Comparison and optimization of DNA Isolation protocols for high throughput genomic studies of Acacia pachyceras Schwartz

Habibi, Nazima; Salameen, Fadila Al; Rahman, Muhammed; Shajan, Anisha; Zakir, Farhana; Abdulrazzack, Nasreem

doi:10.1016/j.mex.2022.101799

Cited by 7 publications

(6 citation statements)

References 16 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Therefore, the development of an optimized DNA extraction method is crucial to ensure accurate and reliable sequencing results. (Healey et al 2014;Habibi et al 2022). We made three critical modifications to the CTAB method.…”

Section: Discussionmentioning

confidence: 99%

“…According to (Ambra et al 2023) polysaccharides are known to obstruct the creation of NGS libraries because of their chemical characteristics, which cause them to coprecipitate with genomic DNA and give solutions a sticky, glue-like look. high-quality DNA is characterized as DNA that is predominantly high molecular weight with an A260/280 ratio between 1.8 and 2.0 and without contaminating substances, such as polysaccharides or phenolics (Healey et al 2014;Habibi et al 2022). Since the prerequisite of NGS starting material is high-quality genomic DNA extract, it is critical to establish a method that delivers such requirements with high reproducibility (Tsaballa et al 2023).…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

High-quality genomic DNA extraction methods of Yellow Spathoglottis Blume complex for next-generation sequencing

JASIM,

OTHMAN,

NORDIN

et al. 2024

Biodiversitas

View full text Add to dashboard Cite

Abstract. Jasim JHM, Othman AS, Nordin FA, Talkah NSM. 2024. High-quality genomic DNA extraction methods of Yellow Spathoglottis Blume complex for next-generation sequencing. Biodiversitas 25: 654-663. Advancements in genomic research have spurred a growing interest in extracting high-quality genomic DNA, particularly from intricate plant species like the Yellow Spathoglottis Blume Complex. The preparation of genomic DNA high-quality from a plant sample is a crucial step for genomic and genetic analysis studies. A variety of genomic DNA extraction methods such as the traditional Hexadecyltrimethylammonium Bromide (CTAB) method and commercially available kits have been reported. Even so, they are either low purity and yield or costly. We develop a good-quality genomic DNA extraction method from fresh and dried leaves of the Yellow Spathoglottis Blume complex in Peninsular Malaysia, specifically tailored for Next-Generation Sequencing (NGS) applications. Three DNA extraction methods were compared traditional CTAB, modified CTAB, and the DNeasy Qiagen plant mini kit methods. The yield and quality of extracted DNA were assessed by PCR amplification using nuclear ETS and ITS genes performed to evaluate the success of DNA extraction. Additionally, the suitability of the modified CTAB method for NGS library preparation and sequencing was tested. It demonstrated better PCR amplification results, yielding high-concentration bands for both ETS and ITS primers. Furthermore, the modified CTAB method proved suitable for high molecular weight DNA extraction, without contamination. The extracted DNA from Spathoglottis aurea exhibited high molecular weight and passed the quality control assessment for NGS library preparation. Sequencing results demonstrated an average quality score of 36.0, with an accurate base call rate of approximately 99.9%. The developed method enables reliable genomic analysis and sequencing of the Yellow Spathoglottis Blume complex, contributing to further research and understanding of this species group.

show abstract

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

High-quality genomic DNA extraction methods of Yellow Spathoglottis Blume complex for next-generation sequencing

JASIM,

OTHMAN,

NORDIN

et al. 2024

Biodiversitas

View full text Add to dashboard Cite

show abstract

“…However, the identification of SSRs and their development using conventional techniques are arduous, expensive, and time-consuming ( Taheri et al., 2018 ; Mathur et al., 2013 ; Habibi et al., 2020 ). High-throughput sequencing technologies have recently made it possible for researchers to identify thousands of microsatellites at a lesser cost with minimal efforts in a matter of few minutes as compared to the traditional BLAST search ( Habibi et al, 2022a , c , Šarhanová et al., 2018 ; Taheri et al., 2018 ). We demonstrated the utility of genome sequencing to filter more than 900,000 SSR motifs.…”

Section: Discussionmentioning

confidence: 99%

“…Loss of plant diversity due to habitat loss, fragmentation, and degradation, overexploitation, pollution, and anthropogenic climate change is infusing worldwide concern ( Corlett, 2016 ). The harsh climatic conditions further intensify the impact in arid lands including the Arabian Peninsula ( Al Salameen et al., 2018 ; Mosa et al., 2019 ; Al Salameen et al., 2020 ; Habibi et al., 2020 ; Habibi et al., 2022a ). Kuwait offers a valuable genetic pool of native flora encompassing genes related to salt and stress tolerance, however the plant diversity faces serious consequences of degradation ( Omar et al., 2001 ; Asem and Roy, 2010 ; Al Salameen et al., 2018b ; Al Salameen et al., 2020 ; Al Salameen et al., 2022 ).…”

Section: Introductionmentioning

confidence: 99%

Genome survey and genetic characterization of Acacia pachyceras O. Schwartz

Habibi

Salameen

Vyas³

et al. 2023

Front. Plant Sci.

