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Next-generation sequencing (NGS) has revolutionized biological research, enabling in-depth analysis of genomes, transcriptomes, and epigenomes. However, analyzing samples with limited material or compromised quality, such as formalin-fixed, paraffin-embedded (FFPE) tissues, remains a challenge. FFPE tissues are invaluable for retrospective studies due to the rapid tissue fixation of formalin and the long-term storage capacity at room temperature. Despite these advantages, FFPE samples are often low-input and degraded due to fragmentation and chemical modifications. To overcome these challenges and maximize data quality, selecting the right library prep kit is crucial.

This article will explore key considerations for selecting the right library prep kit and provide a comparison of select kits ideal for low-input and degraded FFPE samples.

Key Considerations for Library Prep Kits

Several factors influence kit selection:

• Input Amount: Each library prep kit has an optimal range of input, and it’s important to understand this range even before extraction. Although input is specific to kits, in general, if you have between 100-1000 ng, you will likely be able to use a standard library prep kit with no modifications to the protocol. Then for samples with less than 100 ng, a low-input kit or modified protocol may be necessary. It’s also important to note that if your samples are degraded, you may need a higher amount to compensate.¹
• Sample Type: It’s essential to choose a library prep kit that is compatible with your sample type. For example, if you have low-input FFPE samples, it may be important to find a kit with reagents that can help repair some of the DNA damage or a protocol that allows you to skip the fragmentation step in case your samples are too fragmented. Most kits will clearly indicate sample compatibility.
• Workflow: The time a protocol takes and its automation compatibility is another important factor. If a library prep kit is compatible with automation, this can often streamline the process, shorten the time, minimize user error, and maximize reproducibility. Of course, there are different automation devices on the market and compatibility with specific devices will have to be checked.

Comparison of Some Popular Library Prep Kits for FFPE Samples

Below are descriptions of select kits that can be used for low-input and degraded samples, especially those from FFPE.

• Illumina DNA Prep with Enrichment kit (formerly Nextera Flex for Enrichment): It takes about 6.5 hours in total with hands-on time of ~2 hours, can incorporate automation, and requires 10-1000 ng high-quality gDNA or 50-1000 ng FFPE DNA. For the FFPE degraded DNA samples, a ∆Cq value of ≤ 5 needs to be obtained using the Illumina Infinium FFPE QC Kit, and then the PCR cycles for the amplification of tagmented DNA step need to be increased to 12.^{2 3}
• Illumina TruSeq Stranded Total RNA kit: It takes about 11.5 hours in total with a hands-on time of 5.5 hours, can incorporate automation, and requires 0.1-1 ug total RNA. For degraded samples, QC needs to be done to determine the fragment size range and then the fragmentation time will need to be adjusted based on this size range to avoid over fragmentation.^{4 5}
• NEBNext Ultrashear FFPE DNA Library Prep kit: It takes about 3.25-4.25 hours in total, can incorporate automation, and requires 5-250 ng DNA. It uses a specialized enzyme mix for FFPE DNA to increase quantity and quality and also includes reagents that repair damage caused by the FFPE process.^{6 7}
• NEBNext Ultra II Directional RNA Library Prep Kit for Illumina: It takes about 6 hours in total, can incorporate automation, and requires 10 ng-1 ug RNA. It’s a directional kit, which means the researcher will receive data about the original DNA strand that the RNA was transcribed from. This is done through the “dUTP method,” where the second strand of cDNA is labeled with dUTPs and selectively destroyed with subsequent treatment.^{8 9}
• KAPA DNA HyperPrep Kit: It takes about 2-3 hours in total, can incorporate automation, and requires 1 ng-1 ug DNA. The kit removes bead cleanups and combines enzymatic steps into a single-tube to improve library prep efficiency, which is especially useful for degraded FFPE and low-input samples. Additionally, it has both PCR and PCR-free versions.¹⁰
• KAPA RNA HyperPrep Kit: It takes about 4 hours in total, can incorporate automation, and requires 1-100 ng RNA. It’s a stranded kit and enriches the desired transcripts via mRNA capture (polyA-selection) or rRNA-depletion. In addition, it’s optimized for good performance with degraded and low-input samples through single-tube chemistry and high-quality enzymes, such as the engineered KAPA HiFi DNA polymerase, which supports library prep with low GC bias and even coverage.¹¹
• IDT xGen cfDNA & FFPE DNA Library Prep v2 MC Kit: It takes about 4 hours in total, can incorporate automation, and requires 1-250 ng DNA. It’s a kit designed specifically for challenging cfDNA, FFPE, and low-input DNA samples and can support mechanically sheared FFPE DNA samples as well. The kit’s first step is to prepare the DNA for ligation by converting it from sheared DNA into blunt-ended DNA. Then the Ligation 1 Adapter is added to the 3’ end of the insert, where it helps prevent adapter-dimer formation through its blocking group, and the Ligation 2 Adapter is added to the 5’ end of the DNA in order to create a double-stranded product. Finally, PCR amplification is performed.^{12 13}
• IDT xGen Broad-Range RNA Library Preparation Kit: It takes about 4.5 hours in total, can incorporate automation, and requires 10 ng-1 ug RNA or 100 pg-100 ng mRNA. It’s strength is its flexibility in working with a broad range of RNA inputs and its strand-specificity, while also allowing polyA-selection or rRNA-depletion to be done on degraded FFPE samples. The kit uses patented Adaptase technology to directly convert first-strand cDNA into libraries, which saves time by removing the need for second-strand cDNA synthesis or degradation.¹⁴
• Takara ThruPLEX DNA-Seq Kit: It takes about 2 hours in total, cannot incorporate automation, and requires as little as 50 pg fragmented, double-stranded (dsDNA). It builds on their ThruPLEX workflow, which is performed in a single tube and needs no purification steps or sample transfers.¹⁵
• Takara SMARTer Universal Low Input RNA Kit for Sequencing: It takes about 2 hours in total, cannot incorporate automation, and requires 10-100 ng total RNA or 200 pg-10 ng rRNA-depleted RNA. It generates cDNA from very low-input RNA and was created especially for degraded RNA, RNA with a low RIN value and RNA without any polyA-tails. Thus it’s useful for FFPE or laser-captured samples. The SMART technology uses random (universal) priming, which helps ensure the cDNA transcripts maintain the true representation of the original mRNA transcripts. Then once the cDNA is generated, Takara’s ThruPLEX DNA-Seq Kit can be used to finish the library prep process.¹⁶
• Watchmaker DNA Library Prep Kit: It takes about 2 hours, can incorporate automation, and requires 500 pg-1 ug DNA. The kit was designed for low-input samples with automation in mind, and it can convert a higher amount of DNA into library molecules, which is especially important for samples in the pg range. It also has suitable versions for unfragmented and fragmented DNA. The kit with fragmentation is recommended for FFPE samples specifically.¹⁷
• Watchmaker RNA Library Prep Kit: It takes about 3.5 hours, can incorporate automation, and requires only 0.25-100 ng total RNA. The kit is specifically for very low-input samples, and was designed to be easily automated to save both time and consumables. It has a novel, engineered reverse transcriptase that also supports working with degraded FFPE samples.¹⁸

