A comprehensive model of DNA fragmentation for the preservation of High Molecular Weight DNA

Klingström, Tomas; Bongcam‐Rudloff, Erik; Ov, Pettersson

doi:10.1101/254276

Cited by 20 publications

(22 citation statements)

References 63 publications

(95 reference statements)

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“…Several factors influence DNA stability during extraction, including chemical properties of the buffer and the physical forces applied during tissue homogenization, phase separation and pipetting (Klingstrom, Bongcam‐Rudloff, & Pettersson, ). The buffer composition is the least flexible factor, especially for difficult tissues such as field samples of eucalyptus leaves that require complex buffers for DNA extraction (see above).…”

Section: Resultsmentioning

confidence: 99%

Harnessing the MinION: An example of how to establish long‐read sequencing in a laboratory using challenging plant tissue from Eucalyptus pauciflora

Schalamun

Nagar

Kainer

et al. 2018

Molecular Ecology Resources

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Long-read sequencing technologies are transforming our ability to assemble highly complex genomes. Realizing their full potential is critically reliant on extracting high-quality, high-molecular-weight (HMW) DNA from the organisms of interest. This is especially the case for the portable MinION sequencer which enables all laboratories to undertake their own genome sequencing projects, due to its low entry cost and minimal spatial footprint. One challenge of the MinION is that each group has to independently establish effective protocols for using the instrument, which can be time-consuming and costly. Here, we present a workflow and protocols that enabled us to establish MinION sequencing in our own laboratories, based on optimizing DNA extraction from a challenging plant tissue as a case study. Following the workflow illustrated, we were able to reliably and repeatedly obtain >6.5 Gb of long-read sequencing data with a mean read length of 13 kb and an N50 of 26 kb. Our protocols are open source and can be performed in any laboratory without special equipment. We also illustrate some more elaborate workflows which can increase mean and average read lengths if this is desired. We envision that our workflow for establishing MinION sequencing, including the illustration of potential pitfalls and suggestions of how to adapt it to other tissue types, will be useful to others who plan to establish long-read sequencing in their own laboratories.

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Section: Resultsmentioning

confidence: 99%

Harnessing the MinION: An example of how to establish long‐read sequencing in a laboratory using challenging plant tissue from Eucalyptus pauciflora

Schalamun

Nagar

Kainer

et al. 2018

Molecular Ecology Resources

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show abstract

“…The suitability of different preservation buffers remains to be investigated. However, we recommend that samples be cooled at the best possible, and that DNA extraction makes use of methods dedicated to the isolation of HMW DNA (Klingström, Bongcam‐Rudloff, & Pettersson, 2018). To this end, if conducted with great care (that is without vortexing and pipetting up and down to mix), classical phenol‐chloroform DNA extractions may yield DNA quality comparable to bead‐based approaches.…”

Section: Discussionmentioning

confidence: 99%

Linked‐read sequencing enables haplotype‐resolved resequencing at population scale

Lutgen

Ritter

Olsen

et al. 2020

Molecular Ecology Resources

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The feasibility to sequence entire genomes of virtually any organism provides unprecedented insights into the evolutionary history of populations and species. Nevertheless, many population genomic inferences – including the quantification and dating of admixture, introgression and demographic events, and inference of selective sweeps – are still limited by the lack of high‐quality haplotype information. The newest generation of sequencing technology now promises significant progress. To establish the feasibility of haplotype‐resolved genome resequencing at population scale, we investigated properties of linked‐read sequencing data of songbirds of the genus Oenanthe across a range of sequencing depths. Our results based on the comparison of downsampled (25×, 20×, 15×, 10×, 7×, and 5×) with high‐coverage data (46–68×) of seven bird genomes mapped to a reference suggest that phasing contiguities and accuracies adequate for most population genomic analyses can be reached already with moderate sequencing effort. At 15× coverage, phased haplotypes span about 90% of the genome assembly, with 50% and 90% of phased sequences located in phase blocks longer than 1.25–4.6 Mb (N50) and 0.27–0.72 Mb (N90). Phasing accuracy reaches beyond 99% starting from 15× coverage. Higher coverages yielded higher contiguities (up to about 7 Mb/1 Mb [N50/N90] at 25× coverage), but only marginally improved phasing accuracy. Phase block contiguity improved with input DNA molecule length; thus, higher‐quality DNA may help keeping sequencing costs at bay. In conclusion, even for organisms with gigabase‐sized genomes like birds, linked‐read sequencing at moderate depth opens an affordable avenue towards haplotype‐resolved genome resequencing at population scale.

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“…Reducing bead milling times or enzyme digestion temperatures are both possible with Mu-DNA to reduce DNA shearing depending upon user end requirements. Additional measures can be taken to reduce the effects of physical and enzymatic shearing of DNA during sample preparation, extraction and even handling (see Klingstrom et al 2018), yet these could become time-consuming for very large sample numbers.…”

Section: Extracted Dna Integrity and Molecular Weightmentioning

confidence: 99%

Mu-DNA: a modular universal DNA extraction method adaptable for a wide range of sample types

Sellers¹,

Muri²,

Gómez³

et al. 2018

MBMG

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Efficient DNA extraction is fundamental to molecular studies. However, commercial kits are expensive when a large number of samples need to be processed. Here we present a simple, modular and adaptable DNA extraction 'toolkit' for the isolation of high purity DNA from multiple sample types (modular universal DNA extraction method or Mu-DNA). We compare the performance of our method to that of widely used commercial kits across a range of soil, stool, tissue and water samples. Mu-DNA produced DNA extractions of similar or higher yield and purity to that of the commercial kits. As a proof of principle, we carried out replicate fish metabarcoding of aquatic eDNA extractions, which confirmed that the species detection efficiency of our method is similar to that of the most frequently used commercial kit. Our results demonstrate the reliability of Mu-DNA along with its modular adaptability to challenging sample types and sample collection methods. Mu-DNA can substantially reduce the costs and increase the scope of experiments in molecular studies.

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A comprehensive model of DNA fragmentation for the preservation of High Molecular Weight DNA

Cited by 20 publications

References 63 publications

Harnessing the MinION: An example of how to establish long‐read sequencing in a laboratory using challenging plant tissue from Eucalyptus pauciflora

Harnessing the MinION: An example of how to establish long‐read sequencing in a laboratory using challenging plant tissue from Eucalyptus pauciflora

Linked‐read sequencing enables haplotype‐resolved resequencing at population scale

Mu-DNA: a modular universal DNA extraction method adaptable for a wide range of sample types

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