RU2741886C1 - Method for determining influence profile in horizontal oil wells using microbiomic analysis - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention is intended for use in the oil industry to determine the flow profile in horizontal oil wells using microbiome analysis, which helps to determine the level of occurrence of oil-saturated formations. According to the method, samples of drill cuttings carried out from an oil well during drilling are taken. Samples of drilling mud are taken at the inlet and outlet of the well and the well fluid. Next, sample preparation is performed by isolating DNA from drill cuttings samples and well fluid samples. The intended target is amplified, the amplicons are purified, and the libraries are prepared for amplicon sequencing. Next, amplicon sequencing of the samples is performed. Bioinformatic analysis is performed using the QIIME2 software environment by filtering and purifying reads, denoising reads, assembling paired reads, removing chimeras, annotating reads. Then contamination should be eliminated with drilling fluid by subtracting microorganisms isolated from drilling fluid samples. Next, the inflow profile in the well is determined by comparing the well fluid microbiomes with the drill cuttings microbiomes and searching for common representatives in the samples with the determination of oil-saturated zones.

EFFECT: increase in accuracy of determining the flow profile in oil wells.

1 cl, 1 tbl, 1 dwg

Description

Technology area

The invention relates to biotechnology and is intended for use in the oil industry to determine the flow profile in horizontal oil wells using microbiome analysis, which helps to determine the level of occurrence of oil-saturated formations.

State of the art

From the prior art, a method is known for determining downhole fluid inflows (see [1] RF patent No. 2685600, IPC E21B 43/267, publ. 04/22/2019), including obtaining a fluorescent marker in the form of polymer microspheres with the preparation of a dispersion of resin and luminescent substances, combining the obtained marker with the carrier medium supplied into the well, introducing the marker with the indicated carrier medium into the well, sampling from the well and analyzing them with the determination of codes and marker concentrations in the well fluid samples using fluorometry, determination of downhole fluid inflows based on the results of these analyzes.

The disadvantages of this analogue are the significant costs for obtaining markers, the complexity of the process and the limited applicability of the method, since it works in wells where hydraulic fracturing (hydraulic fracturing) is carried out.

A method based on microbiomes for exploration and production of hydrocarbons is known from the prior art (see [2] WO2015103332, IPC E21B 43/22, G16B 10/00, publ. 09.07.2015), including: a) obtaining the first information about the microbiome at the time time ti from hydrocarbons produced from the well, the well having a first production zone and a second production zone; b) performing an assessment of the first information about the microbiome, the assessment includes: relationship-based processing having an associated genetic material component and an industrial environment component; and the bioinformatics stage; whereby a microbiome identifier is obtained for each production zone of the well at time t-i; c) obtaining information about the second microbiome at time t 2 from hydrocarbons produced from the well; d) performing an evaluation of the second microbiome information, the evaluation comprising: relationship-based processing having a related genetic material component and an industrial environment component; and the bioinformatics stage; whereby a second microbiome identifier is obtained for each production zone of the well at time t 2; and, e) comparing the first microbiome identifier and the second microbiome identifier; whereby any change in the amount of hydrocarbons produced in each zone is identified.

The prior art also knows a method based on microbiomes for the exploration and production of hydrocarbons (see [3] US2017139078, IPC G01V 11/00, G16B 10/00, publ. 05/18/2017), including access to derived microbiome data corresponding to one or multiple samples from the field, obtained microbiome data associated with the computational linkage of microbiome data and metadata related to one or more samples; comparing the obtained microbiome data with historical microbiome data corresponding to one or more additional samples from the area or corresponding additional derived microbiome data corresponding to one or more samples; generating forecast data; and displaying predictive information on a display device of the user to aid in resource management in the field.

The general disadvantages of these analogs are the high cost of technology and the lack of a method for quantifying the degree of inflow based on microbiome analysis data.

The essence of the invention

The technical problem facing the invention is to determine the level of occurrence of oil-saturated formations and the degree of inflow of oil-saturated formations along the wellbore.

The technical result of the claimed invention is to improve the accuracy of determining the flow profile in oil wells.

