Methods

HMW-DNA was isolated as per IrysPrep® Plant Tissue DNA Isolation Kit, a summary of which is displayed in Table 2. NGM followed schematic workflow shown in Figure 1.

Abstract

Isolating high-quality high molecular weight (HMW) DNA is key to next-generation mapping (NGM) and other genomic technologies that rely on HMW-DNA to conserve long range genomic information (Anfora et al., 2015). Isolation of HMW-DNA from plant tissue is especially challenging due to the tough cell wall and abundant secondary metabolites that can greatly affect DNA quality. The conventional liquid nitrogen grinding methods with homebrew reagents, used to isolate HMW-DNA from plant species for BAC/BIBAC library construction, usually take a minimum of 3 days to complete, require 20-30 grams of starting material and often result in variable quality DNA. We have developed the IrysPrep® Plant Tissue DNA Isolation Kit to enable successful NGM. It uses a robust and efficient modular workflow employing two alternate homogenization methods and four nuclei isolation strategies, to enable the isolation of high-quality HMW-DNA from a wide variety of plant species in just two days.

The IrysPrep® Plant Tissue DNA Isolation Kit provides a novel density gradient, and essential reagents to break the plant cell wall, lyse organelles and remove/neutralize secondary metabolites such as polyphenols and polysaccharides, while stabilizing and preserving intact nuclei. Microgram amounts of HMW-DNA has been recovered from cotton, soybean, strawberry, Eucalyptus, grapevine, peanut, oak, wheat, oat, tomato, maize, sunflower, brassica, coffee, etc., and successfully used for NGM (Table 1).

Conclusions The IrysPrep® Plant Tissue DNA Isolation Kit allows successful isolation of high-quality HMW-DNA from a wide range of plant species for next-generation mapping (NGM) applications and possibly other technologies that requires preservation of long range information. NGM opens the door for effective reference genome finishing and structural variation studies in complex plant genomes that are polyploid, highly repetitive and/or high heterozygosity.

See other BioNano posters: P0712 , P0957, P0961 and P0958.

References Anfora et al. (2015). Making platinum genomes feasible: purification of chromosomal-length plant DNA molecules. GEN Vol. 35, No. 14 Šimková, et al. (2003). Preparation of HMW DNA from plant nuclei and chromosomes isolated from root tips. Biologia Plantarum (3rd ed., Vol. 46, pp. 369-373). Olomouc. Zhang, et, al. (2012). Preparation of megabase-sized DNA from a variety of organisms using the nuclei method for advanced genomics research. Nature Protocols 7, 467–478

Plant Species Starting Amount

(g) Tissue Type Protocol Used

DNA Yield / 100 mg

Tissue (µg)

Nicking Enzyme / Nick Density / Label Density per

100 Kbp

Est. Genome Size (Mbp) / Assembled Size

(Mbp) / Depth of Coverage (x) / %

Coverage

Assembled Genome Map N50

(Mbp)

Maize B73 1.3 Fresh young leaves General 2.6 Nt.BspQI / 17.8 / 12.5 2,300 / 2,262/ 56x / 98% 2.1

Tomato 0.7 Fresh young leaves General 2.9 Nb.BssSI / 15.1 / 10.4 950 / 983 / 80x / 103% 1.1

Arabidopsis 1.0 Frozen Flower* General 1.5 Nb.BssSI / 28.9 / 19.8 135 / 135 / 100x /100% 1.2

Soybean* Blend 0.5 purify 0.1

Fresh young leaves

High Polysaccharides 10.5 Nt.BspQI / 17.0 / 10.1 1,100 / 1,032 / 150x / 94% 1.0

Diploid strawberry

Blend 0.5 purify 0.3

Fresh young leaves

High Polysaccharides 5.1 Nt.BspQI / 13.8 / 8.7 250 / 250 / 130x / 100% 2.5

Eucalyptus 1.0 Frozen young leaves* High Polyphenols 2.4 Nt.BspQI / 14.2 / 9.0 650 / 673 / 110x / 104% 1.4

AD1 genome cotton 0.5 Fresh young

leaves High Polyphenols 4.5 Nb.BssSI / 23.1 / 12.5 2,400 / 2,366 / 150x / 99% 0.9

AD2 genome cotton 0.5 Fresh young

leaves High Polyphenols 3.6 Nb.BssSI / 23.0 / 11.2 2,500 / 2,334 / 150 / 93% 1.1

Grapevine** 0.5 Fresh young leaves High Polyphenols 1.5 Nb.BssSI / 13.4 / 9.7 485 / 623 / 70x / 128% 0.4

A genome cotton***

Grind 0.5 purify 0.1

Frozen young leaves* LNG 4.8 Nb.BssSI / 23.1 / 12.7 1,700 / 1,653 / 100x / 97% 0.8

Soybean Grind 0.5 purify 0.1

Fresh young leaves LNG 11.8 Nb.BssSI / 17.0 / 10.5 950 / 1,018 / 116x / 93% 1.2

* Refer to Figure 1 for cotton A , and Figure 2 for soybean, genome assembly data metrics. ** Preliminary data. ***.

