CN115011668A

CN115011668A - Cell-free supernatant free DNA second-generation sequencing library building and detecting method for sputum specimen

Info

Publication number: CN115011668A
Application number: CN202210678914.8A
Authority: CN
Inventors: 王征; 刘东戈; 王逸菲; 李琳; 李晓光; 崔娣; 杨丽; 邸婧; 何磊; 孙铭君
Original assignee: Beijing Hospital
Current assignee: Beijing Hospital
Priority date: 2022-06-16
Filing date: 2022-06-16
Publication date: 2022-09-06

Abstract

The invention discloses a cell-free supernatant free DNA second-generation sequencing library building and detecting method for a sputum specimen. The second-generation sputum specimen cfDNA sequencing and library building method comprises the following steps of: 1) extraction and quality control: performing phlegm reduction treatment on the sputum sample, centrifuging, extracting cfDNA of the sputum sample from the supernatant, and performing concentration detection on the cfDNA; 2) constructing a library: and (3) constructing the cfDNA library of the sputum sample by using the gene second-generation sequencing detection kit to obtain the gene sequencing library of the cfDNA of the sputum sample. According to the invention, 83 sputum samples are collected, matched tumor samples are collected at the same time, NGS detection and off-machine data analysis are carried out, a library of NGS of cell-free supernatant cfDNA of the sputum samples is established, and an optimization scheme in the data analysis process before, during and after detection is provided.

Description

Cell-free supernatant free DNA second-generation sequencing library building and detecting method for sputum specimen

Technical Field

The detection technical scheme can be applied to detecting related driving gene changes by a next generation sequencing method (NGS) of cell free DNA (cfDNA) in a sputum sample of a non-small cell lung cancer (NSCLC) patient.

Background

In recent years, the position of targeted drugs in the clinical treatment of NSCLC tumors has been established, so-called "precision therapy". Different gene detection is needed when the targeted drug therapy is applied, and a treatment scheme is selected according to the detection result. After the tumor of a patient with drug resistance treated by the targeted drug progresses, gene change detection is still needed for guiding subsequent treatment. Therefore, national and foreign diagnostic and therapeutic guidelines for NSCLC recommend patients to routinely perform a set of detection of driver gene alterations, including EGFR, ALK, ROS1, BRAF, RET, Her-2, C-MET and other genes.

Traditionally, the specimen required for detecting the relevant driver gene is considered to be a tumor tissue or tumor cytology specimen. However, the NSCLC findings at 2/3 are all advanced, and some patients (especially elderly patients) cannot be tested by invasive tests using tumor specimens. The current liquid biopsy technology, namely, peripheral blood samples are utilized to detect gene changes, but the blood samples have high false negative rate of detection, and the samples cannot be screened by pathological evaluation before detection so as to improve the positive rate of detection.

Cytological specimens are one type of conventional pathological diagnosis specimens, and the value of tumor cell-driven gene detection in cytological specimens has been recognized by clinical diagnostic guidelines [1 ]. However, some cytological specimens have too low tumor content to perform the detection of the driver gene, but studies have confirmed that the detection of cfDNA in cell-free supernatant can be completed in these specimens [2 ]. The vast majority of the clinically collected sputum specimens are low-tumor-content specimens, and the method is suitable for performing gene change detection on cell-free supernatant cfDNA of the sputum specimens by applying a liquid biopsy technology.

The cell-free supernatant of the sputum specimen has more advantages than tumor tissue specimens and blood specimens, and 1) the sputum specimen is convenient to obtain and completely noninvasive; 2) the secretion generated in situ by the tumor has higher detection sensitivity than that of a blood sample; 3) anemia may occur in patients with advanced NSCLC, and the problem of anemia does not need to be considered when sputum samples are collected; 4) the detection sensitivity can be improved by the pathological evaluation of the sputum specimen before detection; 5) the sputum sample detection has no influence of blood sample clonic hematopoiesis on the detection result.

