Method for controlling pathogenic microorganism targeted sequencing data volume distribution

Abstract

The invention relates to a method for controlling pathogenic microorganism targeted sequencing data volume distribution, and belongs to the technical field of gene sequencing. According to the method, a pair of microorganism qPCR primers is used for evaluating the content of microorganisms in a nucleic acid sample during library building (sample processing time and labor are not increased additionally), the entering amount of each library during probe hybridization enrichment is determined according to a Ct value, and relatively uniform data volume distribution is obtained after probe hybridization capture enrichment and sequencing.

Description

Technical Field

[0001] The invention relates to a method for controlling the distribution of pathogenic microorganism targeted sequencing data, and belongs to the technical field of gene sequencing. Background Art

[0002] Targeted metagenomic sequencing (tNGS) technology is a cutting-edge molecular diagnostic method for pathogenic microorganisms. With its high sensitivity and wide detection range, it plays an increasingly significant clinical role in the diagnosis of infectious diseases. Compared with ordinary metagenomic sequencing (mNGS), tNGS can accurately identify and locate these potential pathogens when faced with trace pathogen sequences in clinical samples, providing more accurate diagnostic evidence for clinical workers.

[0003] The current technical paths of tNGS mainly include multiplex PCR and probe hybridization capture. The latter achieves efficient enrichment of pathogens by designing specific probes to complement and bind to pathogen targets in samples. Compared with multiplex PCR, probe hybridization capture can detect more pathogen targets in a single reaction, improving detection efficiency and accuracy.

[0004] However, the probe hybridization capture method also faces some challenges in practical applications. Due to the differences in the content of pathogenic microorganisms in clinical samples, different samples may show different enrichment effects during the multi-sample hybridization capture process. This leads to serious deviations in the amount of data between samples during the subsequent sequencing process. This deviation not only affects the accuracy of the sequencing results, but may also bring great difficulties to data analysis. Although the use of single-sample hybridization can avoid this problem, single-sample hybridization will multiply the sequencing cost and operation complexity of the sample.

[0005] So far, an effective method to completely control the data volume distribution between probe hybridization capture tNGS samples has not been found. This is mainly because the types and contents of pathogens vary greatly between samples, and the binding efficiency and stability of different pathogens and probes are also different. Therefore, how to ensure the balanced distribution of data volume between different samples when performing probe hybridization capture tNGS testing is still a technical problem that needs to be solved urgently. Summary of the invention

[0006] The present invention has developed a method for controlling the data volume distribution of targeted sequencing of pathogenic microorganisms. In this method, a pair of microbial qPCR primers is used to evaluate the content of microorganisms in nucleic acid samples during library construction (without adding additional sample processing time and labor), and the entry amount of each library during probe hybridization enrichment is determined according to the Ct value. After probe hybridization capture enrichment and sequencing, a relatively uniform data volume distribution is obtained.

[0007] A method for controlling the distribution of pathogenic microorganism targeted sequencing data, comprising the following steps:

[0008] Step 1: extract the nucleic acid of the sample and construct a sequencing library. At the same time or after constructing the sequencing library, use universal primers to perform qPCR quantification on the sample to obtain the Ct value in the quantitative process;

[0009] Step 2, according to the relationship between the Ct value and the nucleic acid content in the library, the sample input amount during sample mixing is adjusted to reduce the difference between the input amounts of the samples;

[0010] Step 3, performing hybridization capture on the mixed library obtained in step 2, and performing NGS sequencing;

[0011] The relationship between the Ct value and the nucleic acid content in the library is obtained through the following steps:

[0012] Step 2-1, sequence the enrichment pool containing 2-10 libraries, and then correct the sequencing data volume of each library sample according to the following formula:

[0013]

[0014] Where R i is the amount of sample data after correction, R i0 is the amount of data before correction, n is the total number of samples in the corresponding enrichment pool, k = 1, ... n, A is the expected amount of data;

[0015] Step 2-2, calculate the corrected nucleic acid entry amount in the corrected library according to the following formula:

[0016]

[0017] Among them, M i is the corrected nucleic acid entry amount M value, M i0 is the actual entry amount of the corresponding library;

[0018] Step 2-3, fitting the corrected nucleic acid entry amount M value and Ct value to obtain a relationship.

