Continuing with the Human genome sequencing and analysis course, today I watched the video lesson on “Library preparation: Quality control of input DNA or RNA.” Hazel Johnson, a Technical Services Team member at Oxford Nanopore Technologies, explained that incorrectly quantifying DNA can result in reduced sequencing input. They do not recommend the use of NanoDrop because it is influenced by base composition and contaminants. Johnson noted that the Qubit Broad Range kit is less influenced by RNA than the HS kit. The NanoDrop does help assess purity with A260/280 ratios of 1.8 for pure DNA. Ratios greater than 1.8 indicate possible RNA contamination, and ratios less than 1.8 can indicate possible protein or phenol contamination. The A260/230 ratio should be greater than 2.0. Johnson shared data on on the effect of contaminants on efficiency of DNA and RNA library prep. Assessment of fragment length is also critical. Gel electrophoresis can be used for fragments up to 20 kb. The BioAnalyzer/Tapestation can be used for genomic DNA fragment length. Size of the DNA is important for quantification of how much DNA to load onto the flow cell without overloading. Short fragment libraries tend to have less spread of sizes, and they are therefore easier to quantify. For RNA, RNA Integrity Number (RIN) from the BioAnalyzer/Tapestation correlates with successful library sequencing. In summary, Johnson explained that the Qubit should be used for quantification, and the NanoDrop provides an indication of the purity of the sample based on ratios. For RNA, RIN values are useful for determining if additional purification is useful. If quality is not ideal, Johnson noted that amplification of the library by PCR is possible. For example, for the Ligation Sequencing Kit, Johnson recommended starting with 1 ug of DNA.
