scholarly journals An optimized method for high-quality DNA extraction medicinal fungi Mycoleptodonoides aitchisonii for whole genome sequencing

2021 ◽  
Vol 948 (1) ◽  
pp. 012032
Author(s):  
R Riffiani ◽  
T Wada ◽  
N Shimomura ◽  
T Yamaguchi ◽  
T Aimi
Keyword(s):  
Whole Genome Sequencing ◽  
Dna Extraction ◽  
Genome Sequencing ◽  
Extraction Procedure ◽  
Ammonium Bromide ◽  
Malt Extract ◽  
Whole Genome ◽  
Liquid Media ◽  
High Molecular Weight Dna ◽  
Mycoleptodonoides Aitchisonii

Abstract The extraction of pure high molecular weight DNA and collecting genomic DNA from fungi is difficult. This is because filamentous fungi has a sturdy cell wall, high protein and polysaccharide levels resistant to the usual DNA extraction procedures. A low-cost and reliable DNA extraction method was designed from Mycoleptodonoides. aitchisonii fit for whole-genome sequencing for identification and mapping of the A mating-type gene. Mycoleptodonoides. aitchisonii belongs to the Climacodontaceae family has pharmaceutical activities. In the present study, the mycelia of M. aitchisonii, which grew from the different concentrations of malt extract and minimal liquid media, have been compared systematically to determine the DNA extraction procedure resulted in DNA concentration with excellent purity and quality. The best protocol that resulted in good quality DNA was further validated using polymerase chain reaction amplification with a specific primer to amplify the homeodomain protein (Mahd2-18) gene that encodes a transcription factor. The method proposed DNA extraction using CTAB (Cetyl Trimethyl Ammonium Bromide) method and purify by commercial kit from mycelium grown in the minimal liquid media give the best result with the high concentration of DNA for whole-genome sequencing by Next Generation Sequencing and other applications.

scholarly journals A common protocol for the simultaneous processing of multiple clinically relevant bacterial species for whole genome sequencing

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Kathy E. Raven ◽  
Sophia T. Girgis ◽  
Asha Akram ◽  
Beth Blane ◽  
Danielle Leek ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Dna Extraction ◽  
Genome Sequencing ◽  
Clinical Microbiology ◽  
Bacterial Species ◽  
Outbreak Detection ◽  
Whole Genome ◽  
Library Preparation ◽  
Simultaneous Processing ◽  
Antimicrobial Resistance Gene

AbstractWhole-genome sequencing is likely to become increasingly used by local clinical microbiology laboratories, where sequencing volume is low compared with national reference laboratories. Here, we describe a universal protocol for simultaneous DNA extraction and sequencing of numerous different bacterial species, allowing mixed species sequence runs to meet variable laboratory demand. We assembled test panels representing 20 clinically relevant bacterial species. The DNA extraction process used the QIAamp mini DNA kit, to which different combinations of reagents were added. Thereafter, a common protocol was used for library preparation and sequencing. The addition of lysostaphin, lysozyme or buffer ATL (a tissue lysis buffer) alone did not produce sufficient DNA for library preparation across the species tested. By contrast, lysozyme plus lysostaphin produced sufficient DNA across all 20 species. DNA from 15 of 20 species could be extracted from a 24-h culture plate, while the remainder required 48–72 h. The process demonstrated 100% reproducibility. Sequencing of the resulting DNA was used to recapitulate previous findings for species, outbreak detection, antimicrobial resistance gene detection and capsular type. This single protocol for simultaneous processing and sequencing of multiple bacterial species supports low volume and rapid turnaround time by local clinical microbiology laboratories.

scholarly journals Mycobacterial DNA Extraction for Whole-Genome Sequencing from Early Positive Liquid (MGIT) Cultures

2015 ◽  
Vol 53 (4) ◽  
pp. 1137-1143 ◽  
Author(s):  
Antonina A. Votintseva ◽  
Louise J. Pankhurst ◽  
Luke W. Anson ◽  
Marcus R. Morgan ◽  
Deborah Gascoyne-Binzi ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Dna Extraction ◽  
Genome Sequencing ◽  
Solid Phase ◽  
Clinical Samples ◽  
Reference Laboratory ◽  
Whole Genome ◽  
Mycobacterial Species ◽  
Accurate Identification ◽  
Content Type

We developed a low-cost and reliable method of DNA extraction from as little as 1 ml of early positive mycobacterial growth indicator tube (MGIT) cultures that is suitable for whole-genome sequencing to identify mycobacterial species and predict antibiotic resistance in clinical samples. The DNA extraction method is based on ethanol precipitation supplemented by pretreatment steps with a MolYsis kit or saline wash for the removal of human DNA and a final DNA cleanup step with solid-phase reversible immobilization beads. The protocol yielded ≥0.2 ng/μl of DNA for 90% (MolYsis kit) and 83% (saline wash) of positive MGIT cultures. A total of 144 (94%) of the 154 samples sequenced on the MiSeq platform (Illumina) achieved the target of 1 million reads, with <5% of reads derived from human or nasopharyngeal flora for 88% and 91% of samples, respectively. A total of 59 (98%) of 60 samples that were identified by the national mycobacterial reference laboratory (NMRL) asMycobacterium tuberculosiswere successfully mapped to the H37Rv reference, with >90% coverage achieved. The DNA extraction protocol, therefore, will facilitate fast and accurate identification of mycobacterial species and resistance using a range of bioinformatics tools.

scholarly journals Evaluation of DNA Extraction Methods on Individual Helminth Egg and Larval Stages for Whole-Genome Sequencing