Table of Library Prep Comparisons

Manufacturer	Kit Name	Input Needed	Time Needed	Automation Incorporation
Illumina	Illumina DNA Prep with Enrichment Kit	10-1000 ng gDNA or 50-1000 ng FFPE DNA	6.5 hours	Yes
Illumina	Illumina TruSeq Stranded Total RNA Kit	0.1-1 ug RNA	11.5 hours	Yes
New England Biolabs	NEBNext Ultrashear FFPE DNA Library Prep Kit	5-250 ng DNA	3.25-4.25 hours	Yes
New England Biolabs	NEBNext Ultra II Directional RNA Library Prep Kit for Illumina	10 ng-1 ug RNA	6 hours	Yes
Roche	KAPA DNA HyperPrep Kit	1 ng-1 ug DNA	2-3 hours	Yes
Roche	KAPA RNA HyperPrep Kit	1-100 ng RNA	4 hours	Yes
Integrated DNA Technologies	IDT xGen cfDNA & FFPE DNA Library Prep v2 MC Kit	1-250 ng DNA	4 hours	Yes
Integrated DNA Technologies	IDT xGen Broad-Range RNA Library Preparation Kit	10 ng-1 ug RNA or 100 pg-100 ng mRNA	4.5 hours	Yes
Takara Bio	Takara ThruPLEX DNA-Seq Kit	50 pg fragmented dsDNA	2 hours	No
Takara Bio	Takara SMARTer Universal Low Input RNA Kit for Sequencing	10-100 ng total RNA or 200 pg-10 ng rRNA-depleted RNA	2 hours	No
Watchmaker	Watchmaker DNA Library Prep Kit	500 pg-1 ug DNA	2 hours	Yes
Watchmaker	Watchmaker RNA Library Prep Kit	0.25-100 ng total RNA	3.5 hours	Yes

Table 1: Comparison of Selected Library Prep Kits

How can Genohub help?

Selecting the optimal library prep kit for your low-input or degraded samples is crucial for generating reliable NGS data. We understand this challenge, which is why we combine our expertise with partner NGS facilities who can offer their experience with these kits. Our team can directly connect you with the right partner to ensure your NGS project is successful, even with low-input or degraded samples.

