Methods for ensuring the highest DNA concentration and yield in future and retrospective trace DNA extracts

Dilley, Katherine; Pagan, Felicity; Chapman, Brendan

doi:10.1016/j.scijus.2020.11.005

Cited by 5 publications

(2 citation statements)

References 20 publications

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“…Unusually, this result also seems to show that mechanical-based method did not make excessive shearing of DNA. However, it can also be interpreted with the preference of the silica membrane to capture longer DNA sequences (Doran and Foran, 2014;Dilley et al, 2021), which is also in line with the result of lower DNA yield of mechanical-based method above.…”

Section: Discussionsupporting

confidence: 68%

“…Among the three methods, the DNA concentration of the mechanical-based method was significantly lower compared with the other methods, which may be result from the loss of excessive short DNA sequences during DNA purification on silica membrane ( Dilley et al., 2021 ). However, we performed analysis of correlation to compare DNA yield and microbial proportion within each DNA extraction method, and there were no correlations observed (all P > 0.1) as host DNA accounts for a large proportion in urine samples.…”

Section: Discussionmentioning

confidence: 99%

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Comparison Analysis of Different DNA Extraction Methods on Suitability for Long-Read Metagenomic Nanopore Sequencing

Zhang

Chen

Wang

et al. 2022

Front. Cell. Infect. Microbiol.

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Metagenomic next-generation sequencing (mNGS) is a novel useful strategy that is increasingly used for pathogens detection in clinic. Some emerging mNGS technologies with long-read ability are useful to decrease sequencing time and increase diagnosed accuracy, which is of great significance in rapid pathogen diagnosis. Reliable DNA extraction is considered critical for the success of sequencing; hence, there is thus an urgent need of gentle DNA extraction method to get unbiased and more integrate DNA from all kinds of pathogens. In this study, we systematically compared three DNA extraction methods (enzymatic cell lysis based on MetaPolyzyme, mechanical cell lysis based on bead beating, and the control method without pre–cell lysis, respectively) by assessing DNA yield, integrity, and the microbial diversity based on long-read nanopore sequencing of urine samples with microbial infections. Compared with the control method, the enzymatic-based method increased the average length of microbial reads by a median of 2.1-fold [Inter Quartile Range (IQR), 1.7–2.5; maximum, 4.8) in 18 of the 20 samples and the mapped reads proportion of specific species by a median of 11.8-fold (Inter Quartile Range (IQR), 6.9–32.2; maximum, 79.27]. Moreover, it provided fully (20 of 20) consistent diagnosed results to the clinical culture and more representative microbial profiles (P < 0.05), which all strongly proves the excellent performance of enzymatic-based method in long-read mNGS–based pathogen identification and potential diseases diagnosis of microbiome related.

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Section: Discussionsupporting

confidence: 68%

Section: Discussionmentioning

confidence: 99%