PacBio Sequencing @ FGCZ

From the 1st of May 2022 we will offer PacBio services only on the Sequel IIe.
Please be informed we will stop accepting orders for the Sequel by the end of April 2022.

Would you like to discuss your project, please write to sequencing at fgcz.ethz.ch.
In order to access the FGCZ sequencing services, please register and send a project request via B-Fabric.
Upon project approval, you will be able to submit an order and enter you samples into the system.
To submit samples for PacBio/Oxford Nanopore Sequencing, you must create an order within your existing project.
How to create an order in b-fabric
Our turn around times are from 4 to 6 weeks, upon sample submission.

A comprehensive overview of the currently offered protocols by PacBio can be found here:
Overview-Sequel-Systems-Application-Options-and-Sequencing-Recommendations

Services and Prices

Instruments & Specs:

	Sequel IIe
SMRT Cell Type	8M Cell
Pol. read Number	4-6 M
gDNA library
av. Pol. read length HiFi mode	70+ Kb
expected output HiFi mode	20+ Gb
Amplicons/Iso-Seq library
av. Pol. read length	20+ Kb
number HiFi Reads	1.5-2.5 M

The PacBio av. Read length and output refer to the Polymerase read length.
The final output and the Polymerase read length may vary from sample to sample.

Input Material needed for library prep

Library Protocol and Insert size	Input
Amplicons <3 Kb	300ng
Amplicons 3-10 Kb	500ng
Amplicons >10 Kb	>1000ng
16S rRNA amplicon pool	1000ng
gDNA Standard HiFi 15-18 Kb	3-5µg
gDNA Low Input HiFi 15 Kb	>400ng
gDNA Microbial Multiplexing HiFi 7-10 Kb	300-1000ng
gDNA Shotgun Metagenomics HiFi 10 Kb	>300ng
Iso-Seq	300ng total RNA

The DNA sample must be possibly delivered in a low salt buffer like the Qiagen Elution Buffer (10mM Tris-Cl, pH 8.5-9.0)
Please use a Qubit system or picogreen method in order to quantify your samples.
Spectrophotometric quantitation tends to over estimate the concentration.
1 Library should be enough to sequence from 1-4 Sequel SMRT cells, depending on the recovery yield.

PacBio Recommended DNA extraction methods

Best practices sheets for key PacBio APPLICATIONS

Multiplexing

Protocols and recommendations for multiplexing

Important measures impacting DNA quality

For most applications it is crucial that the extracted gDNA is of HMW (High-molecular-weight) and does not contain any impurities (see below). Please have a look at the following technical note from PacBio first:
DNA Extraction and Quality Control

To maximize read length and quality, it is essential that your DNA sample:
- is double-stranded; single-stranded DNA is not compatible with the library preparation process.
- has not undergone multiple freeze-thaw cycles as they can lead to DNA damage.
- has not been exposed to high temperatures (e.g.: > 65oC for 1 hour can cause a detectable decrease in sequence
quality), pH extremes (< 6 or > 9).
- has an OD260/OD280 ratio of 1.8 to 2.0.
- has an OD260/OD230 ratio of 1.8 to 2.3.
- does not contain insoluble material.
- does not contain RNA contamination.
- has not been exposed to intercalating fluorescent dyes or ultraviolet radiation. SYBR dyes are not DNA
damaging, but do avoid ethidium bromide.
- does not contain denaturants (e.g., guanidinium salts or phenol) or detergents (e.g., SDS or Triton-X100).
- does not contain carryover contamination from the original organism/tissue (e.g., heme, humic acid,
polyphenols, etc.)

These are general recommendations to help maintain high molecular weight DNA.

Before DNA extraction:
a. Avoid incubation in complex or rich media.
b. Harvesting from several cultures rather than a single, high-density culture during early- to mid-
logarithmic growth phase is preferred.
c. Extraction of small volumes is preferred over large volumes to avoid accumulating high concentrations
of potentially inhibiting secondary components.



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