Data from previous studies showed that: the research of extracting cell-free supernatant cfDNA of a sputum specimen to carry out single gene detection (EGFR gene) finds that a plurality of differences exist between the detection of a group of genes (EGFR, KRAS, BRAF, PIK3CA, Her-2, ALK, ROS1, RET and C-MET) by applying an NGS method, and at present, there is no standardized detection scheme for NGS related to the cell-free supernatant cfDNA of the sputum specimen at home and abroad.

Disclosure of Invention

The invention aims to: provides a standardized detection scheme for detecting NGS related to the cfDNA of the sputum specimen so as to ensure the stability of the detection process and the accuracy of the detection result.

The invention provides a sputum specimen cfDNA second-generation sequencing (NGS) library building method, which comprises the following steps:

1) extraction and quality control: performing phlegm reduction treatment on the sputum specimen, centrifuging, extracting cfDNA of the sputum specimen from supernatant, and performing concentration detection on the cfDNA;

2) constructing a library: and constructing the cfDNA library of the sputum specimen by using the second-generation gene sequencing detection kit to obtain the gene sequencing library of the cfDNA of the sputum specimen.

The gene second-generation sequencing detection kit can be a Xiamen Ed 10 gene NGS detection kit (Xiamen Ed biological medicine, Inc.), and the specific steps for constructing the library can be carried out according to the kit instructions.

Wherein the method for the phlegm reduction treatment in the step 1) comprises the following steps: adding 1: sequentially adding 2ml of 0.5mol/L DTT into 2-3 volumes of normal saline until the phlegm is completely reduced; adding 1: 1 normal saline is added with 2ml of 0.5mol/L DTT in turn until complete phlegm reduction.

Wherein, after DTT solution is added each time, shaking is carried out for 5min, and then the phlegm-resolving effect is observed.

Wherein, the sputum sample of the step 1) is at least 3 ml.

Wherein the centrifugal condition is 1500g of rotation speed and 5min of time.

Wherein, the sputum specimen in the step 1) is preserved or transported in a stationary liquid before the sputum specimen is subjected to the phlegm reduction treatment, and the stationary liquid is 3) 50% ethanol + 2% polyethylene glycol 400 (a specific preparation method is as follows: preparing 98ml 50% ethanol, adding 2ml polyethylene glycol 400)

The invention also provides a second-generation sequencing detection method for the sputum specimen cfDNA, and the DNA sequencing library prepared by the library construction method is used for carrying out sequencing detection on the sputum specimen cfDNA.

Wherein the initial input amount of the cfDNA of the NGS constructed library sputum specimen is 150 ng.

The invention also provides a method for extracting the cfDNA of the sputum specimen, which is used for constructing a gene sequencing library of the second-generation sequenced cfDNA and comprises the following steps:

1) performing phlegm reduction treatment on the collected sputum specimen to obtain a sputum specimen completely reduced in phlegm;

2) centrifuging the sputum specimen completely reduced in phlegm at 1500g for 5 min;

3) and taking the centrifuged supernatant, and extracting cfDNA of the sputum specimen.

Wherein the method for the treatment of reducing phlegm comprises the following steps: adding 1: sequentially adding 2ml of 0.5mol/L DTT into 2-3 volumes of normal saline, and observing the phlegm-resolving effect until the phlegm is completely resolved; adding 1: after 1 physiological saline, 2ml of 0.5mol/L DTT is added in turn, and the effect of reducing phlegm is observed until the phlegm is completely reduced.

Before the phlegm reduction treatment, the sputum specimen is stored in a stationary liquid, the stationary liquid is a mixed liquid of 50% ethanol and polyethylene glycol 400, and the volume ratio of the 50% ethanol to the polyethylene glycol 400 is 49: 1.

The invention also provides a second-generation sputum specimen cfDNA sequencing detection kit, which comprises the DNA sequencing library prepared by the library construction method.

The initial input amount of the sputum specimen cfDNA when the kit is used is 150 ng.

The invention has the beneficial effects that: according to the invention, 83 sputum samples are collected, matched tumor samples are collected at the same time, NGS detection and off-line data analysis are carried out, a library of NGS of cell-free supernatant cfDNA of the sputum samples is established, and an optimization scheme in the data analysis process before, during and after detection is provided.

