PacBio's SMRT (single molecule real time) sequencing is one of the most commonly used third-generation sequencing technologies. Compared with the previous two generations, PacBio long-read sequencing enabled by SMRT Sequencing technology requires no PCR amplification and the read length is 100 times longer than that of NGS.
PacBio SMRT sequencing applications: PacBio SMRT sequencing can be used for genomic de novo sequencing to get high quality genome sequences, obtaining full transcriptome information and detecting alternative splicing isoforms, diverse mutations in target regions, and epigenetic modifications and more.
The principle of PacBio SMRT sequencing: Zero-mode waveguides (ZMWs), subwavelength optical nanostructures fabricated in a thin metallic film, are powerful analytical tools that are capable of confining an excitation volume to the range of attoliters, which allows individual molecules to be isolated for optical analysis at physiologically relevant concentrations of fluorescently labeled biomolecules. Arrays of such nanostructures can also be engineered into systems for real-time analysis of a mass of single-molecule reactions or binding events, which is the principle of PacBio SMRT sequencing.
PacBio SMRT Sequencing uses the innovation of ZMW to distinguish the ideal fluorescent signal from the strong fluorescent backgrounds caused by unincorporated free-floating nucleotides. The binding of a DNA polymerase and the template DNA strand is anchored to the bottom glass surface of a ZMW. Laser light travels through the bottom surface of a ZMW and not completely penetrates it, since the ZMW dimensions are smaller than the wavelength of the light. Therefore, it allows selective excitation and identification of light emitted from nucleotides recruited for base elongation.
Library construction:
The workflow for library construction involves the following steps:
Determine the quality of genomic DNA (gDNA)
Shear gDNA using a g-TUBE (Covaris)
Select size and adjust concentration
Repair DNA damage and ends of fragmented DNA
Conduct DNA purification
Blunt-end ligation using blunt adapters
Purify template for submission to a sequencer
The template, called a SMRTbell, is a closed single-stranded circular DNA, which is created by ligating hairpin adapters to both ends of target double-stranded DNA (dsDNA) molecules
Sequencing: As in Figure 3, a SMRTbell (grey) diffuses into a ZMW, and the adaptor binds to a polymerase immobilized at the bottom. Four types of nucleotides are labeled with a different fluorescent dye (indicated in red, yellow, green, and blue, respectively for G, C, T, and A) so that they have distinct emission spectrums. As a nucleotide is held in the detection volume by the polymerase, a light pulse that identifies the base is produced. (1) A fluorescently-labeled nucleotide binds to the template in the active site of the polymerase. (2) The fluorescence output of the color corresponding to the incorporated base (yellow for base C as an example shows here) is elevated. (3) The dye linker-pyrophosphate product is cleaved from the nucleotide and diffuses out of the ZMW to end the fluorescence pulse. (4) The polymerase is translocated to the next position. (5) The next nucleotide binds to the template in the active site of the polymerase and initiates the next fluorescence pulse, which corresponds to base A here.
Bioinformatics Analysis: Bioinformatics analysis, such as de novo assembly, reference genome mapping, genome annotation (pathogenic and susceptibility genes prediction, non-coding RNA prediction, CRISPRs prediction), gene function annotation (COG/ GO/ KEGG), SNP/InDel identification and comparative genomics analysis, evolutionary analysis and estimation of divergence time are viable.
8 ноя 2020
