SEQuoia FAQs

The SEQuoia Complete RNA Library Prep Kit provides all reagents required to construct a library from RNA, with the exception of the SPRISelect beads from Beckman Coulter.

The following reagents are required but not supplied. These materials have been validated with the SEQuoia Complete Stranded RNA Library Prep Kit protocol. Substitutions may not produce ideal results.

• 2 ml RNase, DNase free, low-bind PCR tube strips and cap strips (TBC0802 or TBC1202)
• RNase-free filtered pipet tips: 10 μl, 20 μl, and 200 μl
• SPRISelect beads from Beckman Coulter
• 10mM Tris-HCl pH8 (RNase, DNase Free)
• 80% ethanol (prepare fresh)

Optional Materials Not Provided

• SEQuoia Dual Indexed Primers Set (12011928) or SEQuoia Dual Indexed Primers Plate (12011930)
• rRNA depletion kit
• ddPCR Library Quantification Kit for Illumina TruSeq (1863040)

Equipment List

• Thermal cycler for accurate incubation temperatures
• Calibrated single-channel and multi-channel pipets: 10 μl, 20 μl, and 200 μl
• Magnetic rack for small-scale separation of magnetic beads in 0.2 ml tubes
• Vortex
• Microcentrifuge for 0.2 ml tubes
• 96-well PCR cooler rack
• Ice bucket

The SEQuoia Complete Stranded RNA Library Prep Kit components in Reagent Box A should be stored at –20°C. When using the kit, keep reagents on ice unless otherwise noted in the protocol. The Purification Beads (Reagents Box B) should be stored at 4°C.

SEQuoia Complete Stranded RNA Library Prep Kit is guaranteed for 6 months after the shipping date, if stored properly.

The ideal RNA purification strategy depends on the sample type and experimental objectives. We have tested and recommend using PureZOL RNA Isolation Reagent (7326880) for tissue samples and the SingleShot Cell Lysis Kit (1725080) for cultured cells. If small RNAs are of interest, we do not recommend using a column-based based kit, as RNA fragments under 200 may be lost. Instead, use the SingleShot Cell Lysis Kit.

Additionally, these references provide a good overview of various methods for small RNA isolation;

• Quantification of Small Non-Coding RNAs Allows an Accurate Comparison of miRNA Expression Profiles. Masotti A, et al. Journal of Biomedicine and Biotechnology Volume 2009, Article ID 659028
• A comparison of commercially-available automated and manual extraction kits for the isolation of total RNA from small tissue samples. Sellin Jeffries MK, Kiss AJ, Smith AW, Oris JT. BMC Biotechnology, 201414:94

The SEQuoia Complete Stranded RNA Library Prep kit is compatible with 1 ng to 1 μg of total RNA (pre-depleted of ribosomal RNA)

RNA Fragmentation. Universal Human Reference RNA was incubated at 94°C for the designated amount of time.

Optimum fragmentation depends on several factors including RNA quality. We recommend that the fragmentation protocol be optimized based on experimental conditions. Prior to starting the library construction protocol, assess the quality of the input RNA by running the sample on an Agilent Bioanalyzer RNA 6000 Nano/Pico Chip to determine the RNA Integrity Number (RIN). Vary the temperature and incubation time to achieve the desired fragment size. While this must be determined empirically, the following recommendations can be used as an initial guideline:

Depleting rRNA prior to library construction is optional, but depletion of rRNA or its constructs before sequencing will help to maximize the quantity and quality of sequencing reads. If using a low amount of starting material, it is particularly advantageous to deplete rRNA-specific cDNA constructs after library construction to minimize the risk of losing rare RNA targets. Learn how the SEQuoia RiboDepletion Kit is the optimal solution for post-library depletion.

