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CN115266662B - Hyperspectral sequencing method, hyperspectral sequencing system and gene sequencer - Google Patents

Hyperspectral sequencing method, hyperspectral sequencing system and gene sequencer Download PDF

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CN115266662B
CN115266662B CN202210660961.XA CN202210660961A CN115266662B CN 115266662 B CN115266662 B CN 115266662B CN 202210660961 A CN202210660961 A CN 202210660961A CN 115266662 B CN115266662 B CN 115266662B
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base
light
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CN115266662A (en
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陈龙超
梁倩
张芬
王谷丰
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Shenzhen Sailu Medical Technology Co ltd
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing
    • C12Q1/6874—Methods for sequencing involving nucleic acid arrays, e.g. sequencing by hybridisation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/01—Arrangements or apparatus for facilitating the optical investigation

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Abstract

The embodiment of the invention discloses a hyperspectral sequencing method, a hyperspectral sequencing system and a gene sequencer, wherein the hyperspectral sequencing method comprises the following steps: emitting laser to the sample to be detected so that bases in the sample to be detected generate fluorescence under the excitation of the laser; collecting fluorescence generated after base excitation, diffracting and imaging the fluorescence to obtain a fluorescence image containing a corresponding fluorescence spectrum after diffraction; determining a target fluorescence spectrum in the fluorescence spectrum, and identifying target diffraction light which plays a role in light splitting in the target fluorescence spectrum in a fluorescence image; and obtaining the wavelength of the target base corresponding to the target fluorescence spectrum according to the target diffraction light, and determining the base class of the target base according to the wavelength. The embodiment of the invention has high shooting efficiency and accuracy, can record the fluorescence of four-color bases by one shot in a single channel, can realize gene sequencing without setting a complex optical structure, and has low system cost.

