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PacBio's SMRT System - Single Molecule

Real Time Sequencing
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, the biggest
feature of PacBio's SMRT sequencing is single-molecule sequencing, which requires no PCR
amplification and capable of reading over 100 times longer than NGS.

PacBio SMRT sequencing applications

PacBio SMRT sequencing could be used for genomic de novo sequencing to get high quality genome
sequences, obtaining full transcriptome information and detecting variable shear isomers, detection of
all types of mutations in target gene regions, direct detection of epigenetic modification and so on.

The principle of PacBio SMRT sequencing

Actually, PacBio SMRT sequencing applies the idea of SBS, and uses the SMRT chip as the
sequencing vector, just as flowcell. In simple terms, the principle is: A, C, G, and T is fluorescently
labeled with 4 different colors, and when DNA polymerase binds to the template, in this base pairing
stage, different bases will be irradiated by different fluorescent colors, then according to the wavelength
and peak of fluorescence, the type of base can be judged.

Library construction

The workflow to construct the final DNA libraries for sequencing is shown in Fig. 1 and involves these
steps:

⚫ Determination of the quality of the gDNA

⚫ Fragment gDNA using a g-TUBE (Covaris)

⚫ QC the sizing and adjust the concentration

⚫ Repair DNA damage and repair ends of fragmented DNA

⚫ Purify the DNA

⚫ Blunt-end ligate using blunt adapters

⚫ Purify template for submission to a sequencer

The template, called a SMRTbell, is a closed, single-stranded circular DNA that is created by ligating
hairpin adaptors to both ends of a target double-stranded DNA (dsDNA) molecule.
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Figure 1. PacBio SMRTbell Template Preparation Workflow for PacBio RS II system.

DNA polymerase and fluorescent labeled dNTP

The fluorescent dye is labeled on the nucleotide phosphate chain instead of the base, when the
nucleotide is added to the newborn chain, the labeled group will automatically shed and reduces the
steric hindrance of DNA synthesis, maintains the continuous synthesis of DNA strands, and extends the
PacBio SMRT Sequencing reading length. SMRT sequencing maintains the activity of the polymerase
to the maximum extent and is the most natural polymerase reaction system.

ZMW (zero-mode waveguides)

ZMW, as the holes on the wall of a microwave oven, the diameter of these holes is strictly required. If
the diameter is larger than the wavelength of the microwave, the energy will penetrate the panel under
the diffraction effect and leak out (diffraction effect of light waves), thus interfering with the surrounding
holes (interference of light waves). If the diameter can be smaller than the wavelength, then the energy
will not radiate to the surrounding, but maintain a linear state, which plays a protective role. Similarly,
there are many such circular nanoholes in a reactor tube (SMRTCell: single-molecule real-time reaction
pore), that is ZMW, which diameter is smaller than the detection laser wavelength.

Figure 2. A single SMRT cell.

Each SMRT cell contains 150,000 ZMWs. Approximately 35,000-75,000 of these wells produce a read in a run lasting
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0.5-4 h, resulting in 0.5-1 Gb of sequence.

Sequencing

PacBio SMRT Sequencing uses the ZMW (zero-mode waveguides) principle to distinguish the reaction
fluorescent signal from the strong fluorescent background of the surrounding free base. Laser from the
bottom of SMRTCell will not penetrate the ZMW and enter the solution region above, and the energy
will be constricted to a small area, so that the signal only comes from this small reaction area, excessive
free nucleotide monomers outside remain in the dark, minimizing background noise. This is the key
point of PacBio SMRT Sequencing.

As in Figure 3, a SMRTbell (gray) diffuses into a ZMW, and the adaptor binds to a polymerase
immobilized at the bottom. Each of the four nucleotides is 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 is
produced that identifies the base. (1) A fluorescently-labeled nucleotide associates with 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 here) is elevated. (3) The dye
linker-pyrophosphate product is cleaved from the nucleotide and diffuses out of the ZMW, ending the
fluorescence pulse. (4) The polymerase translocates to the next position. (5) The next nucleotide
associates with the template in the active site of the polymerase, initiating the next fluorescence pulse,
which corresponds to base A here.

Figure 3. Sequencing via light pulses

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 divergence time estimation are possible.

A comparison of RSⅡ and Sequel sequencing platform

Third-generation sequencing has been widely used in genome research since the successful launch of
commercial sequencing instrument PacBio RS II in 2013. After continuous improvement and upgrading,
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PacBio launched its new and upgraded third-generation sequencer PacBio Sequel sequencing system

in October 2015. A comparison of RSⅡ and Sequel sequencing platform is outlined below.

Table 1. The comparison of RSⅡ and Sequel sequencing platform

RSⅡ Sequel

Average read length 10～15kb 8～12kb

ZMWs 150,000 1,000,000

Data size/SMRTCell 500Mb~1Gb 5～10Gb

SMRTCell No./Run 1~16 1~16

Run time/SMRTCell 0.5~6 hours 0.5~6 hours

Multiplex Amplicons 384 1536

Sequel platform has great advantages compared with RS II platform, it has higher throughput, but lower
costs and shorter time-consuming.

Features of PacBio SMRT sequencing

⚫ Single-molecule resolution

PacBio SMRT sequencing requires no PCR amplification, can easily cover high GC and high repeat
areas, and is more accurate in quantifying low abundance/low frequency mutation.

⚫ Long reads

PacBio SMRT sequencing provides very long reads, average read length is 8-15kb, up to 40-70kb.

⚫ rapid

The speed of this sequencing is fast, it could sequence about 10 dNTP per second.

⚫ High accuracy

The rapid sequencing has also brought about some obvious drawbacks: the relatively high error rate of
PacBio SMRT sequencing (which is almost the common fault of current single-molecule sequencing
technology), which can reach 10%-15%, is mostly caused by missing sequences and dislocations. But
the errors are random and do not exist bias as the second-generation sequencing technology does.
Base deviation, therefore, can be effectively corrected through multiple sequencing, and therefore the
accuracy of PacBio SMRT sequencing reached 99.999% (Q50).

⚫ Direct identification of base modification

The base modification can be directly detected when the genome is sequenced.

References:
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Email: contact@cd-genomics.com

1. Kong, N., Ng, W., Thao, K., Agulto, R., Weis, A., & Kim, K. S., et al. (2017) ‘Automation of pacbio smrtbell ngs
library preparation for bacterial genome sequencing’, Standards in Genomic Sciences, 12(1), 27.
2. Rhoads, A., & Au, K. F. (2015) ‘Pacbio sequencing and its applications’, Genomics,Proteomics &
Bioinformatics, 13(5), 278-289.
3. PacBio's website.