RU2785924C1 - Method for simultaneous nanopore sequencing of complete sequences of significant durum wheat genes - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to the field of biotechnology and plant breeding, namely to a method for simultaneous sequencing of the nucleotide sequences of a panel of genes underlying the traits of durum wheat important for the breeding process. This method includes the following steps: isolation of a high-molecular fraction of DNA from 4-day durum wheat seedlings (HMW DNA), enrichment of genomic DNA with high-molecular fragments; designing gRNAs specific to significant durum wheat genes and in vitro synthesis of gRNAs on a double-stranded DNA matrix molecule, assembly of Cas9/gRNA ribonucleoprotein complexes, Cas9-targeted nanopore sequencing, bioinformatic analysis of the data obtained: assembly and alignment on the genome.

EFFECT: expansion of the range of solutions for wheat gene sequencing.

1 cl, 9 dwg, 1 ex

Description

The technical field to which the invention belongs

SUBSTANCE: invention relates to the field of molecular biotechnology and plant breeding, namely to a method for simultaneous sequencing of nucleotide sequences of a panel of genes underlying durum wheat traits important for the breeding process.

Prior Art

Despite the constant improvement of sequencing methods, there is still a need for analyzes of representatives of most plant species important to the agricultural industry. Many plants that have been collected as reference genomes have often been chosen because they are homozygous and generally may not represent the full set of structural variations that are important for breeding (Lopez-Girona E. et al., 2020). The genomes of even closely related forms can often differ significantly, both in sequence and in structure, from the reference ones.

To solve this problem, the researchers focused on classical sequencing methods. These methods provide gene coverage but do not more accurately identify common genomic variations such as single nucleotide polymorphisms (SNPs) that are present in the genomes of other varieties and lines. The loss of various modifications occurs when the sequence is inaccurately assembled, which is more often due to the presence of repeats and mobile elements in the alleles, which is a problem when using short-read sequencing methods. Therefore, new methods for detecting allelic variations were required that could be applied to a wide range of crops (De Coster W et al., 2019).

At present, the most optimal approach in the study of gene sequences has been the use of Oxford Nanopore Technologies. However, in order to reduce sequencing costs and obtain high coverage data in regions of interest in the genome, a targeted sequencing method was developed (Gabrieli T. et al., 2018).

Recently, there has been a growing interest in enrichment in Cas9-mediated nanopore sequencing (NCATS) (Gilpatrick T. et al., 2020). The essence of the method is to create targeted sections of high-molecular-weight genomic DNA using the active ribonucleoprotein complex Cas9-gRNA (RNP) (Sternberg S. H. et al., 2014). The dephosphorylated DNA undergoes selective ligation of adapters to freshly cut regions created by the RNP complex. Synthesis of the Cas9-gRNA complex occurs via pre-designed gRNAs flanking the region of interest.

Such a selective sequencing method is important for solving practical problems in the study of plant genomes, as well as cost-effective for the identification of variants, especially considering long sequences with repeating regions. This approach allows for high coverage of the region of interest, as well as full de novo assembly, which is the best approach to fully characterize genetic diversity. Sequencing coverage of the region of interest is critical for studying patterns of mutation rates and methylation in different samples (Giesselmann P. et al., 2019).

The NCATS method provides dynamic capabilities for assessing genomic rearrangements, including large SVSs and hard-to-display repeating regions. Importantly, since nanopore sequencing examines the DNA strand rather than amplification sequencing, it is possible to simultaneously detect methylation at these loci, providing biological as well as diagnostic insight into the epigenome. This readout allows easy transition from methylation to different allele variations, which facilitates the study of specific epigenetic changes.

The essence of the invention

The objective of the invention is to develop a new method for simultaneous nanopore sequencing of complete sequences of significant durum wheat genes using Cas9/gRNA ribonucleoprotein complexes.

For the extraction of durum wheat genomic DNA by the CTAB method (Kirov I. et. al., 2021), four-day-old seedlings are used. The quality of the isolated genomic DNA is assessed by gel electrophoresis in 1.5% agarose gel at a voltage of 3V/cm. The concentration and purity of genomic DNA was assessed using NanoDrop One UV-Vis Spectrophotometer (Thermo Scientific, USA) and Quantus Fluorometer (Promega, USA). Only pure DNA is suitable for further studies (A260/A280 ~ 1.8 and A260/A230 ~ 2.0 according to NanoDrop data) with practically no differences in concentrations obtained using Nanodrop and Quantus devices.

Next, the genomic DNA is enriched with high molecular weight fragments using the Short Read Eliminator XL Kit (Circulomics, USA). The genomic DNA sample is brought to a total volume of 60 µl at a concentration of 150 ng/. Next, 60 µl of SRE XL buffer is added to the sample and centrifuged at 10,000 g for 30 minutes at room temperature. After removing the supernatant, the precipitate is washed with 200 μl of 70% ethanol, followed by centrifugation at 10,000 g for two minutes at room temperature. The resulting pellet is dissolved in 50 µl of EB buffer.

