WO2017156739A1

WO2017156739A1 - Isolated nucleic acid application thereof

Info

Publication number: WO2017156739A1
Application number: PCT/CN2016/076577
Authority: WO
Inventors: 仲文迪; 张林爽; 郑智俊
Original assignee: 上海锐翌生物科技有限公司
Priority date: 2016-03-17
Filing date: 2016-03-17
Publication date: 2017-09-21
Also published as: CN109072306A

Abstract

An isolated nucleic acid, comprising at least one of the following nucleic acid sequence clusters: a first nucleic acid sequence cluster, the nucleic acid sequences in the cluster corresponding on a one-to-one basis to the sequences shown by SEQ ID NOS: 1-151, the sequence similarity between the nucleic acid sequences in the cluster and the sequences in SEQ 1D NOS: 1-151 corresponding thereto being no less than 90%; and a second nucleic acid sequence cluster, the nucleic acid sequences in the cluster corresponding on a one-to-one basis to the sequences shown by SEQ ID NOS: 152-233, the sequence similarity between the nucleic acid sequences in the cluster and the sequences in SEQ 1D NOS: 152-233 corresponding thereto being no less than 90%. Compared to a group of colorectal cancer patients, the present isolated nucleic acid is significantly enriched in a healthy group, and can be used as a marker to distinguish between a healthy group and a group of colorectal cancer patients.

Description

Isolated nucleic acids and applications

Technical field

The present invention relates to the field of biomarkers, in particular, to isolated nucleic acids and uses of the present invention, and more particularly to the use of a set of isolated nucleic acids, isolated nucleic acids, and a method for determining the state of an individual using the isolated nucleic acids A device for determining the state of an individual using the isolated nucleic acid, a method for classifying a plurality of individuals using the separated nucleic acid, a drug for treating colorectal cancer, and a method for preparing a drug for treating colorectal cancer.

Background technique

Colorectal Cancer (CRC), also known as colon cancer, is a cancer that originates from the colon or rectum (part of the large intestine) because cells grow abnormally and may invade or metastasize to other parts of the body. Patients with colorectal cancer often have symptoms such as blood in the stool, changes in bowel habits, weight loss, and fatigue. Colorectal cancer is the third most common cancer, accounting for about 10%. In 2012, there were 1.4 million newly diagnosed colorectal cancers in the United States, causing 694,000 deaths. Colorectal cancer is more common in developed countries, accounting for 65% of the total number of cases worldwide. Women are less common than men. In recent years, the incidence of colorectal cancer in China has shown a clear upward trend. According to statistics, in 2002, the incidence of colon cancer in China was 7%, and the estimated incidence rate was 13% in 2015. Recently, China's cancer statistics in 2015 were published in the Journal of CA: A Cancer Journal for Clinjicians with an impact factor of 144.8. The data showed that there were 376,300 new cases of colorectal cancer in China in 2015, with a death toll of 191,000 (Chen W et al., 2016). Compared with Westerners, the incidence of rectal cancer in China is higher than that of colon cancer, about 1.5:1; the proportion of young people (<30 years old) is relatively high, accounting for about 15%.

75-95% of patients with colorectal cancer have no or few genetic factors; other risk factors include increased age, male, high fat intake, alcohol or red meat, overweight, smoking, and lack of physical activity; approximately 10% of cases Lack of exercise (Watson A J et al., 2010; Cunningham D et al., 2010). The danger of drinking is gradually increased after more than one cup per day.

Intestinal microorganisms play an important role in intestinal epithelial cells, including the formation of microbial barriers to prevent colonization of pathogenic bacteria, and the implementation of immune regulation and metabolic functions. Studies have shown that intestinal flora imbalance can lead to colorectal cancer through different forms, pathogenic microorganisms will cause intestinal inflammation by activating receptors, adsorbing, secreting enterotoxin or invading. Changes in the number, structure, and stability of intestinal microbes, especially imbalances in the flora, can alter normal physiological functions and cause intestinal diseases, including colorectal cancer.

There are three main types of colorectal cancer diagnostic kits: X-ray examination; sigmoidoscopy and fiberoptic colonoscopy and carcinoembryonic antigen (cea) test; carcinoembryonic antigen (cea) test is of little value in the diagnosis of early cases; High sexuality, but many high-risk groups refused regular screening for 1-2 years because they did not want to accept painful colonoscopy. There are studies to detect Mir-92 in the blood The content of the factor can detect whether the blood tester has colorectal cancer, but the error detection rate of healthy people reaches 30%. With the completion of human genome sequencing and the rapid development of high-throughput sequencing technology, genetic screening has become the direction of colorectal cancer diagnosis. Genetic screening has an advantage in identifying potential populations of colorectal cancer, but colonoscopy is still required for 1-2 years after the discovery of a genetic defect. Colorectal cancer has no obvious symptoms at an early stage, and there is currently no effective early diagnosis method for non-invasive colorectal cancer. Colorectal cancer patients have revealed similar substantial changes in a gut microbiota by metagenomic sequencing studies (Zeller G et al., 2014; Feng Q et al., 2015). However, specific biomarkers related to colorectal cancer in intestinal microbes are still unclear.

Summary of the invention

The present invention is directed to at least one of the above problems or to at least one alternative business means.

According to a first aspect of the invention there is provided a set of isolated nucleic acids comprising at least one of the following two nucleic acid sequence clusters: a first nucleic acid sequence cluster, the nucleic acid sequence in the first nucleic acid sequence cluster and SEQ ID NO: 1-91, wherein the sequence of each of the first nucleic acid sequence clusters has a sequence similarity to the sequence of SEQ ID NO: 1-151 of not less than 90%; the second nucleic acid sequence a cluster, the nucleic acid sequence in the second nucleic acid sequence cluster is in one-to-one correspondence with the sequence shown in SEQ ID NO: 152-233, and the nucleic acid sequence in each of the second nucleic acid sequence clusters corresponds to its corresponding SEQ ID NO: 152 The sequence similarity of the sequences in -233 is not less than 90%.

According to a second aspect of the invention, the invention provides the use of the above isolated nucleic acid for detecting colorectal cancer, and/or for treating colorectal cancer, and/or for preparing a medicament for treating colorectal cancer and/or for preparing a functional food.

According to a third aspect of the present invention, there is provided a method for obtaining an isolated nucleic acid according to one aspect of the present invention, the method comprising: (1) obtaining a first sequencing result and a second sequencing result, the first sequencing result Sequencing results of nucleic acid for a stool sample of a plurality of colorectal cancer patients, comprising a plurality of first reads, the second sequencing result being a sequencing result of nucleic acids of a stool sample of a plurality of healthy individuals, including a plurality of second reads (2) assembling the first read segment and the second read segment respectively, correspondingly obtaining a plurality of first assembly sequences and a plurality of second assembly sequences; (3) respectively supporting the first assembly sequence based on the first read segment and The second read segment supports the second assembly sequence, determining the abundance of the first assembly sequence and the abundance of the second assembly sequence; (4) the abundance of the first assembly sequence determined in (3) and the second Assembling the abundance of the sequence, clustering the first assembly sequence and the second assembly sequence to obtain a plurality of gene clusters, each of the gene clusters comprising a plurality of first assembly sequences and/or second assembly sequences; (5) Statistical test to determine the multiple colorectal cancers A gene cluster that is significantly enriched in the stool sample of the patient and/or the plurality of healthy individuals to obtain the isolated nucleic acid.

According to a fourth aspect of the present invention, there is provided a method for determining the state of an individual using the nucleic acid of one aspect of the invention described above, the method comprising: determining abundance of a cluster of nucleic acid sequences in the nucleic acid in a stool sample of the individual And abundance in the control group; comparing the abundance of the nucleic acid sequence cluster in the fecal sample of the individual with the abundance in the control group, determining whether the individual is statistically significant based on whether the difference is statistically significant State, the control group consists of one or more A stool sample of an individual in the same state is composed of a state including colorectal cancer and no colorectal cancer.

