High-quality, high-molecular-weight genomic DNA is crucial for obtaining long read lengths and optimal sequencing performance The SMART library preparation process does not utilize amplification techniques and resulting library molecules are directly used as templates for the sequencing process. The quality of the DNA and RNA starting material will be directly reflected in the extent of sequencing success or failure. Any unrepaired or irreversible DNA damage present in the input material (e.g., interstrand crosslinks, nicks, etc.) will result in impaired performance in the system.
Required quantitation method:
For DNA, please use pico green or equivalent fluorometric method (e.g., QUBIT).
For RNA, please use either ribo-green or QUBIT.
DNA quality: DNA should be high molecular weight (significantly higher than the desired insert size in the library) without any smear of degradation products.
For 20kb SMRT library: the DNA should be higher than 30Kb, and for 10-15kb SMRT library, the DNA should be larger than 20kb. DNA purity is crucial for successful sequencing. The 260/280 ratio should be around 1.8 (+/- 0.2) and the 260/230 should be in the 2:2.2 range.
RNA quality: Good quality, intact RNA is essential. RIN must ≥8.0 as indicated by the Agilent Bioanalyzer.
Buffer and concentration: DNA or RNA should be in 10 mM Tris, pH 7.5-8.0 at a minimum concentration of 50-100ng/ul.
Required DNA and RNA quantities and concentration:
Sample concentration = 50-100 ng/ul as quantitated by QUBIT
Table 1. Sample Requirements
|Library Size and Type|| ||Amount Required||
|DNA||10-20, 20-30 Kb|
|RNA (IsoSeq)||1-2, 2-3, 3-6, 6-10 Kb cuts|
PacBio Guidelines for Successful SMRTbell Libraries
Q&A for “DNA Quality Requirements for Single Molecule, Real-Time (SMRT) Sequencing”