Yes. Genomic DNA is a common contaminant from RNA preps and it may interfere with accurate quantification of RNA. To remove contaminating gDNA from RNA samples, treat the sample with DNase I (not provided in the kit). After treatment with DNase I, the enzyme should be removed from the sample using phenol/chloroform extraction and ethanol precipitation. This is a critical step, as any residual activity of DNase I will degrade single-stranded DNA probes.

Yes. In fact, the SEQuoia Complete Stranded RNA Library Prep Kit works exceptionally well with degraded RNA from FFPE liquid biopsies. The unique properties of SEQzyme bypass many of the limitations of enzymes used in other commercial kits, enabling it to accommodate a broad range of input amounts and RNA quality range. FFPE-RNA-specific fragmentation recommendations are included in the manual.

Typically, SEQuoia Complete Stranded RNA Library Prep Kit requires 3–5 fewer cycles compared to other commercially available kits, due to a high conversion rate. This is advantageous because over amplification can result in artifacts such as amplification bias, increased duplicate rates, and chimeras. To minimize artifacts, optimize the number of PCR amplification cycles so that the final library concentration is between 200–1,000 ng. The number of PCR cycles is dependent upon the amount and quality of input RNA. The following table provides general guidelines and can be used as a starting point for optimization.

PCR Cycle Recommendations

SEQuoia Complete Stranded RNA Library Prep Kit captures both short and long RNA biotypes, including mRNA, miRNA, lncRNA, snoRNA. Because this kit efficiently captures small RNAs, it is recommended that sequencing depth be 20M–30M reads to ensure the accuracy of the results.

Yes. Strandedness is maintained through the unique properties of SEQzyme, a proprietary, engineered retrotransposon. During library construction, RNA fragments undergo polyadenylation, creating a synthetic poly(A) tail on each fragment. This tail hybridizes to poly(dT) primers, which contain the R2 sequence, creating a complex that is recognized by SEQzyme. SEQzyme binds the complex and synthesizes cDNA through reverse transcription using its strand displacement and high-processivity properties. When SEQzyme reaches the 5’ end of the RNA fragment, it binds to and adds the second adapter. This highly efficient continuous synthesis reaction results in a sequencing-ready, 99% stranded library that represents all RNA biotypes.

Molecular Structure of RNA Library prepared with the SEQuoia Complete Stranded RNA Library Prep Kit. SEQuoia Complete libraries will retain >99% of strand information. The "second strand" should be used during alignment and analysis.

Following the fragmentation step, all fragments undergo polyadenylation. The synthetic poly(A) tails anneal to poly(dT) primers, creating a complex recognized by SEQzyme. SEQzyme binds to the complex and synthesizes cDNA through reverse transcription using its strand-displacement and high-processivity properties.

This is the result of the unique properties of SEQzyme. When SEQzyme reaches the 5’ end of an RNA fragment, it binds to and adds a second adapter. This highly efficient continuous synthesis reaction results in a sequencing-ready, 99% stranded library that represents all RNA biotypes.

To achieve the highest quality sequencing data, it is important to accurately quantify libraries to create optimum cluster densities. It is also imperative the quality of the library be assessed to confirm insert size and minimal adapter-adapter products. For highly precise quantification and qualification of a library preparation use the ddPCR Library Quantification Kit for Illumina TruSeq (1863040). This kit enables the assessment of library quality by viewing fluorescence amplitude plots of droplet populations that discern features such as well-constructed libraries and adapter-adapter species.

Alternatively, use Qbit or a qPCR library quantification kit to assess library concentration and Bioanalyzer to assess size distribution. These methods, however, are subject to bias due non-specificity.

Following library construction, assess the library quality on an Agilent Bioanalyzer DNA chip. Check that the electropherogram shows a narrow distribution with a peak size approximately 400 bp. If a peak at ~80 bp (primers) or 128 (adapter-dimer) is visible, repeat the SPRISelect Bead clean-up step.