Drawings

Fig. 1 shows that there is a correlation between the concentration of cfDNA in sputum and the success rate of second-generation sequencing detection and the amount of collected sputum, and the amount of collected sputum is less than 3ml and the total amount of cfDNA extracted is less than the initial amount required for second-generation sequencing library construction. The triangular dots represent the second generation of sequencing failure cases.

FIG. 2 shows comparison of the concentration of cfDNA of sputum obtained by pre-and post-improvement sputum collection and treatment, with the mean concentration increased to 70.1ng/uL after improvement, and with a significant difference from the concentration obtained by the modified pre-method (p 0.047).

FIG. 3 is a scattergram of correlation between double-strand proofreading mutant read number and mutant abundance. The abscissa is the number of double-strand proofreading mutations and the ordinate is the mutation abundance. And displaying the corresponding mutation abundance of the sample with the double-chain proofreading mutation read number less than 10 by using a scatter diagram at the upper left corner, wherein the mutation can be detected when the double-chain proofreading mutation read is more than or equal to 1, and the lowest mutation abundance is 0.03%.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.

The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Definitions of terms involved in the present invention:

1) driver gene: the gene playing a key role in the occurrence and development of tumors, and the mutation of a driving gene is an important reason for the development or development of tumors, so that the process of normal cell malignant transformation is driven. Common driver genes in NSCLC patients are EGFR gene mutation, ALK gene fusion, ROS1 gene fusion, and the like.

2) Targeted therapy: the method is a treatment means for designing corresponding treatment medicines aiming at the change of a tumor driving gene or the change of a protein level, and the medicines enter a body and specifically select carcinogenic sites to combine and act so as to control the specific death of tumor cells and the development of tumors; 3) detection of gene alteration: detecting the change of a driving gene carried by a NSCLC patient, and aiming at being applied to clinical tumor gene targeted therapy;

4) liquid biopsy: performing gene change detection by using blood sample plasma, serum sample or non-blood sample (tumor cytology sample cell-free supernatant);

5) next Generation Sequencing (NGS) technique: is the mainstream detection method for detecting the driving gene change of NSCLC patients at present. Nucleic acid (mainly DNA at present) is extracted from a tumor patient specimen, a library is built by utilizing a hybrid capture technology or an amplicon method and the like, a second-generation sequencer is used for sequencing, and data after sequencing is analyzed to obtain a driving gene change result. The gene mutation detection involved in the present invention is the NGS technique.

The invention relates to a second-generation sequencing detection method for cell-free supernatant free DNA of a sputum specimen, which comprises the following steps:

1) NSCLC patient selection suitable for genetic alteration detection using sputum specimens: patients with advanced stage (IIIb-IV) NSCLC can naturally discharge sputum (without inducing sputum by atomization).

2) And (3) collecting sputum: the sputum sample of the NSCLC patient at the late stage (sputum sample is naturally removed without induction), the amount of the collected sputum sample is more than or equal to 3ml, the sputum is required to be placed in a fixed solution (50% ethanol + 2% polyethylene glycol 400) by a remote sample, and the sputum sample is sent to a pathology department or a clinical gene detection laboratory.

3) And (3) sputum specimen transportation: sputum specimens involving out-of-hospital (remote) transport are placed in a fixative (50% ethanol + 2% polyethylene glycol 400) and transported to a pathology department or a clinical gene testing laboratory.

4) And (3) phlegm reduction treatment: put the sputum sample that the volume is 1, 2, 3, 5, 6, 8, 10ml in 50ml centrifuging tube respectively, observe sputum sample property, glue thick type sputum sample and add 1: 2-3 volumes of normal saline, then 2ml of 0.5mol/L DTT is added (the prepared DTT solution is added when the phlegm reduction treatment is carried out, the normal saline and DTT mixed solution are prepared in advance), and the sputum sample is added with 1: 1, adding 2ml of 0.5mol/L DTT after the physiological saline; the remote fixed sputum specimen can be added with normal saline or directly added with 2ml of 0.5mol/L DTT according to the situation. Wherein, the thick sputum specimen is thick sputum specimen without fluidity; the water sample sputum specimen is a thin sputum specimen with fluidity.

