Optimized extraction of insect genomic DNA for long-read sequencing

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Alysha Monk

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Agricultural Research Service Center for Grain and Animal Health Research, United States Department of Agriculture, 1515 College Street, Manhattan, KS 66502

Agricultural Research Service of the United States Department of Agriculture American Meat Animal Research Center, Clay Center, Northeast United States 67432

The author to be contacted.

Method agreement. 2019 , 2 (4), 89 points; https://doi.org/10.3390/mps2040089

Received: October 24, 2019 / Revision: November 15, 2019 / Accepted: November 19, 2019 / Released: November 23, 2019

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abstract

Long-read sequencing technology continues to increase read length. At present, the average read length can be> 20 kb, up to 60-80 kb. The challenge now is to extract genomic DNA of sufficient fragment size and quality to support longer read lengths. We have developed a successful method to continuously obtain high-quality long genomic DNA from insects . It is determined that the best developmental stage of insects for genomic DNA extraction is the stage, which can eliminate DNA from ingested food and reduce the pollution of chitin substances that may interfere with the extraction. Improved results were obtained through improved procedures of commercial genomic DNA extraction kits. The soft p tissue of the red flour beetle Tribolium castaneum was originally destroyed in the lysis buffer of the kit using Teflon micronized powder. Modifications to the kit protocol also include gentle mixing by inverting the test tube instead of a harsh vortexing step, and the use of wide-bore pipette tips to transfer the fraction containing genomic DNA. Data from one sample is provided as an example of successful downstream library production and sequencing. Although the technology has been optimized for insects, using these improved procedures to extract from tissues of other organisms can also improve sequencing results for long-term reading.

Keywords: genomic the DNA ; long sequence ; Oxford nanopore ; the PacBio ; red beetles ; BOL quinoa

1 Introduction

Long-read sequencing is increasingly used in many biological applications, especially genome assembly. De novo assembly of complex genomes, especially those with highly repetitive sequences characteristic of many insect genomes, can be improved by incorporating long-read (> 10 kb) sequences. A solution that can separate long segments of genomic DNA while minimizing damage (such as DNA strand nicking) is needed to improve long-term read sequencing data.

Historically, the first report of DNA extraction was called "nucleoprotein" from the nucleus, which was described by Friedrich Miescher [ 1 ]. Since the report, there have been many arrangements of basic separation schemes. These schemes start with the use of surfactants or detergents to lyse cells, the removal of proteins by general proteases, and the removal of RNA by RNase. Protein, RNA and cell membrane lipids are removed by salt precipitation and centrifugation. It is also possible to separate DNA from the aforementioned contaminants by adding alcohol (ethanol or isopropanol), phenol/chloroform and/or binding to a solid phase (such as silica) and eluting by changing pH and salt concentration.

Blin and Stafford [ 2 ] proposed a method for isolating eukaryotic high-molecular-weight DNA, which involves homogenizing tissues in liquid nitrogen in Waring Blender and obtaining 200×10 6 Da (about 300,000) without gaps DNA. bp). Recently, another low-cost and rapid method of extracting genomic DNA from plant materials has been demonstrated [ 3 ]. However, this method also uses an initial process of grinding tissue in liquid nitrogen, and we found that this process is not always suitable for insects, because of the loss of limited materials and the destruction of genomic DNA.

Many commercial kits have been developed for the rapid and efficient isolation of genomic DNA. For insects, Chen et al. [ 4 ] evaluated five different time, efficacy and cost methods for isolating genomic DNA from western corn rootworm (a major corn pest). These methods include using SDS or CTAB, or using the kit DNAzol (Molecular Research Center, Cincinnati, Ohio, USA), Puregene (Gentra Systems, Minneapolis, Minnesota, USA) and DNeasy (Hilgen, Kilgen, Germany) ). Although all five methods can produce sufficient quantities of genomic DNA for the intended molecular application, SDS and CTAB extracts can produce more quantities with less degradation. The advantage of all kits is that they will not produce harmful phenol and chloroform waste, but the DNeasy kit has the shortest extraction time, and the Puregene kit has the least protein contamination. These researchers also found that using up to 8 times the volume of ethanol and 4°C can increase the amount of extracted DNA.

We have evaluated many commercial kits to improve methods for extracting genomic DNA from stored insects for long-term sequencing (data not shown). We have discovered a kit that can be used to extract high-quality long genomic DNA reproducibly, and here we prove our improved method with the common storage product harmful organism Tribolium castaneum (red flour beetle).

2. Experimental design

Genomic DNA extraction. Among the commercially available kits we evaluated, we found that the EZNA Insect DNA Kit (Omega BioTek, Norcross, Georgia, USA) provides the most consistent and high-quality method for extracting genomic DNA from insects. However, as described in Section 3 , the protocol accompanying the kit has been modified to provide higher quality and longer genomic DNA.