All or part of the steps of the above-described method of the present invention for determining the state of an individual using a nucleic acid of one aspect of the invention may be performed using a device/system comprising a detachable corresponding unit functional module, or the method may be programmed Stored on a machine readable medium, implemented by a machine running the readable medium.

According to a fifth aspect of the invention, there is provided a device for determining the state of an individual using the nucleic acid of one aspect of the invention described above, the device for carrying out the method for determining the state of an individual in accordance with one aspect of the invention described above, the device comprising: abundance a determining unit for determining abundance of a nucleic acid sequence cluster in the nucleic acid in the fecal sample of the individual and abundance in a control group; an individual state determining unit for comparing the nucleic acid sequence cluster in the individual The abundance in the stool sample is different from the abundance in the control group, and the state of the individual is determined based on whether the difference is statistically significant, the control group being the stool of one or more groups of individuals in the same state A sample consisting of having colorectal cancer and not having colorectal cancer.

According to a sixth aspect of the invention, there is provided a system for determining the state of an individual using the nucleic acid of one aspect of the invention described above, the system for performing all or part of the steps of the method for determining the state of an individual in accordance with one aspect of the invention described above, The system comprises: a data input module for inputting data; a data output module for outputting data; a processor for executing an executable program, the executing the executable program comprising performing the above-described determination of an individual state of the invention All or part of the steps of the method; a storage module coupled to the data input module, the data output module, and the processor for storing data, including the executable program.

According to a seventh aspect of the present invention, there is provided a method of classifying a plurality of individuals using the nucleic acid of one aspect of the present invention, the method comprising: determining a state of each individual using the method of determining an individual state according to an aspect of the present invention, respectively. The individual individuals are classified according to the status of each individual obtained. The method can distinguish a plurality of individuals according to different states of the individual or distinguish a plurality of unknown stool samples, which is convenient for classification and label management.

According to an eighth aspect of the present invention, the present invention provides a medicament for treating colorectal cancer, characterized in that the medicament promotes an increase in the abundance of the nucleic acid of the above aspect of the invention in the intestinal tract of a patient.

According to a ninth aspect of the present invention, the present invention provides a method for producing or screening a medicament for treating colorectal cancer according to one aspect of the present invention, which comprises screening a substance which promotes an increase in abundance of a nucleic acid of one aspect of the present invention as a The steps of the drug.

The above-mentioned isolated nucleic acid of one aspect of the present invention is a difference in the abundance of the intestinal microbial sequence of the colorectal cancer patient population and the healthy population by comparing the sequencing data of the intestinal microbial sample by the inventor, and then verified by a large number of sample tests. Determined. The isolated nucleic acid of this group can be used as a marker for colorectal cancer. Compared with the group of patients with colorectal cancer, the so-called isolated nucleic acid is significantly enriched in a healthy population, and the so-called significant enrichment refers to the abundance in the disease control group. Compared to the above The abundance of the nucleic acid sequence clusters contained in the colorectal cancer markers in the healthy group was statistically higher than or significantly higher than the abundance in the disease group. The set of isolated nucleic acids can be used to determine the probability of an individual having a high or low probability of being in a state of colorectal cancer, and can be used for non-invasive early detection or for the detection of colorectal cancer.

Furthermore, a substance capable of increasing the abundance of the isolated nucleic acid can be used for treating colorectal cancer or for patients with colorectal cancer, and a substance capable of increasing the abundance of the isolated nucleic acid is not limited to a drug for treating colorectal cancer and A functional food that is beneficial to the balance of the intestinal flora. The nucleic acid identified by the method of one aspect of the present invention, that is, a colorectal cancer marker, can be used for the preparation of a medicament for treating colorectal cancer and/or for the preparation of a functional food, a health care drug or the like which is advantageous for balancing the intestinal flora.

Moreover, the method, apparatus and/or system for determining the state of an individual in an aspect of the invention described above is based on detecting the abundance of nucleic acid sequence clusters in a stool sample of an individual, and detecting the abundance of the nucleic acid sequence cluster in the determined nucleic acid against it. The abundances in the group were compared, and based on the obtained comparison results, the relative probability of the individual being a colorectal cancer individual or a healthy individual can be determined. Provides a non-invasive adjunct test for early detection of colorectal cancer.

DRAWINGS

The above and/or additional aspects and advantages of the present invention will become apparent and readily understood from

BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a flow chart showing the experimental analysis of screening and identifying colorectal cancer markers in an embodiment of the present invention.

2 is a abundance heat map of the clustered CAG16610 in an embodiment of the present invention; wherein the left side is the abundance heat map in the found data set, and the right side is the abundance heat map in the verification data set.

detailed description

The embodiments of the present invention are described in detail below, and the examples of the embodiments are illustrated in the drawings, wherein the same or similar reference numerals are used to refer to the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are intended to be illustrative of the invention and are not to be construed as limiting. It should be noted that the terms "first" or "second" and the like as used herein are merely for convenience of description, and are not to be construed as indicating or implying relative importance, nor to be construed as having between the first and second Sequential relationship.

In the description of the present invention, "a plurality" means two or more unless otherwise stated. In this document, the terms "connected", "connected" and the like shall be understood broadly, and may be, for example, a fixed connection, a detachable connection, or an integral connection; it may be mechanical, unless otherwise explicitly defined and defined. The connection may also be an electrical connection; it may be directly connected, or may be indirectly connected through an intermediate medium, and may be internal communication between the two elements.

A biological marker is a cellular, biochemical, or molecular alteration that can be detected from a biological medium. Biology The quality includes various body fluids, tissues, cells, feces, hair, exhalation and the like.

By abundance is meant the degree of abundance of such a microorganism or sequence in a population of microorganisms or nucleic acid sequences. For example, the richness of the microorganism in the intestinal microbial population can be expressed as the content of the microorganism in the population; and, for example, the degree of enrichment of a nucleic acid sequence in a set of nucleic acid sequences can be expressed as the nucleic acid sequence. The number of the proportions of the total number of sequences in the group.

The identity of the sequence and the similarity of the sequences refer to the degree of similarity or similarity between the sequences, respectively.

A set of isolated nucleic acids according to one embodiment of the invention, comprising at least one of the following clusters of nucleic acid sequences: a first nucleic acid sequence cluster, the nucleic acid sequence in the first nucleic acid sequence cluster and SEQ ID NO: 1- The sequences shown in 151 correspond one-to-one, and the sequence similarity between the nucleic acid sequence in each of the first nucleic acid sequence clusters and the corresponding sequence in SEQ ID NO: 1-110 is not less than 90%; the second nucleic acid sequence cluster, The nucleic acid sequence in the second nucleic acid sequence cluster is in one-to-one correspondence with the sequence shown in SEQ ID NO: 152-233, and the nucleic acid sequence in each of the second nucleic acid sequence clusters corresponds to its corresponding SEQ ID NO: 152-233 The sequence similarity of the sequences in the sequence is not less than 90%.

