Small RNA Sequencing NGS Library Preparation Kits, Single Adapter Ligation and Circularization-based
Accuracy through circularization: Bias-free NGS library preparation
• Elimination of incorporation bias
• Detect all important biomarkers
• Gel-free highly efficient protocol yielding superior quality sequencing libraries
• Lower sequencing costs by reducing the number of irrelevant reads
• Kits for low total RNA inputs from either cells/tissue or biofluids such as plasma/serum
SomaGenics´ RealSeq® is a novel method for preparing small-RNA sequencing libraries that nearly eliminates incorporation bias in Next-Generation- Sequencing (NSG). This technology solves the problem of commonly used sequencing library preparations that lead to underdetection of many miRNAs, some by as much as 10,000-fold.
Under-representation in sequencing libraries can obscure the presence of some RNAs, including potential biomarkers. Accurate quantification of microRNAs (miRNA) and other small RNAs is important for understanding their biology and for developing new biomarkers and therapeutic targets.
Most bias stems from sequence-dependent variability in the enzymatic ligation reactions that attach the two adapters to the 3’ and 5’ ends of the miRNAs /small RNAs during preparation of sequencing libraries. By ligating the miRNA/small RNA to a novel single adapter and circularization of the ligation product, RealSeq® greatly reduces library preparation and sequencing bias, allowing the identification of the largest variety of miRNAs and other small RNAs and their robust quantification in biological samples across a wide range of RNA input levels.
Read more about SomaGenics´ RealSeq® Technology:
Genome Biology 19:105 (2018)
Decreasing miRNA sequencing bias using a single adapter and circularization approach. Sergio Barberán-Soler, Jenny M. Vo, Ryan E. Hogans, Anne Dallas, Brian H. Johnston and Sergei A. Kazakov.
3´ ends of miRNAs or any other 5’-p and 3’-OH small RNAs are ligated to RealSeq® adapters which contain standard adapter sequences (shown in blue and green) that are compatible with Illumina/Solexa sequencing.
Unligated adapters are blocked before circularization of the miRNA-adapter product and then removed. This intramolecular ligation is significantly more efficient than intermolecular ligation of 5’-adapters in standard two-adapter ligation schemes.
Reverse transcription of the circularized miRNA-adapter products yields monomer cDNAs with the targets sequences flanked by Illumina 5’- and 3’-adapter sequences. PCR amplification of the RT product with the extended, bar-coded PCR primers, yields amplicons ready for size selection using SPRI select technology (Beckman Coulter) which is included in RealSeq®-AC and RealSeq®-biofluids kits.
RealSeq® accurately quantifies small RNAs
One pmole of miRXPlore Universal Reference pool (Miltenyi Biotec) was used to compare incorporation bias in six different library preparation kits. Purified libraries were sequenced on the Illumina MiSeq platform. Trimmed sequencing reads were aligned to a custom miRNA reference. Reads mapping to miRNAs were counted and fold-deviations from the equimolar input were calculated and plotted as log2 values. Measurements of miRNA levels within a factor of two of the expected values (between vertical lines) are considered unbiased (Fuchs et al, 2015). The method of adapter attachment to the miRNAs is noted in the legend. Libraries prepared using RealSeq®-AC have substantially fewer miRNAs with bias than the ones prepared using any of five other commercially available library preparation kits.
The RealSeq® workflow is efficient and gel-free
Library preparation takes approximately 8 hours for sequence-ready samples
You can order RealSeq®-AC kit for Total RNA inputs as low as 1 ng from cells or tissue or RealSeq®-biofluids kits for cf-miRNA/small RNA sequencing from serum/plasma volumes as low as 50 ul.
Additional reverse index primers are available separately.
Size selection beads are included in RealSeq®-biofluids kits BUT NOT in RealSeq®-AC kits. RealSeq®-AC size selection beads are available seperately.
|RealSeq-AC Kit for small RNA Library Construction from total RNA samples from tissue or cells (Illumina Platform) without Size Selection Beads||500-00012-SOM||12 rxns||1121 €||DETAILS||Add to Cart|
|RealSeq-AC Kit for small RNA Library Construction from total RNA samples from tissue or cells (Illumina Platform) without Size Selection Beads||500-00048-SOM||48 rxns||3609 €||DETAILS||Add to Cart|
|Size Selection Beads for RealSeq-AC Kit||510-00012-N-SOM||12 rxns||100 €||DETAILS||Add to Cart|
|Size Selection Beads for RealSeq-AC Kit||510-00048-N-SOM||48 rxns||233 €||DETAILS||Add to Cart|
|RealSeq-Biofluids NGS Library Preparation Kit for miRNAs and small RNAs, for total RNA samples from biofluids (Illumina Platform), includes size selection reagent||600-00012-SOM||12 rxns||1172 €||DETAILS||Add to Cart|
|RealSeq-Biofluids NGS Library Preparation Kit for miRNAs and small RNAs, for total RNA samples from biofluids (Illumina Platform), includes size selection reagent||600-00048-SOM||48 rxns||3660 €||DETAILS||Add to Cart|
|RealSeq Reverse Primers, Index 1-12||501-00024-SOM||12 rxns||201 €||DETAILS||Add to Cart|
|RealSeq Reverse Primers, Index 13-24||502-00024-SOM||12 rxns||201 €||DETAILS||Add to Cart|
|RealSeq Reverse Primers, Index 25-36||503-00024-SOM||12 rxns||201 €||DETAILS||Add to Cart|
|RealSeq Reverse Primers, Index 37-48||504-00024-SOM||12 rxns||201 €||DETAILS||Add to Cart|