Quality control strategies. (A) To determine the overall efficiency at which medium-sized DNA fragments are recovered in DNA extraction, a 65-bp double-stranded DNA fragment was added to each lysate prior to DNA extraction. The concentration of the fragment was then measured before and after DNA extraction using digital PCR. (B) The conversion rate of library preparation was determined by comparing library yields measured with qPCR obtained from 3, 9, or 27 µL of extract to those obtained from 1 µL of extract. (C) In an alternative approach, a 40-nt library control oligonucleotide was added to each aliquot of DNA extract used for library preparation and to the TT buffer used as input in library negative controls. The number of library molecules generated from the control oligonucleotide was determined using a probe-based qPCR assay specific to successfully converted oligonucleotides. Note that no selection is necessary on the P7 adapter sequence, as molecules without P7 adapters lack the biotin required for bead binding in library preparation. The efficiency of library preparation is determined by comparing the number of oligonucleotide library molecules generated in the sample libraries to those in extraction and library blanks.
