Polynucleotide adapter design for reduced bias
A polynucleotide, target polynucleotide technology, applied in the field of polynucleotide adaptor design for reducing bias, can solve problems such as sequencing and discovery difficulties
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Embodiment 1
[0082] Example 1: Splint Ligation for Generation of RNA-Seq Libraries in a Single Reaction Vessel
[0083]In this example, the input RNA was a pool of microRNAs comprising 962 synthetic miRNAs (MiRXplore from Miltenyi Biotec (Auburn, CA)) with equimolar concentrations. TM library). All total RNA samples were obtained from BioChain, Inc. (Newark, CA). All oligonucleotides were synthesized by Integrated DNA Technologies, Inc. (Coralville, IA). use figure 1 The workflow shown in first ligates DNA adapters to the 3' end and then ligates RNA adapters to the 5' end of each input RNA in the cohort to form a sequencing library. 5' RNA adapters (double-stranded molecules with 5' single-stranded extensions) can be replaced with RNA hybrids, where the RNA is the upper strand suitable for hybridization to the 5' end of the target RNA using T4 RNA ligase, and the lower strand can be is DNA or RNA.
[0084] To reduce the secondary structure of the input RNA, heat the RNA to 70°C and th...
Embodiment 2
[0099] Example 2: Splint Ligation Enables Improved Library Yield Generation from Human Brain Total RNA
[0100] Three different methods for constructing RNA libraries were compared using the same amount of starting material (500 ng of human brain total RNA). These are (1) Illumina Small RNA Library Prep Kit (RS-200-0012, Illumina, SanDiego, CA), (2) Bioo Scientific Small RNA-seq kit V3 (NOVA-5132-05, BiooScientific, Austin, TX), and (3) the RNA library preparation method based on splint ligation described in Example 1. Prepare libraries according to manufacturer's instructions. Assess library yield with a bioanalyzer. Data shown are averages of 6-8 technical replicates. Yields were normalized to 9 PCR cycles. As a result, splint ligation-based RNA library preparation methods yield higher yields than Illumina and Bioo Scientific methods (see Figure 5 ).
Embodiment 3
[0101] Example 3: Demonstrating the benefit of cutting 3' single-stranded extensions at the 3' adapter prior to the second ligation step
[0102] RNA libraries were generated as described in Example 1, where the cleavage site in the 3' adapter was deoxyuridine, using (NEB M5505) to remove the single strand extension on the lower strand. Input RNA: 50 fmol of miRXplore input RNA and 2.5 pmol of Adapter 2 were ligated. Cleavage was performed either before ("pre-cut") or after ("post-cut") the second ligation (where 5.0 pmol of Adapter 1 was ligated to the 5' end of the RNA). The results are shown in Figure 3C middle. When USER cleavage was performed before the second ligation, primer dimer formation was reduced while target miRNA yield was increased when compared to USER cleavage after the second ligation. When comparing libraries from the two methods, cleavage before the second ligation resulted in a target-to-adaptor-dimer ratio of 7:1, while cleavage after the second li...
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