Bio-On-Magnetic-Beads (BOMB): Open platform for high-throughput nucleic acid extraction and manipulation

Oberacker, Phil; Stepper, Peter; Bond, Donna M.; Höhn, Sven; Focken, Jule; Meyer, Vivien; Schelle, Luca; Sugrue, Victoria J.; Jeunen, Gert‐Jan; Moser, Tim; Hore, Steven R.; Meyenn, Ferdinand von; Hipp, Katharina; Hore, Timothy A; Jurkowski, Tomasz P.

doi:10.1371/journal.pbio.3000107

Cited by 186 publications

(148 citation statements)

References 33 publications

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“…We compared DNA extraction performance across seven protocols: (a) Qiagen DNeasy Blood & Tissue Kit (Qiagen GmbH, Hilden, Germany), (b) MO BIO PowerWater DNA Isolation Kit (MO BIO Laboratories, Inc., Carlsbad, CA, USA), (c) MO BIO PowerMax Soil DNA Isolation Kit (MO BIO Laboratories, Inc., Carlsbad, CA, USA), (d) Presto™ Mini gDNA Bacteria Kit (Geneaid Biotech Ltd., Taiwan), (e) PCI alcohol DNA extraction protocol (Renshaw et al, ), (f) Silica extraction protocol (Alawi, Schneider, & Kallmeyer, ; Ogram, Sayler, & Barkay, ), and (g) Magnetic Beads extraction protocol (Oberacker et al, ). A detailed description of each procedure can be found in Supplement .…”

Section: Methodsmentioning

confidence: 99%

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Species‐level biodiversity assessment using marine environmental DNA metabarcoding requires protocol optimization and standardization

Jeunen

Knapp

Spencer

et al. 2019

Ecology and Evolution

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DNA extraction from environmental samples (environmental DNA; eDNA) for metabarcoding‐based biodiversity studies is gaining popularity as a noninvasive, time‐efficient, and cost‐effective monitoring tool. The potential benefits are promising for marine conservation, as the marine biome is frequently under‐surveyed due to its inaccessibility and the consequent high costs involved. With increasing numbers of eDNA‐related publications have come a wide array of capture and extraction methods. Without visual species confirmation, inconsistent use of laboratory protocols hinders comparability between studies because the efficiency of target DNA isolation may vary. We determined an optimal protocol (capture and extraction) for marine eDNA research based on total DNA yield measurements by comparing commonly employed methods of seawater filtering and DNA isolation. We compared metabarcoding results of both targeted (small taxonomic group with species‐level assignment) and universal (broad taxonomic group with genus/family‐level assignment) approaches obtained from replicates treated with the optimal and a low‐performance capture and extraction protocol to determine the impact of protocol choice and DNA yield on biodiversity detection. Filtration through cellulose‐nitrate membranes and extraction with Qiagen's DNeasy Blood & Tissue Kit outperformed other combinations of capture and extraction methods, showing a ninefold improvement in DNA yield over the poorest performing methods. Use of optimized protocols resulted in a significant increase in OTU and species richness for targeted metabarcoding assays. However, changing protocols made little difference to the OTU and taxon richness obtained using universal metabarcoding assays. Our results demonstrate an increased risk of false‐negative species detection for targeted eDNA approaches when protocols with poor DNA isolation efficacy are employed. Appropriate optimization is therefore essential for eDNA monitoring to remain a powerful, efficient, and relatively cheap method for biodiversity assessments. For seawater, we advocate filtration through cellulose‐nitrate membranes and extraction with Qiagen's DNeasy Blood & Tissue Kit or phenol‐chloroform‐isoamyl for successful implementation of eDNA multi‐marker metabarcoding surveys.

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Section: Methodsmentioning

confidence: 99%

“…We compared DNA extraction performance across seven proto- (Renshaw et al, 2015), (f) Silica extraction protocol (Alawi, Schneider, & Kallmeyer, 2014;Ogram, Sayler, & Barkay, 1987), and (g) Magnetic Beads extraction protocol (Oberacker et al, 2018). A detailed description of each procedure can be found in Supplement 2.…”

Section: Extraction Performancementioning

confidence: 99%

Species‐level biodiversity assessment using marine environmental DNA metabarcoding requires protocol optimization and standardization

Jeunen

Knapp

Spencer

et al. 2019

Ecology and Evolution

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“…The resulting purified products were amplified in a reaction containing 25uL Q5 2× Mastermix (NEB), 2uL template, 0.5uL 50uM primer mixture (Supplemental sequences), 2.5uL Evagreen dye (Biotium), and 22.5uL ddH2O. Amplification was monitored in the SYBR channel on an Eppendorf Realplex4 real-time PCR system until several cycles of productive amplification were observed, typically less than 10 cycles, and were purified using DNA-binding magnetic beads 3 .…”