[0019] A takes 1-10M.

[0020] The universal primer is determined by the type of microorganism species to be detected in the sample.

[0021] The nucleotide sequence of the universal primer is shown in SEQ ID NO.1-2, or a sequence with the same function.

[0022] The sequences with the same function refer to sequences with at least 90% of their bases being identical.

[0023] The Ct value needs to be normalized.

[0024] The homogenization process is calculated by the following formula:

[0025]

[0026] Ct i is the Ct value of sample i after normalization, Ct i0 is the Ct value obtained by qPCR quantification, C i is the DNA concentration in sample i.

[0027] The methods of the present invention are used for non-therapeutic and diagnostic purposes.

[0028] A computer-readable medium records a program capable of running the above-mentioned method for controlling the distribution of pathogenic microorganism targeted sequencing data.

[0029] A system for controlling the distribution of pathogenic microorganism targeted sequencing data, comprising:

[0030] A nucleic acid extraction module, used to extract nucleic acid from a sample;

[0031] A library construction module, used to construct a sequencing library;

[0032] The qPCR module is used to perform qPCR quantification of samples using universal primers at the same time or after constructing the sequencing library to obtain the Ct value in the quantification process;

[0033] The sample input amount calculation module is used to adjust the sample input amount when mixing samples according to the relationship between the Ct value and the nucleic acid content in the library, so as to reduce the difference between the input amounts of the samples;

[0034] The sequencing module is used to perform hybridization capture on the obtained mixed library and perform NGS sequencing. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 It is an experimental flow chart of the present invention;

[0036] Figure 2 is a flow chart for constructing an entry volume and Ct value model using clinical samples;

[0037] Figure 3 It is the correlation analysis between Ct value and theoretical enrichment entry amount (M value);

[0038] Figure 4 It is a comparison of the data volume between equal quality entry enrichment and entry enrichment adjusted according to Ct value;

[0039] Figure 5It is a comparison of data volume distribution under two entry methods. DETAILED DESCRIPTION

[0040] The experimental process of the sample of the present invention is as shown in the attached Figure 1 As shown in the figure, while building the library, it is necessary to perform quantitative PCR sequencing on the pathogens in each nucleic acid sample to determine its Ct value, and determine the appropriate enrichment input amount based on the relationship between the Ct value and the input amount. Then, after each library is mixed and enriched, NGS sequencing is performed to obtain a more uniform data volume distribution.

[0041] Example 1: Constructing an input volume and Ct value model using clinical samples

[0042] 1. Sample information

[0043] The samples to be tested were nucleic acids extracted from 154 clinical samples, including 63 samples of alveolar lavage fluid, 48 samples of peripheral blood, 18 samples of cerebrospinal fluid, and a small amount of sputum, pleural effusion, tissue and other types of samples.

[0044] 2. Library construction

[0045] NGS library construction was performed using the Hieff NGS OnePot II DNA Library Prep Kit for MGI.

[0046] Take 5μL Smerase mix and add it to 25μL diluted 10ng nucleic acid sample, and run DNA fragmentation / end repair / add A reaction in the PCR instrument. The reaction program is 4℃1min; 20℃10min, 72℃20min. After the end, prepare the ligation reaction system, Fast T4 DNA Ligase 2.5μL, Ligation Enhancer 15μL, Adapter 2.5μL, and run it on the PCR instrument at 20℃15min. Purify the ligation product with magnetic beads. Take 10μL of the adapter ligation and purification product, add 2×UltimaAmplification Mix 12.5μL, Primer Mix 2.5μL, and run it on the PCR instrument at 98℃1min; 98℃15s, 60℃15s, 72℃30s, 15 cycles; 72℃5min. Purify the PCR product with magnetic beads and collect the purified product for use.

[0047] 3. Pathogen qPCR

[0048] The following primers were used for pathogen qPCR detection. The following qPCR primers are universal primers for common pathogens summarized by relevant existing technologies, which can cover major common pathogens, such as: Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, Acinetobacter baumannii, Mycobacterium tuberculosis, Klebsiella pneumoniae, etc.

