How does PacBio sequencing work?

How does PacBio sequencing work?

PacBio sequencing captures sequence information during the replication process of the target DNA molecule. 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 (Figure 1) [2].

What is ISO-seq?

The Iso-Seq method produces full-length transcripts using Single Molecule, Real-Time (SMRT) Sequencing [3]. Long read lengths enable sequencing of full-length transcripts up to 10 kb or longer, eliminating the need for transcript assembly or inferencing.

How much does PacBio sequencing cost?

Table 1 Technical specifications of Next Generation Sequencing platforms utilised in this study

Platform Illumina MiSeq PacBio RS
Instrument Cost* $128 K $695 K
Sequence yield per run 1.5-2Gb 100 Mb
Sequencing cost per Gb* $502 $2000
Run Time 27 hours*** 2 hours

Can PacBio detect methylation?

Further, PacBio sequencing simultaneously detects epigenetic modifications without requiring a specialized library preparation step like bisulfite conversion. This ability is commonly used to characterize epigenetic marks in bacterial genomes.

How does PacBio sequencing methodology identify nucleotides?

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.

How does long-read sequencing work?

Long-read, or third-generation, sequencing involves reading sequences of between 10,000 and 100,000 base pairs in one go (although much longer reads have also been reported), without the need to cut up and amplify DNA samples.

What is the meaning of the isoform?

Definition of isoform : any of two or more functionally similar proteins that have a similar but not an identical amino acid sequence.

What is the difference between NovaSeq and HiSeq?

Novaseq Speed: NovaSeq is fast, but for core lab users the reduction in run time compared to HiSeq 4000 is unlikely to have a dramatic impact on total turnaround times for a typical RNA-Seq or Exome project. Larger projects will finish more quickly especially with an S2 PE150 run time of just 40 hours.

How much is a NovaSeq machine?

The machines themselves are still costly: The NovaSeq 5000 and 6000 cost $850,000 and $985,000, respectively.

What is 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. Therefore, it allows selective excitation and identification of light emitted from nucleotides recruited for base elongation.

How long are PacBio reads?

The PacBio® HiFi sequencing method yields highly accurate long-read sequencing datasets with read lengths averaging 10–25 kb and accuracies greater than 99.5%.

Why is long-read sequencing better?

They sequence a single molecule, eliminating amplification bias, and generate a reasonable length to overlap a sequence for better sequence assembly. This is then used to determine the base sequence. A downside to long-read sequencing is that the accuracy per read can be much lower than that of short-read sequencing.

What is PacBio’s iso-seq service?

Transcriptome and Isoform sequencing with PacBio’s Iso-Seq protocol. This service includes cDNA generation from PolyA+ or total RNA and SMRTbell addition. *Maximum number of genomes per pool is dependent on expected genome size and desired coverage level.

Is PacBio compatible with single-cell iso-seq library preparation?

Although PacBio does not have a specific single-cell partner or system recommendation, in principle, practically any single-cell platform should be compatible with single-cell Iso-Seq library preparation so long as that platform generates cDNA.

Where can I get a quote for PacBio sequencing services?

University of Washington PacBio Sequencing Services. For a consultation and quote, email us at [email protected] or call +1-206-616-5117. Single-molecule, real-time (SMRT) sequencing is a third generation sequencing technology that generates long-read (ten thousand to a hundred thousand bases) length DNA and cDNA sequences.

How can I obtain full-length cDNAs using iso-seq?

-For the Iso-Seq method to achieve full-length cDNAs, it is recommended to use a template-switching oligo (TSO). This is a common technique and is currently used in single-cell platforms and PacBio’s current bulk Iso-Seq methods.