2019 ◽  
Vol 10 ◽  
Author(s):  
Stephen R. Doyle ◽  
Geetha Sankaranarayanan ◽  
Fiona Allan ◽  
Duncan Berger ◽  
Pablo D. Jimenez Castro ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Dna Extraction ◽  
Genome Sequencing ◽  
Extraction Methods ◽  
Whole Genome ◽  
Larval Stages ◽  
Dna Extraction Methods

scholarly journals Direct DNA Extraction from Mycobacterium tuberculosis Frozen Stocks as a Reculture-Independent Approach to Whole-Genome Sequencing

2015 ◽  
Vol 53 (8) ◽  
pp. 2716-2719 ◽  
Author(s):  
K. Bjorn-Mortensen ◽  
J. Zallet ◽  
T. Lillebaek ◽  
A. B. Andersen ◽  
S. Niemann ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Whole Genome Sequencing ◽  
Dna Extraction ◽  
Genome Sequencing ◽  
Sequence Data ◽  
Whole Genome ◽  
Content Type ◽  
Direct Dna Extraction ◽  
Slow Growing

Culturing before DNA extraction represents a major time-consuming step in whole-genome sequencing of slow-growing bacteria, such asMycobacterium tuberculosis. We report a workflow to extract DNA from frozen isolates without reculturing. Prepared libraries and sequence data were comparable with results from recultured aliquots of the same stocks.

scholarly journals DNA extraction from primary liquid blood cultures for bloodstream infection diagnosis using whole genome sequencing

2018 ◽  
Vol 67 (3) ◽  
pp. 347-357 ◽  
Author(s):  
Luke W. Anson ◽  
Kevin Chau ◽  
Nicholas Sanderson ◽  
Sarah Hoosdally ◽  
Phelim Bradley ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Bloodstream Infection ◽  
Dna Extraction ◽  
Genome Sequencing ◽  
Blood Cultures ◽  
Whole Genome ◽  
Infection Diagnosis

scholarly journals DNA Extraction Method Optimized for Nontuberculous Mycobacteria Long-Read Whole Genome Sequencing

2018 ◽  
Author(s):  
Jennifer M. Bouso ◽  
Paul J. Planet
Keyword(s):  
Whole Genome Sequencing ◽  
Dna Extraction ◽  
Genome Sequencing ◽  
Nontuberculous Mycobacteria ◽  
Systemic Disease ◽  
Matrix Material ◽  
Whole Genome ◽  
Oxford Nanopore ◽  
Long Read ◽  
Oxford Nanopore Technologies

AbstractNontuberculous mycobacteria (NTM) are a major cause of pulmonary and systemic disease in at-risk populations. Gaps in knowledge about transmission patterns, evolution, and pathogenicity during infection have prompted a recent surge in genomic NTM research. Increased availability and affordability of whole genome sequencing (WGS) techniques, including the advent of Oxford Nanopore Technologies, provide new opportunities to sequence complete NTM genomes at a fraction of the previous cost. However, extracting large quantities of pure genomic DNA is particularly challenging with NTM due to their slow growth and recalcitrant cell wall. Here we report a DNA extraction protocol that is optimized for long-read WGS of NTM, yielding large quantities of highly pure DNA. Our refined method was compared to 6 other methods with variations in timing of mechanical and enzymatic digestion, quantity of matrix material, and reagents used in extraction and precipitation. We also demonstrate the ability of our optimized protocol to produce sufficient DNA to yield near-complete NTM genome assemblies using Oxford Nanopore Technologies long-read sequencing.

scholarly journals Evaluation of DNA extraction methods on individual helminth egg and larval stages for whole genome sequencing

2019 ◽  
Author(s):  
Stephen R. Doyle ◽  
Geetha Sankaranarayan ◽  
Fiona Allen ◽  
Duncan Berger ◽  
Pablo D. Jimenez Castro ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Dna Extraction ◽  
Genome Sequencing ◽  
De Novo ◽  
Strongyloides Stercoralis ◽  
Extraction Methods ◽  
Whole Genome ◽  
Life Stages ◽  
Trichuris Muris ◽  
Low Input

AbstractWhole genome sequencing is being rapidly applied to the study of helminth genomes, including de novo genome assembly, population genetics, and diagnostic applications. Although late-stage juvenile and adult parasites typically produce sufficient DNA for molecular analyses, these parasitic stages are almost always inaccessible in the live host; immature life stages found in the environment for which samples can be collected non-invasively offer a potential alternative, however, these samples are typically yield very low quantities of DNA, can be environmentally resistant, and are susceptible to contamination, often from bacterial or host DNA. Here, we have tested five low-input DNA extraction protocols together with a low-input sequencing library protocol to assess the feasibility of whole genome sequencing of individual immature helminth samples. These approaches do not use whole genome amplification, a common but costly approach to increase the yield of low input samples. We first tested individual parasites from two species spotted onto FTA cards - egg and L1 stages of Haemonchus contortus and miracidia of Schistosoma mansoni - before further testing on an additional six species - Ancylostoma caninum, Ascaridia dissimilis, Dirofilaria immitis, Dracunculus medinensis, Strongyloides stercoralis, and Trichuris muris - with an optimal protocol. Whole genome sequencing followed by analyses to determine the proportion of on- and off-target mapping revealed successful sample preparations for six of the eight species tested with variation between species, and within species but between life stages, described. These results demonstrate the feasibility of whole genome sequencing of individual parasites, and highlight a new avenue towards generating sensitive, specific, and information-rich data for the diagnosis and surveillance of helminths.

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