PacBio Guidelines

Input Sample requirements for PacBio sequencing:

Target Library Insert Size / Recommended DNA Quantity for Submission

250bp/600ng

500bp/600ng

1kb/1.2ug

2kb/1.2ug

5kb/2.4ug

10kb/2.4ug

10-20kb (Ampure)/15ug

20kb (Blue Pippin)/15ug

Sample Requirements for PacBio Sequencing:

The quality of the DNA starting material will be directly reflected in the sequencing results.  Any irreversible DNA damage present in the input material will result in impaired performance in the system.  High-quality, high-molecular weight genomic DNA is imperative for obtaining long read lengths and optimal sequencing performance.

Important Measures impacting DNA Quality:

For optimal sequencing performance, it is essential that your DNA sample:

- Is double - stranded DNA; single stranded DNA is not compatible with the library preparation process

- Has not undergone multiple freeze-thaw cycles as they 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 between 1.8 and 2.0

- Has an OD260/OD230 ratio between 2.0 and 2.2

- Does not contain insoluable material

- Does not contain RNA contamination

- Has not been exposed to intercalating fluorescent dyes or ultraviolet radiation. SYBR dyes are not DNA damaging, but avoid ethidium bromide

- Does not contain denaturants (e.g., guanidinium salts or phenol) or detergents (e.g., SDS or TritonX-100)

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

Guidelines for DNA Extraction to Obtain High Molecular Weight and Clean Genomic DNA:

Before DNA extraction:

- Avoid incubation in complex or rich media

- Harvesting from several cultures rather than a single, high density culture in logarithmic phase is preferred

- Extraction of small volumes is preferred over large volumes to avoid accumulation of potentially inhibitory secondary components