CN109239029B

CN109239029B - Efficient and accurate quality evaluation method for high-throughput detection sample

Info

Publication number: CN109239029B
Application number: CN201810858187.7A
Authority: CN
Inventors: 段飞蝶; 韩维; 汉雨生; 揣少坤
Original assignee: Guangzhou Burning Rock Dx Co ltd
Current assignee: Guangzhou Burning Rock Dx Co ltd
Priority date: 2018-07-31
Filing date: 2018-07-31
Publication date: 2022-06-14
Anticipated expiration: 2038-07-31
Also published as: CN109239029A

Abstract

The invention discloses a method for evaluating the degradation condition of a DNA sample, which respectively measures the DNA concentration of the sample by using a fluorescent dye method and an ultraviolet absorption method and calculates the ratio of the two measurement methods. The method for evaluating the degradation condition of the DNA sample provided by the invention has the advantages that the accuracy and the performance meet the requirements of subsequent NGS and even other fragment evaluation, and compared with the conventional method, the method saves time and space, has good economy and small human error. Therefore, the method provided by the invention can replace the conventional method for evaluating the high-throughput sequencing DNA fragment, greatly shortens the quality control link and the detection period of the high-throughput sequencing, brings great advantages in cost and is worthy of large-area popularization and application.

Description

Efficient and accurate quality evaluation method for high-throughput detection sample

Technical Field

The invention relates to the technical field of high-throughput sequencing, in particular to a high-efficiency and accurate quality evaluation method for a high-throughput detection sample.

Background

High throughput sequencing technology, also known as Next-generation sequencing technology (NGS), allows millions of DNA molecules to be sequenced simultaneously, allowing researchers to study biological systems at levels previously unavailable due to the unparalleled throughput, scalability, and speed of Next-generation sequencing (NGS). NGS technology can rapidly sequence entire genomes, amplifying to deeply sequence target regions. High throughput sequencing technology has been widely used in life sciences. However, the NGS detection process is relatively long, starting with sample collection, pathology assessment, nucleic acid extraction, library construction, on-board, and data analysis. In order to ensure that each sample for clinical examination can obtain high-quality and accurate results, quality control standards, especially initial sample extraction, need to be set in each process of high-throughput assay in order to achieve the best detection result.

Tissue biopsy samples are always used as the gold standard for molecular detection of precise medical NGS tumors, but in order to facilitate the preservation of tissues and the convenience of subsequent sampling, Formalin-Fixed and paraffin-Embedded (FFPE) tumor tissue samples are a preservation method widely used for analyzing cell tissues of gene expression, and although Formalin can preserve samples for a long time, the Formalin also has a non-negligible serial influence on experiments. In the preparation and storage process of FFPE tissue specimens, the problems of biomolecule cross-linking, DNA degradation and the like are easily caused after the tissues are fixed by formalin and embedded by paraffin. The molecular cross-linking and DNA degradation bring about low DNA extraction amount and fragmentation after nucleic acid extraction.

DNA fragmentation is a crucial factor in the NGS detection process, the requirement on fragments in the NGS-library building process is strict, and the first step of library building is as follows: the DNA fragments are mechanically broken into fragments of about 200bp, and the parameters for breaking the fragments need to be selected according to the result of the degradation condition of the sample DNA sample. Inappropriate disruption conditions can result in DNA fragments that are too long or too short (sequencing length requires 150bp x 2) for NGS pooling, leading to final data quality failures.

At present, the conventional methods for evaluating the degradation of DNA fragments comprise agarose gel electrophoresis and QPCR, and the two methods are commonly used but have certain disadvantages. (1) Agarose gel electrophoresis-method of assessing fragment degradation: risk of contamination: when DNA is electrophoresed, the DNA is exposed to an electrophoresis buffer, and heat is generated along with the electrophoresis process, so that the risk of aerosol pollution exists. It is therefore necessary that the electrophoresis apparatus is in one room alone, and at the most downstream of the NGS flow, which is also well defined at the PCR room setup; the occupied space is as follows: the electrophoresis system has pollution risk and needs to be in one room independently, which also increases burden on space, and particularly, the electrophoresis system has fatal defect that the space is extremely limited when an NGS platform is built at a hospital end; time consumption and human error are large: DNA electrophoresis process: preparing glue, adding sample, performing electrophoresis, photographing and evaluating for about 1.5 hours in one process; in addition, during electrophoresis evaluation, since the DNA bands are all the fragment images with certain degradation, certain evaluation technology is needed, and artificial differences also exist. Toxic: the electrophoresis reagent is particularly a DNA dye which has certain toxicity to human bodies. (2) QPCR judged fragment: the operation is complex and time-consuming: the QPCR method is a more accurate method compared with electrophoresis, but the QPCR method is not generally used due to high operation requirement, complex operation and long required time of 3-4 h, and the clinical report period brings great pressure; the economic efficiency is as follows: the reagent cost of QPCR is high.