The isolated nucleic acid is determined by the inventors by analyzing the sequencing data of the intestinal microbial sample, comparing the abundance of the intestinal microbial sequence of the colorectal cancer patient population and the healthy population, and then determining by a large number of sample tests. The nucleic acid sequence contained in each nucleic acid sequence cluster is a non-redundant sequence. The isolated nucleic acid can be used as a colorectal cancer marker, and the colorectal cancer marker is significantly enriched in a healthy population compared with the colorectal cancer patient group, and the significant enrichment refers to the abundance in the disease control group. In comparison, the abundance of the nucleic acid sequence clusters contained in the above-mentioned colorectal cancer markers in the healthy group was statistically higher than or significantly higher than the abundance in the disease group. The set of isolated nucleic acids can be used to determine the probability of an individual having a high or low probability of being in a state of colorectal cancer, and can be used for non-invasive early detection or for the detection of colorectal cancer. It should be noted that SEQ ID NO: 1-151 and SEQ ID NO: 152-233 are all sequence clusters determined by the inventors based on sample nucleic acid sequencing data and assembly cluster analysis, and the sequences in each sequence cluster are from The same species, and it is generally believed that the success rate of DNA/DNA hybridization is as high as 80% or more in the same species, so the inventors hereby define that the sequence contained in the nucleic acid sequence cluster and the corresponding SEQ ID NO: 1-151 or The sequences in SEQ ID NOS: 152-233 have a sequence similarity of not less than 90%, all belonging to the same sequence cluster, and are capable of functioning as markers for colorectal cancer sequences. The inventors repeatedly changed the nucleotides of the sequence in the nucleic acid sequence cluster such that the sequence in the nucleic acid sequence cluster and the sequence of the corresponding sequence in the SEQ ID NO are not less than 90%, and the test is verified, not less than 90%. Sequence similarity is supported by experiments, and the resulting sequence clusters can also be used as markers for colorectal cancer.

According to an embodiment of the invention, the set of isolated nucleic acids consists of one or both of the first nucleic acid sequence cluster and the second nucleic acid sequence cluster.

According to an embodiment of the invention, the nucleic acid comprises the first nucleic acid sequence cluster. According to an embodiment of the present invention, the sequence similarity between the nucleic acid sequence in each of the first nucleic acid sequence clusters and the corresponding sequence in SEQ ID NO: 1-151 is not less than 95%. According to an embodiment of the invention, the nucleic acid further comprises the second sequence cluster. According to one embodiment of the invention, the sequence identity of the nucleic acid sequence in each of said second nucleic acid sequence clusters to its corresponding SEQ ID NO: 152-233 is not less than 95%.

According to an embodiment of the invention, the nucleic acid comprises the second nucleic acid sequence cluster. According to one embodiment of the invention, the sequence identity of the nucleic acid sequence in each of said second nucleic acid sequence clusters to its corresponding SEQ ID NO: 152-233 is not less than 95%. According to an embodiment of the invention, the nucleic acid further comprises the first sequence cluster. According to an embodiment of the present invention, the sequence similarity between the nucleic acid sequence in each of the first nucleic acid sequence clusters and the sequence in its corresponding SEQ ID NO: 152-233 is not less than 95%.

Use of the isolated nucleic acid of any of the above embodiments according to another embodiment of the present invention for detecting colorectal cancer, and/or for treating colorectal cancer, and/or for preparing a drug for treating colorectal cancer and/or for preparing a functional food .

The so-called isolated nucleic acid is determined by the inventors by analyzing the sequencing data of the intestinal microbial sample, comparing the abundance of the intestinal microbial sequence of the colorectal cancer patient population and the healthy population, and then determining by a large number of sample tests. . The isolated nucleic acid can be used as a colorectal cancer marker, and the colorectal cancer marker is significantly enriched in a healthy population compared to the individual group of colorectal cancer patients, and the so-called significant enrichment refers to abundance in the disease control group. In contrast, the abundance of the nucleic acid sequence clusters contained in the above-described colorectal cancer markers in the healthy group was statistically higher than or significantly higher than the abundance in the disease group. The set of isolated nucleic acids can be used to determine the probability of an individual having a high or low probability of being in a state of colorectal cancer, and can be used for non-invasive early detection or assisted detection of colorectal cancer; The increased substance can be used for treating colorectal cancer or for patients suffering from colorectal cancer, and the substance capable of increasing the abundance of the isolated nucleic acid is not limited to a drug for treating colorectal cancer and a functional food for benefiting the intestinal flora. The isolated pair of nucleic acids provided by this embodiment can be used for the preparation of a medicament for the treatment of colorectal cancer and/or for the preparation of functional foods, health care drugs and the like which are beneficial to the balanced intestinal flora.

According to another embodiment of the present invention, a method for obtaining the isolated nucleic acid of any of the above embodiments of the present invention, the method comprising: (1) obtaining a first sequencing result and a second sequencing result, the first The sequencing result is a sequencing result of a nucleic acid sequence of a stool sample of a plurality of colorectal cancer patients, comprising a plurality of first reads, the second sequencing result is a sequencing result of a nucleic acid sequence of a stool sample of a plurality of healthy individuals, including a plurality of a second read segment; (2) assembling the first read segment and the second read segment respectively, correspondingly obtaining a plurality of first assembly sequences and a plurality of second assembly sequences; and (3) respectively performing the first assembly sequence based on the first read segment Supporting condition and support of the second assembly sequence for the second assembly, determining the abundance of the first assembly sequence and the abundance of the second assembly sequence; (4) the abundance of the first assembly sequence determined in (3) Degree and second assembly sequence Abundance of the column, clustering the first assembly sequence and the second assembly sequence to obtain a plurality of gene clusters, each of the gene clusters comprising a plurality of first assembly sequences and/or second assembly sequences; (5) statistics A test is performed to determine a gene cluster that is significantly enriched in the stool samples of the plurality of colorectal cancer patients and/or the plurality of healthy individuals to obtain the nucleic acid. By this method, a cluster of nucleic acid sequences capable of being a marker of colorectal cancer can be efficiently determined.

According to an embodiment of the present invention, (2) includes: after assembling the first read segment and the second read segment respectively, respectively de-redunding the obtained assembly result of the first read segment and the assembly result of the second read segment, The plurality of first assembly sequences and the second assembly sequence are obtained, and the assembly sequences defining the identity of the paired sequences are not less than 95% and the sequence coverage is more than 90%, and the assembly sequences are the same sequence. According to one embodiment of the invention, two or more sequences are paired/aligned, utilizing Blat.

According to an embodiment of the present invention, (3) includes: comparing the first read segment and the second read segment to the first assembly sequence and the second assembly sequence, respectively, to obtain a first comparison result and a corresponding Two alignment results; determining the abundance of the first assembly sequence and the second assembly sequence based on the obtained first alignment result and the second alignment result respectively: the abundance Ab(S) of the assembly sequence S =Ab(U _S )+Ab(M _S ), where Ab(U _S )=U _S /l _S , U _S is the only number of reads of the assembly sequence S, l _{S is} the assembly sequence S length,

M _S is the number of reads that are non-uniquely aligned with the assembly sequence S, i represents the number of reads of the assembly sequence S that are not uniquely aligned, and Co _{i is a} non-unique comparison of the reads of the assembly sequence S The abundance coefficient of i,

N1 is the non-unique comparison of the total number of assembly sequences on the read alignment of the assembly sequence S, and j is the number of the assembly sequence on the read alignment of the assembly sequence S that is non-unique, U _j is The number of reads that uniquely align the assembly sequence j. The abundance determination formula described above is based on the contribution of the readout of the assembled sequence to the assembly sequence of the unique and non-unique alignment in the comparison result, and the abundance determined by the use of the sequencing data is very accurate.

According to an embodiment of the invention, prior to (4), the first assembly sequence and the second assembly sequence that are present only in less than one-fifth of the stool sample of the total number of stool samples are removed. In this way, the nucleic acid sequence which can be finally obtained as a marker has practical significance and can be used for detection determination of unknown samples.

According to an embodiment of the present invention, (4) includes: performing first clustering on the first assembly sequence and the second assembly sequence to obtain a first clustering result; and removing the cluster including only one assembly sequence The first clustering result performs a second clustering to obtain the plurality of gene clusters. Based on the difference in abundance, secondary clustering is performed, and the cluster containing only one assembly sequence in the first clustering result is eliminated, which facilitates the finally obtained nucleic acid sequence cluster to effectively serve as a colon cancer sequence marker.