5) Screwing a tube cover of the centrifugal tube, placing on a vortex oscillation instrument, oscillating for 5min, observing the phlegm reduction effect (whether the phlegm reduction is complete or not, and whether mucus substances which are not fully decomposed still exist, such as incompletely-reduced phlegm is added in turn by taking physiological saline with one volume and 2ml of 0.5mol/L DTT as units), and continuing the vortex oscillation process until the phlegm reduction is complete. (complete phlegm-resolving: a state in which the specimen is completely hydrated after the phlegm-resolving treatment and no visible components).

6) Centrifuging at 1500g for 5min at room temperature.

7) Collecting cell-free supernatant, transferring into another centrifuge tube, subpackaging and freezing in a refrigerator at-20 deg.C; the centrifugal tube bottom sediment is added with cell preservation solution for cytology pathological diagnosis after liquid-based cytology slides. Pathological diagnosis is divided into 3 cases, a finding cancer cells; phagocytes can be seen, and no cancer can be seen; c, deep sputum was not observed, and the next step was performed in the first 2 cases.

8) Extracting cf DNA: taking out 4ml of cell-free supernatant of the sputum specimen at-20 ℃, centrifuging at 13000g for 10 minutes at 4 ℃, and transferring the supernatant into another centrifuge tube for later use. Using a circulating DNA extraction kit (

Serum/plasma free DNA separation kit-centrifugal column type, Xiamen Edd biomedical science and technology, Inc.; record number: 20150006) extracting cfDNA in the cell-free supernatant specimen of the sputum, and the subsequent cfDNA extraction process is carried out according to the instruction.

9) Determination of the DNA concentration of sputum cf: the concentration and quantification of cf DNA are detected by using a Promega Quantus reagent and instrument or a Qubit method.

10) The NGS detection kit is used for the establishment and detection of the sputum cfDNA, wherein the NGS detection kit can be a human 10 gene mutation joint detection kit (reversible end termination sequencing method), Xiamen Ed biomedical science and technology Co., Ltd., registration number: national mechanical Standard 20183400507. Wherein, in the operation of detecting the sputum cfDNA NGS: the initial input amount of the sputum cfDNA is recommended to be 150ng, the interruption is needed before the NGS detection is carried out and the library is built, the reagents and the operation are used in the interruption process, the tumor genome DNA is recommended to be cut by enzyme, and the interruption process and the time are carried out according to the kit operation steps. And after interruption, constructing a pre-library and a final library according to the selected NGS detection kit, and carrying out detection operation steps according to the kit instructions.

11) Analyzing data after detecting the sputum cfDNA NGS: the positive interpretation standard of the hot spot mutation of the driving gene of the sputum cfDNA is as follows: the mean original depth (MeanDepth) should be greater than 10000 ×, the mean effective depth (DSBCDepth) should be greater than 500 ×, the DSBC supported reads number is at least 1 pair, and the allele frequency (MAF) is 0.03%. (Positive mutation indicating or candidate for NSCLC)

Example 1: extraction of specimen before DNA detection of sputum supernatant cf

Selecting NSCLC patients suitable for genetic alteration detection using sputum specimens: selecting a patient with advanced NSCLC and simultaneously discharging sputum naturally to collect the sputum; the extraction of the sample before DNA detection from the supernatant cf was carried out as follows.

1) And (3) collecting sputum: the sputum specimen of the NSCLC patient in the late stage is collected respectively (the sputum specimen is naturally removed without induction), the amount of the collected sputum specimen is more than or equal to 3ml, the sputum of the remote specimen needs to be fixed by 50% ethanol and 2% polyethylene glycol, and the sputum specimen is sent to a pathology department or a clinical gene detection laboratory after reaching a sufficient amount.

