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Why PACBio NGS Libraries?

Jul 1, 2016 | Focus Of The Month

PACBio’s SMRT is a parallelized single molecule DNA sequencing method with sequencing-by- synthesis technology based on real-time imaging of fluorescently tagged nucleotides as they are synthesized along individual DNA template molecules. As the technology uses a DNA polymerase to drive the reaction and because it images single molecules, there is no degradation of signal over time.

    • PacBio’s long reads are that makes it a good fit for number of applications which are not well served by other sequencing platforms: de novo genome assembly, genome finishing, structural variations, HLA typing, full length transcriptomes, etc.
    • The Iso-Seq protocol developed by Pacific Biosciences for transcript sequencing allows direct sequencing of transcripts upto 10 kb without the use of reference genome.
    • The real time nature of PacBio sequencing show advantages –
      1. Fast reads.
      2. Longer reads when compared to other sequencing platforms with a mean of ~ 15 kb.
      3. PacBio captures information about the nucleotide incorporation, which can be used further to determine the modification status of template nucleotide.
    • PacBio- SMRT sequencing is able to sequence regions of high GC content leading to much more uniform coverage of the genome.

The power of SMRT sequencing data lies both in its long read lengths and in the random nature of the error process .It is true that individual reads contain a higher number of errors: approximately 11% to 14% or Q12 to Q15, compared with Q30 to Q35 from Illumina and other technologies. However, given sufficient depth (8x or more, say), SMRT sequencing provides a highly accurate statistically averaged consensus perspective of the genome, as it is highly unlikely that the same error will be randomly observed multiple times.

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