The clustering can utilize known methods, which are not limited in the present invention. According to one embodiment of the invention, sequences are clustered using the canopy algorithm based on the abundance of the sequences. Unlike traditional clustering algorithms such as K-means, The biggest feature of Canopy clustering is that it does not need to specify the k value in advance, that is, the number of clustering (clustering), so it has great practical value. Moreover, compared with other clustering algorithms, Canopy clustering has a lower precision, but it has a great advantage in speed. Therefore, Canopy clustering can be used to first perform “rough” clustering of data, and then use Canopy or K- Means and so on for further "fine" clustering.

According to still another embodiment of the present invention, there is provided a method for determining the state of an individual using the isolated nucleic acid of any of the above embodiments, the method comprising the steps of:

The abundance of the cluster of nucleic acid sequences contained in the isolated nucleic acid is determined.

The abundance of the nucleic acid sequence cluster in the nucleic acid in the fecal sample of the individual and the abundance in the control group is determined.

According to an embodiment of the present invention, the abundance of the nucleic acid sequence cluster in the fecal sample of the individual and/or in the control group is determined by obtaining the nucleic acid in the fecal sample of the individual and the sequencing data of the nucleic acid in the control group Sequencing data from any source includes a plurality of reads; determining the nucleic acid sequence at the source based on the support of each of the nucleic acid sequences in the sequence of nucleic acid sequences based on reads in sequencing data from any source Abundance in the stool sample; determining the abundance of the cluster of nucleic acid sequences in which the nucleic acid sequence is located in the same stool sample based on the abundance of the nucleic acid sequence in the stool sample of the source.

The so-called sequencing data is obtained by sequencing the nucleic acid in the sample. The sequencing can be selected according to the selected sequencing platform, but is not limited to the semiconductor sequencing technology platform such as PGM, Ion Proton, BGISEQ-100 platform, and synthetic sequencing. Technology platforms such as Ilhexa's Hiseq, Miseq sequence platform and single molecule real-time sequencing platforms such as the PacBio sequence platform. The sequencing method can be either single-ended sequencing or double-end sequencing, and the obtained offline data is a segment read out, which is called a read.

According to one embodiment of the invention, the nucleic acid sequence is determined to be in the stool sample of the source, based on the support of the read sequence in the sequencing data from any source for each nucleic acid sequence in the nucleic acid sequence cluster. Abundance, comprising: aligning the reads to the nucleic acid sequence, based on the obtained alignment results, determining the abundance of the nucleic acid sequence using the following formula: Abundance of nucleic acid sequence G Ab(G)=Ab (U _G )+Ab(M _G ), wherein Ab(U _G )=U _G /l _G , U _G is the number of reads of the nucleic acid sequence G uniquely aligned, and l _{G is} the length of the nucleic acid sequence G ,

M _G the only number than the non-read period of the nucleic acid sequence of G, x represents the ratio of non-unique number on the read segment of the nucleic acid sequence G, Co _x nonunique than on the nucleic acid sequence of the G reads The abundance coefficient of x,

N2 is the non-uniquely aligned total number of nucleic acid sequences on the read alignment of the nucleic acid sequence G, and y is the number of the nucleic acid sequence on the read alignment of the nucleic acid sequence G that is non-unique, U _y is The number of reads that are unique to the nucleic acid sequence j.

The comparison can be performed using known comparison software, such as SOAP, BWA, and TeraMap. In the comparison process, the parameters are generally compared, and one or a pair of reads are allowed to allow up to k base errors. Mismatch, for example, setting k ≤ 2, if more than k bases in the reads are mismatched, it is considered that the reads cannot be aligned (aligned) to the nucleic acid sequence. The so-called obtained alignment results include the alignment of each read with each nucleic acid sequence, including whether the read can match a certain nucleic acid sequence or a certain nucleic acid sequence, only uniquely aligned to a nucleic acid sequence or aligned Information such as a plurality of nucleic acid sequences, alignment to a nucleic acid sequence, alignment to a unique position of the nucleic acid sequence, or multiple positions. According to one embodiment of the invention, the alignment is performed using SOAPalign 2.21 with the parameter set to –r 2–m 100–x 1000. The reads are aligned with the nucleic acid sequences in the nucleic acid sequence cluster, and the alignment can be divided into two parts: a) a unique read of the previous nucleic acid sequence, said reads are Unique reads (U); b) ratio For multiple nucleic acid sequences, these reads are referred to as Multiple reads (M). For a given nucleic acid sequence G, the abundance is Ab(G), which is related to Unique reads and Multiple reads. Ab(U) and Ab(M) in the above formula are the Unique reads and Multiples of the assembly fragment G, respectively. The abundance of reads. For each multiple reads, there is a unique abundance coefficient Co. Assuming multiple reads of the N nucleic acid sequences, the Co of the multiple reads can be calculated using the following formula:

That is, for such multiple reads, the sum of the abundances of the unique reads of the N sequences on which they are compared is used as the denominator.

The abundance of a cluster of nucleic acid sequences is related to the abundance of the nucleic acid sequences contained therein, which according to one embodiment of the invention, the abundance of the cluster of nucleic acid sequences is the mean or median of the abundance of the nucleic acid sequences contained therein.

Determine the status of the individual.

Comparing the abundance of the nucleic acid sequence cluster in the fecal sample of the individual with the abundance in the control group, determining the state of the individual based on whether the difference is statistically significant, the control group consisting of a group or A plurality of sets of stool samples of individuals of the same state, including those having colorectal cancer and not having colorectal cancer.

According to an embodiment of the present invention, the control group is composed of a stool sample of a plurality of individuals suffering from colorectal cancer, when the nucleic acid sequence cluster is abundant in the stool sample of the individual and its abundance in the control group When there is no statistical difference, the state of the individual is determined to have colorectal cancer.

According to an embodiment of the invention, the control group consists of a stool sample of a plurality of healthy individuals, when the abundance of the nucleic acid sequence cluster in the individual's stool sample is statistically lower than it is in the control When the abundance in the group is determined, the state of the individual is determined to have colorectal cancer.

The so-called nucleic acid sequence cluster abundance of the individual with or without statistical significance is determined to fall within or fall within a predetermined confidence interval for its abundance in the control group. The nucleic acid sequence cluster abundance of a statistically lower than determined individual is less than the lower bound of a predetermined confidence interval for its abundance in the control group. The so-called confidence interval refers to the sample The estimated range of the overall parameters constructed by the statistic. In statistics, the Confidence interval of a probability sample is an interval estimate of a population parameter for this sample. The confidence interval shows the extent to which the true value of this parameter has a certain probability of falling around the measurement. The confidence interval gives the degree of confidence in the measured value of the measured parameter, ie the "certain probability" required previously. This probability is called the confidence level. According to an embodiment of the invention, the so-called predetermined confidence interval is a 95% confidence interval, i.e., the use of this embodiment to determine that the individual is in the determined state, 95% is reliable. It should be noted that, depending on the purpose or requirement, there may be different requirements for determining the degree of credibility of the individual state result, and those skilled in the art may select different levels of significance (α), that is, select different probability of making mistakes. Thus, the determined degree of identity of the individual is 1-α.

According to an embodiment of the present invention, when the control group is a sample from a colorectal cancer patient, the so-called non-statistical meaning means that the abundance of the determined nucleic acid sequence cluster falls into the first of its abundance in the control group. Predetermined confidence interval. According to an embodiment of the present invention, the first predetermined confidence interval is a 95% confidence interval, and the first predetermined confidence interval of the abundance of the first nucleic acid sequence cluster in the control group is 2.72E-08 to 7.02E-07, The first predetermined confidence interval for the abundance of the second nucleic acid sequence cluster in the control group is 4.84E-07 to 1.61E-06.