Therefore, a simple, quick, accurate, non-toxic and pollution-free method for judging the DNA degradation degree is lacked at present.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide an efficient and accurate quality evaluation method for a high-throughput detection sample.

In order to achieve the purpose, the invention is realized by the following technical scheme:

a method for evaluating the degradation condition of a DNA sample comprises the steps of measuring the DNA concentration of the sample by a fluorescent dye method and an ultraviolet absorption method respectively, and calculating the ratio of the two measurement methods.

Preferably, the sample DNA concentration is measured using a Nanodrop spectrophotometer using an ultraviolet absorption method.

Preferably, the sample DNA concentration is measured using a fluorescence dye method using a Qubit fluorometer.

Preferably, the order number: the Qubit dsDNA HS Assay Kit of Q32854 is combined with the Qubit fluorometer to detect the DNA concentration.

Preferably, the method for calculating the ratio of the two measurement methods is:

N/Q ratio = sample DNA concentration measured using Nanodrop spectrophotometer/sample DNA concentration measured using a Qubit fluorometer.

Preferably, when the ratio of 0< N/Q < 3.5, the integrity of the sample DNA is good; when the N/Q ratio is more than 3.5 and less than or equal to 6.5, the integrity of the sample DNA is better; when the N/Q ratio is >6.5, the sample DNA integrity is not good.

Preferably, when the ratio of 0< N/Q < 3.5, the DNA of the sample is more than or equal to 2500 bp; when the N/Q ratio is more than 3.5 and less than or equal to 6.5, the DNA of the sample is 1000-2500 bp; when the N/Q ratio is greater than 6.5, the DNA of the sample is less than or equal to 1000 bp.

Q/N ratio = sample DNA concentration measured using a Qubit fluorometer/sample DNA concentration measured using a Nanodrop spectrophotometer.

Preferably, when the Q/N ratio >0.29, the sample DNA integrity is good; when the Q/N ratio is more than 0.15 and less than or equal to 0.29, the integrity of the sample DNA is better; when the Q/N ratio is <0.15, the sample DNA integrity is not good.

Preferably, when the ratio is greater than 0.29, the sample DNA is more than or equal to 2500 bp; when the ratio is less than or equal to 0.29 and 0.15, the DNA of the sample is 1000-2500 bp; when the ratio is less than 0.15, the sample DNA is less than or equal to 1000 bp.

Compared with the prior art, the invention has the following beneficial effects:

the method for evaluating the degradation condition of the DNA sample provided by the invention has high evaluation accuracy, and can be used for integrally evaluating the length of the DNA fragment of the sample. The accuracy of the method can meet the requirements of subsequent NGS detection and other requirements of DNA fragment degradation condition or length distribution evaluation, and compared with the conventional method, the method has the following advantages/meanings:

1. the time is saved: the method provided by the invention can be used for quickly evaluating without additional experiments. The evaluation time is greatly shortened, which brings great advantages to the clinical detection period.

2. The space is saved: the method provided by the invention does not need other equipment and does not need additional independent space. A great advantage in space is brought about.

3. The economic efficiency is as follows: compared with the conventional electrophoresis method or QPCR method, the method provided by the invention does not need any reagent, consumable material and equipment, and the cost is greatly reduced.

4. The human error is little, and the technical requirement is low: compared with other evaluation methods, the method provided by the invention directly evaluates according to specific numerical values, has no error of artificial evaluation and has extremely low technical requirements.

