High-quality, high-molecular-weight genomic DNA is crucial for obtaining long read lengths and optimal sequencing performance The SMRT 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.) or contaminants 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 without any smear of degradation products. For best results it is imperative that the input DNA size is significantly higher than the desired insert size of the final library. For 20 Kb SMRT libraries, the DNA should be higher than 30 Kb, and for 10-15Kb SMRT library, the DNA should be larger than 20 kb. Longer read lengths can be achieved with higher input amounts of sufficiently high molecular weight input.
DNA purity is especially crucial for successful sequencing with PacBio. The 260/280 ratio should be around 1.8 (+/- 0.2) and the 260/230 should be in the 2:2.2 range. Significant contaminants that cannot be removed by bead cleanup can cause library prep and/or sequencing to fail.
RNA quality: Good quality, intact RNA is essential. RIN must be ≥8.0 as indicated by the Agilent Bioanalyzer.
Buffer: DNA or RNA should be in 10 mM Tris, pH 7.5-8.0. Water is okay if it is nuclease free, but buffer is preferred.
Required DNA and RNA quantities and concentration:
Standard DNA library prep: a minimum of 4 ug of DNA per sample is required. Please submit samples in single lo-bind tubes in a volume of 100 uL or less. Higher volumes will require concentration into a smaller volume.
Standard DNA library prep with aggressive size selection for the longest possible read lengths (>30 kb): minimum10 ug of DNA per sample is required, higher amounts are recommended. Please submit samples in single lo-bind tubes in a volume of 100 uL or less. Higher volumes will require concentration into a smaller volume. If your DNA is sufficiently high molecular weight and you can provide enough input material, we recommend providing significant excess DNA for aggressive size selection to achieve longer read lengths.
Low input DNA library prep: a minimum of 150 ng of DNA per sample is required. Please note that size selection cannot be done with this low input protocol, and we recommend providing more material for standard library prep if possible. This option is only recommended for samples types for which it is difficult to acquire higher amounts of DNA, i.e. single insects.
RNA for IsoSeq: 10 uL of RNA at least 250ng/uL is required. Lower input amounts will require several replicates and increased amplification.
If you are submitting 24 or more samples, please submit samples in a 96-well plate and include an Excel file with the sample layout in your order.
PLEASE USE V BOTTOM PLATES DESIGNED FOR MOLECULAR BIOLOGY AND NOT FLAT BOTTOM PLATES DESIGNED FOR OTHER PURPOSES.
Table 1. Sample Requirements
|Library Type||Library Insert Size||Amount Required
|DNA||10 kb, 15kb, 20 kb, 30 kb||4 µg
|DNA||Low Input, no size selection||4 µg
|DNA||Aggressive size selection, > 30kb||10 µg or more
PacBio Guidelines for Successful SMRTbell Libraries
Q&A for “DNA Quality Requirements for Single Molecule, Real-Time (SMRT) Sequencing”
Note on the 260/280 and 260/230 Ratios
DNA Extraction Protocols: www.ExtractDNAforPacBio.com