Protocol: a simple method for extracting next-generation sequencing quality genomic DNA from recalcitrant plant species

Abstract

Next-generation sequencing technologies rely on high quality DNA that is suitable for library preparation followed by sequencing. Some plant species store large amounts of phenolics and polysaccharides within their leaf tissue making genomic DNA extraction difficult. While many DNA extraction methods exist that contend with the presence of phenolics and polysaccharides, these methods rely on long incubations, multiple precipitations or commercially available kits to produce high molecular weight and contaminant-free DNA. In this protocol, we describe simple modifications to the established CTAB- based extraction method that allows for reliable isolation of high molecular weight genomic DNA from difficult to isolate plant species Corymbia (a eucalypt) and Coffea (coffee). The simplified protocol does not require multiple clean up steps or commercial based kits, and the isolated DNA passed stringent quality control standards for whole genome sequencing on Illumina HiSeq and TruSeq sequencing platforms.

Keywords: CTAB; Coffea; Corymbia; DNA extraction; Next-generation sequencing.

Figure 1

Genomic DNA preparation of Corymbia citriodora subsp. variegata resolved by electrophoresis. 1 kb DNA ladder (1, 14), 100 and 200 ng λ DNA standards (2, 3 and 12, 13) respectively. DNA extractions using the traditional CTAB-based method with no PVP (4), 1% PVP (5), and 4% PVP (6). DNA extractions using the NGS protocol with no PVP (7), 1% PVP (8), and 4% PVP (9). Endonuclease digestions of DNA extracted without PVP with EcoRI (10) and High-Fidelity HindIII (11). Results from six grams of leaf tissue finely ground using a mortar and pestle, then aliquoted (1 g) for each extraction. DNA was resolved by electrophoresis in a 0.7% agarose gel and visualized using SYBR Safe DNA gel stain. Percentages are represented as w/v. CTAB: hexadecyltrimethylammonium bromide; PVP: polyvinylpyrrolidone.

Figure 2

NanoDrop measurement profile of genomic DNA extractions from Corymbia citriodora subsp. variegata. DNA extractions using a traditional CTAB-based method with (A) no PVP, (B) 1% PVP and (C) 4% PVP. DNA extractions using the NGS protocol with (D) no PVP, (E) 1% PVP, and (F) 4% PVP. Results from six grams of leaf tissue finely ground using a mortar and pestle, then aliquoted (1 g) for each extraction. Percentages are represented as w/v. CTAB: hexadecyltrimethylammonium bromide; PVP: polyvinylpyrrolidone.

Figure 3

DNA quality and yield assessment for Coffea brassii genomic DNA using the NGS extraction protocol. A) Genomic DNA preparation of Coffea brassii resolved by electrophoresis. 1 kb DNA ladder (1), 100 ng and 200 ng λ DNA standards respectively (3, 4), and DNA extraction using the modified NGS extraction protocol (2). DNA was separated by electrophoresis in a 0.7% agarose gel and visualized using SYBR Safe DNA gel stain. The gel image was cropped to exclude an unrelated sample. B) NanoDrop measurement profile of Coffea brassii leaf extracted genomic DNA using the modified NGS extraction protocol.

Figure 4

Paired end read quality distributions of Corymbia and Coffea samples from Illumina HiSeq and TruSeq library preparations. (A) Corymbia citriodora subsp. citriodora, (B) Corymbia henryi, (C) Corymbia torelliana, (D) Corymbia citriodora subsp. variegata, and (E) Coffea brassii. The x-axis represents the PHRED quality score and the y-axis represents the percentage of sequences with a particular score, normalized to the total number of sequences. The distribution graph was generated using CLC Bio software (Version 5.5.2).