Libraries were prepared and hybrid captured using the Nextera Rapid Capture Exome kit (Illumina) with 25 ng of DNA input.
DNA inputs in fragmentation step were 30 100 ng in the case of a downstream manual preparation, or 250 ng for semi-automatized protocol, more demanding in DNA quantity.
Assay performance was equivalent across the input DNA range (<2 ng (limit of detection) to 100ng dsDNA, 41.4ng median DNA input, p = 0.852, chi-squared test) (Fig. 4d,e,f).
Based on availability, 2.24 4.92 µg gDNA were used in the initial PCR with paired samples having the same total DNA input.
The DNA input and bound fractions were purified, amplified and labeled using Whole Genome Amplification (Sigma-Aldrich) and CGH Enzymatic Labeling (Agilent Technologies) kits.
Genomic DNA (Input) was prepared by treating aliquots of chromatin with RNase, proteinase K and heat for de-crosslinking, followed by ethanol precipitation.
Rojas-Bracho, however, noted that researchers at NOAA's South West Fisheries Science Centre are working with old and degraded vaquita DNA to map out a genetic library "that accurately reflect[s] the original DNA input".
Analyses of the dilution series of control DNA 9948 yielded reproducible results down to 1 ng of DNA input.
However, application of CHA has been hindered by the need of specially redesigned sequences for each single-stranded DNA input.
In this study, we describe a TaqMan real time PCR allelic discrimination assay requiring minimal DNA input for TPMT genotyping.
