Core Facilities

SAMPLE REQUIREMENTS FOR PACBIO SEQUENCING

PacBio library preparation utilizes native DNA templates for sequencing and does not include amplification techniques. The quality of the input DNA will be directly reflected in the sequencing results, and any irreversible damage present in the DNA will result in impaired sequencing results.  Both high quality, high molecular weight genomic DNA is imperative for obtaining long read lengths and optimal sequencing performance.

 For Iso-Seq, total RNA with a RIN value >8 is required.

*At this time, we still recommend Illumina sequencing for differential expression experiments. PacBio Iso Seq is highly suitable for whole transcriptome or isoform sequencing.

HOW TO SUBMIT SAMPLES

1. Contact Katie directly if you need DNA extraction to discuss the most appropriate method for sample collection.
2. Extract high molecular weight DNA or total RNA from your sample. Quantify your sample with the Qubit or similar assay to see if you have enough material for a library preparation. Run this sample on the Nanodrop to see if 260/280 and 230/260 values are within recommended ranges. Submit this sample for a genomic Tape Station assay if you don’t have access to a pulsed field electrophoresis machine. Provide Qubit and Nanodrop readings when the sample is submitted. Provide as much DNA as possible. Higher input DNA allows for more aggressive size selection, which can improve the N50 read length. Contact Katie when you are ready to drop off samples.

IMPORTANT MEASURES IMPACTING DNA QUALITY

• DNA must be double stranded; single-stranded DNA cannot be used to generate sequencing template.
• DNA cannot undergo multiple freeze-thaw cycles, as that can lead to DNA damage.
• DNA cannot be exposed to high temperatures (>65C for 1 hour) or pH extremes (<6 or >9).
• DNA should have an OD₂₆₀/OD₂₈₀ ratio of 1.8 to 2.0.
• DNA should have an OD₂₆₀/OD₂₃₀ ratio of ~2.0
• DNA should not contain insoluble material or carryover contamination from the organism/tissue.
• DNA should not contain RNA contamination
• DNA should not been exposed to intercalating dyes, UV radiation, or ethidium bromide.
• DNA should not contain denaturants or detergents

DNA SAMPLE QUALITY ASSESSMENT

A DNA quality check is imperative before submitting sample for PacBio sequencing and/or starting the library preparation.  The following recommendations for determining DNA integrity, purity, and concentration are highly recommended.

• Genomic DNA integrity can be assessed by pulse field gel electrophoresis; the presence of one predominant high molecular weight band with no degradation is optimal. If this is not available, DNA integrity can also be evaluated with a genomic Tape Station assay at the CQLS.

PURITY OF YOUR DNA SAMPLE

Readings of both A260/A280 and A260/230 ratios on a Nanodrop instrument need to be obtained to make a determination of purity. A nanodrop instrument is available for customer use in the Multi-user room (link to this?).

• The ratio of absorbance at 260 nm and 280 nm is used to assess purity of DNA.  A ratio of ~1.8 is generally accepted as pure for DNA.
• The 260/230 ratios provide a secondary measurement of DNA purity to assess the quality of the extraction.  Expected 260/230 values are commonly in the range of 2.0-2.2.  Abnormal 260/230 values may indicate a problem with the extraction procedure.

CONCENTRATION OF DNA SAMPLE

• For PacBio library preparation, it is critical to determine the concentration of the double-stranded DNA present in the sample.  Spectrophotmetric assays do not distinguish between different types of nucleotides, therefore it is highly recommended to use an intercalating dye on a fluorometer, like the Qubit or Picogreen assays, for quantitation purposes.