only the products we recommend for your experiment.”
PCR vs PCR-free
Another consideration when selecting a DNA library prep is deciding whether to utilize a kit with or without an amplification step. The workflows of PCR-free library preps are frequently used with applications like whole genome sequencing because they reduce the potential of amplification errors caused during the preparation.
There are many kits designed to handle this type of workflow, like those mentioned by Pinet. “Because of the high conversion efficiency offered by the Ultra II DNA kits, they are ideal for sensitive applications such as PCR-free and low-level mutation detection.”
However, PCR-free workflows are not necessary for every application and most contemporary library preps use high-fidelity polymerases, which significantly reduce the likelihood of PCR-induced errors. Library preparations with an amplification step can also include unique molecular identifiers (UMIs) that are used to identify duplicates and PCR-induced errors and filter them from the analysis.
Workflow and multiplexing
Streamlined workflows and multiplexing capabilities are also critical factors for sequencing labs of any size. Utilizing library preps with improved workflows significantly reduces prep time and allows for increased levels of multiplexing, which in turn leads to higher data generation. Therefore, it’s necessary to examine the workflow and the level of multiplexing supported when selecting library preps.
The scale and efficiency of library prep workflows is one of the top considerations for Joe Mellor, the Chief Scientific Officer and Co-Founder of seqWell. “seqWell at its core is a multiplexed workflow company, focused on how to unlock the throughput and scale of modern sequencing instrument using innovative library prep products and technologies,” says Mellor. “We’re creating purpose-built multiplexing products for improving sequencing workflows of a variety of applications, helping our users scale their projects from 10s to 100s or 1000s of samples quickly and easily.” Plate-based library preps from seqWell are one example of kits designed to simplify the workflow and increase the scale of multiplexing.
Higher multiplexed library preps have been enabled by the diminishing cost of sequencing and the increased throughput of modern-day sequencers1. While the need to multiplex many samples may be less of a concern for smaller labs or experiments requiring only a few samples, workflow is still a key area for future developments in library prep technology. “Researchers are also able to use our products on a low-plex level,” adds Mellor, “and benefit from having a very simple, predictable, and robust workflows.”
Input DNA
The input material can also have a big impact on library prep selection. “Most library prep kits work well across a range of DNA inputs, but the source (tissue/liquid) and quality (degraded/fragmented) of the DNA can affect the complexity of libraries,” says Nick Downey, NGS Collaborations Lead, Integrated DNA Technologies.
In addition, many library prep kits have minimum requirements for DNA concentrations and the quality of the starting material. Special kits for low-input libraries are often necessary as they are optimized to process low concentrations of DNA, which would otherwise lead to sample loss on normal kits. “IDT’s xGen ssDNA & Low-Input DNA Library Prep Kit is suitable for researchers looking to sequence difficult-to-process sample types,” adds Downey. “This kit enables the preparation of libraries from sample types that might be degraded, and for samples with very low DNA input, from as low as 10 pg.”
The sample compatibility should be checked for each kit since the fragmentation, amplification, and addition of adapters are performed differently and have separate requirements. Due to their fragmented state and lower yields, DNA isolated from formalin-fixed paraffin-embedded (FFPE) tissue specimens and cell-free DNA (cfDNA) are common examples of sample types that require extra considerations for library preps.
Final considerations
The cost of each kit is another top consideration for library prep selection. Users should examine the cost per sample and account for the costs of additional reagents and equipment needed to perform the entire protocol. Some prep kits may require reagents or supplies from other vendors that should be factored into the final costs per sample.
After narrowing down several appropriate kits, the selection process can be simplified by examining the unique properties of the enzymes and adapters available in each of the library preps. High-efficiency enzymes can help reduce prep time, sequence bias, and the number of required sample manipulations. Some of the offered enzymes can also enhance DNA fragmentation, inhibit adapter dimers, and assist with library normalizations. Kits containing adapters with valuable elements such as unique dual indexes (UDI) and unique molecular identifiers (UMI) are often recommended as they assist with correcting errors during data analysis.
When asked about final considerations, Downey says, “Don’t be afraid to request a consultation. A good genomics provider will have experts that are ready and happy to assist and answer any of your research questions to help you find the right solution for your project or workflow needs.”
References
1. Wetterstrand KA. DNA Sequencing Costs: Data from the NHGRI Genome Sequencing Program (GSP). Published November 1, 2021. Accessed January 12, 2023. https://www.genome.gov/about-genomic...ing-Costs-Data