All or part of the steps of the method for determining the state of an individual using the separated nucleic acid in any of the above embodiments of the present invention may be performed using a device/system comprising a detachable corresponding unit function module, or the method may be programmed, It is stored on a machine readable medium and is implemented by a machine running the readable medium.

An apparatus for determining the state of an individual using the isolated nucleic acid of any of the above embodiments, according to an embodiment of the present invention, the apparatus for performing all or part of the steps of any of the methods for determining the state of the individual, the apparatus The method includes: abundance determining unit, configured to determine abundance of a nucleic acid sequence cluster in the nucleic acid in the septic sample of the individual and abundance in a control group; and an individual state determining unit for comparing the nucleic acid sequence cluster The difference between the abundance in the individual's stool sample and the abundance in the control group, and determining the status of the individual based on whether the difference is statistically significant, the control group being one or more sets of the same state A stool sample of an individual consisting of having colorectal cancer and not having colorectal cancer. The above description of the technical features and advantages of the method for determining the state of an individual using the separated nucleic acid in any of the embodiments of the present invention is equally applicable to the apparatus of this aspect of the present invention and will not be described herein.

According to an embodiment of the present invention, the following is performed in the abundance determining unit: obtaining nucleic acid in the fecal sample of the individual and sequencing data of the nucleic acid in the control group, and sequencing data of any source includes a plurality of reads Determining the abundance of the nucleic acid sequence in the fecal sample of the source according to the support of the nucleic acid sequence in the nucleic acid sequence cluster by the reads in the sequencing data from any source; according to the nucleic acid sequence at the source The abundance in the stool sample determines the abundance of the cluster of nucleic acid sequences in which it is located in the same stool sample.

According to an embodiment of the invention, the abundance of the nucleic acid sequence in the stool sample of the source is determined according to the support of the read sequence in the sequencing data of any source to the nucleic acid sequence in the nucleic acid sequence cluster, comprising: The reads are aligned to the nucleic acid sequence, and based on the obtained alignment results, the abundance of the nucleic acid sequence is determined using the following formula: Abundance of nucleic acid sequence G Ab(G)=Ab(U _G )+ Ab(M _G ), wherein Ab(U _G )=U _G /l _G , U _G is the only number of reads of the nucleic acid sequence G, and l _{G is} the length of the nucleic acid sequence G,

According to an embodiment of the invention, the abundance of the cluster of nucleic acid sequences is the mean or median of the abundance of the nucleic acid sequences contained therein.

According to an embodiment of the present invention, the control group is composed of a stool sample of a plurality of individuals suffering from colorectal cancer, and the abundance of the nucleic acid sequence cluster in the stool sample of the individual has its abundance in the control group When there is no statistically significant difference in abundance, it is determined that the state of the individual is suffering from colorectal cancer.

According to an embodiment of the invention, the control group consists of a stool sample of a plurality of healthy individuals, when the abundance of the nucleic acid sequence cluster in the individual's stool sample is statistically lower than it is in the control When there is no statistical difference in abundance in the group, it is determined that the state of the individual is suffering from colorectal cancer.

The so-called nucleic acid sequence cluster abundance of the individual with or without statistical significance is determined to fall within or fall within a predetermined confidence interval of its abundance in the control group. The nucleic acid sequence cluster abundance of a statistically lower than determined individual is less than the lower bound of a predetermined confidence interval for its abundance in the control group. The so-called confidence interval refers to the estimated interval of the population parameters constructed by the sample statistic. In statistics, the Confidence interval of a probability sample is an interval estimate of a population parameter for this sample. The confidence interval shows the extent to which the true value of this parameter has a certain probability of falling around the measurement. The confidence interval gives the degree of confidence in the measured value of the measured parameter, ie the "certain probability" required previously. This probability is called the confidence level. According to an embodiment of the invention, the so-called predetermined confidence interval is a 95% confidence interval, i.e., the use of this embodiment to determine that the individual is in the determined state, 95% is reliable. It should be noted that, depending on the purpose or requirement, there may be different requirements for determining the degree of credibility of the individual state result, and those skilled in the art may select different levels of significance (α), that is, select different probability of making mistakes. Thus, the determined degree of identity of the individual is 1-α.

According to one embodiment of the present invention, there is provided an isolated nucleic acid according to any of the above embodiments of the present invention A system for determining the state of an individual, the system for performing all or part of the steps of the method for determining the state of an individual using the isolated nucleic acid of any of the above embodiments in any of the above embodiments of the invention, the system comprising: data input a module for inputting data; a data output module for outputting data; a processor for executing an executable program, the executing the executable program comprising the method of determining the state of the individual in any of the embodiments of the present invention described above; a storage unit, coupled to the data input module, the data output module, and the processor, for storing data, including the executable program. The above description of the technical features and advantages of the method for determining the state of an individual using the isolated nucleic acid in any of the embodiments of the present invention is equally applicable to the system of this aspect of the present invention and will not be described herein.

According to one embodiment of the present invention, there is provided a medicament for treating colorectal cancer which promotes an increase in the abundance of the isolated nucleic acid in any of the above embodiments in the intestinal tract of a patient. The so-called isolated nucleic acid is determined by the inventors by analyzing the sequencing data of the intestinal microbial sample, comparing the abundance of the intestinal microbial sequence of the colorectal cancer patient population and the healthy population, and then determining by a large number of sample tests. Each nucleic acid sequence cluster it contains is a non-redundant sequence. The isolated nucleic acid can be used as a colorectal cancer marker, and the colorectal cancer marker is significantly enriched in a healthy population compared with the colorectal cancer patient group, and the so-called significant enrichment refers to abundance in the disease control group. In comparison, the abundance of the nucleic acid sequence clusters contained in the above-mentioned colorectal cancer markers in the healthy individual group is statistically higher than or significantly higher than the abundance in the disease group. The set of isolated nucleic acids can be used to determine the probability of an individual having a high or low probability of being in a state of colorectal cancer, and can be used for non-invasive early detection or assisted detection of colorectal cancer; The increased substance can be used for treating colorectal cancer or for patients suffering from colorectal cancer, and the substance capable of increasing the abundance of the isolated nucleic acid is not limited to a drug for treating colorectal cancer and a functional food for benefiting the intestinal flora. The isolated pair of nucleic acids provided by this embodiment can be used for the preparation of a medicament for the treatment of colorectal cancer and/or for the preparation of functional foods, health care drugs and the like which are beneficial to the balanced intestinal flora.

By using the drug or functional food of this embodiment, the determined colorectal cancer sequence marker can be reasonably and effectively applied, the growth of beneficial bacteria or sequences of the intestinal tract can be supported, and/or the intestinal pathogenic bacteria or sequence can be inhibited, and the intestinal tract can be blocked. Defects in the barrier, improving and restoring the intestinal micro-ecological structure are important for assisting in reducing blood endotoxin levels and/or reducing the clinical symptoms of colorectal cancer.

According to an embodiment of the present invention, there is further provided a method of producing or screening the above-described medicament, which comprises the step of screening for a substance which causes an increase in the abundance of the isolated nucleic acid in any of the above embodiments as the medicament.

By using the method for producing or screening for colorectal cancer in the embodiment of the present invention, by appropriately and effectively applying the determined colorectal cancer biomarker for screening, it is possible to obtain growth capable of supporting intestinal beneficial bacteria and/or inhibit intestinal tract. Drugs with potential pathogens can prevent intestinal barrier defects, improve and restore intestinal micro-ecological structure, and are important for assisting in reducing blood endotoxin levels and/or reducing the clinical symptoms of colorectal cancer.

