Optimization of insect genomic DNA and total RNA extraction protocols for high fidelity gene sequencing

doi:10.21203/rs.3.pex-1928/v1

Method Article

Optimization of insect genomic DNA and total RNA extraction protocols for high fidelity gene sequencing

https://doi.org/10.21203/rs.3.pex-1928/v1

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There is a strong demand for sustainable, durable, safe, and specific solutions against agricultural pests due to breeding limitations and chemical control constraints. Biotechnological approaches to insect pests require phylogenetic information at the molecular level for RNAi application and other tailored strategies, such as insecticide resistance molecular markers. Sequencing technologies have advanced in resolution, speed, and cost-effectiveness to offer striking impact on genomics and transcriptomics. To provide deep and reliable data to high-throughput studies, state-of-the art technologies require high purification and integrity of the submitted samples. However, the limited number of existing protocols to obtain high mass nucleic acids from insects render difficult to obtain elevated purity and integrity standards. Species characteristics, as scales covered bodies of Lepidoptera and mining eating habits, hinder the quality of nucleic acid samples. Additionally, small size individuals are limited to yield sufficient amounts to robust base sequence coverage. The coffee leaf miner (CLM) Leucoptera coffeella is microlepdoptera insect responsible for severe leaf damage and huge yield losses to the coffee crop. Here we present simple and reproducible procedures we used to successfully obtain the whole-genome long-read PACBIO and Illumina (DNA) and transcriptome (RNA) of L. coffeella for de novo assembly. Therefore, we developed customized protocols applicable to insects to the extraction of genomic High Molecular Weight DNA and total RNA suitable for downstream high standard sequencing applications and other sensitive molecular analyses.

DNA

Illumina

insect

PacBio

RNA

sequencing.

Among the insects of economic importance are the miners, which feed on the mesophyll of agricultural species leaves. The most remote occurrence records of miner insects date back to the Triassic, the first mesozoic period, approximately 250 million years ago1. In this group, flies such as Liriomyza bryoniae (Tomato Leaf Miner) 2,3, beetles such as Taphrocerus cocois (Coconut Leaf Miner)4 and moths such as Leucoptera coffeella (Coffee Leaf Miner)5 are highlighted. They are phytophagous insects whose damage to the target host is related to the way they feed in the larval stage. In this case, the larva feeds on the region amidst the two epidermis of the leaves forming tunnels here called mines, causing necrosis and defoliation, which compromise the photosynthetic capacity of the plant, affecting its viability 6–8.

The phytosanitary department of the European Food Safe Authority (ESFA), published in 2020 a panel concerning species threats and among them there are mining insects 3. Although being economically relevant in several production chains, miner insects aren’t widely studied due to their cryptic habits9, which makes it difficult to obtain adequate samples for phylogenetic, epidemiological and phytosanitary studies. An initiative to sequencing 100 pest genomes has generated genomic data from arthropod pests of agricultural species. The selection of insects followed predetermined criteria. One of them was the existence of high-quality nucleic acid extraction methods, where the miner insects were not contemplated 10.

Our group has been dedicated to studying the Coffee Leaf Miner (CLM) (Leucoptera coffeella) (Guérin-Mèneville & Perrottet) (Lepidoptera: Lyonetiidae) from several approaches such as life cycle, morphological features and, more recently, the genome and transcriptome. CLM is an exclusive pest of coffee trees where it could cause losses up to 87% in yield 11,12. Coffee is grown by millions of producers in more than 60 countries, which reflects in a wide production chain, considered one of the most profitable in the world, which is threatened by pests such as CLM 13,14. The control of CLM with chemical, pesticides is necessary, however it entails disadvantages such as the selection of resistant populations, high costs of production and harmful effects on the environment and human health 8,15,16. For this reason, it is important to develop strategies for the implementation of integrated pest management systems (IPM), which balance the increase and quality of grain production and the protection of the environment 8,17.