For gRNA design, the FlashFry program with the “discover” tool and reference genome sequences is used. All gRNA positions matched to the genome are searched. These gRNAs are then filtered out using the scoringMetrics function using a number of parameters, the main limiting factor of which is the minimization of off-target sequences. Next, the conservatism of gRNA positions among the genomes of different durum wheat varieties is assessed by mapping the selected gRNAs to known sequences of different durum wheat varieties. All gRNAs are designed to target homeologous genes located in both the A and B genomes.

gRNA synthesis is carried out on DNA templates by PCR of four partially overlapping oligonucleotides: a specific region of the target crRNA gene, a universal tppRNA that binds to the Cas-9 protein sequence, and two primers T7 F and T7 R, which are the promoter and terminator for T7 RNA polymerase used for in vitro transcription. Synthesis of DNA templates for each reaction is carried out in the following reaction mixture: 2 μl of a unique crRNA oligonucleotide (1 μM); 2 µl tracrRNA (1 µM); 2 µl forward T7 and 2 µl reverse primers (100 µM each); 2 µl 50x dNTP (10 µM); 10 µl 10x Encyclo buffer (Evrogen, Moscow, Russia); 1 µl encyclopolymerase (Evrogen, Moscow, Russia); 79 µl RNase-free water. Oligonucleotides are amplified according to the PCR program: initial denaturation at 98°C (2 min); 30 cycles of denaturation at 98°C (30 sec), annealing at 60°C (30 sec), elongation at 72°C (30 sec); final elongation at 72°C (1 min). Then in vitro transcription is carried out at 37°C for two hours using a kit for high-performance in vitro RNA synthesis (Bioloabmix, Novosibirsk, Russia) according to the manufacturer's protocol.

Preparation of the DNA library for Caz9-targeted nanopore sequencing on the MinlON device (Oxford Nanopore Technologies, UK) is carried out according to the protocols described in the article by Kirov I. et. al., 2021 (Fig. 1). First, the analyzed DNA is dephosphorylated by the Quick CIP enzyme (New England Biolabs, UK), then the dephosphorylated DNA is cleaved using the Cas9/rRNA ribonucleoprotein complexes. Then, protein adapters for sequencing ((Oxford Nanopore Technologies, UK) are ligated to the ends of the DNA free from the Cas9/rRNA ribonucleoprotein complexes (Oxford Nanopore Technologies, UK) using the SQK-LSK109 kit (Oxford Nanopore Technologies, UK). ), with flow cells R9.4.1 or R10.3.

Next, a bioinformatics analysis of the obtained data is carried out: assembly and alignment for the genome.

The proposed method shows the possibility of using nCATS sequencing to determine the photoperiod genes in durum wheat.

Brief description of the drawings

Figure 1. Library preparation for targeted nanopore sequencing.

Figure 2. DNA electrophoresis of durum wheat varieties. 1 - Koshelevskaya; 2 - Cyprida; 3 - Kermen; 4 - Golden; M - Step 100 molecular weight size marker (Biolabmix, Novosibirsk, Russia).

Figure 3. High molecular weight DNA concentration measurements on Qubit 4 and Opes Nanodrop.

Figure 4. The location of gRNA relative to the studied sequences of genes for vernalization and sensitivity to photoperiod.

Figure 5. Designed gRNA sequences.

Figure 6. Separation of individual gRNAs into 2 pools

Figure 7. Targeted sequencing of the VRN-A gene with nCATS technology

Figure 8. Targeted sequencing of the VRN-B gene using nCATS technology

Figure 9. Targeted sequencing of the Ppd-A gene using nCATS technology

Example 1

Determination of complete sequences of significant durum wheat genes: vernalization (VRN) (vernalization requirement) and photoperiod (Ppd-1) (photoperiod sensitivity) by simultaneous nanopore sequencing.

In many plants, in the course of evolution, mechanisms have been developed that determine the need for prolonged exposure to low temperatures (vernalization) for the subsequent transition to flowering. This is necessary so that the plants pass to the generative stage of development only at the right time of the year. Vernalization is genetically controlled by at least five loci, VRN-A1, VRN-B1, VRN-D1, VRN4 and VRN-B3. Wheat has three major vernalization genes VRN-A1, VRN-B1, and VRN-D1, which are located on homeologous chromosomes 5A, 5B, and 5D, respectively.

In cereals, including wheat, the central regulator of the photoperiodic response is the Ppd-1 gene, which in turn is represented by three homologous genes, Ppd-A1, Ppd-B1, Ppd-D1, located on the short arm of chromosome 2.