[0049] SEQ ID NO. Sequence(5' to 3') 1 GTGCCNGCNGCNGCGGTAA 2 GGACTACNCGGGTNTCTAAT

[0050] qPCR used the SYBR Green dye method, and a single reaction system included 5 μL of PCRmix carrying SYBR Green dye, 4 μL of the above-mentioned PCR primers (working solution concentration was 5 μM), and 1 μL of extracted nucleic acid from the sample.

[0051] The reaction program was 95°C for 5 min; (95°C for 10 s, 60°C for 30 s) for 45 cycles.

[0052] 4. Probe hybridization capture

[0053] Probe enrichment was performed using the xGen Hybridization and Wash Kit (IDT) and a customized probe combination of 3,500 pathogens. During enrichment, the entry amount of all libraries was 100 ng, and all samples with less than 100 ng of library were enriched. Multiple sample hybridization was performed, and each enrichment included 2-10 samples, totaling 36 tNGS enriched libraries. The probe hybridization capture operation refers to the reagent instructions of the xGen Hybridization and Wash Kit (IDT). In order to improve timeliness, the hybridization capture time was reduced to 1 hour.

[0054] 5. Sequencing

[0055] The enriched library was sequenced on the MGI200 platform, and each sample was given 3M reads.

[0056] 6. Data fitting between data volume and Ct value

[0057] As attached Figure 2 As shown, the relationship between Ct value and library input amount under consistent data volume was fitted for 154 samples from 36 tNGS batches.

[0058] (1) Due to the difference in data volume between different sequencing batches, the data volume of each sample is first corrected according to the following formula. In this step, the amount of data obtained from each tNGS batch is different due to many factors such as library construction, nucleic acid content in the sample, amplification, etc., so the amount of data from each batch can be normalized to an expected value through this step. In the calculation process of this step, the data volume of each sample in the same enrichment pool is first averaged, and then normalized to the theoretical expected value, so that the data volume before correction can be adjusted to make it uniform.

[0059]

[0060] Where R i is the amount of sample data after correction, R i0 is the data volume before correction, n is the total number of samples in the corresponding enrichment pool, k=1,...n, and A is the expected data volume (3M reads can be taken in this embodiment).

[0061] (2) Further, the amount of data needs to be associated with the nucleic acid content in the sample, and the injection amount can be regulated and corrected by this method. The required amount of sample input is calculated based on the actual amount of sample input and the corrected data amount when the data amount of all samples in the enrichment pool is consistent (3M reads). The library input amount (hereinafter referred to as M value) when the data amount is consistent is calculated according to the following formula.

[0062]

[0063] Among them, M i is the corrected nucleic acid entry amount M value, M i0 is the actual entry amount of the corresponding library.

[0064] (3) Furthermore, it is necessary to construct the relationship between the Ct value and the actual input amount, and then calculate the sample input amount required for sequencing based on the obtained relationship. Since the library input amount is 10 ng and the pathogen qPCR input amount is 1 μL, the Ct value obtained by pathogen qPCR needs to be normalized. The specific calculation is according to the following formula.

[0065]

[0066] Among them, Ct i is the Ct value of sample i after normalization, Ct i0 is the Ct value obtained by qPCR quantification, C i is the DNA concentration in sample i.

[0067] (4) Fitting the normalized Ct value and log2 The relationship between the M values ​​is shown in the following table. Figure 3 As shown in the figure, the normalized Ct value and the corrected M value showed a negative correlation trend and had a high linear relationship. The final fitting result was the following formula.

[0068] log 2 M=0.1944×Ct+1.1494 (4)

[0069] Thus, the rule of determining the amount of sample enrichment input according to the Ct value is obtained. The above formula can be used to control the actual amount of each sample by combining the relationship between the Ct value and the amount of sample input. In subsequent embodiments, an equal amount of input can be adopted, or other proportions of the amount of addition can be adopted, which can be determined in combination with actual conditions.

[0070] Example 2 Verification of the method for determining the enrichment entry amount based on the Ct value

[0071] Libraries were constructed for 14 samples according to the method described in Example 1, and enrichment was performed using the method of equal quality input library and the method of adjusting the input amount of the library according to the Ct value.