Table 2. Selected plant species used for next-generation mapping (NGM).

Results

Protocol Items General High

Polysaccharides High

Polyphenols Liquid Nitrogen

Grinding Tissue input amount 0.5 – 3.0 g 0.5 g 0.5 g 0.5 g

Tissue disruption Rotor stator Rotor stator Rotor stator Liquid nitrogen

Fix in formaldehyde / chop / blend Transfer 0.1-0.3g tissue eq. Filter nuclei / pellet based on genome size

Low speed spin Optional Four washes Optional

Density gradient nuclei purification

Plug lysis purification

Table 1. IrysPrep® Plant Tissue DNA Isolation Kit user guide summary.

IrysPrep kit extraction of long DNA molecules

IrysPrep reagents label DNA at specific sequence motifs

IrysChip linearizes DNA in NanoChannel arrays

Irys automates imaging of single molecules in

NanoChannel arrays

Molecules and labels detected in images by instrument

software

IrysView software assembles optical maps

(1) Long molecules of DNA are labeled with IrysPrep® reagents by (2) incorporation of fluorophore labeled nucleotides at a specific sequence motif throughout the genome. (3) The labeled genomic DNA is then linearized in the IrysChip® using NanoChannel arrays and single molecules are imaged by Irys. (4) Single molecule data are collected and detected automatically. (5) Molecules are labeled with a unique signature pattern that is uniquely identifiable and useful in assembly into genome maps. (6) Maps may be used in a variety of downstream analysis using IrysView® software.

1 2 3 4 5 6

Blood Cell Tissue Microbes

Free DNA Solution DNA in a Microchannel DNA in a Nanochannel

Gaussian Coil Partially Elongated Linearized

Free DNA Displaced Strand

Polymerase Nick Site Nickase Recognition

Motif

Position (kbp)

1

10

100

1000

0 5 10 15 20 25 30 35 40 45 50 55 60 65 70

%/Kb

Figure1.Singlemoleculesmetricsofindependentprepsofseveralco;onspeciesisolatedbyHighPolyphenolsprotocol

N50>20Kb

N50>150Kb

Labels/100Kb

FalsePosi8ve

FalseNega8ve

Mappingrates

a.HighPolyphenolsprotocol(prep1-35) b.LiquidNirtogenGrindingprotocol(prep36-70)

preps1

10

100

1000

0 5 10 15 20 25 30

%/Kb

Figure2.SinglemoleculesmetricsofindependentsoybeanprepsisolatedbyHighPolysaccharidesprotocol

N50>20Kb

N50>150Kb

label/100kb

FalsePosi8ve

FalseNega8ve

Mappingrates

preps

The IrysPrep® Plant Tissue DNA Isolation kit has been used to isolate dozens of high-quality HMW-DNA from a wide variety of plant species for successful NGM producing genome map N50 1.2± 0.6 Mbp at genome coverage depth ranging from 50x-150x. Selected plant species are listed in Table 2. Diploid strawberry produces a genome map N50 of 2.5 Mbp while grapevine genome map N50 is about 0.4 Mbp most likely due to genome complexity - nick site and fragile site uneven distribution, challenging repetitive elements architecture - requiring more depth of genome coverage. Figures 1 and 2 show representative single molecules metrics for multiple independent preparations of different cotton varieties and one soybean variety using the appropriate IrysPrep protocols. Performance is assessed by mapping single molecules to NGM maps generated on Irys System. A total of 146 preparations have been carried out on different cotton varieties using the two different isolation strategies - High Polyphenols protocol and Liquid Nitrogen Grinding protocols, with a subset of data shown in Figure 1. On average, cotton A genome DNA yield is 4.8± 2.1 µg per 100 mg young leaves (Table 2). Molecule N50 (>150 kbp) is over 230 kbp with median mapping rates of 85%. The assembled genome map is 1,653 Mbp covering >97% of estimated genome size with a genome map N50 >800 kb at 100x depth of genome coverage. Figure 2 shows representative data metrics for soybean preparations. DNA yield is 11.8± 5.4 µg per 100 mg young leaves The molecule N50 (>150 kbp is 249 kb with median mapping rates of 71%. The assembled soybean genome map is 1,032 Mbp covering ~94% of the estimated genome size with a genome map N50 >1.0 Mbp at 150x depth of coverage.

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Yang Zhang, J Shi and M Saghbini 1BioNano Genomics, Inc., San Diego, CA

High Molecular Weight DNA Isolation from Plant Tissue for Next-Generation Mapping Applications