As an object, a variety of hard winter wheat was chosen: Koshelevskaya ((Originator / Patentee: selection of Koshelevskiy Posad LLC, now it is at the stage of registration and reproduction), the seeds were provided by the Kurchatov Genomic Center - VNIISB.

Seed germination was carried out in Petri dishes on moistened filter paper, in 2 replications, 10 seeds in each Petri dish. Healthy four-day-old seedlings were selected for the isolation of high-molecular-weight DNA. Isolation of genomic DNA was carried out in a fume hood using the CTAB method with the following modifications. The plant material was homogenized to a powder state using mortars and pestles with the addition of liquid nitrogen. To the resulting powder was immediately added preheated to 75°C lysis buffer (2% CTAB; 100mM Tris-HCl, pH=8.0; 20mM EDTA, pH=8.0; 1.4M NaCl, 0.25% PVP) in a proportion of 500 μl per 100 µg of plant material. The resulting mixture was transferred into 1.5 ml tubes with the addition of 30 μl of p-mercaptoethanol, then incubated at 75°C for 30 minutes, periodically turning the tubes over. After incubation, an equal volume of a mixture (500 μl) of chloroform:isoamyl alcohol (24:1) was added to the samples for deproteinization. After centrifugation at room temperature for 30 minutes at 13400 g, transfer the aqueous phase into new 1.5 ml tubes. Precipitation of the CTAB-DNA complex was performed by adding 2 volumes of precipitating buffer (1% CTAB; 50mM Tris-HCl, pH=8.0; 10mM EDTA, pH=8.0; 0.125% PVP) followed by centrifugation at room temperature for 30 minutes at 13400g. After removal of the supernatant, the resulting precipitate was dissolved in 200 μl of 1M NaCl. DNA precipitation was carried out with the addition of 200 μl of isopropanol and subsequent centrifugation at room temperature for 30 minutes at 13400 g. The obtained precipitate and the walls of the test tubes were washed by adding 500 µl of 75% ethanol to the precipitate and centrifuging at room temperature for 10 minutes at 13400 g. After the alcohol was removed to evaporate its residues, the tubes were kept open for 2 minutes. The pellet was dissolved in 100 µl of elution buffer (10 mM Tris-HCl, pH=8.0; 0.1 mM EDTA, pH=8.0) with 1 µl of RNase A (10 U/µl) added to the sample. Complete dissolution of the precipitate and elimination of contaminating RNA was achieved by incubation for 1 hour at 37°C. Next, purification from RNase A was carried out by adding an equal volume (100 μl) of a mixture of chloroform:isoamyl alcohol and subsequent centrifugation at room temperature for 15 minutes at 13400 g. The aqueous phase was transferred to new 1.5 ml tubes. DNA precipitation was carried out with the addition of 10 μl of 3M sodium acetate and 100 μl of isopropanol, followed by centrifugation at room temperature for 15 minutes at 10,000 g. The sediment and walls of the test tubes were washed by adding 500 µl of 80% ethanol and further centrifugation for 5 minutes at 10,000 g. The DNA was eluted in 50 μl of elution buffer.

The quality of the isolated genomic DNA was assessed by electrophoresis (Fig. 2). Separation of DNA molecules was performed by gel electrophoresis in 1.5% agarose gel at a voltage of 3V/cm.

Measurement of the concentration and purity of genomic DNA was performed using a Nanodrop Opes spectrophotometer and a Qubit 4 fluorimeter (Fig. 3).

Due to the minimal discrepancy between the indicators obtained using the Nanodrop Opes spectrophotometer and the Qubit 4 fluorometer, the genomic DNA of the durum wheat seedling of the Koshelevskaya variety is ideal for further research.

Enrichment in long fragments was performed using the Short Read Eliminator Kit (Circulomics, USA).

For the annotation of the studied genes in the durum wheat genome, their known sequences found from the literature sources of databases were used. These sequences were compared with a reference wheat genome taken from the GrainGenes database (Triticum turgidum subsp.durum cv. Kronos Earlham Inst, vl scaffolds) using the BLAST program.

The annotated sequence taken from the genomic browser was used for gRNA design (Fig. 4, Fig. 5). Guide RNAs (gRNAs) were obtained by in vitro transcription on a double-stranded DNA template molecule containing the T7 promoter sequence and obtained by combining two oligonucleotides, specific and universal. After the synthesis of ssRNA, the assembly of the ribonucleoprotein (RNP) complex was carried out, which is a complex of the RNA-binding protein Cas-9 with ribonucleic acid - ssRNA. The Cas9/crRNA/tracrRNA ribonucleoprotein complex is capable of recognizing DNA regions identical to 20 bp at the 5' end of crRNA and can cut both DNA strands with Cas9 nuclease to form blunt ends.