According to the invention, the technical problem is solved, and the technical result is achieved due to the method for determining the flow profile in horizontal oil wells, including:

• sampling of drill cuttings removed from an oil well during drilling;

• sampling of drilling mud at the inlet and outlet of the well;

• sampling of borehole fluid;

• then perform sample preparation by isolating DNA from samples of drill cuttings and well fluid samples, amplify the target target, clear amplicons, and prepare libraries for amplicon sequencing;

• then perform amplicon sequencing of the samples;

• then bioinformatic analysis is performed using the QIIME2 software environment by filtering and cleaning reads, denoising reads, assembling paired reads, removing chimeras, annotating reads;

• exclude contamination with drilling mud by subtracting microorganisms isolated from drilling mud samples;

• Next, the flow profile in the well is determined by comparing the well fluid microbiomes with the drill cuttings microbiomes and searching for common representatives in the samples with the determination of oil-saturated zones.

Implementation of the invention

FIG. 1 shows the general scheme for determining the oil-saturated intervals in the well, as well as the flow rate.

The claimed method for determining the flow profile can be called DNA logging technology, which consists in comparing the genetic data of bacteria isolated from fluid samples (well fluid) (i.e. crude oil) with the genetic data of bacteria isolated from the produced drill cuttings during the drilling of a well in oil fields to determine oil-saturated zones.

To carry out “DNA logging” in the process of drilling a well, samples of drill cuttings are taken along the entire section with different steps in depth (for example, 5-10 m), removed from an oil well during drilling, from which, using molecular biology methods, all total DNA contained in the sample.

Next, the target target is amplified (for example, the small subunit of 16S rRNA) and the sequencing libraries are prepared. The resulting genomic libraries are sequenced using second-generation sequencers and, using modern methods of microbiome analysis, the taxonomic composition of microorganisms inhabiting the sludge is determined, and the relative abundance of identified microorganisms is also determined. Comparison of fluid and sludge samples is based on similarities or differences in taxonomic composition and their relative abundance. Information on the taxonomic affiliation of microorganisms makes it possible to associate some of them with the presence of hydrocarbons in certain intervals of the well due to the participation of these microorganisms in the metabolism of hydrocarbons in oil-saturated deposits. Some of them are associated exclusively with the presence of hydrocarbons in the penetrated formations.

To exclude contamination, microorganisms isolated from the drilling fluid samples are subtracted in the analyzed samples. The main advantages of the developed technology are the absence of downhole interventions, which makes it possible to avoid accidents during the installation of equipment on the well; high measurement accuracy due to technical replicas, which makes it possible to reliably judge the results of “DNA logging”; efficiency in obtaining results and the possibility of long-term monitoring of the inflow profile and the absence of additional environmental impact on the environment during well operations.

The process of carrying out “DNA-logging” in a well consists of the following stages:

• sampling of drill cuttings removed from an oil well during drilling;

• sampling of drilling mud at the inlet and outlet of the well;

• sampling of well fluid (crude oil);

• transportation to the laboratory or preparation for on-site work;

• sample preparation (DNA isolation from sludge and oil samples; amplification of the target target; purification of amplicons; preparation of libraries for amplicon sequencing);

• sequencing (receiving data, demultiplexing data);

• bioinformatics analysis using the QIIME2 software environment, or packages for the R programming language, such as DADA2, phyloseq and vegan (filtering and cleaning reads; denoising reads; assembling paired reads; removing chimeras; annotating reads). Elimination of contamination with drilling fluid;

• Interpretation of DNA logging data. Identification of the most promising oil-saturated intervals in a well based on the similarity of microbiomes of cuttings and fluids using beta-diversity metrics.

On the basis of DNA logging data in directional or horizontal wells, it is proposed to construct the distribution of characteristic biomarkers in depth (in this case, microorganisms are biomarkers). When the well is put into operation, production samples are taken with the required time step, the samples of which are also sequenced. Production samples may include cuttings samples, drilling mud samples, well fluid samples. Statistical comparison and correlation of genetic sets of fluids and cuttings makes it possible to plot the dependence of the intensity of inflow to the wellbore on depth, as well as the nature of the inflow (oil / water). This study, in contrast to the field geophysical surveys (PLT) generally accepted in the industry and the use of chemical tracers, does not require additional intervention in technological processes, well interventions, production shutdowns, changes in the well design, injection of specialized proppant during hydraulic fracturing.

The following types of samples are taken to determine the flow profile from an oil well.