2) And (3) phlegm reduction treatment: put the sputum sample that the volume is 1, 2, 3, 5, 6, 8, 10ml in 50ml centrifuging tube respectively, observe sputum sample property, glue thick type sputum sample and add 1: 2-3 volumes of normal saline, then 2ml of 0.5mol/L DTT is added (the prepared DTT solution is added when the phlegm reduction treatment is carried out, the normal saline and DTT mixed solution are prepared in advance), and the sputum sample is added with 1: 1, adding 2ml of 0.5mol/L DTT after the physiological saline; the remote fixed sputum specimen can be added with normal saline or directly added with 2ml of 0.5mol/L DTT according to the situation.

3) Screwing a tube cover of the centrifugal tube, placing on a vortex oscillation instrument, oscillating for 5min, observing the phlegm reduction effect (whether the phlegm reduction is complete or not, and whether mucus substances which are not fully decomposed still exist, such as incompletely-reduced phlegm is added in turn by taking physiological saline with one volume and 2ml of 0.5mol/L DTT as units), and continuing the vortex oscillation process until the phlegm reduction is complete.

4) Centrifuging at 1500g for 5min at room temperature.

5) Collecting cell-free supernatant, transferring into another centrifuge tube, subpackaging and freezing at-20 deg.C in refrigerator; the sediment at the bottom of the centrifugal tube is added with cell preservation solution for cytological pathological diagnosis after liquid-based cytology slides. Pathological diagnosis is divided into 3 cases, a finding cancer cells; phagocytes can be seen, and no cancer can be seen; c, deep sputum was not observed, and the next step was performed in the first 2 cases.

6) Extracting cf DNA: taking out 4ml of cell-free supernatant of the sputum specimen from-20 ℃, centrifuging at 13000g for 10 minutes, and transferring the supernatant into another centrifuge tube for later use at 4 ℃. The kit for extracting the cfDNA of the plasma specimen is applied (the specific type is the same as the above) to extract the cfDNA in the cell-free sputum supernatant specimen, and the subsequent cfDNA extraction process is carried out according to the instruction.

7) Determination of the DNA concentration of sputum cf: the concentration and quantification of cf DNA are detected by using a Promega Quantus reagent and instrument or a Qubit method.

8) And (3) performing NGS detection on the obtained cf DNA by using an NGS detection kit. The results are shown in FIG. 1.

As can be seen from FIG. 1, the amount of cfDNA extracted from 1ml sputum specimen is mostly lower than 150ng, which causes low success rate of NGS detection. The obtained cfDNA amount is obvious when the sputum volume is more than or equal to 3ml and less than 3ml (1.2 ng/mu L and 75.1 ng/mu L, p is 0.006), the sputum volume is more than or equal to 3ml, the NGS detection success rate of the sample can reach 100%, and the NGS detection success rate of the sputum volume less than 3ml is 28.6% (the triangle points represent sequencing failure cases).

In this embodiment, the unfixed sputum specimen may cause pollution and specimen deterioration during transportation, but the previous research results show that the use of methanol fixation may cause crosslinking of cfDNA in the sputum supernatant, which affects the concentration of extracted nucleic acids. The use of freezing conditions to transport specimens increases transportation costs and may not guarantee freezing results. Meanwhile, the evaluation concentration of the cfDNA extracted from the sputum supernatant after the fixing by using the 50% ethanol and 2% polyethylene glycol 400 fixing agent is 42.4 ng/mu l, and the evaluation concentration has no significant difference with the statistical analysis of the cfDNA concentration obtained by the sputum reducing treatment of the normal saline, which shows that the cell morphology in the sputum specimen is not only preserved by using the fixing agent 50% ethanol and 2% polyethylene glycol 400, but also the extracted cfDNA concentration has no significant difference with the cfDNA concentration obtained by the sputum reducing treatment of the normal saline under the condition of the same volume.

Example 2

The amount of Sputum specimen was set to 3ml or more, and the Sputum supernatant cfDNA was performed according to the procedures 1 to 7 in the examples and the methods in the literature (WangZheng, et al; Sputum Cell-Free DNA valuable surface Sample for Detection of EGFR Mutation in Patents with Ad variant Lung Adenoccanconism, Journal of Molecular Diagnostics,2020,22(7):934-942), respectively, and the results are shown in FIG. 2.