To increase the efficiency of sequencing, the gRNA complex was divided into 2 pools, each of which contained one pair of gRNA flanking the gene sequence (Fig. 6).

For transcription, we used the T7-transcription kit for high-performance in vitro RNA synthesis (Biolabmix, Novosibirsk, Russia) in accordance with the manufacturer's recommendations. Analysis of the concentration and quality of the obtained gRNAs was carried out using a Nanodrop OneC (Thermo Scientific, USA), Qubit 4 (Thermo Scientific, USA) spectrophotometer and separation in a 2% agarose gel. Prior to assembly of the RNP complex, gRNA was denatured to destroy secondary structures. To do this, individual gRNAs (50 ng) in each of the mixtures were combined in a molar ratio of 1:1 in a volume of 11 μl of RNase-free water, then the resulting mixtures were incubated at 95°C for 3 minutes and transferred to ice.

To assemble the RNP complex, 1 µl (20 pmol) of EnGen Spy dCas9 (New England Biolabs, UK) and 3 µl (5x) of NEB buffer (New England Biolabs, UK) were added to 11 µl of each gRNA mixture, and the resulting mixture was incubated for 30 minutes at room temperature.

Library preparation for targeted nanopore sequencing consists of the following steps:

1. Phosphatase dephosphorylation of the 5' ends of DNA fragments in the original sample. DNA was dephosphorylated by treatment with alkaline phosphatase (CIP). For this, 4 µl of CutSmart buffer (10x), 8 µl of Quick CIP were added to high molecular weight genomic DNA (concentration not less than 210 ng/µl) and incubated for 45 minutes at 37°C. Enzyme inactivation was carried out by heating the mixture to 80°C for 2 minutes.

2. Addition of Cas9/crRNA/tracrRNA ribonucleoprotein complexes, which bind to sequences at opposite ends of the studied genes, into the sample.

3. Excision of target genes with 5'-phosphate groups at the ends and adenylation with Taq DNA polymerase and dATP 3' ends. In this case, Cas9 proteins usually remain associated with non-target fragments that have been cleaved off from the target gene. To the dephosphorylated DNA was added 15 μl of the preassembled RNP complex, 1.5 μl of dATP (10 μM), and 1 μl of Taq polymerase. The mixture was incubated at 37°C for 30 min to make cuts in the DNA, then 5 min at 72°C to construct an A-tail at the cut sites.

4. Ligation of adapters with motor proteins. Since most of the ends of non-target fragments are "hidden" from ligase during dephosphorylation and Cas9 nucleases, adapters are generally ligated only with target fragments. Nanopore sequencing (AMPS) adapters were ligated to Cas9 digested DNA by mixing the following components: 25 µl LNB ligation buffer (LSK109 kit, Oxford Nanopore Technologies, UK), 5 µl nuclease water, 12.5 µl T4 DNA ligase (NEBnext Quick T4 Ligase) and 5 µl adapters (AMX). The mixture was added to the digested Cas9 DNA and incubated for 20 min at room temperature.

5. Purification on magnetic particles.

6. Apply the library to the flow cell and start sequencing. Sequencing was run on a MinlON flow cell (R9 Version).

Run sequences were performed using the MinKNOW software (version 19.2.2).

The final part of the sequencing process is the bioinformatic analysis of the obtained sequences. As a result, sequences of target gene sequences were determined. The enrichment of the allelic variant of the vernalization gene VRN-A is 84x (Fig. 7), the allelic variation of the vernalization gene belonging to the B genome -VRN B has 118x enrichment (Fig. 8). The photoperiod sensitivity gene Ppd-A has the largest coverage of all the genes studied in the study, which is 43 8x (Fig. 9).

The work on the creation of the present invention was financed from the funds of Agreement No. 075-15-2019-1667 dated October 31, 2019 on the provision of grants from the federal budget in the form of subsidies in accordance with paragraph 4 of Article 78.1 of the Budget Code of the Russian Federation for state support creation and development of the world-class center for genomic research "Kurchatov Genomic Center" within the framework of the federal project "Development of scientific and scientific-industrial cooperation" of the national project "Science".

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Claims (7)

A method for simultaneous nanopore sequencing of complete sequences of significant durum wheat genes, including: isolation of the high molecular weight fraction of durum wheat DNA (HMW DNA), enrichment of genomic DNA with high molecular weight fragments; design of gRNA specific for significant durum wheat genes and in vitro gRNA synthesis on a double-stranded DNA template molecule, assembly of ribonucleoprotein Cas9/gRNA complexes, Cas9-targeted nanopore sequencing, bioinformatics analysis of the obtained data - assembly and alignment for the genome.

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