In the process of drilling a well, samples of cuttings (drill cuttings) are taken, including biological replicas at a distance of 5 and 10 meters. The collection of samples of drill cuttings was carried out in accordance with the penetration. Each sample was collected on so-called shaker screens. To determine the exact depth of each sample, the time delay of the cuttings emergence was determined. Each sample can be no more than 50 grams. Each sample should be appropriately marked for depth, mass and sampling time. Samples are collected in sterile plastic containers or sterile plastic bags. Samples are stored at a temperature not exceeding + 4 ° С.

Also, samples of well fluid containing oil at different stages of well operation are taken. The volume of each sample is no more than 70 ml. Well fluid samples were taken at the stage of development, sampling and operation.

To eliminate contamination, drilling fluid samples are taken during the drilling phase of the formation. Each sample volume does not exceed 100 ml and samples are collected at the inlet and outlet of the well. Samples at the outlet may contain a certain amount of sludge in the form of sediment.

Description of working with samples

The “DNeasy Powersoil kit” (Qiagen) is used to isolate total DNA from cuttings samples. Vinogradsky buffer is used to isolate samples from well fluid samples (Weid et al., 2008). After DNA isolation, PCR (polymerase chain reaction) is used to amplify target DNA regions, in our case, these are hypervariable regions of 16S rRNA using the NEB-Phusion master mix kit. PCR was performed using a Bio-rad T100 or Eppendorf personal cycler.

Primers:

16S-Next-F (TCG TCG GCA GCG TCA GAT GTG TAT AAG AGA CAG) CCT ACG GGN GGC WGC AG

16S-Next-R (GTC TCG TGG GCT CGG AGA TGT GTA TAA GAG ACA G) GA CTA CHV GGG TAT CTA ATC C

The adapter for the Illumina HiSeq2500 and MiSeq is shown in parentheses.

Ampure XP (Beckman) magnetic particles are used to remove byproducts in the amplicons. To determine the concentration of DNA, a kit for determining the concentration of DNA - Qubit dsDNA HS is used on a Qubit 3.0 spectrofluorite meter.

List of equipment required for the study:

• homogenizer Tissue Lyser II (Qiagen),

• tabletop centrifuges eppendorf mini spin,

• Bio-san vortex centrifuges,

• automatic Eppendorf pipettes (0.5-10 μl, 10-100 μl, 100-1000 μl),

• Eppendorf refrigerated centrifuge 5804R,

• thermocycler Bio-rad T100,

• thermocycler Eppendorf personal cycler,

• chamber for horizontal electrophoresis Bio-rad mini,

• Bio-rad current source (any for 300 volts),

• Qubit 1.0 and 2.0 spectrofluorimeters,

• Solid-state thermostat Termit (DNA technology) and Eppendorf thermomixer compact.

Implementation example

Isolation and purification of DNA from samples of cuttings (drill cuttings):

Collection of total DNA from downhole samples is carried out as follows in accordance with the DNeasy Powersoil kit protocol:

Under sterile conditions, 300 mg of drill cuttings are placed in a PowerBead tube containing destructive particles and a buffer that promotes homogenization, dissolves humic acids and protects nucleic acids from degradation. Then gently mix by shaking. Then add 60 μl of solution C1 and mix by inverting 4 times. Solution C1 contains sodium dodecyl sulfate and other detergents necessary for cell lysis. Power Bead tubes are placed in a Tissue Lyser II homogenizer rack (Qiagen, Germany), which is well secured with screws. For the most complete homogenization of samples and lysis of cells, homogenize for 10 minutes at a frequency of 30 Hz.

After homogenization, centrifuge the tubes for 1 minute at room temperature at 13000 rpm. Next, transfer 600 μl of the supernatant into a clean 2 ml tube. The supernatant may contain sludge particles, which is not critical at this stage. Add 250 μl of C2 solution to the supernatant and shake for 5 seconds and incubate for 5 minutes on ice. Solution C2 contains reagents that contribute to the deposition of destroyed cell walls, humic acids and proteins, which must be removed from the sample as they can inhibit the subsequent PCR reaction. The tubes are centrifuged for 1 minute at room temperature at 13000 rpm. Without touching the pellet, transfer 600 μl of the supernatant into a 2 ml tube; then add 200 µl of C3 solution to the supernatant and mix. Next, incubation takes place for 5 minutes on ice. At this stage, additional purification of the sample from substances of a non-nucleic nature occurs. The tubes are centrifuged for 1 minute at room temperature at 13000 rpm. Transfer 750 μl of supernatant to a clean 2 ml tube. Add 1.2 ml of C4 solution to the supernatant and vortex for 5 seconds.