The test results in fig. 2 show that the water sample sputum specimen 1: 1 adding sputum to make sputum specimen 1: 2-3 adding the sputum sample to completely dissolve the sputum sample. The method for treating the sputum is modified by increasing the amount of the sputum (more than or equal to 3ml), so that the concentration of cfDNA is increased to 70.1ng/uL, and a significant difference (p is 0.047) exists between the cfDNA amount obtained by modifying the treatment scheme (as shown in figure 2)

Example 3

The construction and detection of NGS were performed according to the following procedure using different initial input amounts of cfDNA (50ng, 100ng, and 150ng), respectively:

1) the sputum cfDNA needs to be interrupted before the NGS detection is applied to the construction of a reservoir. After extracting the sputum cfDNA, analyzing the distribution of the sputum cfDNA fragments by using a high-sensitivity chip of the Aglient 2100Bioanalyzer, and finding that the sputum cfDNA fragments are from 100bp to 10380bp, and the main peak is positioned near 1500 bp. The result shows that genome DNA components exist in the sputum cfDNA, and the characteristics of the method are different from those of the plasma cfDNA (the plasma cfDNA fragment is about 160 bp), so that the method for constructing the library of the plasma cfDNA is not completely suitable for the sputum cfDNA, the cfDNA fragment breaking is required before constructing the library, and the breaking method is the same as that of the tumor specimen DNA (the enzyme cutting breaking method is recommended). The specific operation steps of the enzyme digestion interruption method are as follows: and (3) carrying out fragmentation treatment by an enzyme cutting method: a total amount of 150ng of sputum cfDNA sample with a volume of 35ul was taken and put into a 200ul EP tube, and 5ul of fragmentation buffer and 10ul of fragmentation enzyme were added to perform an enzyme digestion reaction on a PCR instrument. After the reaction is finished, purifying by using a certain proportion of purified magnetic beads (1.1X, 55ul of magnetic beads, and 50ul of fragmented products), wherein the purified products are fragmented DNA, and the total amount is not less than 30 ng.

2) The library construction method adopts a hybridization capture method. After interruption, the sputum cfDNA was purified and eluted at 23uL with a concentration of 3-5 ng/uL. Constructing a pre-library of all cfDNA, including end repairing, adaptor connection, purification, library amplification and re-purification, measuring the concentration of the pre-library (approximate concentration is 100ng/uL, and 23uL elution), wherein the amount of the pre-library is about 2ug, taking 1ug, mixing samples (1-4 samples, one system and 1ug-4ug), concentrating, dissolving 15uL in concentrated dry powder (5uL probe +10uL hybridization buffer), carrying out hybridization capture (one system is added with 10uL capture magnetic beads), and carrying out overnight hybridization at 48 ℃. And washing at 48 ℃ for 45min (adding washing buffer, shaking, placing a magnetic frame, washing for 6 times, and gradually reducing the concentration). Carrying out amplification and purification with magnetic beads, and constructing a final library. Sputum cfDNA captured about 200bp (same as tumor genomic DNA) for the fragment of interest (insert) and about 160bp for the plasma insert.

The quality control data after sequencing among the three groups with the initial cfDNA input amount of 50ng, 100ng and 150ng respectively in this example respectively show that: the average target area coverage (the mean on-target coverage rate) is 97.1%, 100%, 100% respectively; the average insert sizes (lengths) are respectively 210.5bp, 216.2bp and 204.9 bp; the sequencing depths are 22249.9X,26775.0X and 32675.1X respectively; the average effective double-stranded base pair calibration (DSBC) depth is 831.0X,1487.4X and 2177.1X respectively, most importantly, the original sequencing depth of all samples in 150ng group is more than 10000X in quality control evaluation, and the DSBC sequencing depth is more than 500X. The results of plotting the reads number and the MAF value (minimum allele frequency) of the off-line sequencing data of NGS are shown in fig. 3, which is a scatter plot of correlation between the read number of double-strand proofreading mutations and the abundance of mutations. The abscissa is the number of double-strand proofreading mutations and the ordinate is the mutation abundance. And displaying the corresponding mutation abundance of the sample with the double-chain proofreading mutation read number less than 10 by using a scatter diagram at the upper left corner, wherein the mutation can be detected when the double-chain proofreading mutation read is more than or equal to 1, and the lowest mutation abundance is 0.03%.