Self Cite

View full text Add to dashboard Cite

Acacia pachyceras O. Schwartz (Leguminoseae), a woody tree growing in Kuwait is critically endangered. High throughput genomic research is immediately needed to formulate effective conservation strategies for its rehabilitation. We therefore, performed a genome survey analysis of the species. Whole genome sequencing generated ~97 Gb of raw reads (92x coverage) with a per base quality score above Q30. The k-mer analysis (17 mer) revealed its genome to be 720Mb in size with an average guanine-cytosine (GC) ratio of 35%. The assembled genome was analyzed for repeat regions (45.4%-interspersed repeats; 9%-retroelements; 2%-DNA transposons). BUSCO assessment of completeness of genome identified 93% of assembly to be complete. Gene alignments in BRAKER2 yielded 34,374 transcripts corresponding to 33,650 genes. Average length of coding sequences and protein sequences were recorded as 1,027nts and 342aa, respectively. GMATA software filtered a total of 901,755 simple sequence repeats (SSRs) regions against which 11,181 unique primers were designed. A subset of 110 SSR primers were PCR validated and demonstrated for its application in genetic diversity analysis of Acacia. The SSR primers successfully amplified A. gerrardii seedlings DNA depicting cross transferability among species. The principal coordinate analysis and the split decomposition tree (bootstrapping runs of 1000 replicates) distributed the Acacia genotypes into two clusters. The flow cytometry analysis revealed the A. pachyceras genome to be polyploid (6x). The DNA content was predicted as 2.46 pg, 1.23 pg, and 0.41 pg corresponding to 2C DNA, 1C DNA and 1Cx DNA, respectively. The results provide a base for further high throughput genomic studies and molecular breeding for its conservation.

show abstract

“…The S1 and S2). Home-made DNA extraction methods have been calculated to cost between 1.2 and 2.56 USD while the most commonly used commercial kits (Qiagen blood and tissue kit, and Mobio power water kits cost 4.3 and 12.75 USD, respectively) (Chacon-Cortes et al, 2012;Habibi et al, 2022;Qiagen, 2023aQiagen, , 2023bTakahashi et al, 2023). Cost is crucial for…”

Section: Cost Comparisonmentioning

confidence: 99%

Improving the recovery for dissolved eDNA state: A comparative analysis of isopropanol precipitation, magnetic bead extraction, and centrifugal dialysis

Kirtane,

Deiner

2024

Environmental DNA

View full text Add to dashboard Cite

Environmental DNA consists of multiple states including but not limited to membrane‐bound, adsorbed, and dissolved states. Because of their chemical and physical properties, each of these states may have different degradation and transport potential. Essential to the study of eDNA states is being able to isolate them from an environmental sample. Here, we focus on improving the DNA recovery of the dissolved state of eDNA from water. We compared three recovery methods, namely isopropanol precipitation, magnetic bead extraction, and centrifugal dialysis. We evaluated the effectiveness of these methods based on the measured DNA recovery of two different species' DNA of different sizes. DNA recovery was assessed with qPCR. We also checked for the effect of inhibitor removal, and lastly, we estimated the cost of reagents and consumables (not labor). The DNA recovery varied among extraction methods, with isopropanol precipitation yielding the highest recovery (52.47 ± 19.69%), followed by centrifugal dialysis (12.58 ± 7.15%) and magnetic bead extraction (9.92 ± 3.89%). However, isopropanol precipitation's efficacy was influenced by humic acid concentration in the water matrix. The presence of humic acids significantly affected isopropanol precipitation, with higher humic acid concentrations leading to increased DNA recovery. This suggests that humic acids act as co‐precipitators during isopropanol precipitation. We observed that longer DNA fragments (chicken) had lower recovery compared to shorter fragments (salmon) across all extraction methods. Magnetic bead extraction effectively removed inhibitors, while centrifugal dialysis and isopropanol extraction required an additional inhibitor removal step. Isopropanol, magnetic bead extraction, and centrifugal dialysis methods have estimated costs of 7.74, 8.53, and 23.62 USD in 2023 respectively. Overall, isopropanol precipitation was the least expensive and most effective with the highest measured recoveries, but it was dependent on humic acid concentration in the environmental sample.

show abstract

Comparison and optimization of DNA Isolation protocols for high throughput genomic studies of Acacia pachyceras Schwartz

Cited by 7 publications

References 16 publications

High-quality genomic DNA extraction methods of Yellow Spathoglottis Blume complex for next-generation sequencing

High-quality genomic DNA extraction methods of Yellow Spathoglottis Blume complex for next-generation sequencing

Genome survey and genetic characterization of Acacia pachyceras O. Schwartz

Improving the recovery for dissolved eDNA state: A comparative analysis of isopropanol precipitation, magnetic bead extraction, and centrifugal dialysis

Contact Info

Product

Resources

About