Pacific Biosciences Sequencing
Cold Spring Harbor Laboratory is an HHMI designated Pacific Biosciences (PacBio) sequencing site. We provide PacBio sequencing services to all HHMI investigators located at nearby non-profit research institutions.
We also provide fee-for-service sequencing to non-HHMI investigators around the world, Our users includes scientists from the US, UK, Italy, Russia, South Africa and Australia.
We offer full service sequencing on the Pacific Biosciences RS instrument. We will QC the DNA, make the PacBio sequencing library and sequence the library on the appropriate number of SMRT cells.
Furthermore, we will help you design your sequencing experiment (insert size, type of run, number of SMRT cells, etc.) to help ensure that you will receive the data you need for your research.
Because PacBio sequencing libraries are made from double stranded DNA molecules with hairpin loops (adapters) ligated at both ends, the final product is a circular molecule. Therefore, the polymerase can sequence both strand of the insert multiple times, separated by the adapter sequences. We remove the adapter sequences, breaking up the polymerase reads into subreads. If the polymerase read did not reach the end of the insert, then you get one subread = polymerase read. If the polymerase reach the end of the insert, go through the circular adapter and start sequencing the opposite strand, then you get two subreads. If the polymerase goes N times around the circular molecule, then you get N subreads from one polymerase read. A read_of_insert (aka CCS read) is the consensus sequence obtained by doing a multi-sequence alignment using all the subreads coming from one polymerase read.
The subread sequences are provided in a fastq file on our FTP server.
PacBio technology revealed:
Current performance of the Pacific Biosciences RS sequencer:
A typical SMRT Cell will generate between 40,000 and 50,000 reads. After filtering (using a 20 kbp insert library), the maximum read length is 45 Kbp. The average read length is around 5,000 bp with the P4 enzyme and around 12,000 bp with the new P6 enzyme. Subread N50 sequence length are around 15Kb with the P6 enzyme.
The following figures show typical polymerase read length (fig 1) and subread length (fig 2) histograms for a 20kb library size selected on the BluePippin (15-50kb) ) and sequenced using the P6 enzyme on 8 SMRT cells (680Mb/SMRT cell).
|Figure 1||Figure 2|
The error rates are as advertised by the company, about 12% with a single pass over a template. Hence, in the long read mode, your DNA molecule would be sequenced a single time with this overall error rate. In the circular consensus mode (CCS), a molecule would be sequenced several times, and the consensus sequence (CCS) error rate will be considerably lower (exact error rate depends on the number of individual sequences used to generate the CCS sequence).
Details of the full range of applications can be found at the PacBio applications page; http://www.pacb.com/applications/overview/index.html
For understanding accuracy in SMRT sequencing, see the following document; http://www.pacb.com/pdf/Perspective_UnderstandingAccuracySMRTSequencing.pdf
|Per sample QC
|Sequencing Library Prep
|SMRT cell sequencing
|BluePippin size selection
|Cost Per Library
(for large insert library)
|Iso_seq library prep
|Cost Per Sample
(for cDNA sequencing)