Example 4

83 NSCLC patient sputum samples and matched tumor samples are collected, sputum cell-free supernatant is prepared, cfDNA of the cell-free supernatant is extracted, 2 samples of common driver gene changes of lung cancer are detected by a human 10-gene NGS method respectively, and detection sensitivity and specificity of the sputum cfDNA in driver gene detection are determined. The results show that: compared with paired tumor specimens, the total detection sensitivity of the sputum cfDNA was 81.3% (95% CI: 69.2%, 89.5%), and the specificity was 100% (95% CI: 79.1%, 100%). The sensitivity of the IIIB-IV stage sputum specimen is 87.0% (95% CI: 74.5%, 94.1%), and the detection sensitivity of the sputum specimen of the patient in IIIB-IV stage when the cancer cells are found is 94.1% (95% CI: 78.9%, 99.0%). The above results show that: 83 matched sputum and tumor specimens are collected, and the feasibility, the detection success rate and the detection accuracy of the scheme are verified.

In the aspect of sample treatment before NGS detection, research shows that various sequencing quality control data are optimal when the initial input DNA amount is 150ng, the sputum collection amount (more than or equal to 3ml) needs to be increased to the maximum extent of 10ml in order to ensure the sufficient cfDNA amount, and the success rate and the accuracy of NGS detection of the cfDNA of the sputum sample are ensured. Meanwhile, the scheme for reducing phlegm is improved, and the treatment is respectively carried out according to different properties (viscous type and 2 types of water sample phlegm) of the sputum specimen so as to improve the cfDNA content (concentration) in unit volume to the maximum extent. Through the 2 measures, 83 cases of sputum specimen cfDNA are successfully detected in the group. This also illustrates the importance of pretreatment of the specimen for NGS detection.

Because the distribution of the sputum cfDNA fragments is similar to that of the tumor gDNA, the process of breaking treatment is the same as that of the tumor specimen gDNA before constructing the reservoir, so as to ensure the smooth operation of the subsequent reservoir constructing process. From the analysis of sequencing off-machine data, the target fragment (insert) captured by the cfDNA of the sputum specimen is about 200bp, which is the same as the tumor genome DNA (about 160bp of plasma cfDNA insert), and the characteristics of the cfDNA of the sputum specimen are proved to be closer to the tumor gDNA. The subsequent construction method of the pre-library and the final library is the same as that of a tumor gDNA sample, and only 1GB sequencing data is given to one sample.

Sputum cfDNA detection is also different from tumor and plasma cfDNA specimens in the aspects of off-line analysis of results and interpretation of positive results of gene mutation. Research shows that the sputum cfDNA is more similar to the plasma cfDNA in the abundance and positive interpretation of the driver gene mutation detection, but when the plasma cfDNA is subjected to high-depth sequencing and low-frequency mutation interpretation, the false positive caused by clonal hematopoiesis needs to be carefully interpreted, and the sputum specimen does not have the influence factor in the aspect, so that the detection specificity is still 100% in 83 patients in the group when the average original depth (MeandeDepth) is more than 10000 times, the average effective depth (DSBCDepth) is more than 500 times, the number of DSBC supported reads is at least 1 pair, and the allele frequency (MAF) is 0.03%.

