SEQuoia RiboDepletion Kit

Most rRNA depletion kits available on the market use a strategy that removes rRNA and mt rRNA from total RNA prior to library preparation. This strategy is not ideal when working with low-quality RNA or with low input amounts because the risk of degrading rare or low abundant transcripts is higher when working with RNA, which is susceptible to degradation.

By employing a post-library prep strategy to remove cDNA library fragments derived from rRNA or mt rRNA, the SEQuoia kit reduces this risk. In addition, removal of rRNA sequences after library preparation avoids the loss of small RNA or small RNA fragments from degraded RNA samples, a problem inherent to pre-library rRNA depletion kits.

The efficiency of a hybridization and capture depletion method depends on library fragment size. Larger fragments hybridize to the probes more efficiently, resulting in greater depletion. For maximum depletion efficiency with SEQuoia Complete Library Kit preps, the average library fragment size should be greater than 380 bp. Depleting a library composed of smaller fragments may result in a higher percentage of sequencing reads mapping to rRNA.

Yes. This kit uses a proprietary blend of synthetic biotinylated oligos that have homology to cDNA produced from human, mouse, and rat cytoplasmic (5S rRNA, 5.8S rRNA, 18S rRNA and 28S rRNA) and mitochondrial ribosomal RNA (12S rRNA and 16S rRNA). Target cDNA sequences hybridize to complementary biotinylated oligonucleotides through a PCR-mediated reaction. These complexes are captured using streptavidin-coated paramagnetic beads and removed from the library. Small library fragments may not hybridize as efficiently to the probes, thus will be left behind in the library, resulting in a higher percentage of reads mapping to rRNA.

The kit depletes rRNA from human, mouse, and rat samples. Depending on sequence similarity of rRNA in the sample, depletion rates may vary.

The SEQuoia RiboDepletion Kit is compatible with libraries generated using most common on-market library prep kits and of course, our SEQuoia Complete Stranded Library Prep Kit. The commercial kits we validated in our study include the NEBNext Ultra II Directional Kit, the Illumina TruSeq Stranded Total Kit, and the KAPA RNA HyperPrep Kit.

Yes, this kit will deplete rRNA-derived fragments from libraries prepared using degraded or fragmented RNA.

The input material used with the SEQuoia RiboDepletion Kit is RNA-Seq library fragments and not total RNA. This kit is designed to remove RNA-Seq library fragments that originated from rRNA. The input range is 0.1 ng to 20 ng of library fragments (cDNA).

The percent of reads that map to rRNA will vary depending on the library preparation kit used to construct the RNA-Seq library. Preparation kits that capture long RNAs (>200 bp) will typically have less than 10% of reads mapping to rRNA. A library prepared using the SEQuoia Complete Stranded Library Prep Kit will have more reads mapping to rRNA (typically less than 20%) because of the efficient capture of small RNA fragments.

Yes, so long as the libraries are generated using consistent indexing strategies. Do not pool a mixture of multiple single-indexed and dual-indexed libraries.

One of the benefits of a post-library preparation depletion strategy is that indexed libraries can be pooled and depleted in a single reaction. The SEQuoia RiboDepletion kit can be used to multiplex up to 96 libraries in one reaction. To maximize depletion efficiency for each indexed library, combine equimolar ratios of the indexed libraries to not exceed 20 ng of cDNA (combined) per ribodepletion reaction.

If the library is constructed using the SEQuoia Complete Stranded Library Prep Kit, use SeqSense NGS Data Analysis Software to process the FASTQ file and generate a SEQuoia Analysis Report. The percentage of rRNA reads present in the dataset can be calculated by dividing the Ribosomal Bases by PF Bases and multiplying by one hundred. Ribosome Bases and PF Bases are both reported in the Alignment section of the SEQuoia Analysis Report.

If the analysis pipeline reports bases or reads mapping to rRNA, divide this number by the total number of input bases and multiply by one hundred.

If the analysis pipeline does not report bases or reads mapping to rRNA, the percent residual rRNA can be assessed using the BAM output file produced when aligning the dataset against a reference genome and a third-party application.