DNA Assessment

CHAPTER 10 DNA Assessment Carolina Corrales*, Emily Veltjen*, Péter Poczai, Jackie Mackenzie-Dodds, Elisabeth Haring, María Paz Martín, Daniel Mulcahy, and Jonas J. Astrin * These authors contributed equally to this chapter. Corrales, Veltjen et al. 141 Introduction The technical value of a DNA sample, as opposed to its biological value (i.e., taxon, collection data), can be expressed in DNA quantity and DNA quality. The latter can be measured in terms of DNA purity and DNA integrity (although some sources also subsume concentration under DNA quality). These measurements are fundamentally important to estimate the probability of success for planned downstream molecular techniques, to evaluate DNA isolation procedures, and to validate DNA extracts, which in turn, will allow for a better sample management. Note that DNA quantity, purity and integrity are influenced by several factors such as the sampling method and selected tissue (Casas-Marce et al. 2010; Nowland and Southgate 2015), the organism´s biology (Spooner and Ruess 2014), the preservation method (Nagy 2010; Wong et al. 2012; Allison et al. 2021), the DNA extraction and storage procedures (Lee et al. 2010), and the subsequent DNA sample manipulation (e.g., freeze-thaw cycles). Measurements taken right after DNA extraction will predict the quality of the sampled material and the extraction method, whereas measurements taken at a later point will assess DNA storage conditions (e.g., temperature and preservation fluid). Contamination can also affect the DNA´s technical value, and so measures should be taken to minimise the risk of contamination during sample handling, and the production of aliquots should be considered. Refer to Ruvira and Ruiz Arahal (2012) for DNA quality control validation guidelines. RECOMMENDATION All technical values available should be fully recorded in the collection and/or laboratory database, including gel images, used measurement methods, and for which procedure they were intended (e.g., DNA extraction, library preparation). DNA purity DNA purity can be assessed by measuring the absorbance using a UV spectrophotometer (e.g., NanoDrop, DeNovix, bioDrop Duo, GeneQUant Pro). Spectrophotometry methods are convenient because they can be carried out quickly, instruments are rather inexpensive and easy to use, do not require additional reagents and data analysis is relatively simple (Boesenberg-Smith et al. 2012). Ideally, Tris buffer should be preferred over water when eluting the sample to prevent inaccurate and highly variable measurements (Koetsier and Cantor 2019). It is important that the spectrophotometer is placed in a temperature-controlled area to avoid sample evaporation, due to the small sample aliquot (1–2 μl) taken for measurement (Green and Sambrook 2018). In general, purity values are optimal when ranging from 1.8 to 2.0 at a 260/280 ratio (or 2.0 to 2.4 at a 260/230 ratio) (Lucena-Aguilar et al. 2016; Peñafiel et al. 2019; ERGA 2021; PacBio 2022), when DNA preparation have little absorbance at 320 or 230 nm, and when the spectral profile shows a smooth peak shape at 260 nm, which indicates that the DNA extraction has produced an ample nucleotide quantity (Green and Sambrook 2018). Values below 1.8 will mean that the DNA might be contaminated (e.g., with proteins, salts, ethanol, phenol) or that the sample shows a very low DNA concentration (PacBio 2022). Higher values are usually not an issue, but they may indicate that some oversight has occurred during the procedure, such as, a wrong solu- CHAPTER 10 DNA Assessment 142 tion having been used as negative control, the instrument not being well-calibrated, or that the pedestal being dirty (Sambrook and Russell 2001; GTF 2020). In any case, the spectral profile should be checked for abnormalities (PacBio 2022). Note that diluted nucleic acid samples may look highly contaminated, whereas concentrated ones may appear to be clean (Koetsier and Cantor 2019). RECOMMENDATION It is best practice to measure the absorbance at different wavelength ratios (230, 260, 280, and 320 nm) to confirm the presence of other compounds within the sample that cannot be absorbed at 260 nm, for which DNA absorbs strongly. This method is more appropriate for pure DNA concentrations ranging between 3.5 and 90 ng/μl (Sambrook and Russell 2001; García-Alegría et al. 2020), although measurements can be unstable as slow as 20 ng/ μl (Koetsier and Cantor 2019). Spectrophotometry should not be used for low concentrations, as found in small-sized organisms or impure DNA (Hodkinson et al. 2007; Wilding et al. 2009), because spectrophotometric methods are sensitive to contaminants (e.g., proteins, nucleotides, phenol) (Green and Sambrook 2018). DNA quantification or concentration DNA quantification is an essential calculation because specific targets are required for optimal downstream application performance (Boesenberg-Smith et al. 2012). DNA quantity is expressed as either DNA concentration (e.g., ng/µl) or DNA weight/yield (e.g., ng), which can also be assessed using spectrometry. However, it is not especially recommended because it often overestimates DNA concentrations (Green and Sambrook 2018) and it is not informative concerning degradation of DNA. RECOMMENDATION If there is enough DNA available, it is a good practice to take aliquots (2 µl) from the top, middle, and bottom of each DNA sample to obtain an average estimate of the DNA concentration (Dahn et al. 2022). Another way to measure DNA quality and quantity is using agarose gels. Gels are stained, for instance, with ethidium bromide or GelRed and visualised under UV