In conclusion, the method provided by the invention can replace the conventional method (gel electrophoresis method) for evaluating the DNA fragment in high-throughput sequencing, so that the quality control and detection period of high-throughput detection are greatly shortened, great advantages are brought to the cost, and the method is worthy of large-area popularization and application.

Drawings

FIG. 1 shows evaluation criteria of nucleic acid extraction gel electrophoresis.

Detailed Description

The invention is described in further detail below with reference to the drawings and specific examples, which are provided for illustration only and are not intended to limit the scope of the invention. The test methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are, unless otherwise specified, commercially available reagents and materials.

In the following comparative examples:

the database building method comprises the following steps: SureSelectXT Target entity System for Illumina Pair-End Sequencing Library

The upper machine sequencing platform comprises: miseq, Nextseq 500, Miniseq, Novaseq 6000

The sample to be tested is: daily clinical detection FFPE sample

Rating scale for genomic DNA quality:

a level: the average length of the sample DNA fragment is more than 2500bp, namely the DNA is more than or equal to 2500 bp;

b stage: the average length of the sample DNA fragments is between 1000 and 2500 bp;

c level: the average length of the sample DNA fragments is 500-1000 bp;

d stage: the average length of the sample DNA fragments is below 500 bp; namely DNA less than or equal to 500 bp.

EXAMPLE 1 evaluation method of N/Q ratio

First, experiment operation

1. Extraction of sample DNA

Extraction of DNA was carried out using commercially available tissue or cell DNA extraction kits.

2. Determining breaking conditions and performing mechanical breaking

The DNA concentration of the samples was measured using a Nanodrop spectrophotometer and a Qubit fluorometer, respectively, (DNA concentration measurements were performed using the Qubit dsDNA HS Assay Kit with the cat number Q32854 in combination with the Qubit fluorometer) and the ratio of their concentrations was calculated according to the following formula:

The sample grading annotation and interruption conditions are shown in table 1.

Table 1:

3. analysis of broken segments

And then, carrying out a normal NGS flow, establishing a library of the fragments obtained after interruption, carrying out on-machine sequencing, carrying out QC (quality control) analysis on off-machine data by using a bioinformatics method, judging the size of insert size (the fragments after interruption), and judging the accuracy of the sample grade evaluation method according to the size of the insert size, namely the corresponding relation (goodness of fit) between the theoretical insert size of the N/Q ratio evaluation method and the actual insert size, so as to determine the accuracy of the evaluation of the N/Q ratio evaluation method.

Second, experimental results

The relationship between the evaluation method and the QC analysis insert goodness of fit is shown in table 2.

Table 2:

the method rates 1114 samples with A, of which 1008 samples actually have 200-250bp inserts, and can confirm that the 1008 sample evaluation is accurate, i.e., the coincidence rate is 90.2% (1008/1114). Similarly, the anastomotic rates of B and C were calculated to be 36.1% and 37.1%, respectively, which were able to meet daily requirements.

Comparative example gel electrophoresis evaluation method

First, experiment operation

1. Extraction of sample DNA

The same as in example 1.

2. Gel electrophoresis determination of interrupting conditions

The samples were subjected to agarose gel electrophoresis.

The criteria for the disruption parameters according to gel electrophoresis are now shown in Table 3 and FIG. 1.

Table 3:

3. analysis of broken segments

And then, carrying out a normal NGS flow, establishing a library of the fragments obtained after interruption, carrying out on-machine sequencing, carrying out QC (quality control) analysis on off-machine data by using a bioinformatics method, judging the size of insert fragments (namely the fragments after interruption), and judging the accuracy of the sample grade evaluation method according to the size of the insert fragments, namely the corresponding relation (goodness of fit) between the theoretical insert fragment size and the actual insert fragment size of the gel electrophoresis evaluation method so as to determine the accuracy of the evaluation of the gel electrophoresis evaluation method.

Second, experimental results

The relationship between the electrophoretic evaluation and the goodness of fit of the insert is shown in table 4. The total number of 1053 samples with the electrophoresis rating of A, among which 952 samples had the actual insert of 200 and 250bp, confirmed that the 952 samples were evaluated accurately, i.e., the anastomosis rate was 90.4% (952/1053). Similarly, the calculation of the coincidence rate of B and C is calculated, and the coincidence rate of B and C with the insert is currently evaluated by electrophoresis to be relatively low.

