Fig. 4

Influence of genomic DNA (gDNA) extraction protocols on sequencing quality. We assessed and modified methodologies to establish a high-throughput protocol to extract gDNA from white clover, a species prone to yielding degraded DNA. Chemistry based on a CTAB protocol [6] was modified for gDNA extraction using 96-well plates (a). Adaptation of the Whitlock method [2] to a 96-well plate protocol (b), and development of a streamlined inexpensive protocol described in this paper (c). In these examples, gDNA was extracted from freeze-dried leaves and aliquots (2 μL from 100 μL gDNA extraction/elution) were resolved and visualised by electrophoresis in an agarose lithium borate buffer (0.8% w/v) gel containing 25 μg ethidium bromide. The samples were flanked at either end by 1 kb Plus size ladders (www.thermofisher.com). Sequence quality of the extracted gDNA was assessed by producing a genotyping-by-sequencing (GBS) library [1] comprising 95 individuals from each of the white clover gDNA extractions shown above (a–c). The GBS libraries were single-end sequenced (100 bp) on a single lane each of an Illumina 2500 Hi-Seq sequencer. Sequencing quality assessment using FastQC version 0.10.1 [9] for GBS libraries made from the gDNA shown a–c is represented in graphs describing quality across all bases from every sequence read at each position (d–f, respectively). Sequence quality is based on phred scores [10], an exponential scale where, for example, 20 = one incorrect sequence base-call in 100, and 30 = one incorrect base-call in 1000. The y-axis shows the quality scores, and the higher the score the greater confidence in the base-calls at that position. The background of the graph divides the y-axis into very good quality calls (green), reasonable quality (orange), and poor quality (red)