During the last decade, next-generation sequencing (NGS) has exploded in both popularity and routine usage, transforming how biological systems are studied. While the technology has improved, it has also become very complex; there are many steps within the NGS’ workflow, and each one can be critical to obtaining high-quality data. Fortunately, some things have been simplified and kits are now available to help with different steps of library preparation, including target selection, library construction, and template preparation.
There are a number of kits on the market, and determining which one to use can be overwhelming. To help untangle how these different kits perform, we’ve put together a compilation of NGS library prep kit reviews from the Biocompare product review team.
There are a few crucial insights you should consider before selecting an NGS library prep kit:
- Sample inputs — The quality and quantity of your sample inputs are one of the most critical parts of any NGS workflow. For example, if you’re selecting a tagmentation-based library prep workflow, the resulting fragment sizes depend directly upon the input materials’ mass. Adding too much input DNA can lead to “undertagmentation,” and too much can lead to “overtagmentation,” so read through the kits’ preferred input size and make sure the applied volume of DNA is appropriate to avoid errors in downstream protocols.
- PCR protocols — PCR plays an essential role in targeted NGS sequencing, allowing multiple NGS libraries to be generated and multiple targeted regions to be sequenced simultaneously. However, several prep kits do not require PCR but include reagents, sample purification beads, and indexes for multiplexing. If you don’t want to prepare your libraries completely PCR-free, you can utilize a single PCR cycle to create double-stranded library molecules without any subsequent problems in library QC and with no introduced PCR bias.
- Qualitative and quantitative QC methods — Library QC can help verify insert size and check for adapter dimer contaminants before sequencing. However, it’s important to assess both qualitative and quantitative QC methods. qPCR can assist with quantitative assessment, and analyzing fragment size and distribution is helpful for qualitative QC. Methods like electrophoresis, MiSeq, or UV-Vis spectroscopy are popular for library QC. However, before choosing, consider the average fragment size of your NGS library, as it might influence your ability to use fluorometric or electrophoresis methods. When in doubt, follow your kit’s advice regarding library QC and any quantitation steps!
After making these decisions about your workflow, you can start researching which kit meets your requirements. To start, check out some of what Biocompare reviewers have to say about kits from various vendors and how they’re utilized for their research.
Revvity
NEXTflex PCR-Free DNA-Seq Kit
The NEXTflex PCR-Free DNA-Seq Kit from Revvity is a step-by-step PCR-free DNA library preparation kit for DNA samples ranging from 500 ng to 3 ug in starting material. According to Biocompare reviewer Annie Kessler, a Research Scientist from New York University, this kit is ideal for high-concentration genomic DNA samples that don’t require PCR amplification during library preparation. Annie states that, though they must be purchased separately, the kit can be multiplexed using Revvity's PCR-free barcodes and requires access to a non-enzymatic fragmentation method for best results. Additionally, Annie boasts how the kits’ color coding makes it very easy to follow an already well-laid out instruction manual and has consistently produced high-quality results for her work.
NEXTflex Rapid DNA-Seq Kit 2.0
Annie also reviewed another DNA prep kit from Revvity, their NEXTflex Rapid DNA-Seq Kit 2.0. She states that this kit is excellent for preparing Illumina-compatible libraries from low-concentration gDNA samples. The kit requires access to a non-enzymatic fragmentation platform and incorporates various PCR cycles to amplify libraries prepared from low concentrations of starting material (range of 1 ng to 1 ug) before sequencing applications. Annie notes that PCR cycling can be different by library/sample if the starting concentration of samples being prepped is varied, and the incorporation of PCR amplification may introduce bias. However, Annie also notes that the workflow is easy to follow due to the reagent color coding system, and safe stopping points are well defined within the manual. Plus, this kit works with many different barcodes or kits for multiplexing.
New England Biolabs
NEBNext Ultra II DNA Library Prep Kit
Biocompare reviewer Dr. Thippeswamy Sannasiddappa, a Post-Doctoral Fellow from the University of Birmingham regularly uses New England Biolabs’ NEBNext Ultra II DNA Library Prep Kit for whole bacterial genome sequencing using Illumina’s Miseq platform. They started with 100 ng of sheared S. aureus genomic DNA for the input and stated that the kit’s protocols for subsequent steps are easy to follow. After sequencing, Dr. Sannasiddappa noted that 3.5 million reads were obtained using this library prep kit, with more than 90% of reads mapping to their genome of interest. They also note that they have used this kit at lower sample levels, going down to as little as 500 picograms of sheared DNA.
Takara
ThruPLEX® DNA-Seq Kit
Biocompare reviewer Sibiao Yue from Carnegie Mellon University’s Embryology Department utilizes Takara’s ThruPLEX® DNA-Seq Kit for epigenetic work in mice. Sibiao uses this kit to prepare samples for chromatin immunoprecipitation followed by sequencing, or ChIP-Seq, analysis. Sibiao states that this easy-to-use kit includes a simple protocol that’s easy for new users without experience preparing ChIP-seq libraries and it only takes three hours to go from start to finish. They only mention that you must handle small volumes of enzyme and buffer mixtures during the template preparation and synthesis steps, so pipette with care.
Agilent
HaloPlex Custom Kits
The Agilent HaloPlex custom kits offer the flexibility to create custom panels for any genomic regions of interest, which reviewer Rachel Crossland from Newcastle University states that this kit works well for genomics research of B-NHL. The HaloPlex system allows Rachel to prepare DNA libraries to sequence fresh, frozen and formalin-fixed paraffin-embedded (FFPE) tumor samples using a custom-designed targeted sequencing approach. However, she notes that the protocol is rather lengthy, requiring two dedicated days since it includes a 16-hour-long hybridization step. Despite the extended workflow, library preparations were successful using this kit for both FF and FFPE material, although Rachel stated that FFPE required a higher input DNA concentration. According to Rachel, this is a good DNA library prep kit for downstream targeted sequencing that can be applied to both FF and FFPE, with some optimization necessary.
Roche
Library Quantification Kit
Reviewer Triveni Mahendrappa from the University of Birmingham uses Roche's Library Quantification Kit to research Salmonella enterica DNA. Triveni states that the kit performed very well, following protocols for cycling conditions, data collection and quantification analysis. They note consistent quantification values among libraries and optimal cluster generation in MiSeq.
Conclusion
Before selecting any NGS library prep kit, you must consider your experimental goals. There are many possible methods for DNA amplification, each with its strengths and weaknesses, but planning out your workflow ahead of time can give you the best idea about which kit suits your research needs. Considering run time, PCR vs. PCR-free, cost and availability are just a few considerations you should make before selecting an NGS library prep kit. Once you’ve run your experiments, consider leaving a Biocompare review detailing your experience for a $20 Amazon gift code!