A solution of concentrated salts in C4 helps the DNA to bind to the filter in the next step. Apply 675 μL of the mixture to the column (spin filter) and centrifuge for 1 minute at room temperature at 13000 rpm. At this stage, the DNA binds to the filter on the column and impurities pass through it. Liquid containing impurities is poured from the collection tube and an additional 675 μL of mixture is applied to the column. Centrifuged for 1 minute at room temperature at 13,000 rpm, after which the liquid is poured out. The remaining volume of the mixture is applied to the column. Centrifuge for 1 minute at room temperature at 13000 rpm, and the supernatant is decanted. Next, 500 μl of C5 solution is added and centrifuged for 45 seconds at room temperature at 13000 rpm. Solution C5 is a 70% ethanol wash solution that removes residual salts, humic acids, and other contaminants from the sample while the DNA remains bound to the filter. The liquid is removed from the bottom of the tube, and the washing is repeated as described above. The columns are centrifuged for another 1 minute at room temperature at 13,000 rpm. This is necessary to completely remove the residual C5 solution, which can inhibit the subsequent PCR reaction.

The filter column is placed in a clean 2 ml tube. Add 100 μl of C6 solution, preheated to 60 ° C, to the center of the membrane. Centrifuged for 1 minute at room temperature at 13000 rpm. In this step, a salt-free C6 solution (10 mM Tris-HCl, pH 8.0) removes the pure DNA from the filter. The concentration of the obtained DNA is determined, DNA storage is carried out at -20 ° C.

Isolation and purification of DNA from well fluid samples

Samples of oily fluid (10 ml) (well fluid) are mixed with an equal volume of Vinogradsky's buffer and incubated for 10 min at 80 ° C. The aqueous phase is removed with a sterile pipette. This procedure is repeated five times, collecting a total volume of 50 ml of the aqueous phase for each sample. The total volume of the aqueous phase for each sample is precipitated by centrifugation at 1200 × g for 20 minutes at 4 ° C. An aliquot (2 ml) of TE buffer (10 mM Tris; 1 mM EDTA) was added to the pellet and DNA extraction was carried out further as previously described. To exclude the possibility of bacterial contamination from the used reagents, buffer or sand, DNA extraction is additionally performed from an empty tube without a sample.

Amplification of 16S rRNA:

Mix the PCR mix in 0.2 ml Eppendorf on ice:

• sterile water - 7 μl,

• mix 2xPCR (NEB) - 10 μl,

• primer 1 - 1 μl (10 pmol),

• primer 2 - 1 μl;

• DNA of the genome - 1 μl.

The total volume of the PCR mixture is 20 μl. The amplification program is shown in Table 3.

Table 3. - Amplification program

Stage number Stage Temperature Time one Denaturation 95 ° C 2 minutes. 2 Denaturation 95 ° C 30 sec. 3 Annealing primers 58 ° C 30 sec 4 Synthesis of 2 DNA strands 72 ° C 40 sec five Steps 2 to 4 were repeated 24 times 6 Additional synthesis 72 ° C 5 minutes

Purification of 16S fragments (amplicons)

Add to PCR a mixture of magnetic particles AMPure XP Beads (Agencourt) in the presence of 1: 0.9 (18 μl per 20 μl PCR mixture). Incubated for 5 minutes at room temperature. Samples are placed on a magnetic stand and held until fully illuminated. The supernatant is removed, the particles are washed with 80% alcohol 2 times. The magnetic particles are air dried until the alcohol has completely evaporated. Add 15 μl of sterile water, mix with a pipette. The magnetic particles collect on the side of the tube facing the magnet until the supernatant is completely clear. Without affecting the particles, the supernatant is collected in a clean test tube. The concentration of the purified amplicons is measured on a Qubit series fluorometer according to the manufacturer's protocol.

Preparing libraries for sequencing

Sample preparation for sequencing was performed according to the Illumina recommended protocol (https://support.illumina.com/documents/documentation/chemistry_documentation/16s/16s-metagenomic-library-prep-guide-15044223-b.pdf) using the kit indexed primers Nextera XT index kit (Illumina).