We did not follow the recommended liquid nitrogen pulverization program because we found that we lost sample quantity and quality (data not shown). Instead, use 10 male, female or mixed Castanea henryi T (approximately 30 mg) as starting material and grind in the kit lysis buffer. Protein is degraded by proteinase K; DNA is extracted with 24:1 chloroform:isoamyl alcohol and digested with RNAse to remove RNA. Isolate and purify the genomic DNA, and analyze the quality and quantity of a 1μL aliquot of the genomic DNA sample from cast wood T by digital nanophotometer and TapeStation .

The samples were transported on ice to the USDA ARS American Meat Research Center in Clay Center, Nebraska for library construction and PacBio Sequel I sequencing. The library was prepared using the SMRTbell template preparation kit 1.0-SPv3 according to the manufacturer's recommendations, using the 15 kb lower limit for insert size selection on BluePippin.

2.1. material

insect. Established a colony of Red piranhas in the Center for Food and Animal Health Research (CGAHR), Manhattan, KS, and kept feeding to maintain 95% wheat flour and 5% brewer’s yeast, 28°C feeding stage (larvae and adults) , Relative humidity 75%, completely dark. The colony used for genomic DNA extraction and genome sequencing is GA-2, which is the same as the colony used in the original genome sequencing project [ 5 ]. We extract genomic DNA from all stages of storing insects, and determined that the stage stage is the best choice for extracting high-quality long genomic DNA because of the lack of food contamination and chitin that may clog the column (data not shown). Therefore, early p (about 3 mg) was used for genomic DNA extraction.
EZNA insect DNA kit (Omega BioTek, Norcross, Georgia, USA; catalog number: D0926-02).
Sterile blue pellet pestle (Kimble Chase, obtained from Labsource, Northlake, Illinois, USA; catalog number: 749520-0000).
24:1 chloroform: isoamyl alcohol (Acros Organics, obtained from ThermoFisher Scientific, Waltham, Massachusetts, USA; catalog number: AC327155000).
200 and 1000 µL wide bore micropipette tips (ART TM, available through ThermoFisher Scientific).
200 molecular biology identification level of 100% ethanol (Decon Laboratory, King of Prussia, Pennsylvania, USA; catalog number: 3916).
SMRTbell template preparation kit (Pacific Biosciences, Menlo Park, California, USA, version 1.0-SPv3).

2.2. equipment

Shake the incubator (5436 Thermomixer, Eppendorf, Hauppauge, New York, USA).
Benchtop centrifuge (Sorvall Legend MicroCL 21R, ThermoFisher Scientific).
NP80 digital nanophotometer (Implen, Westlake Village, California, USA).
Tapestation 2200 model (Agilent, Santa Clara, California, USA).
PacBio Sequel I (Pacific Biosciences).
BluePippin (Sage Science, Beverly, Massachusetts, USA).

2.3. cost

The cost of samples that can be extracted ranges from $2.29 to $3.60 per sample, depending on the size of the kit. Other costs not included include consumables (pestle and needle tip) and 24:1 chloroform:isoamyl alcohol ($309.50 for a 500ml bottle).

3. Procedure

3.1. Destroy the organization (5 minutes)

Combine the tissue (less than 25 mg) with the 350μLCTL buffer in the EZNA insect DNA kit in a 1.5 mL sterile microcentrifuge tube, and grind by hand with a sterile blue sediment pestle for about 2 minutes.

3.2. Digest protein (1-12 hours)

Add 25 μL of proteinase K solution from the kit to the tissue in CTL buffer, gently mix by carefully inverting the tube 10 times, and incubate at 60°C for 1 h in a shaking incubator set to a low speed. Incubation overnight on an orbital mixer at room temperature can increase yield.

3.3. Extract DNA (10 minutes)

Add an equal volume (350 μL) of 24:1 chloroform:isoamyl alcohol to the sample. Instead of the recommended vortex, gently invert 20 times to mix thoroughly. Centrifuge in a benchtop centrifuge at 10,000× g for 2 minutes at room temperature . Carefully transfer the upper water layer to avoid using 100 µL wide-mouth pipette tips (approximately 250 µL each time) to form any milky precipitate on the interface at a time, and transfer 100 µL each time to a clean 1.5 mL microcentrifuge tube. Transferred).

3.4. RNase digestion (15 minutes)

Add a volume of BL buffer and 2 µL RNaseA from the kit. Do not vortex, but gently invert the sample 20 times and incubate at 70°C for 10 minutes.

3.5. Extract DNA (5 minutes)

Add a volume (500 µL in this example) of 100% ethanol to the sample to prove molecular biology grade. Do not vortex, but gently invert the sample 20 times. At this point, you may be able to see floating strands of translucent DNA.