In this way, the molecular characterization of CLM with the sequencing of its reference genome and transcriptome, are required for identification of genetic based tools. Moreover, those data will allow further studies such as the exploration of target genes for gene silencing via interfering RNA (RNAi) and molecular markers. These approaches will enable the development of biotechnological solutions aimed at controlling this important pest in coffee parks. The genomic sequencing technique chosen for this approach was PacBio technology, which allows the reading of long fragments, reaching up to 60–80 kb 18. However, genomic DNA (gDNA) samples of high molecular weight, which allows the reading of longer lengths were not obtained by methods already described due to the high chitin content in the structure of CLM and also to the feeding habits that impairs the extraction. These restrictions also limit obtaining high quality RNA samples. Here we present a new method of extraction of high quality and repeatable total gDNA and RNA, from different stages of development of CLM for Long-Read sequencing (PacBio) and MiSeq Illumina, respectively.

Kit E.Z.N.A insect DNA (Omega BioTek, Cat. No.: D0926-02)

Kit ReliaPrep RNA Tissue Miniprep System (Promega, Cat.No.: PRZ6111).

Proteinase K (Invitrogen, Cat. No.: AM2544)

Chloroform (Sigma-Aldrich, Cat. No.: C2432)

Isoamyl alcohol (Sigma-Aldrich, Cat. No.: W205702)

RNAse A (Invitrogen, Cat. No.: 12091021)

Ethyl alcohol, Pure (Sigma-Aldrich, Cat. No.: E7023)

TRI Reagent® (Invitrogen, Cat.No.: 15596026).

Isopropyl alcohol (Sigma-Aldrich, Cat. No.: I9516)

Incubator Shaker Ecotron (Infors HT)

Refrigerated Centrifuge (Eppendorf - 5415R)

MultiTemp®III (PharmaciaBiotech)

NanoDrop 2000/2000c Spectrophotometer (Thermo Fisher Scientific)

Qubit Fluorometric Quantification (Thermo Fisher Scientific)

Femto Pulse (Agilent)

Bioanalyzer 2100 system (Agilent)

Genomic DNA extraction

Extractions were performed from CLM pupae collected in Coffea arabica leaves during the dry and hot season, from April to September 2021, in the brazilian savanna region of the central plateau (experimental field of Embrapa Cerrados, Planaltina - DF, latitude: 15° 35' 30' 'S, longitude 47° 42' 30'' W).

The E.Z.N.A insect DNA kit (Omega BioTek, Cat. No.: D0926-02) was used to obtain the insect gDNA. However, considerable modifications of the protocol provided by the kit was required in order to obtain a higher amount and quality of gDNA extracted.

1- Cell lysis: The standard kit protocol provided low yield for our samples. Therefore, in order to increase the yield of the samples we suggest: macerate 20 pupae (around 10 mg) in 700 uL of lysis buffer (CTL) for 2 minutes in a Dounce tissue grinder (or homogenizer) instead at liquid nitrogen as recommended by the kit manufacturer. Transfer the lysate to a sterile 1.5 mL microcentrifuge tube with the aid of a 1 mL automatic pipette with a wide terminus tip.

2- Protein Digestion: add 100 mg of proteinase K (Invitrogen, Cat. No.: AM2544) to the tube containing the macerated tissue in CTL buffer, slowly invert 10 times and incubate for 16 hours at 37 °C under mild agitation (100 rpm) (Incubator Shaker Ecotron - Infors HT).

3- DNA extraction: add 700 μL of chloroform:isoamyl alcohol (24:1) to the microtube from the previous step and carefully invert 20 times. In order to avoid genome DNA shearing, the use of homogenizers like vortex are not recommended. Centrifuge at a 10,000 g speed in an Eppendorf® 5415R Microcentrifuge apparatus for 2 minutes, room temperature (RT). After centrifugation, transfer approximately 500uL of the aqueous phase (upper one) to a 2 mL fresh tube using a 1 mL automatic pipette with a wide terminus tip. Cellular debris deposited at the interface between the polar and apolar phases should be discarded.