Data analysis

The metadata file includes the linking of cuttings samples to the depth of its extraction, its type and reservoir. Oily fluid samples (wellbore fluid) are classified by sampling time. Drilling fluid samples are also classified by sampling time and by categories prior to entering the oil well and at the stage of exiting the oil well.

Most of the data analysis is performed by the QIIME2 program. The received reads (reads) are translated into the ".qza" format, which is suitable for QIIME2 using their internal script.

Converting reads to the ".qza" format, assessing the number and quality of read operations, and deleting reads with low quality is performed using QIIME2.

Trimming reads by quality, merging paired reads, correcting sequencing errors, removing chimmers and obtaining the final OTU table are performed using the DADA2 program (built into QIIME2). DADA2 clusters reads at a 100% similarity level.

OTUs contained in the drilling fluid before it enters the well are considered potential sources of contamination for cuttings samples. These OTUs are optionally subtracted from the cuttings samples, for example using the DECONTAM package for the R programming language.

To use further analysis of the similarity metrics of samples that take into account data on the phylogeny of OTU, multiple alignment of representative DNA sequences from different OTUs is constructed using the MAFFT program, on the basis of which a phylogenetic tree is constructed using the FastTree program (both programs can be included in the QIIME2 program).

To take into account the differences in the number of reads obtained between the samples, before evaluating the beta and / or alpha diversity, the number of reads in different samples is equalized by selecting a random subsample of reads of a fixed size from each sample, or the relative OTU content in each sample is used for analysis. The Shannon index is used to assess alpha diversity in samples.

The taxonomy is defined using a naive Bayesian classifier trained on the SILVA database.

During the analysis, the relative abundance of different OTUs and / or different taxonomic groups is determined in each sample of cuttings, and using the data on the depth of sampling of each sample, a profile of the representation of OTU and / or different taxonomic groups along the wellbore is built. The flow profile is constructed by assessing the similarity of OTUs and / or different taxonomic groups from samples obtained at different depths and samples of fluid coming from the well. The sample similarity metric can be any metric used to assess beta diversity, such as the Bray-Curtis index, weighted and unweighted UNIFRAC, or the number of OTUs present simultaneously in both samples. To reduce the effect of noisy data, it is advisable to compare simultaneously several fluid samples with several, for example, five, cuttings samples obtained from adjacent depths and calculate the average value of the similarity metrics between them. It is assumed that the higher the inflow rate from certain depths, the higher the level of similarity will be between the fluid samples obtained at the exit from the well and the cuttings obtained from these depths.

Additionally, when simultaneously comparing several samples of drill cuttings with fluid samples, it is possible to assess the significance of the obtained inflow profile using the method of multiple random mixing of data on the depth of obtaining samples of drilled rocks with the subsequent assessment of the flow profile on such mixed data, which makes it possible to estimate how often observed in real data, the level of similarity is also achieved with random mixing of samples.

Additionally, drill cuttings and fluid samples can be clustered. To do this, one of the above similarity metrics is calculated between all samples and the samples are clustered using hierarchical clustering methods and / or the data dimensionality is reduced by the method of principal coordinate analysis. It is assumed that the fluid samples will form clusters with the cuttings obtained from the depths that make the main contribution to the flow. Additionally, in this way it is possible to find clusters of drill cuttings with similar characteristics.

All scripts were written using open metagenomic data analysis protocols available on the Qiime and DADA2 websites.

Claims (9)

A method for determining the flow profile in horizontal oil wells, including: sampling of drill cuttings removed from an oil well during drilling; sampling of drilling mud at the inlet and outlet of the well; borehole fluid sampling; then, sample preparation is performed by isolating DNA from drill cuttings samples and well fluid samples, amplifying the target target, purifying amplicons and preparing libraries for amplicon sequencing; then perform amplicon sequencing of the samples; then bioinformatic analysis is performed using the QIIME2 software environment by filtering and cleaning reads, denoising reads, assembling paired reads, removing chimeras, annotating reads; exclude contamination with drilling fluid by subtracting microorganisms isolated from drilling fluid samples; then the flow profile in the well is determined by comparing the well fluid microbiomes with the drill cuttings microbiomes and searching for common representatives in the samples with the determination of oil-saturated zones.

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