Journal: Nature Communications

Article Title: Simultaneous and stoichiometric purification of hundreds of oligonucleotides

doi: 10.1038/s41467-018-04870-w

Figure Lengend Snippet: Characterization of high-plex SNOP product purities using next-generation sequencing (NGS). a To minimize the ligation and sequencing bias of library preparation and sequencing steps, the oligos used for this set of experiments all possess a common 3′ sequence. Using the complement of the common 3′ sequence as a PCR primer generates double-stranded product oligos. Afterwards, Illumina sequencing adaptors and P5/P7 sequences are appended using the NEBnext kit. In all NGS experiments, overlapped paired-end sequencing was performed, and any sequence in which the forward and reverse reads do not perfectly agree were discarded bioinformatically, in order to minimize the effects of NGS intrinsic error. b . The purity distributions for two replicate libraries are plotted. SNOP products exhibit significantly higher purities than commercial oligos individually purified via PAGE; median purity values are displayed in the figure. c Summary of NGS reads aligned to each oligo, by the number of nucleotides matched to the designed sequence. For most oligos, the three most dominant classes of reads are Perfect (green), single-base truncations or deletions (cyan), or gross (6+) errors. d Summary of NGS reads analysis for the desalted, PAGE, and SNOP libraries. N = 64 precursor oligonucleotides; error bars show standard deviation across different oligos. e Distribution of purities observed for 256-plex SNOP products, as compared to commercial desalted oligos

Article Snippet: In Step 1, 100 pmol of the common reverse primer was added to a total of 2.5 pmol of the SNOP products (or desalted/PAGE reference oligos), in 60 μL PCR buffer (Sigma Aldrich).

Techniques: Next-Generation Sequencing, Ligation, Sequencing, Polymerase Chain Reaction, Purification, Polyacrylamide Gel Electrophoresis, Standard Deviation

Journal: PLoS ONE

Article Title: Automated Genotyping of Biobank Samples by Multiplex Amplification of Insertion/Deletion Polymorphisms

doi: 10.1371/journal.pone.0052750

Figure Lengend Snippet: An overview of the experimental design and procedure. A shows the role of the selector probe and complementary vector. The target DNA fragment containing the insertion/deletion is cut with restriction enzymes and ligated to a complementary probe to form a circle. The circular ligation product is again cut to form a linear fragment with universal primer binding site. B shows the MLGA reaction scheme. Genomic DNA is restriction digested; ligated to specific selector probes and these products are amplified by multiplex PCR using fluorescent labels. The fragments can then be separated by capillary electrophoresis and analyzed. C is a schematic representation of the process, from design to analysis.

Article Snippet: The enzymes were subsequently inactivated at 80°C for 20 min. Circularization and ligation of restriction digested fragments was performed in a 20 µl reaction by adding 2.2 nM vector oligonucleotide, 0.1 nM of each Selector probe, 9.67 mM MgCl2 , 0.8 mM NAD, 4 U Ampligase (Epicentre) and 1× Taq DNA Polymerase PCR Buffer (Invitrogen) to the DNA.

Techniques: Plasmid Preparation, Ligation, Binding Assay, Amplification, Multiplex Assay, Polymerase Chain Reaction, Electrophoresis