3.6. Purified DNA (30 minutes)

Add 500 μL of the sample with a 1 mL wide-mouth pipette tip to the HiBand DNA Mini Column inserted into the 2 mL collection tube (all provided in the kit), and centrifuge at 15,000× g for 1 minute. Discard the filtrate and repeat with the remaining sample (500 μL in our case) and the same HiBand column.

After transferring all samples, transfer the column to the sterile 2.0 mL collection tube also provided in the kit, and add 500 μL of HBC buffer from the kit (make sure to dilute the HBC buffer with isopropanol according to the kit instructions) . Centrifuge at 15,000 × g for 1 minute. Discard the filtrate, and add 700 μL DNA Wash buffer (diluted with 100% ethanol according to the kit instructions) from the kit. Centrifuge at 15,000× g for 1 minute, then repeat the washing step once.

After the washing step, the column was rotated at a speed of 15,000× g for another 2 minutes to dry the column matrix. Transfer the column to a sterile 1.5 mL microcentrifuge tube, carefully add 50 μL of kit elution buffer (preheated to 70°C) to the center of the column membrane, and incubate at room temperature for 5 minutes. The genomic DNA is eluted from the column by spinning at 15,000 x g for 1 minute; repeat the elution process once. Evaluate the quality and quantity of genomic DNA and store it at room temperature for immediate use. If necessary, samples can be frozen at -80°C, but the length and quality of genomic DNA may be affected.

4. Results

The modified method using commercial kits successfully obtained genomic DNA from T grass . DNA has the best A 260 / A 280 and A 260 / A 230 ratios recommended for long-read sequencing ( Table 1 ). From the female Castanea henryi. The concentration of genomic DNA obtained was approximately twice the concentration of genomic DNA obtained from males. The genomic DNA extracted from T wood was measured by electrophoresis , and its peak length was greater than 50 kb ( Figure 1 ).

About 40 μg of genomic DNA extracted from sample B1 was used for library production and sequencing. The average insertion length of the library is 8731, and the number of insertions N 50 = 14,750 ( Figure 2 ). Sequel v2.1 chemical method was used for sequencing, and a total of 3,764,395 sub-reads were obtained from 30.0 Gb, with an average length of 7970 ( Figure 3 ).

5. Discussion

We have proposed a fast and reproducible method to obtain high-quality long genomic DNA from insect p. After extracting from different life stages of insects, we found that p provides the best quality sequence without food, and extraction using a column is less likely due to chitin material.

The program is easy to implement, it took more than two hours to complete, and there is no overnight incubation period. The main changes we made to the recommended kit protocol are:

Homogenize the tissue in CTL buffer with a pestle instead of grinding in liquid nitrogen;
Always use a wide-mouth pipette to move any samples containing genomic DNA;
Never vortex, always mix by gently inverting.

The kit includes a variety of methods to increase DNA yield, such as incubating the elution buffer on the chromatographic column for 5 minutes before elution, and then repeating the elution step. Both methods are used in our procedures. It is also recommended to increase the elution volume to more than 100 μL, which we did not choose to avoid diluting the sample.

There are some considerations when modifying protocols to increase yield and improve the quality of genomic DNA. It is important to grind the sample sufficiently to maximize the yield of genomic DNA, but we have found that grinding and transferring in liquid nitrogen can result in lower yield and sometimes lower quality. Manually grind the micronized powder in the lysis buffer for a few minutes to obtain higher quantity and quality of genomic DNA. Although the kit suggests that 4 hours of proteinase K incubation may be sufficient to remove contaminated proteins, we have found that overnight incubation can improve the absorbance ratio in quality assessment. However, violent shaking must be avoided during incubation. The use of wide-mouth pipette tips is essential for transferring genomic DNA to avoid shearing. If there is no wide hole tip, use a sterile razor to slice and widen the opening at the end of the tip. Another important step to avoid shearing DNA is to always gently invert the test tube and never vortex. According to the recommendations of the kit protocol, a new aliquot of ethanol must be used to fully precipitate the DNA. These suggestions provide an improvement for the successful purification of genomic DNA from T. castaneum for long-term reading and sequencing, and it has also been successfully used in other insect species.

Author contribution

Conceptualization, BO; Methodology, BO, AM, SS, TS; Formal Analysis, BO, AM, SS, TS; Investigation, BO, AM, SS, TS; Resources, BO; Data Management, BO and TS; Writing-Original Draft , BO; Writing-review and editing, BO, AM, SS, TS; visualization, BO; supervision, BO; project management, BO; fund acquisition, BO

funds

This research was internally funded by the U.S. Department of Agriculture ARS CRIS 3020-43000-032-00D, Ecology, Genomics, and Management of Stored Product Insects. No external funds were used.

Thanks

Thanks to Tom Morgan, Kristen Kuhn and Ken Friesen for their technical assistance. Mention of trade names or commercial products in this publication is only to provide specific information and does not imply a recommendation or endorsement by the USDA. USDA is an equal opportunity provider and employer.

Conflict of interest

The author declares no conflict of interest.