The C2 chemistry for Pac Bio Third Generation Sequencing has arrived at the UCSD BIOGEM Core.  Single Molecule Real Time (SMRT™) DNA sequencing technology enables, for the first time, the observation of natural DNA synthesis by a DNA polymerase as it occurs.  The approach is based on eavesdropping on a single DNA polymerase molecule working in a continuous, processive manner.  Distinguished by long reads, fast time to results, more informative data, and lower overall costs, SMRT DNA sequencing promises to be a transformative technology that will enable a new paradigm in genomic analysis.

There are two sequencing modes utilized on the PacBio RS. With the new C2 chemistry insert sizes range from 250 bp up to 10kb 

Standard sequencing (up to 10 Kb). Standard SMRT sequencing generates single pass, long reads. Long insert lengths ensure continuous polymerase synthesis along a single strand. As with all protocols, this process runs in parallel across thousands of individual polymerases in each SMRT Cell. The library prep is divided into 250 to 3Kbp prep and 3-10Kbp preps.

Circular consensus sequencing (250 bp).  The circular consensus sequencing protocol uses a circular DNA template to enable multiple reads across a single molecule.  This approach provides both forward and reverse reads with a double stranded template.

BIOGEM offers C2 sequencing on the PacBio RS. We are accepting projects on a first come first serve basis.  Priority is given to UCSD and HHMI investigators.

Step to perform a Pac Bio sequencing run at BIOGEM:

Firstly please note that BIOGEM will provide all the necessary sequencing reagents and SMRT Cells so purchase directly from Pacific Biosciences is not required.

There are three steps involved in Pacific Biosciences sequencing

1) Sample QC and Validation

2) Sequencing DNA library preparation

3) Binding of the SMRTbell™ DNA library to the polymerase and running the samples on the instrument.

Before you begin please contact:

Kristen Jepsen

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BIOGEM will validate the nucleic acid samples and if they pass QC will then prepare the sequencing libraries for Pacific Biosciences. If there is a problem with your samples we will notify you.

Great care needs to be taken with DNA samples for this single molecule based technology.

Please ensure that the DNA:

•Is double-stranded. Single-stranded DNA will not be made into a SMRTbell template in this template preparation process and can interfere with
quantitation and polymerase binding.

• Has undergone a minimum of freeze-thaw cycles.

• Has not been exposed to high temperatures (> 65ºC for 1 hour can cause a detectable decrease in sequence quality).

• Has not been exposed to pH extremes (> 9).

• Has an OD260/280 ratio of approximately 1.8 to 2.0.

• Does not contain insoluble material.

• Does not contain RNA.

• Has not been exposed to intercalating fluorescent dyes or ultraviolet radiation.

• Does not contain chelating agents (e.g., EDTA) divalent metal cations (e.g., Mg2+), denaturants (e.g., guanidinium salts, phenol),

or detergents (e.g., SDS, Triton-X100).

• Does not contain carryover contamination from the starting organism/tissue (e.g., heme, humic acid, polyphenols)

Please coordinate samples drop-off with Colleen Ludka. DNA must be in water or Tris buffer (i.e. EB buffer). DNA is lost during the fragmentation and initial bead concentration steps -  after which the following amount are needed for the library prep, based on the insert size:

(initial shearing/cleanup steps can lose up to 80% of the input)

1ug input for 250-500bp inserts

2ug input for 1 to 3kbp inserts

5-10ug input for 4 to 6kbp inserts

10-20ug input for 8 to 10kbp inserts

We will return what we do not use.

The sequencing takes place on a group of SMRT cells, and there are 8 per group – so the preferred multiple is 8 samples (although we can accommodate less than 8 samples). The estimated turn around time for library preparation and sequencing is 2 weeks.

Once the raw sequencing data has been generated it will be run through Pacific Biosciences proprietary software for evaluation of read length and quality. All run related files including the sequence text files will be copied to your account on the BIOGEM SERVER. If you don't have an account please contact Kristen Jepsen to create one.

Researchers desiring additional help with data analysis on a fee for services basis can obtain support for analysis of their data through the BIOGEM Core.

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Please note the following BIOGEM Policy for Pacific Biosciences Sequencing:

The Investigator agrees that they solely are responsible for the quality of the DNA samplessubmitted. They are required to provide BIOGEM with the concentration of the sample and the OD260/280 ratio. If the DNA is not of sufficient quality for sequencing, the investigator will be notified.