4- RNAse digestion: add 0,06 ng of RNAse A (Invitrogen, Cat. No.: 12091021) to the microtube from the previous step containing the aqueous phase. Carefully invert 10 times and keep at 37 °C for 30 minutes. Add 500 μL of BL buffer and carefully invert the tube 20 times and incubate for 10 minutes 70 °C.

5- DNA Precipitation: add 1 μL of ethanol 100% to the microtube from the previous step and mix by inverting the tube slowly 20 times. In order to avoid genome DNA shearing the use of homogenizers like vortex are not recommended.

6- DNA Purification: for DNA binding, load 750 μL of the microtube content of the previous step on the top of a Mini Column HiBand DNA already placed in a collect tube of 2 mL (column and tube provided by the kit) with the aid of a 1 μL automatic pipette with a wide terminus tip. Centrifuge the set of column plus collect tube in a 15,000 g speed for 30 seconds and discard the flow-through and repeat this step until sample is over. Carry on the last centrifugation by 1 minute. Clean the DNA by adding 500 μL of HBC buffer. Centrifuge at a 15,000 g speed for 1 minute. Discard the flow-through and load 700 μL of wash buffer (provided by the kit). Repeat the centrifugation and cleaning for a best result. Place the column in a fresh microtube.

7- DNA elution: For DNA elution, centrifuge the column where the DNA was bound in a 15,000 g speed during 2 minutes to eliminate any trace of binding buffer and place in a fresh 1,5 mL microtube. Carefully load 15 uL of the elution buffer (provided by the kit) on the middle of the column membrane. Incubate at RT for 5 min, and after centrifuge at a 15,000 g speed for 30 s to collect the eluted DNA from the column. Repeat the elution step to maximum yield.

8- Quality control and storage: evaluate the eluted DNA for its concentration and quality in devices such as NanoDrop, Qubit, agarose gel electrophoresis and Femto Pulser. The samples must be stored at 4°C. If necessary, the samples can be frozen at -20°C, however the quality might be compromised.

The main steps of gDNA extraction are illustrated in Figure 1.

Total RNA extraction

The extractions were performed of 7 stages of development from CLM: 4 larval instars (L1, L2, L3 and L4) (Motta et al., 2021), pupae, males and females. TRIzol® method was used, followed by RNA cleanup with ReliaPrep RNA Tissue Miniprep System kit (Promega, Cat.No.: PRZ6111).

1- Cell lysis: with the aid of a mortar and pestle, macerate separately the different stages of development (approximately 20 μL of powder per tube) with liquid nitrogen. Dissolve the material in a sterile 1,5 mL microfuge tube containing 1 mL of TRIzol® Reagent (Invitrogen, Cat.No.: 15596026). Invert the tube carefully 10 times and incubate it for 5 minutes at RT.

2- Organic extraction: centrifuge the tube at 10,000 g speed (Refrigerated Centrifuge Eppendorf - 5415R) for 10 minutes at 4 °C for precipitation of impurities and other undissolved materials. Transfer the supernatant to a new sterile 1,5 mL microfuge tube containing 200 mL of chloroform. Agitate the tube vigorously by manual inversion for 15 s. Incubate for 3 minutes at RT and centrifuge at 10,000 g speed for 15 minutes at 4°C.

3- RNA precipitation: carefully transfer the aqueous phase (upper one) from the previous step to a new microtube containing 500 μL of isopropanol. Slowly invert the tube 10 times and incubate for 10 minutes at RT. After this, centrifuge RNA at 10,000 g speed for 10 minutes at 4 ºC to sediment and discard the supernatant.

4- RNA cleaning: after obtaining the precipitated, discard the supernatant and add 1 mL of ethanol 75%. Carefully invert 5 times the tube containing the pellet and centrifuge at 10,000 g for 5 minutes at 4 ºC. Discard the Ethanol and dry the tube containing the pellet at RT.

