Intact DNA purified from flow-sorted nuclei unlocks the potential of next-generation genome mapping and assembly in Solanum species

Kód výsledku v IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F61389030%3A_____%2F18%3A00499675" target="_blank" >RIV/61389030:_____/18:00499675 - isvavai.cz</a>

Výsledek na webu

<a href="http://dx.doi.org/10.1016/j.mex.2018.03.009" target="_blank" >http://dx.doi.org/10.1016/j.mex.2018.03.009</a>

DOI - Digital Object Identifier

<a href="http://dx.doi.org/10.1016/j.mex.2018.03.009" target="_blank" >10.1016/j.mex.2018.03.009</a>

Jazyk výsledku

angličtina

Název v původním jazyce

Intact DNA purified from flow-sorted nuclei unlocks the potential of next-generation genome mapping and assembly in Solanum species

Popis výsledku v původním jazyce

Next-generation genome mapping through nanochannels (Bionano optical mapping) of plant genomes brings genome assemblies to the 'nearly-finished' level for reliable and detailed gene annotations and assessment of structural variations. Despite the recent progress in its development, researchers face the technical challenges of obtaining sufficient high molecular weight (HMW) nuclear DNA due to cell walls which are difficult to disrupt and to the presence of cytoplasmic polyphenols and polysaccharides that co-precipitate or are covalently bound to DNA and might cause oxidation and/or affect the access of nicking enzymes to DNA, preventing downstream applications. Here we describe important improvements for obtaining HMW DNA that we tested on Solanum crops and wild relatives. The methods that we further elaborated and refined focus on nnImproving flexibility of using different tissues as source materials, like fast-growing root tips and young leaves from seedlings or in vitro plantlets. nnObtaining nuclei suspensions through either lab homogenizers or by chopping. nnIncreasing flow sorting efficiency using DAPI (4',6-diamidino-2-phenylindole) and PI (propidium iodide) DNA stains, with different lasers (UV or 488 nm) and sorting platforms such as the FACSAria and FACSVantage flow sorters, thus making it appropriate for more laboratories working on plant genomics. nnThe obtained nuclei are embedded into agarose plugs for processing and isolating uncontaminated HMW DNA, which is a prerequisite for nanochannel-based next-generation optical mapping strategies.

Název v anglickém jazyce

Intact DNA purified from flow-sorted nuclei unlocks the potential of next-generation genome mapping and assembly in Solanum species

Popis výsledku anglicky

Next-generation genome mapping through nanochannels (Bionano optical mapping) of plant genomes brings genome assemblies to the 'nearly-finished' level for reliable and detailed gene annotations and assessment of structural variations. Despite the recent progress in its development, researchers face the technical challenges of obtaining sufficient high molecular weight (HMW) nuclear DNA due to cell walls which are difficult to disrupt and to the presence of cytoplasmic polyphenols and polysaccharides that co-precipitate or are covalently bound to DNA and might cause oxidation and/or affect the access of nicking enzymes to DNA, preventing downstream applications. Here we describe important improvements for obtaining HMW DNA that we tested on Solanum crops and wild relatives. The methods that we further elaborated and refined focus on nnImproving flexibility of using different tissues as source materials, like fast-growing root tips and young leaves from seedlings or in vitro plantlets. nnObtaining nuclei suspensions through either lab homogenizers or by chopping. nnIncreasing flow sorting efficiency using DAPI (4',6-diamidino-2-phenylindole) and PI (propidium iodide) DNA stains, with different lasers (UV or 488 nm) and sorting platforms such as the FACSAria and FACSVantage flow sorters, thus making it appropriate for more laboratories working on plant genomics. nnThe obtained nuclei are embedded into agarose plugs for processing and isolating uncontaminated HMW DNA, which is a prerequisite for nanochannel-based next-generation optical mapping strategies.

Druh

J_imp - Článek v periodiku v databázi Web of Science

CEP obor

—

OECD FORD obor

10603 - Genetics and heredity (medical genetics to be 3)

Projekt

—

Návaznosti

I - Institucionalni podpora na dlouhodoby koncepcni rozvoj vyzkumne organizace

Rok uplatnění

2018

Kód důvěrnosti údajů

S - Úplné a pravdivé údaje o projektu nepodléhají ochraně podle zvláštních právních předpisů

Název periodika

MethodsX

ISSN

2215-0161

e-ISSN

—

Svazek periodika

5

Číslo periodika v rámci svazku

2018

Stát vydavatele periodika

NL - Nizozemsko

Počet stran výsledku

9

Strana od-do

328-336

Kód UT WoS článku

000454531500035

EID výsledku v databázi Scopus

2-s2.0-85046028003