PacBio SMRT Sequencing

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CD Genomics is providing PacBio SMRT sequencing to complement our NGS facility. By taking advantage of the long-read and single molecular sequencing capability developed by PacBio, we are proud to offer advanced genome de novo assembly solutions and full-length gene/transcript sequencing strategy to suit your project needs.

The Introduction of PacBio SMRT Sequencing

Single Molecular Real-Time (SMRT) sequencing employs a specialized flow cell with many thousands of individual picolitre wells with transparent bottoms — zero-mode waveguides (ZMW). The polymerase is fixed to the bottom of the well and allows the DNA strand to progress through the ZMW. As a result, the system can focus on a single molecular. SMRT sequencing allows for real-time imaging of fluorescently tagged nucleotides that are synthesized along with individual DNA template molecules. The sequencing reaction ends when the template and polymerase dissociate. The average read length from the PacBio  instrument is approximately 2 kb, and some reads may be over 20 kb. Longer reads are especially useful for de novo assemblies of novel genomes that can span many more repeats and bases.

Highly repetitive elements found in both eukaryotic and prokaryotic genomes pose a challenge for genome assembly and make the detailed study of repetitive sequences difficult. Long-read sequencing delivers reads in excess of several or dozens of kilobases (kbs), which can span complex or repetitive regions with a single continuous read, allowing for the resolution of these large structural features. Besides considerably longer and highly accurate DNA sequences from individual unamplified molecules, it can also exhibit where methylated bases occur, thereby providing functional information about DNA methyltransferases encoded by the genome. PacBio SMRT sequencing has unique advantages in studies of de novo genomics, metagenomics, transcriptomics and epigenetics.

Advantages of PacBio SMRT Sequencing

  • Longest average read lengths
  • Highest consensus accuracy
  • Uniform coverage
  • Simultaneous epigenetic characterization
  • Single-molecule resolution
  • Rapid and affordable

Our PacBio SMRT Sequencing services

We utilize the advanced PacBio SMRT instruments (PacBio SR II and PacBio Sequel) for several research purposes including whole-genome de novo genome assembly, full-length target sequencing, metagenomics studies, full-length transcripts sequencing, and genome-wide DNA modification analysis. Our highly experienced expert team executes quality management following every procedure to ensure confident and unbiased results.

Our bioinformatics pipeline includes de novo assembly, base modification detection, single molecule consensus generation, transcript analysis, amplicon analysis, sequence alignment with variant detection. And more data mining are available based on your specific needs.

Supported by our experienced scientists and advanced platforms, CD genomics can assist you in the studies on genomics, transcriptomics, epigenomics, microbial genomics, and single-cell sequencing. with unmatched read lengths, uniform coverage, and high accuracy. If you have additional requirements or questions, please feel free to contact us.

1. What are the differences between PacBio RS II and PacBio Sequel?

RS II system was released by PacBio in 2013, based on the SMRT technology, of which the average read length reaches 10 kb with the longest one beyond 20 kb. Sequel is the newest platform of PacBio that is also based on SMRT technology but has a large increase in throughput and data quality.

Table 1. The comparison between PacBio RS II and Sequel.

Parameters RS II Sequel
principles SMRT SMRT
Average read length 10-15kb 8-12kb
Data sizes/SMRTcells 500Mb-1Gb (750Mb-1.5Gb) 5-10Gb
Max Output/Run 18Gb (24Gb) 160Gb
Run time/SMRTcell 0.5-6 hours 0.5-6 hours
Multiplex Amplicons 384 1536
2. What is size selection?

PacBio allows for the sequencing of full-length cDNA libraries at single-molecule resolution. However, most transcripts are 1-1.5kb. Longer transcripts are hard to detect due to low abundance, amplification bias, and preferential loading of smaller SMRTbell constructs. Therefore, size selection is a powerful way to dramatically increase the number of transcripts>1.5kb. This is especially essential for transcripts>3kb. As for degraded samples, shearing is not necessary, which may further reduce library insert size, and size selection is important to remove shorter fragments that will be much less beneficial in assembly. Size selection can be performed with agarose gels or BluePippin system. Additionally, size selection can  be used for whole-genome de novo sequencing.

3. The requirements of DNA samples.

In general, the following precautions need to be taken when submitting DNA samples:

  • Make sure the purification and integrity of the DNA samples.
  • Avoid overdrying of DNA.
  • Avoid ethidium/UV based visualization when using gel purification.
  • Avoid overheating and vortexing DNA.
  • Undergo a minimum of freeze-thaw cycles.
  • Double-stranded DNA.

Table 2. The estimated yield and recommended amounts of DNA samples.

Library insert size

Recommended quantity for submission

Min concentration required (Post-shearing)

Est. Total yield (range)

Min

Max

250bp 600ng 250ng 60Gb 125Gb
500bp 600ng 250ng 10 Gb 20Gb
1kb 1.2μg 500ng 90Gb 180Gb
2kb 1.2μg 500ng 45Gb 90Gb
5kb 2.4μg 1μg 45Gb 91Gb
10kb 2.4μg 1μg 20Gb 45Gb
10kb (AMPure kit) 10μg 5μg 90Gb 182Gb
20kb (AMPure kit) 15μg 5μg 45Gb 91Gb
20kb (BluePippin kit) 15μg 5μg 9Gb 18Gb

4. The requirements for RNA samples.

Please provide integrated and purified RNA samples (RNA ≥ 5 µg; concentration ≥ 300 ng/µl) for full-length transcripts sequencing.

Reference:
PacBio’s website

For Research Use Only. Not for use in diagnostic procedures.

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