According to a last embodiment of the present invention, there is provided a method of classifying a plurality of individuals using the isolated nucleic acid of any of the above-described embodiments of the present invention, the method comprising: utilizing the determined individual in any of the above embodiments of the present invention, respectively The state of the method determines the state of each individual; each individual is classified according to the state of each individual obtained. The method can distinguish a plurality of individuals according to different states of the individual or distinguish a plurality of unknown stool samples, which is convenient for classification and label management. In addition, the above description of the technical features and advantages of the method for determining the state of an individual using the isolated nucleic acid of any of the embodiments in any of the embodiments of the present invention is equally applicable to the method of this aspect of the present invention, and no further description is provided herein. .

The method and/or apparatus of the present invention is described in detail below in conjunction with the specific embodiments. Unless otherwise stated, the reagents, sequences (linkers, tags, and primers), software, and instruments not specifically addressed in the following examples are conventionally commercially available or open source, such as the Illumina transcriptome library construction kit. .

The following embodiments include a first phase and a second phase, namely a corresponding discovery phase and a verification phase. The discovery phase consisted of analyzing and comparing data sets of 53 colorectal cancer patients, 42 colon adenoma patients, and 61 healthy controls to determine intestinal microbial composition and functional changes to identify species markers; the validation phase included: A data set of colorectal cancer patients and 19 healthy controls was used to verify the accuracy of the first-stage predictions.

Example 1

In this example, the inventors performed a cohort analysis of the entire gut microbiota from 53 colorectal cancer patients, 42 colon adenoma patients, and 61 healthy control stool samples to describe fecal microbial community and functional component characteristics. In total, the inventors downloaded high-quality sequencing data of approximately 1084.87 Gb to construct a colorectal cancer reference gene set. Quantitative metagenomic analysis showed that 2,464,280 genes could be clustered into 258 genes representing bacterial species (Co-abundance gene groups, CAG) in a large number of patients and healthy controls, while those in colorectal cancer patients were compared with healthy controls. There were 26 differences in the performance of the group, of which 17 CAG were enriched in the colorectal cancer patient group and 9 CAG were enriched in the healthy group, as shown in Table 1. Of the 26 CAGs, only 9 were annotated, and 17 were unspecified species, which were unidentified new species.

1. Acquisition of sequencing data

1.1 Sample collection and DNA extraction

The sequencing data of the first stage of colorectal cancer patients, colon adenoma patients and healthy human stool samples were obtained from the EBI database, data number: ERP005534 (Zeller G et al., 2014), of which 53 patients with colorectal cancer, colon adenoma There were 42 patients and 61 healthy people, all from France. On average, each sample produced 5Gb high-quality sequencing results, totaling 1084.87Gb of sequencing data.

The sequencing data of the second stage colorectal cancer patients and healthy human stool sample DNA were derived from the EBI database. Data compilation No.: ERP005534 (Zeller G et al., 2014), including 38 patients with colorectal cancer and 5 healthy people from Germany; data number: ERA000116 (Qin et al., 2010), of which 14 were healthy people from Spain.

Referring to the experimental protocol of Figure 1, the relevant microbial markers of colorectal cancer are identified, with the omitted steps or details being well known to those skilled in the art, and several important steps are described in the following examples.

2. Identification of biomarkers

2.1 Basic processing of sequencing data

EBI raw data has been processed by quality control and de-hosted data, but there are many short reads in the data. After downloading from EBI to data, pairs of raw data in the original data are paired with less than 60 reads.

2.2 Obtaining the colony of the colorectal cancer microbial group

The genome of the metagenomic biomarker is a gene and a corresponding function, so it is necessary to assemble and predict the sequence of the sequence, to redundantly, and to construct a non-redundant reference gene set. All sample reads were assembled into contigs (assembly fragments or contigs) using SOAPdenovo software. Finally, 8.98 million contigs were generated from 64.06% of the total number of reads (the minimum fragment length was 500 bp). These contigs have a total length of 18.8 Gb, and the N50 has a length ranging from 1,253 to 18,741 bp and an average length of 4,773 bp.

To predict each sample microbial gene in 156 samples, the inventors used the method in the MetaHIT Human Intestinal Genome Study. The MetaGeneMark program predicted from 26,039,803 open reading frames (ORFs) that were greater than 100 bp in length. The predicted total length of ORFs was 16,095,621,987 bp, accounting for 85.61% of the total length of contigs. A non-redundant "CRC gene set" is created by removing excess ORFs, defining that the identity of the paired sequence is over 95% and the short ORFs with a sequence coverage of more than 90% are the same sequence, removing the excess ORFs. De-redundancy, that is, one of them is randomly reserved for the same sequence. The final non-redundant colorectal cancer gut gene set contains 6,585,575 ORFs with an average length of 609.70 bp.

2.3 Gene abundance analysis

The paired paired-end reads treated in step 2.1 were aligned (matched) to the non-redundant reference gene set in 2.2 using SOAPalign 2.21 with the parameter –r 2–m 100–x 1000. The alignment of Reads with non-redundant reference gene sets may be divided into two parts: a) Unique reads (U): reads are aligned only with one gene in a non-redundant gene set; these reads are defined as unique reads. b) Multiple reads (M): If reads compares more than one gene in a non-redundant gene set, it is defined as multiple reads.

For a given gene G, its abundance is Ab(G), associated with U reads and M reads, and the abundance is calculated as follows:

Ab(S)=Ab(U)+Ab(M)

Ab(U)=U/l

Ab (U) and Ab (M) are the abundances of the unique reads and multiple reads of the gene G, respectively, and l represents the length of the gene G. For each multiple reads, there is a unique gene abundance coefficient Co; assuming that a multiple reads match the N genes, calculate the Co of the multiple reads as follows:

That is, for multiple reads, the inventor uses the sum of the unique reads abundance of the N genes on the comparison as the denominator.

2.4 Cluster analysis / screening of colorectal cancer biomarkers

In order to conduct intestinal metagenomics studies in healthy individuals (88 patients, including patients with small colon adenomas) and colorectal cancer patients (53 patients), a study to identify unknown species was performed in the de-redundant gene set. In order to explore and understand the known and unknown species associated with colorectal cancer, genes with different abundances were first identified based on the gene set of 156 samples, and then the genes were clustered according to the abundance table. The gene abundance of the same species in the same individual is similar, and the genes of the same species in different individuals are different, so the genes of the same species can be clustered by abundance consistency. The resulting gene cluster represents the metagenomic species (CAG). Based on the CAG found, a known or unknown species associated with colorectal cancer is identified.

Briefly, genes detected in at least 10 samples are first screened from the gene abundance table as input to the cluster. The first clustering was performed using the canopy algorithm. The T1 threshold was Pearson correlation coefficient >0.95 and the Spearman correlation coefficient was >0.6. The T2 threshold was Pearson correlation coefficient >0.9.

After the first clustering, in order to correct the fragmentation of the first cluster, the canopy with only one gene was removed and the second cluster was performed. The input used in the second clustering is the average abundance of canopy after the first clustering. The algorithm used is the canopy-like algorithm. The condition that the new element (gene sequence) is added to the original class is 70% of the class. The above elements satisfy the threshold Pearson correlation coefficient > 0.97. After obtaining the results of the quadratic clustering, it is necessary to remove the overlapping parts of the cluster, and for each gene existing in the plurality of classes, the class with the largest correlation coefficient with Pearson is added.

The actual operation and results of the example: 2,464,280 genes selected from the gene abundance table were added to the cluster, and 1,110,070 canopy were obtained after the first clustering, of which 254,018 canopy had more than one gene, and participated in the first Secondary clustering. After the second cluster is completed, 170,203 CAGs are obtained, of which CAG has more than 100 genes. There were 1,546 CAG with 285 genes with more than 700 genes. Based on the average abundance of 285 CAGs with a gene number greater than 700, the rank sum test using the Benjamini Hochberg multiple test was used, the threshold was fdr<0.05, and 9 CAGs were found to be enriched in healthy individuals, and 17 CAGs were found in patients with colorectal cancer. Enriched, which contained 51,472 and 8,374 genes, respectively.