5- RNA purification with ReliaPrep RNA Tissue Miniprep System: dissolve the pellet in 250 μL of LBA/TG solution (supplied by the kit) and add 85 μL of isopropanol. Insert the sample into a ReliaPrep™ Mini Column (supplied by the kit) and centrifuge at 10,000 g for 1 minute at RT. Discard the flowthrough and add 500 μL of RNA Wash Solution on the top of the column. Centrifuge at 10,000 g for 30 s at RT. Meanwhile, prepare a DNAse mix containing 24 μL de Yellow Core Buffer + 3 μL MnCl2 + 3 μL DNAse (reagents supplied by the kit). Add the DNAse mix on the top of the column and centrifuge at 10,000 g for 15 minutes at RT. Add 200 μL of Column Wash solution and centrifuge at 10,000 g for 15 minutes at RT. Add 500 μL of RNA Wash Solution to the column and centrifuge at 10,000 g for 30 s at RT. Discard the flowthrough, add 300 μL of RNA Wash Solution to the column and centrifuge at 10,000 g for 2 minutes at RT. Transfer the column to a fresh microtube, add 15 μL of RNAse free water on the top of the column and centrifuge at 10,000 g for 1 minute at RT.

6- Quality control and storage: after extraction, evaluate the RNA concentration and quality in NanoDrop, Qubit and Bioanalyzer. RNA should be stored immediately in a -80°C freezer to prevent degradation.

The main steps of RNA extraction are illustrated in Figure 2.

DNA

Several features of insects such as high amount of chitin in its structure and feeding habits which bring contaminants to the samples disturb the obtaining of high-quality genetic material 19. Some described methods of gDNA extraction used in other insects19 have not been successfully applied to our goal, even those recommended by PacBio such as Anopheles coluzzii, Tribolium castaneum and Hyles vespertilio 18,20,21. After carrying out different tests of extractions of different stages of development (pupae, larval stages and adult), we concluded that pupae provided better quality DNA because they had less chitin in their structure and almost no content in their guts.

Key changes were performed in the E.Z.N.A insect DNA kit protocol: 1) the material was not macerated in liquid nitrogen. To promote cell lysis, pupae were macerated in Dounce tubes with CTL buffer (provided by the kit); 2) to avoid gDNA degradation, automatic pipette with a wide terminus tip were used for any manipulation of the sample; 3) The samples were not homogenized by vortexing. Instead, slow movements were performed by inversion; 4) During cell lysis and protein digestion, samples were incubated longer at a lower temperature and under gentle agitation to increase yield and reduce DNA degradation; 5) instead eluting DNA with pre-heated buffer, a higher integrity was revealed using RT e 6) the volume of the elution buffer was reduced to obtain more concentrated samples. These recommendations provided significant improvements for obtaining L. coffeella gDNA and may be useful for other insect species.

RNA

In general, the extraction of total RNA for sequencing presents methodological challenges to keep stability of the molecules. RNA is susceptible to degradation since the initial steps with cell lysis until final quantification 22. To avoid degradation and loss of transcriptome information, RNA extraction protocols suggest strict procedures during manipulation, such as the use of exclusive equipment for RNA and isolated and decontaminated areas of RNase 23.

The protocol established here provided for obtaining total RNA from the different developmental stages of L. coffeella. In addition to the common steps of conventional protocols, we added the step of purification of the samples extracted using a commercial extraction kit, as a differential of the methodology. In comparison to other RNA extraction methods, this protocol showed a higher degree of purity and concentration of the samples.

Although there are relevant ribosomal RNA (rRNA) profiles for various model organisms 24, there are still few recent studies that assess metrics and quality standards for non-model organisms 25. Most analyzes of insect RNA profiles document the “hidden denaturation” of the 28S subunit as an apparently degraded profile 26–29. However, with increased sequencing of different organisms for RNA investigations in recent years, more invertebrate rRNA profiles have been generated and suggest that denaturation of the 28S subunit is indeed very common 25,28,29.