In addition, it was found that sputum cfDNA was similar to tumor specimen gDNA in fragment distribution and total amount available, but similar to plasma cfDNA in terms of driver gene assay abundance and positive interpretation. And (3) carrying out filtration annotation on NGS sequencing sites of the sputum cfDNA sample by referring to a matched tumor sample gene hotspot mutation positive judgment standard of grouping research, and finding that the sputum sample has higher false negative rate when positive cut off values (tumor gDNA and plasma cfDNA) are judged according to the kit by referring to a matched tumor sample driving gene detection result. The reason for the occurrence of false negative is analyzed, and the driving gene mutation hot spot mutation site existing in the matched tumor specimen is detected in the sputum cfDNA, but the driving gene mutation site is judged as background noise and filtered out because the driving gene mutation site does not pass the positive judgment value, so that the false negative condition occurs. Therefore, we re-demarcate the sputum cfDNA mutation positive judgment value: the average original depth (MeanDepth) should be greater than 10000 ×, the average effective depth (DSBCDepth) should be greater than 500 ×, and the number of DSBC supported reads is at least 1 pair. According to the new interpretation standard, the hot spot Mutation Allele Frequency (MAF) of the sputum specimen cfDNA can reach 0.03 percent at least, and the specificity of the sputum cfDNA mutation detection under the condition is 100 percent according to the detection result of the matched tumor sample driving gene.

The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific examples, it will be appreciated that the invention may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.

Claims

1. A sputum specimen cfDNA second-generation sequencing and library building method is characterized by comprising the following steps:

2) constructing a library: and constructing the cfDNA library of the sputum specimen by using the second-generation gene sequencing detection kit to obtain the gene sequencing library of the cfDNA of the second-generation sequenced sputum specimen.

2. The sputum specimen cfDNA next-generation sequencing and library building method according to claim 1, wherein the sputum reduction treatment in the step 1) comprises: adding 1: sequentially adding 2ml of 0.5mol/L DTT into 2-3 volumes of normal saline, and observing the phlegm-resolving effect until the phlegm is completely resolved; adding 1: after 1 physiological saline, 2ml of 0.5mol/L DTT is added in turn, and the effect of reducing phlegm is observed until the phlegm is completely reduced.

3. The secondary sputum specimen cfDNA sequencing and library construction method according to claim 2, wherein the DTT solution is added each time, and then the sputum specimen is shaken for 5min and then the expectorant effect is observed.

4. The secondary sequencing and library-building method for the cfDNA of the sputum specimen according to claim 2, wherein the sputum specimen in the step 1) is at least 3 ml.

5. The secondary sputum specimen cfDNA sequencing and library building method according to claim 2, wherein the centrifugation condition is 1500g for 5 min.

6. The secondary sequencing and library construction method for the sputum specimen cfDNA according to claim 2, wherein the sputum specimen in the step 1) is preserved or transported in a fixing solution before being subjected to the sputum reducing treatment, the fixing solution is a mixture of 50% ethanol and polyethylene glycol 400, and the volume ratio of the 50% ethanol to the polyethylene glycol 400 is 49: 1.

7. The method for extracting the cfDNA of the sputum specimen is characterized in that the gene sequencing library of the cfDNA of the sputum specimen for second-generation sequencing is constructed, and comprises the following steps:

1) performing phlegm reduction treatment on the collected sputum specimen to obtain a sputum specimen completely reducing phlegm;

8. The method according to claim 7, wherein the method for reducing sputum comprises: adding 1: sequentially adding 2ml of 0.5mol/L DTT into 2-3 volumes of normal saline, and observing the phlegm-resolving effect until the phlegm is completely resolved; adding 1: after 1 physiological saline, sequentially adding 2ml of 0.5mol/L DTT, and observing the phlegm-resolving effect until the phlegm is completely resolved; before the phlegm reduction treatment, the sputum specimen is stored in a stationary liquid, the stationary liquid is a mixed liquid of 50% ethanol and polyethylene glycol 400, and the volume ratio of the 50% ethanol to the polyethylene glycol 400 is 49: 1.

9. A sputum specimen cfDNA second-generation sequencing detection kit, characterized in that the kit comprises a DNA sequencing library prepared by the library construction method according to any one of claims 1 to 6.

10. The second-generation sputum specimen sequencing detection kit according to claim 9, wherein the initial input amount of the sputum specimen cfDNA is 150ng when the kit is used.