Contact Genohub today to discuss your specific needs for your next low-input NGS experiment: https://genohub.com/ngs/ .

References

1. “Standard vs Low Input Workflow Selection FAQ for Illumina DNA PCR Free Prep | Illumina Knowledge.” Illumina.com, 2019, knowledge.illumina.com/library-preparation/dna-library-prep/library-preparation-dna-library-prep-faq-list/000003460. Accessed 31 Jan. 2025. ↩︎
2. “Illumina DNA Prep with Enrichment | Fast, Flexible Targeted Resequencing.” Illumina.com, 2020, http://www.illumina.com/products/by-type/sequencing-kits/library-prep-kits/illumina-dna-prep-enrichment.html. Accessed 31 Jan. 2025. ↩︎
3. “Recommended Quality Control of FFPE Samples for Illumina FFPE Supported Library Preparation Kits | Illumina Knowledge.” Illumina.com, 2019, knowledge.illumina.com/library-preparation/general/library-preparation-general-reference_material-list/000001941. Accessed 31 Jan. 2025. ↩︎
4. “TruSeq Stranded Total RNA | a Complete View of the Transcriptome.” Illumina.com, 2019, http://www.illumina.com/products/by-type/sequencing-kits/library-prep-kits/truseq-stranded-total-rna.html. ↩︎
5. TruSeq ® Stranded Total RNA Sample Preparation Guide. 2013. ↩︎
6. Front Line Genomics, and Ashleigh Davey. “Which NGS DNA Library Prep Kit Should You Choose? – Front Line Genomics.” Front Line Genomics, 19 Mar. 2024, frontlinegenomics.com/which-ngs-dna-library-prep-kit-should-you-choose/.
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7. New England Biolabs. “NEBNext Ultrashear® FFPE DNA Library Prep Kit.” New England Biolabs, 2025, http://www.neb.com/en-us/products/e6655nebnext-ultrashear-ffpe-dna-library-pep-kit. ↩︎
8. New England Biolabs. Neb.com, 2020, http://www.neb.com/en-us/products/e7760-nebnext-ultra-ii-directional-rna-library-prep-kit-for-illumina. Accessed 31 Jan. 2025. ↩︎
9. Obtain Superior NGS Library Performance with Lower Input Amounts Using the NEBNext ® Ultra TM II Directional RNA Library Prep Kit for Illumina ®. ↩︎
10. “KAPA HyperPrep Kits.” Diagnostics, 2025, sequencing.roche.com/us/en/products/group/kapa-hyperprep-kits.html#productSpecs. Accessed 31 Jan. 2025. ↩︎
11.  “KAPA RNA HyperPrep Kits.” Diagnostics, 2020, sequencing.roche.com/us/en/products/group/kapa-rna-hyperprep-kits.html#productSpecs. Accessed 31 Jan. 2025. ↩︎
12. “CfDNA & FFPE DNA Library Prep Kit | IDT.” Integrated DNA Technologies, 2024, http://www.idtdna.com/pages/products/next-generation-sequencing/workflow/xgen-ngs-library-preparation/dna-library-preparation/cfdna-ffpe-prep-kit#faqs. Accessed 31 Jan. 2025. ↩︎
13. Integrated DNA Technologies. XGenTM CfDNA & FFPE DNA Library Prep v2 MC Kit. 2024, sfvideo.blob.core.windows.net/sitefinity/docs/default-source/protocol/xgen-cfdna-and-ffpe-dna-library-prep-mc-kit-protocol.pdf?sfvrsn=f7a6e007_12. ↩︎
14. Optimizing RNA-Seq Data Quality and Costs for FFPE Samples with the XGen. ↩︎
15. “All-Purpose Library Prep for Fragmented DNA from Any Sample Type.” Takarabio.com, 2025, http://www.takarabio.com/products/next-generation-sequencing/dna-seq/ngs-library-prep/general-purpose-library-prep-(50-pg%e2%80%9350-ng). Accessed 31 Jan. 2025. ↩︎
16. “SMARTer Universal Low Input RNA Kit.” Takarabio.com, 2025, http://www.takarabio.com/products/next-generation-sequencing/rna-seq/universal-low-input-cdna-synthesis. Accessed 31 Jan. 2025. ↩︎
17. “DNA_LPK.” Watchmaker Genomics, 5 Nov. 2024, watchmakergenomics.com/portfolio/dna-seq-solutions/dna_lpk/. Accessed 31 Jan. 2025. ↩︎
18. “Watchmaker RNA Library Prep Kits.” Watchmaker Genomics, 2 May 2024, watchmakergenomics.com/portfolio/rna-solutions/rna-prep/. Accessed 31 Jan. 2025. ↩︎