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.

Determination of the Population Structure of Fig Genotypes from Algeria and Turkey Using Inter Primer Binding Site-Retrotransposon and Simple Sequence Repeat Markers

“…The total genomic DNA was isolated following the CTAB DNA isolation protocol described by Healey et al [20] with minor modifications. For this purpose, 150 mg grind leave sample was used for each genotype.…”

Efficient Strategies for Elimination of Phenolic Compounds During DNA Extraction from Roots of Pistacia vera L.

“…The A260/A230 ratio of extracted DNAs by introduced method varied between 1.55-1.80 whereas the ratio for extracted DNAs using commercial kit was very low between 0.02-0.25, indicating the presence of organic contaminants (Table 1). Healey, Furtado, Cooper, & Henry (2014) tried to raise the quality of extracted DNA from recalcitrant plant species (Corymbia and Coffea) by adding β-mercaptoethanol to a CTAB based method and the centrifugation step after 65 °C incubation. In this investigation the high concentration of phenolic compounds accumulated in pistachio roots was eliminated using AlNH 4 (SO 4 ) 2 .12H 2 O during DNA extraction.…”

“…In this study DNA extraction from Pistachio roots via commercial DNA extraction kits resulted in dark color DNA with low spectral quality ( Table 1). The problem was relevant to dark browncolored compounds in root cells, called polyphenols substances that have a similar size and charge with DNA, tending to co-precipitate with extracted DNA, interfering with downstream enzymatic applications (Schrader, Schielke, Ellerbroek, & Johne, 2012;Borse, Joshi, & Chaphalkar, 2011;Healey, Furtado, Cooper, & Henry, 2014). Once the plant cells are broken apart, polyphenols become exposed to oxygen and reacted with polyphenol oxidases.…”

Isolation of high quality RNA from pistachio (Pistacia vera L.) and other woody plants high in secondary metabolites

“…The amplification slope value for cDNA samples of interest was determined to be −3.43 and −3.614 for actin and β-tubulin primers, respectively, which is within the range of acceptable slope, further confirming the quality of extracted RNA. Providing high quality RNA is a critical requirement of any RNA-seq experiment as the isolated RNA should pass stringent quality control standards for sequencing on the available platforms (Healey et al 2014). The quality of sequencing data were assessed by CLC Bio Genomics WorkBench software, version 6.5 (CLC Bio, Denmark) and illustrated in Fig.…”

“…Currently, RNA-sequencing (RNA-seq) is being used as an efficient tool to investigate transcriptome of plants, especially non-model species (Healey et al 2014). However, applying these technologies for recalcitrant plants are challenging as a result of high polyphenols and polysaccharides amounts that lead to poor quality and quantity of isolated RNA (Xiao et al 2012).…”