Typical Bioanalyzer Trace of a SEQuoia Complete Stranded RNA Library. Following library construction, library quality was assessed on an Agilent Bioanalyzer DNA chip. Note that the electropherogram shows a narrow distribution, with a peak size of approximately 400 bp. If a peak at ~80 bp (primers) or 128 (adapter-dimer) is visible, repeat the SPRISelect Bead clean-up step.

Yes. The first 8 bases on Read 2 are a random tag sequence which can be used as a UMI. With the SEQuoia Complete Stranded RNA Library Prep Kit, we recommend only the first 8 bp are acquired 8 bp of Read 2 and have longer Read 1.

Yes. We offer a set of 12 or a set of 96 unique dual-indexed primers for multiplexing of libraries. Primers are added in the final amplification step, resulting in sequencing-ready libraries at the completion of the final clean up step. We strongly recommend using SEQuoia Dual Indexed Primers for multiplexing, as these have been optimized to reduce sequencing artifacts. Visit bio-rad.com/SEQDIP for more information.

The SEQuoia Dual Indexed Primers are available as a set of 12 or a plate of 96 unique dual indexed primers. The vial in the set of 12 DIPs and each well of the 96-well DIP plate contain pairs of primers that contain one i5 indexed adapter and one i7 indexed adapter. The primers in the set of 12 DIPs are identical to the DIPs plated in wells A1 through B4 on the 96-well plate.

Each option provides sufficient volume for 96 total reactions. Though volumes provided on the 96-well plate may exceed that which is required, to avoid the possibility of cross-contamination it is not recommended that left over volume be saved and reused. 

The proprietary design of the Dual Indexed Primers ensures the addition of two different adapters in the correct orientation to opposing ends of a DNA fragment during the PCR amplification step. This results in a final construct that is ready for sequencing.

This is not recommended as the cluster density and run quality may be compromised.

We recommend sequencing with single-end reads to save on sequencing cost.

• The first 8 bases on R2 are a random tag sequence which can be used as a UMI.
• Recommended read length: 100 x 8 or 75 x 8 of Read1 x Read2
• Read depth should be determined based on the application and prior experience. The typical range is 20–30M single-end reads due to the number of small RNA fragments captured, which increases the library complexity.
• With single reads, using SEQuoia Complete Stranded RNA Library Prep Kit, you can get coverage of full transcript as shown with all the data on this page.
• For pair-end reads, there may be a significant drop in read quality for R2 because of the presence of the poly(T) sequence.

Sequencing depth depends on your specific sample type and application. Because of the complexity of libraries constructed using SEQuoia Complete Stranded RNA Library Prep Kit, a read depth of 20M–30M reads is recommended.

Pair-end reads are not recommended. The presence of the poly(T) sequence may decrease the read quality for Read 2 after the 8 base UMI.

Yes, a read trimming step is recommended before proceeding with any quality metric calculations or downstream analysis. The SEQuoia Complete adapters contain 5 random bases that appear immediately upstream of the insert and a poly(A) sequence immediately following the insert. To trim the 5 random bases and the poly(A) tail use cutadapt, which runs on Linux, Mac OS, and Windows. See the cutadapt user guide for more information. Work with the reads in a FASTQ file (compressed or uncompressed). The reads can be trimmed running cutadapt from the command line:

cutadapt -u 1 -a A{10} -m 15 -o output_file.fastq.gz input_file.fastq.gz

Here is what is happening:

No. The adapters in the SEQuoia Complete Stranded RNA Library Prep Kits are compatible with flow cells used with Illumina platforms.

The range of mapped reads will vary from library to library, depending on several factors, but you may see a higher fraction of unmapped (unaligned) reads with SEQuoia Complete libraries compared to other products. This is linked to the fact that the SEQuoia workflow captures a larger variety of transcripts (several of which are as yet unmapped) than other kits.

In addition to traditional analysis tools for fastseq data files, SEQuoia Complete data can be processed by a set of Bio-Rad applications. These are available as a Docker container or as a web-hosted portal.
Visit the SeqSense NGS Analysis Software page for details and downloads.