Section: Methodsmentioning

confidence: 99%

High throughput functional variant screens via in-vivo production of single-stranded DNA

Schubert

Goodman

Wannier

et al. 2020

Preprint

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5Tremendous genetic variation exists in nature, but our ability to create and characterize 6 individual genetic variants remains far more limited in scale. Likewise, engineering proteins and 7 phenotypes requires the introduction of synthetic variants, but design of variants outpaces 8 experimental measurement of variant effect. Here, we optimize efficient and continuous 9 generation of precise genomic edits in Escherichia coli, via in-vivo production of single-stranded 10 DNA by the targeted reverse-transcription activity of retrons. Greater than 90% editing 11 efficiency can be obtained using this method, enabling multiplexed applications. We introduce 12 Retron Library Recombineering (RLR), a system for high-throughput screens of variants, 13 wherein the association of introduced edits with their retron elements enables a targeted deep 14 sequencing phenotypic output. We use RLR for pooled, quantitative phenotyping of synthesized 15 variants, characterizing antibiotic resistance alleles. We also perform RLR using sheared 16 genomic DNA of an evolved bacterium, experimentally querying millions of sequences for 17 antibiotic resistance variants. In doing so, we demonstrate that RLR is uniquely suited to utilize 18 non-designed sources of variation. Pooled experiments using ssDNA produced in vivo thus 19 present new avenues for exploring variation, both designed and not, across the entire genome. 20 21 Introduction: 22Constructing genotypes of interest and observing their effect on phenotype critically aids 23 our understanding of genetics and genome function. As methods for editing genomes have 24 progressed, this "reverse genetics" approach has expanded in breadth and scale, from knockout 25 libraries 1 to refactored genomes 2,3 . These experiments can now be performed within multiplexed 26 pools, which allow an ever greater number of mutations to be explored across varied conditions. 27Critically, both creating genotypes and observing phenotype within pools has necessitated 28 2 development of new techniques. Transposon insertions 4 , marked integrations 5 and CRISPR-29 inhibition 6,7 can create thousands of variants simultaneously within pooled experiments, and 30 targeted sequencing of these elements enables pooled measurement of variant phenotypes. These 31 advancements in experimental scale have fundamentally transformed our understanding of 32 genome function 8 . 33However, these current high-throughput genetic techniques remain limited, in that they 34 typically introduce, ablate, or regulate kilobases of DNA to create variation. This contrasts with 35 point mutations, which are ubiquitous in natural variation 9 , and are indispensable for engineering 36 proteins 10 and metabolic pathways 11 . While modifying kilobases of DNA can add and subtract 37 functional elements such as genes and regulatory sequences from the genome, point mutations 38 can alter the function of these elements, accessing a larger phenotypic landscape. 39Point mutations and other precision edits can be performed by oligonucleo...

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“…Samples dissolved in DNAzol direct can be directly added to PCR reactions because the pH of DNAzol Direct dramatically drops upon a ten-fold or greater dilution [Chomczynski and Rymaszewski, 2006]. The PCR product was purified using magnetic beads (Oberacker et al, 2019) and then submitted for Sanger sequencing (UC Berkeley DNA…”

Section: Establishing Clonal Strainsmentioning

confidence: 99%

Genome editing enables reverse genetics of multicellular development in the choanoflagellateSalpingoeca rosetta

Booth

King

2020

Preprint

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In a previous study, we established a forward genetic screen to identify genes required for multicellular development in the choanoflagellate, Salpingoeca rosetta (Levin et al., 2014). Yet, the paucity of reverse genetic tools for choanoflagellates has hampered direct tests of gene 25 function and impeded the establishment of choanoflagellates as a model for reconstructing the origin of their closest living relatives, the animals. Here we establish CRISPR/Cas9-mediated genome editing in S. rosetta by engineering a selectable marker to enrich for edited cells. We then use genome editing to disrupt the coding sequence of a S. rosetta C-type lectin gene, rosetteless, and thereby demonstrate its necessity for multicellular rosette development. This 30 work advances S. rosetta as a model system in which to investigate how genes identified from genetic screens and genomic surveys function in choanoflagellates and evolved as critical regulators of animal biology.

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Bio-On-Magnetic-Beads (BOMB): Open platform for high-throughput nucleic acid extraction and manipulation

Cited by 186 publications

References 33 publications

Species‐level biodiversity assessment using marine environmental DNA metabarcoding requires protocol optimization and standardization

Species‐level biodiversity assessment using marine environmental DNA metabarcoding requires protocol optimization and standardization

High throughput functional variant screens via in-vivo production of single-stranded DNA

Genome editing enables reverse genetics of multicellular development in the choanoflagellateSalpingoeca rosetta

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