DNA

The extraction of gDNA is performed in two days. The first consists of overnight cell lysis and digestion for approximately 16 hours. The second day, which includes the remaining stages, takes approximately 1 hour and 30 min.

RNA

Trizol® RNA extraction takes approximately 1 hour and 45 min. Purification of RNA with the commercial kit takes approximately 45 min.

DNA

We present a new, fast and reproducible methodology for obtaining high quality CLM gDNA for Long-Read (PacBio) and Illumina sequencing. The extracted gDNA samples showed concentrations, absorbance ratios (A260/A280 and A260/A230) and integrity recommended for Long-Read sequencing (PacBio). Evaluations of these parameters were conducted in NanoDrop and Qubit (Table 1), agarose gel (Figure 3) and Femto Pulse (Figure 4).

Total RNA

Samples extracted using this protocol were evaluated on NanoDrop and Qubit spectrophotometers and met the specific concentration and ratio criteria (A260/A280 and A260/A230) required for high-throughput transcriptome sequencing (Table 2). RNA integrity was confirmed by agarose gel electrophoresis (Figure 5) and Bioanalyzer (Figure 6), currently the two methods used to evaluate RNA integrity and degradation after extraction. The Bioanalyzer calculates the level of RNA integrity (RNA integrity number - RIN) from the distribution peaks of RNA fragments, which varies from 1 to 10. Values above 7 are considered high quality and suitable for sequencing ³⁰. Another way of assessing RNA integrity considers the size of the rRNA subunits: between 4400 and 5000 base pairs (bp) for the 28S subunit and 2000 bp for the 18S subunit ³¹.

The migration profile of the samples on non-denaturing agarose gel showed an rRNA band of approximately 850 bp (Figure 5) while the electropherogram generated by the Bioanalyzer device shows the presence of a rRNA band with approximately 2000 bp (Figure 6A). This discrepancy in the height of the RNA band can be explained by intramolecular interactions where RNA molecules can bend and change the secondary structure of the molecules, affecting migration in the non-denaturing agarose gel ³². More accurate size estimates can be obtained using denaturing gel electrophoresis in which hydrogen bonds are broken allowing the RNA to migrate as a single-stranded molecule ³².

At first these results were interpreted as a degradation profile, as traditionally the high quality RNA presentes the subunits 28S and 18S ³². However, a particularity is described for invertebrates, where the subunit 28S is highly prone to denaturation, resulting in two smaller fragments that overlaps with subunit 18S in a single band ^28,29,33, as observed here. Moreover, the distribution peaks of RNAr fragments showed a high RIN, as an indicator of the high quality of the samples (Figure 6B).

The protocol optimization of RNA extraction from CLM contributes to the attainment of biological material highly purified that allows the use of sequencing methods that generate more robust data. The methodology described here allows the surrender of significant results for the generation of knowledge in the areas of genome and transcriptome and can be useful in other molecular sequencing strategies, such as microRNAs, epigenomics, among others. In addition, molecular techniques of PCR, qPCR and others can benefit from these protocols.

More broadly, the application of the data generated from these protocols will enable the targeting of specific and sustainable insect control strategies, such as gene silencing via RNAi and the development of molecular markers of insecticide resistance. Is expected it will contribute to studies of insecticidal bio inputs of species harmful to plants for forest, ornamental, medicinal and agricultural use.

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The authors thank the researcher Adriano Veiga from Embrapa Cerrados, for providing access to the coffee plantations and for helping in insect collection and to Genomix Data, by the scientific advisory during the development of work. Work supported by the Consortium Brazilian Research and Development of Coffee - CBP & D Exchange / Coffee No. 10.18.20.004.00.00.

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Optimization of insect genomic DNA and total RNA extraction protocols for high fidelity gene sequencing

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Optimization of insect genomic DNA and total RNA extraction protocols for high fidelity gene sequencing

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