Comparison of SYBR Enzymes and Standards in Illumina Library Quantification
Year: 2011
Type: article
Abstract: Accurate quantification of libraries generated for Illumina next generation sequencing is critical to prevent under- and over clustering of the flowcell. Success of qPCR library quantifications depend on the accuracy and reproducibility of standards used in addition to the ability of DNA polymerase to efficiently amplify all adaptor-flanked libraries. We compared the (i) Roche SYBR Green and (ii) KAPA SYBR Fast polymerases and the PhiX standards and KAPA pre-diluted standards to determine the relative importance of these two factors in library quantification by qPCR. We evaluated the success of each method by comparing variation in the number of reads and absolute yield generated from sequencing. Our results indicate that the DNA polymerase from the KAPA SYBR Fast kit is better suited to library quantification applications while the Roche SYBR Green kit showed significant variability, possibly related to sample type or insert size. No differences were observed between the two types of standards used, making DNA polymerase the determining factor in the successful amplification of each library.
Source: Europe PMC (PubMed Central)
Authors: Stuart S. Levine, Khanh K. Thai
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W2243586571