Quantification of massively parallel sequencing libraries - a comparative study of eight methods

Abstract Quantification of massively parallel sequencing libraries is important for acquisition of monoclonal beads or clusters prior to clonal amplification and to avoid large variations in library coverage when multiple samples are included in one sequencing analysis. No gold standard for quantification of libraries exists. We assessed eight methods of quantification of libraries by quantifying 54 amplicon, six capture, and six shotgun fragment libraries. Chemically synthesized double-stranded DNA was also quantified. Light spectrophotometry, i.e. NanoDrop, was found to give the highest concentration estimates followed by Qubit and electrophoresis-based instruments (Bioanalyzer, TapeStation, GX Touch, and Fragment Analyzer), while SYBR Green and TaqMan based qPCR assays gave the lowest estimates. qPCR gave more accurate predictions of sequencing coverage than Qubit and TapeStation did. Costs, time-consumption, workflow simplicity, and ability to quantify multiple samples are discussed. Technical specifications, advantages, and disadvantages of the various methods are pointed out

Increasing the reference populations for the 55 AISNP panel: the need and benefits

Ancestry inference for an individual can only be as good as the reference populations with allele frequency data on the SNPs being used. If the most relevant ancestral population(s) does not have data available for the SNPs studied, then analyses based on DNA evidence may indicate a quite distantly related population, albeit one among the more closely related of the existing reference populations. We have added reference population allele frequencies for 14 additional population samples (with >1100 individuals studied) to the 125 population samples previously published for the Kidd Lab 55 AISNP panel. Allele frequencies are now publicly available for all 55 SNPs in ALFRED and FROG-kb for a total of 139 population samples. This Kidd Lab panel of 55 ancestry informative SNPs has been incorporated in commercial kits by both ThermoFisher Scientific and Illumina for massively parallel sequencing. Researchers employing those kits will find the enhanced set of reference populations useful