using a transilluminator, and HMW can be easily assessed with a large-range ladder, like Hind III (Mulcahy et al. 2016). DNA size bands are compared with a standard DNA ladder, and concentrations can be measured using the densitometry function of any imaging system software (Wang et al. 2017). Impurities such as detergents or probes, can usually be seen as a smear, whereas RNA impurities are often visible at the bottom of the gel, due to their faster migration. Note that classic gel electrophoresis can give inaccurate values due to dye intensity problems (Green and Sambrook 2018). A more reliable alternative to assess concentration of nucleic acids is fluorometric determination (e.g., Qubit Fluorometer, Quantus, Tecan Genios), because it is not affected by the presence of contaminants, and because low concentrations of DNA can easily be detected (Wilding et al. 2009; Green and Sambrook 2012). It is also the preferred method for long-read sequencing. This method uses dyes (e.g., Quant-iT PicoGreen, Hoechst 33258, SYBR dyes) to stain DNA and a lambda DNA standard to help determine the DNA concentration of the samples (Green and Sambrook Corrales, Veltjen et al. 143 2012; Wilding et al. 2009). Keep in mind that the Hoechst 33258 dye does not recognise single-stranded DNA, and hence quantitation results may be overestimated (Invitrogen-MP 2008). If working with HMW DNA, each sample should be measured in triplicate to check for reproducibility, as HMW DNA is usually not perfectly dissolved. Spectrophotometric and fluorometric measurements should usually agree on estimated concentration values. If values are very different (≥50% difference), a bead purification step should be carried out to get a cleaner sample and hence, similar values (PacBio 2022). For a comparison of fluorescence and absorbance methods, see the Invitrogen technical note (2016) or Leggate et al. (2006). Additionally, RT-PCR and ddPCR are sensitive methods that can be performed using fluorometric probes to detect PCR inhibitors and DNA quantities, even in small amounts, and thus are also suitable for aDNA studies (Boesenberg-Smith et al. 2012; Robin et al. 2016). These amplification methods, however, require a larger investment in resources compared to other options, and DNA integrity will not be assessed in qualities needed for some NGS methods. It is essential to pipette correctly to measure DNA concentration, especially when dealing with uHMW DNA, which is very viscous and sticky, as opposed to HMW. Koetsier and Cantor (2021) provide a shearing approach based on vortexing to produce fragments in the range of 50–100 kb, allowing easy uHMW DNA sample handling and reliable concentration measures. Furthermore, accurate pipetting is essential for DNA assessment, so in addition to proper sterilisation, pipettes should always be calibrated and should not be used at the extreme of volume specifications. DNA integrity DNA integrity measurements examine DNA fragmentation, meaning the length of DNA, which can be visualised using agarose gels or capillary gel instruments / DNA screen tapes (e.g., Bioanalyzer, Fragment Analyzer, Agilent TapeStation) (Forrest et al. 2019; Akinwole and Babarinde 2019). The latter can perform electrophoresis, imaging, and analysis, enabling a more automated workflow. DNA purity can also be determined using pulsed-field gel electrophoresis (PFGE), minigel electrophoresis, or AFLP (Clermont et al. 2014; Green and Sambrook 2012). The PFGE (e.g., Agilent Femto, Bio-Rad CHEF Mapper XA, Sage Science Pippin) is a technique used to separate very large DNA molecules, and hence, it is used for long-read sequencing technologies. However, the set-up for PFGE can be expensive and time-consuming. For a cheaper alternative, Mulcahy et al. (2016) proposed a standardised method to assess the quality and size of DNA by scoring gel images in the ImageJ software. A slow, concentrated gel is run with a large-range ladder (Hind III), and a size can be chosen for standard comparison (e.g., 23–9 kb). The size marker ~ “9 kb” (= 9,416 bp) was proposed as a standard for genomic quality in biobanking samples, and it is suitable for both shortand longread sequencing. The detailed protocol can be found in Mulcahy et al. (2016, supplementary information). When using analytical quantification equipment, DNA degradation can be determined by calculating (from 1 to 10) the DNA integrity number (DIN). A high DIN indicates intact DNA, whereas a low DIN a very degraded sample (Agilent Genomic DNA 2015). Note that DNA integrity can be compromised if DNA samples are not correctly handled. The following recommendations will minimise shearing and degradation (GTF 2020): • Use wide-bore tips (~3 mm in diameter). • Prevent or minimise vortexing. CHAPTER 10 DNA Assessment 144 • Pipette slowly. • Use low-binding microfuge tubes. • Avoid freeze-thaw cycles, overdrying and overheating. • Samples should not contain insoluble material, denaturants, (e.g., phenol), detergents (e.g., SDS) nor RNA or carryover contamination (e.g., humid acid). • DNA should not be exposed to high temperatures (>65°C) nor extreme pH (<6 or >9). RECOMMENDATION The choice of DNA assessment method will depend on the cost, time, and available equipment, as well as on the aim of the laboratory/project. In general, an inexpensive standardised method should be used for all DNA samples, except for those that demand more information for downstream analyses (e.g., candidates for NGS sequencing). If necessary, quality control can be performed by third-party companies, such as Rapid Genomics, Macrogen, Edinburgh Genomics, Genomics Core, BGI, or Imagene.