[0072] When using the enrichment method to adjust the library entry amount based on the Ct value, it is necessary to perform pathogen qPCR detection on the nucleic acid while the sample library is being built. After the library is built, the Ct value of the sample is corrected according to formula (3), and then the enrichment entry amount M of each sample is determined according to formula (4).

[0073] The data volume distribution of the fourteen samples under two different methods is shown in the attached figure. Figure 4 and attached Figure 5 As shown in the figure, under the condition of equal quality entering the library, the maximum value of the sample data volume accounts for ~25.2% of the total data volume, the minimum value accounts for only ~0.2%, and the coefficient of variation is 118%, proving that there are indeed large differences in the data volume between samples. In comparison, when the input volume is adjusted according to the Ct value, the difference between samples is greatly reduced. On the one hand, this avoids the waste of data caused by excessive sequencing of samples with high pathogen content, and on the other hand, it provides more data for samples with low pathogen content, thereby improving the detection sensitivity.

Claims

1. A method for controlling the distribution of pathogenic microorganism targeted sequencing data, characterized in that: The steps include: Step 1: extract the nucleic acid of the sample and construct a sequencing library. At the same time or after constructing the sequencing library, use universal primers to perform qPCR quantification on the sample to obtain the Ct value in the quantitative process; Step 2, according to the relationship between the Ct value and the nucleic acid content in the library, the sample input amount during sample mixing is adjusted to reduce the difference in the input amount between samples; Step 3, performing hybridization capture on the mixed library obtained in step 2, and performing NGS sequencing; The relationship between the Ct value and the nucleic acid content in the library is obtained through the following steps: Step 2-1, sequence the enrichment pool containing 2-10 libraries, and then correct the sequencing data volume of each library sample according to the following formula: Where R i is the amount of sample data after correction, R i0 is the amount of data before correction, n is the total number of samples in the corresponding enrichment pool, k = 1, ... n, A is the expected amount of data; Step 2-2, calculate the corrected nucleic acid entry amount in the corrected library according to the following formula: Among them, M i is the corrected nucleic acid entry amount M value, M i0 is the actual entry amount of the corresponding library; Step 2-3, fitting the corrected nucleic acid entry amount M value and Ct value to obtain a relationship.

2. The method for controlling the distribution of pathogenic microorganism targeted sequencing data according to claim 1, characterized in that: A takes 1-10M.

3. The method for controlling the distribution of pathogenic microorganism targeted sequencing data according to claim 1, characterized in that: The universal primer is determined by the type of microorganism to be detected in the sample.

4. The method for controlling the distribution of pathogenic microorganism targeted sequencing data according to claim 1, characterized in that: The nucleotide sequence of the universal primer is shown in SEQ ID NO.1-2, or a sequence with the same function.

5. The method for controlling the distribution of pathogenic microorganism targeted sequencing data according to claim 1, characterized in that: The sequences with the same function refer to sequences with at least 90% of their bases being identical.

6. The method for controlling the distribution of pathogenic microorganism targeted sequencing data according to claim 1, characterized in that: The Ct value needs to be normalized.

7. The method for controlling the distribution of pathogenic microorganism targeted sequencing data according to claim 1, characterized in that: The homogenization process is calculated by the following formula: Ct i is the Ct value of sample i after normalization, Ct i0 is the Ct value obtained by qPCR quantification, C i is the DNA concentration in sample i.

8. The method for controlling the distribution of pathogenic microorganism targeted sequencing data according to claim 1, characterized in that: For non-therapeutic and diagnostic purposes.

9. A computer readable medium, characterized in that It records a program capable of running the method for controlling the distribution of pathogenic microorganism targeted sequencing data as described in any one of claims 1-8.

10. A system for controlling the distribution of pathogenic microorganism targeted sequencing data, characterized in that: include: A nucleic acid extraction module, used to extract nucleic acid from a sample; A library construction module, used to construct a sequencing library; The qPCR module is used to perform qPCR quantification of samples using universal primers at the same time or after constructing the sequencing library to obtain the Ct value in the quantification process; The sample input amount calculation module is used to adjust the sample input amount when mixing samples according to the relationship between the Ct value and the nucleic acid content in the library, so as to reduce the difference between the input amounts of the samples; The sequencing module is used to perform hybridization capture on the obtained mixed library and perform NGS sequencing.

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