In order to prove that the genes in the gene cluster belong to the genome and are consistent with the CAG classification annotation, blat analysis was performed on 6006 genomes, with reference to the effective reference genome in the third edition of NCBI as of August 2012 and the DACC intestinal tract of HMP and MetaHIT. Genome. When there is only one species, more than 90% of the genes in the CAG are aligned to the genome, and the alignment is 90% of the shorter ORF, and the similarity is 95%. The known genome is noted by CAG. . Of the 26 CAGs with a gene number greater than 700, 9 were classified at the species level, and 17 were unable to annotate the species, indicating that these were unpublished new species, as shown in Table 1. Marker gene uniform annotation validates cluster quality and applies to the entire CAG gene.

Table 1 Species annotation of differential CAG

2,464,280 genes were partially clustered into 285 CAGs, and the abundance of 26 CAGs was significantly different between healthy and colorectal cancer patients. Nine CAGs contained 51,472 genes enriched in healthy individuals, and 17 CAGs contained 8,374 genes enriched in colorectal cancer patients. Of these, 17 were unable to annotate the species and were unidentified new species.

Example 2

Taking the significance level α=0.05, the partial nucleic acid sequence of the marker significantly enriched in the colorectal cancer patient or healthy population determined in Example 1 represents the CAG in which it is located, and the partial marker in the healthy group of the validation group and The difference in abundance of the disease group was also significant (P<0.05), and the results of the verification are shown in Table 2 and Figure 2.

In the validation set, it was found that all 258 CAGs maintained abundance consistency, and these gene clusters were considered to represent all genes or partial genes of a certain strain. For the 26 CAGs that were associated with colorectal cancer in the experimental set, this example still found significant differences between the two (p < 0.05), as shown in Table 2 and Figure 2. The abundance of these two CAGs is the average of the abundances of all the genes they contain, as shown in Table 3.

Table 2

table 3

Example 3

57 fecal samples were used to detect the individual status of the sample source.

The abundance of at least one CAG (strain) of Table 2 in each stool sample was determined according to the method of Example 2, and it was judged whether the abundance of the cag(s) in each sample was significantly lower than that in the control group (health) The abundance in the group), for example, in the 95% confidence interval of CAG16610 and/or CAG28666 in the colorectal cancer patient group in Table 3 above, the individual was judged to be a colorectal cancer patient, and in the above Table 3 CAG16610 and/or CAG28666 were judged to be non-colorectal cancer patients in the 95% confidence interval of the healthy group.

The results show that for the detection of judgment using only any of the cag in Table 2, about 55 samples can be judged for individual state, and more than 80% of the samples of 55 samples correspond to the state of the individual. , consistent with the state of the recorded sample source individuals; for the individual state detection using all two cags in Table 2, 48 individual samples can be judged for individual status, and, for more than 48 samples 90% of the samples correspond to the judgment of the individual's state, consistent with the recorded state of the sample source individual. That is, the inventors found that the joint detection of all the species in Table 2, that is, the detection of all the two markers in Table 2 in the sample to be tested are not enriched, can more accurately determine the colorectal cancer patients or easy to find Feeling the crowd.

In the treatment of colorectal cancer using markers, the inventors found that all of the two markers in Table 2 were enriched and the therapeutic effect was excellent.

In the description of the present specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" and the like means a specific feature described in connection with the embodiment or example. A structure, material or feature is included in at least one embodiment or example of the invention. In the present specification, the schematic representation of the above terms does not necessarily mean the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be in any One or more embodiments or examples are combined in a suitable manner.

While the embodiments of the present invention have been shown and described, the embodiments of the invention may The scope of the invention is defined by the claims and their equivalents.

Claims

A set of isolated nucleic acids comprising at least one of the following two clusters of nucleic acid sequences:

a first nucleic acid sequence cluster, the nucleic acid sequence in the first nucleic acid sequence cluster is in one-to-one correspondence with the sequence shown in SEQ ID NO: 1-151, and the nucleic acid sequence in each of the first nucleic acid sequence clusters and its corresponding SEQ The sequence similarity of the sequence in ID NO: 1-151 is not less than 90%;

a second nucleic acid sequence cluster, the nucleic acid sequence in the second nucleic acid sequence cluster is in one-to-one correspondence with the sequence shown in SEQ ID NO: 152-233, and the nucleic acid sequence in each of the second nucleic acid sequence clusters and its corresponding SEQ The sequence similarity of the sequences in ID NO: 152-233 is not less than 90%.
The nucleic acid of claim 1 wherein said nucleic acid comprises said first nucleic acid sequence cluster.
The nucleic acid according to claim 2, wherein the nucleic acid sequence in each of said first nucleic acid sequence clusters has a sequence similarity to a sequence in SEQ ID NO: 1-151 corresponding thereto of not less than 95%.
The nucleic acid of claim 1 wherein said nucleic acid comprises said second nucleic acid sequence cluster.
The nucleic acid according to claim 4, wherein the nucleic acid sequence in each of said second nucleic acid sequence clusters has a sequence similarity to the sequence of SEQ ID NOS: 152-233 of not less than 95%.
The nucleic acid according to claim 2 or 4, characterized in that the nucleic acid further comprises the other of the two clusters of nucleic acid sequences.
Use of a nucleic acid according to any of claims 1-6 for detecting colorectal cancer, and/or for treating colorectal cancer, and/or for preparing a medicament for treating colorectal cancer and/or for preparing a functional food.
A method of obtaining a nucleic acid according to any one of claims 1 to 6, characterized in that it comprises:

(1) obtaining the first sequencing result and the second sequencing result,

The first sequencing result is a sequencing result of nucleic acid of a stool sample of a plurality of colorectal cancer patients, including a plurality of first readings,

The second sequencing result is a sequencing result of nucleic acid of a stool sample of a plurality of healthy individuals, including a plurality of second reads;

(2) assembling the first read segment and the second read segment respectively, correspondingly obtaining a plurality of first assembly sequences and a plurality of second assembly sequences;

(3) determining the abundance of the first assembly sequence and the abundance of the second assembly sequence based on the support of the first assembly sequence for the first read sequence and the support of the second assembly sequence for the second read sequence, respectively;

(4) clustering the first assembly sequence and the second assembly sequence according to the abundance of the first assembly sequence determined in (3) and the abundance of the second assembly sequence, to obtain a plurality of gene clusters, each of which is The gene cluster includes a plurality of first assembly sequences and / Or a second assembly sequence;

(5) A statistical test to determine a gene cluster significantly enriched in the stool samples of the plurality of colorectal cancer patients and/or the plurality of healthy individuals to obtain the nucleic acid.
The method of claim 8 wherein (2) comprises:

After assembling the first read segment and the second read segment respectively, respectively, the obtained assembly result of the first read segment and the assembly result of the second read segment are respectively de-redunded to obtain the plurality of first assembly sequences and the second The assembled sequence defines an assembly sequence in which the sequence identity after pairing is not less than 95% and the sequence coverage exceeds 90%.
The method of claim 8 wherein (3) comprises:

Comparing the first read segment and the second read segment to the first assembly sequence and the second assembly sequence respectively, respectively obtaining a first comparison result and a second comparison result;

Abundance of the first assembly sequence and the second assembly sequence is determined using the following formula based on the first alignment result and the second alignment result, respectively:

Abundance Ab(S)=Ab(U S )+Ab(M S ) of the assembly sequence S, wherein

Ab(U S )=U S /l S ,

U S is the only number of reads that align the assembly sequence S,

l S is the length of the assembly sequence S,

M S nonunique than the read section of the assembly of the sequence number S,

i represents the number of the read of the assembly sequence S that is not uniquely aligned,