Table 4:

it is believed that the main causes of this are: the differences of artificial factors and samples with slightly poor quality after detection are more obvious during electrophoresis evaluation.

The results are combined, the number of accurate samples evaluated by the two evaluation methods is compared, and the results are shown in table 5.

Table 5:

as shown in the table, the accuracy of the evaluation of the N/Q evaluation method and the gel electrophoresis evaluation method in a large-fragment sample with good quality (A grade) is similar, but the evaluation result of the N/Q evaluation method is far better than that of the gel electrophoresis in the evaluation of B grade and C grade samples.

Claims

1. A method for evaluating the degradation condition of DNA sample features that the fluorescent dye method and ultraviolet absorption method are used to respectively measure the DNA concentration of sample and calculate the ratio of two measuring methods,

the calculation method of the ratio comprises the following steps: N/Q ratio ═ sample DNA concentration measured using Nanodrop spectrophotometer/sample DNA concentration measured using the Qubit fluorometer;

when the ratio of N/Q is more than 0 and less than or equal to 3.5, the average length of the DNA fragments of the evaluation sample is more than or equal to 2500 bp; when the ratio of N/Q is more than 3.5 and less than or equal to 6.5, the average length of the DNA fragments of the evaluation sample is 1000-2500 bp; when the N/Q ratio is greater than 6.5, the average length of the sample DNA fragments is estimated to be less than or equal to 1000 bp.

2. The method of claim 1, wherein the DNA concentration is measured using a Qubit dsDNA HS Assay Kit with a cat number Q32854 in combination with a Qubit fluorometer.

3. The method according to claim 1 or 2, wherein the breaking conditions are selected according to the degradation condition of the DNA sample, and when the N/Q ratio is less than or equal to 3.5, mechanical breaking is carried out for 60 seconds; when the N/Q ratio is greater than 3.5, the machine is interrupted for 30 seconds.

4. The method according to claim 3, wherein the expected size of the fragmented insert of the sample DNA fragment is estimated to be 200-250bp when the ratio N/Q is 0< 3.5; when the N/Q ratio is more than 3.5 and less than or equal to 6.5, the expected size of the insert after the sample DNA fragment is judged to be 170-200 bp; the expected size of the insert after disruption of the sample DNA fragments was evaluated to be <170bp when the N/Q ratio > 6.5.

5. A method for evaluating the degradation condition of DNA sample features that the fluorescent dye method and ultraviolet absorption method are used to respectively measure the DNA concentration of sample and calculate the ratio of two measuring methods,

the calculation method of the ratio is as follows: Q/N ratio ═ sample DNA concentration measured using a Qubit fluorometer/sample DNA concentration measured using a Nanodrop spectrophotometer;

when the Q/N ratio is greater than 0.29, the average length of the DNA fragments of the evaluation sample is more than or equal to 2500 bp; when the Q/N ratio is more than 0.15 and less than or equal to 0.29, the average length of the DNA fragments of the evaluation sample is 1000-2500 bp; when the Q/N ratio is <0.15, the average length of the sample DNA fragments is evaluated to be less than or equal to 1000 bp.

6. The method of claim 5, wherein the DNA concentration is measured using a Qubit dsDNA HS Assay Kit with a cat number Q32854 in combination with a Qubit fluorometer.

7. The method according to claim 5 or 6, wherein the breaking conditions are selected according to the degradation condition of the DNA sample, and when the Q/N ratio is more than 0.29, mechanical breaking is carried out for 60 seconds; when the Q/N ratio is less than or equal to 0.29, the machine is interrupted for 30 seconds.

8. The method according to claim 7, wherein the expected size of the fragmented insert of the sample DNA fragment is estimated to be 200-250bp when the Q/N ratio is > 0.29; when the Q/N ratio is more than 0.15 and less than or equal to 0.29, the expected size of the insert after the sample DNA fragment is judged to be 170-200 bp; when the Q/N ratio is <0.15, the expected size of the insert after fragmentation of the sample DNA fragment is assessed to be <170 bp.