Co i is a non-unique comparison of the abundance coefficients corresponding to the read i of the assembly sequence S,

N1 is the non-unique comparison of the total number of assembled sequences on the read alignment of the assembly sequence S,

j is the number of the assembly sequence on the read alignment of the assembly sequence S that is not uniquely aligned,

U j is the number of reads that uniquely align the assembly sequence j.
The method of claim 8 wherein prior to (4), removing the first assembly sequence and the second assembly sequence that are only present in less than one-fifth of the stool sample of the total number of stool samples.
The method of claim 8 wherein (4) comprises:

Performing a first clustering on the first assembly sequence and the second assembly sequence to obtain a first clustering result;

Performing a second clustering on the first clustering result after removing the cluster containing only one assembly sequence to obtain the plurality of gene clusters.
A method for determining the state of an individual using the nucleic acid of any of claims 1-6, comprising:

Determining the abundance of the nucleic acid sequence cluster in the nucleic acid in the fecal sample of the individual and the abundance in the control group;

Comparing the abundance of the nucleic acid sequence cluster in the fecal sample of the individual with the abundance in the control group, determining the state of the individual based on whether the difference is statistically significant,

The control group consists of one or more sets of stool samples from individuals of the same state,

Such conditions include having colorectal cancer and not having colorectal cancer.
The method of claim 13 wherein the abundance of the nucleic acid sequence cluster in the individual's stool sample and/or in the control group is determined by:

Obtaining sequencing data of the nucleic acid sequence in the fecal sample of the individual and the nucleic acid sequence in the control group, and the sequencing data of any source includes a plurality of reads;

Determining the abundance of the nucleic acid sequence in the stool sample of the source based on the support of the reads in the sequencing data from any source for each nucleic acid sequence in the nucleic acid sequence cluster;

Based on the abundance of the nucleic acid sequence in the stool sample of the source, the abundance of the cluster of nucleic acid sequences in which it is located is determined in the same stool sample.
The method of claim 14 wherein determining the abundance of said nucleic acid sequence based on the support of a read in said sequence of sequencing data for each nucleic acid sequence in said sequence of nucleic acid sequences comprises:

Comparing the reads to the nucleic acid sequence,

Based on the obtained alignment results, the abundance of the nucleic acid sequence is determined using the following formula:

Abundance of nucleic acid sequence G Ab(G)=Ab(U G )+Ab(M G ), wherein

Ab(U G )=U G /l G ,

U G is the only number of reads that align the nucleic acid sequence G,

l G is the length of the nucleic acid sequence G,

M G than the read section of the non-unique nucleic acid sequences of the G number,

x represents the number of the read of the nucleic acid sequence G that is not uniquely aligned,

Co x is a non-unique comparison of the abundance coefficient corresponding to the read x of the nucleic acid sequence G,

N2 is the total number of nucleic acid sequences in the nucleic acid sequence cluster on the alignment of the nucleic acid sequence G that is not uniquely aligned,

y is a non-unique number that aligns the nucleic acid sequence on the read alignment of the nucleic acid sequence G,

U y is the number of reads that uniquely align the nucleic acid sequence j.
The method of claim 14 wherein the abundance of said sequence of nucleic acid sequences is the mean or median of the abundance of the nucleic acid sequences contained therein.
The method of claim 13 wherein said control group consists of a stool sample of a plurality of individuals having colorectal cancer,

When the abundance of the nucleic acid sequence cluster in the fecal sample of the individual is not statistically different from its abundance in the control group, it is determined that the state of the individual is suffering from colorectal cancer.
The method of claim 13 wherein said control group consists of stool samples from a plurality of healthy individuals.

When the abundance of the nucleic acid sequence cluster in the fecal sample of the individual is statistically lower than its abundance in the control group, the state of the individual is determined to be suffering from colorectal cancer.
A device for determining the state of an individual using the nucleic acid of any of claims 1-6, comprising:

An abundance determining unit for determining abundance of a nucleic acid sequence cluster in the nucleic acid in the fecal sample of the individual and abundance in a control group;

An individual state determining unit configured to compare a difference in abundance of the nucleic acid sequence cluster in a stool sample of the individual with abundance in a control group, and determine a state of the individual based on whether the difference is statistically significant,

The control group consists of one or more sets of stool samples from individuals of the same state,

Such conditions include having colorectal cancer and not having colorectal cancer.
The apparatus of claim 19, wherein said abundance determining unit performs the following:

Obtaining sequencing data of the nucleic acid sequence in the fecal sample of the individual and the nucleic acid sequence in the control group, and the sequencing data of any source includes a plurality of reads;

Determining the abundance of the nucleic acid sequence in the stool sample of the source based on the support of the read sequence in the sequencing data of any source for the nucleic acid sequence in the nucleic acid sequence cluster;

Based on the abundance of the nucleic acid sequence in the stool sample of the source, the abundance of the cluster of nucleic acid sequences in which it is located is determined in the same stool sample.
The device of claim 20, wherein said nucleic acid is read according to a read in sequencing data from any source The support of the nucleic acid sequence in the sequence cluster determines the abundance of the nucleic acid sequence in the stool sample of the source, including:

Comparing the reads to the nucleic acid sequence,

Based on the obtained alignment results, the abundance of the nucleic acid sequence is determined using the following formula:

Abundance of nucleic acid sequence G Ab(G)=Ab(U G )+Ab(M G ), wherein

Ab(U G )=U G /l G ,

U G is the only number of reads that align the nucleic acid sequence G,

l G is the length of the nucleic acid sequence G,

M G than the read section of the non-unique nucleic acid sequences of the G number,

x represents the number of the read of the nucleic acid sequence G that is not uniquely aligned,

Co x is a non-unique comparison of the abundance coefficient corresponding to the read x of the nucleic acid sequence G,

N2 is the total number of nucleic acid sequences in the nucleic acid sequence cluster on the aligned pair of non-uniquely aligned pairs of the nucleic acid sequence G,

y is a non-unique number that aligns the nucleic acid sequence on the read alignment of the nucleic acid sequence G,

U y is the number of reads that uniquely align the nucleic acid sequence j.
18. Apparatus according to claim 18 wherein the abundance of said sequence of nucleic acid sequences is the mean or median of the abundance of the nucleic acid sequences contained therein.
The device of claim 17, wherein said control group consists of a stool sample of a plurality of individuals having colorectal cancer,

When the abundance of the nucleic acid sequence cluster in the fecal sample of the individual is not statistically different from its abundance in the control group, it is determined that the state of the individual is suffering from colorectal cancer.
The device of claim 17 wherein said control group consists of stool samples from a plurality of healthy individuals.

When the abundance of the nucleic acid sequence cluster in the fecal sample of the individual is statistically less than its abundance in the control group, the state of the individual is determined to be suffering from colorectal cancer.
A system for determining the state of an individual using the nucleic acid of any of claims 1-6, comprising:

a data input module for inputting data;

a data output module for outputting data;

a processor for executing an executable program, the executing the executable program comprising performing the method of any one of claims 13-18;

a storage module coupled to the data input module, the data output module, and the processor for storing data, including the executable program.
A medicament for treating colorectal cancer, characterized in that the medicament promotes an increase in the abundance of the nucleic acids of any of claims 1-6 in the intestinal tract of a patient.
A method of producing or screening a medicament according to claim 26, which comprises the step of screening for a substance which promotes an increase in abundance of the nucleic acid of any of claims 1-6 as said medicament.
A method of classifying a plurality of individuals using the nucleic acid of any of claims 1-6, comprising:

Determining the status of each individual using any of claims 13-18;

Each individual is classified according to the state of each individual obtained.