Description

Hyperspectral sequencing method, hyperspectral sequencing system and gene sequencer
Technical Field
The invention relates to the field of gene sequencing, in particular to a hyperspectral sequencing method, a hyperspectral sequencing system and a gene sequencer.
Background
Microscopic imaging techniques have found widespread use in sample detection, for example, gene sequencers require fluorescent imaging of bases during sequencing, after which the sequence of bases in DNA is obtained from the imaging results.
In the related art, the gene sequencing needs to be imaged in a multi-channel mode, and is commonly a four-channel imaging system or a two-channel imaging system, but no matter the four-channel imaging system or the two-channel imaging system is used, when the gene is detected, crosstalk can be generated between base fluorescent signals, so that the shooting efficiency is low, the accuracy is low, a plurality of sleeve lenses are required to be arranged for imaging respectively, and the cost of the system is increased.
Disclosure of Invention
The embodiment of the invention provides a hyperspectral sequencing method, a hyperspectral sequencing system and a gene sequencer, which have high shooting efficiency and accuracy and low system cost.
In a first aspect, embodiments of the present invention provide a hyperspectral sequencing method, including: emitting laser to a sample to be detected so that bases in the sample to be detected generate fluorescence under the excitation of the laser; collecting fluorescence generated after base excitation, diffracting and imaging the fluorescence to obtain a fluorescence image containing a corresponding fluorescence spectrum after diffraction; determining a target fluorescence spectrum in the fluorescence spectrum, and identifying target diffraction light which plays a role in light splitting in the target fluorescence spectrum in the fluorescence image; and obtaining the wavelength of a target base corresponding to the target fluorescence spectrum according to the target diffraction light, and determining the base class of the target base according to the wavelength.
In some embodiments, the sample to be tested is provided with a plurality of rows of DNA cluster arrays arranged in sequence, including a first DNA cluster array and a second DNA cluster array, the bases including bases in the first DNA cluster array and bases in the second DNA cluster array; collecting fluorescence generated after base excitation, diffracting and imaging the fluorescence to obtain a fluorescence image containing a corresponding fluorescence spectrum after diffraction, wherein the method comprises the following steps: sequentially collecting fluorescence generated after the base in the first DNA cluster array and the base in the second DNA cluster array are excited; diffracting and imaging the fluorescence generated by the base in the first DNA cluster array to obtain a diffracted fluorescence image, wherein the fluorescence image comprises a fluorescence spectrum corresponding to the luminous base in the first DNA cluster array; and diffracting and imaging the fluorescence generated by the bases in the second DNA cluster array in sequence to obtain a diffracted fluorescence image, wherein the other fluorescence image comprises a fluorescence spectrum corresponding to the luminous bases in the second DNA cluster array.
In some embodiments, the DNA cluster array includes a target DNA cluster, the target DNA cluster includes a plurality of the bases, including a first target base and a second target base, sequentially arranged on the target DNA cluster, the method further comprising: before the first target base is subjected to one-round base class detection, the first target base is dyed through fluorescent dye to obtain a corresponding first fluorescent group, and the corresponding first fluorescent group is synthesized into the target DNA cluster; and after the detection of the base class of the first target base is completed, carrying out another round of base class detection on the second target base, cutting off the first fluorescent group before the detection of the base class of the second target base, dyeing the second target base through fluorescent dye to obtain a corresponding second fluorescent group, and synthesizing the second fluorescent group into the target DNA cluster.
In some embodiments, in determining the target fluorescence spectrum to which the second target base corresponds, determining a target fluorescence spectrum in the fluorescence spectra comprises: obtaining synthetic spectrum characteristic information of a fluorescence spectrum corresponding to the second target base and sub-spectrum morphological characteristic information of the fluorescence spectrum; linear spectrum unmixing is carried out on the fluorescence spectrum according to the synthesized spectrum characteristic information and the sub-spectrum morphological characteristic information, and a plurality of sub-spectrums are obtained through analysis, wherein the sub-spectrums comprise a first sub-spectrum and a plurality of second sub-spectrums, and the brightness of the first sub-spectrum is higher than that of the second sub-spectrum; and determining the first sub-spectrum as the target fluorescence spectrum.
In some embodiments, the target diffracted light is a1 st order light in the target fluorescence spectrum that is spectrally active; the obtaining the wavelength of the target base corresponding to the target fluorescence spectrum according to the target diffraction light comprises the following steps: identifying level 0 light in a target fluorescence spectrum in the fluorescence image; calculating a relative distance between the level 0 light and the level 1 light of the target fluorescence spectrum; and obtaining the wavelength of the corresponding target base according to the relative distance.
In a second aspect, embodiments of the present invention also provide a hyperspectral sequencing system, comprising: the light source module is used for emitting laser; the optical path module comprises a dichroic mirror, an objective lens and a beam splitter; the dichroic mirror is arranged on a light path of the laser emitted by the light source module, and is used for receiving the laser and reflecting the laser; the objective lens is arranged on a light path of the laser reflected by the dichroic mirror, the objective lens is used for converging the laser and forming an illumination light spot on the sample to be detected so that bases in the sample to be detected generate fluorescence under the excitation of the laser, the objective lens is also used for receiving the fluorescence generated after the excitation of the bases and transmitting the fluorescence to the dichroic mirror, and the dichroic mirror is also used for transmitting the fluorescence; the beam splitter is arranged on the light path of the fluorescence transmitted by the two directions, and the beam splitter is used for diffracting the fluorescence; the imaging module is arranged on a light path of the fluorescence diffracted by the beam splitter, and is used for receiving the diffracted fluorescence and obtaining a fluorescence image containing a corresponding fluorescence spectrum; the processing module is used for determining a target fluorescence spectrum in the fluorescence spectrum and identifying target diffraction light which plays a role in splitting in the target fluorescence spectrum in the fluorescence image; and the method is used for obtaining the wavelength of the target base corresponding to the target fluorescence spectrum according to the target diffraction light, and determining the base class of the target base according to the wavelength.
In some embodiments, the sample to be tested is provided with a plurality of rows of DNA cluster arrays arranged in sequence, including a first DNA cluster array and a second DNA cluster array, the bases including bases in the first DNA cluster array and bases in the second DNA cluster array; the imaging module is further configured to sequentially image fluorescence excited by the bases in the first DNA cluster array and the bases in the second DNA cluster array, and includes: imaging the fluorescence generated by the base in the first DNA cluster array to obtain a diffracted fluorescence image, wherein the fluorescence image comprises a fluorescence spectrum corresponding to the luminous base in the first DNA cluster array; imaging the fluorescence generated by the bases in the second DNA cluster array in sequence to obtain another diffracted fluorescence image, wherein the other fluorescence image comprises a fluorescence spectrum corresponding to the luminous bases in the second DNA cluster array.
In some embodiments, the device further comprises a displacement table, wherein the displacement table is arranged on a light path of the objective lens converging the laser, and is used for placing the sample to be tested; the displacement table is also used for moving the sample to be detected, so that the imaging module images fluorescence excited by the base in the first DNA cluster array and the base in the second DNA cluster array in sequence.
In some embodiments, the target diffracted light is a1 st order light in the target fluorescence spectrum that is spectrally active; the processing module is further used for identifying 0-level light in a target fluorescence spectrum in the fluorescence image; and further for calculating a relative distance between the level 0 light and the level 1 light of the target fluorescence spectrum; and is also used for obtaining the wavelength of the corresponding target base according to the relative distance.
In some embodiments, the device further comprises a beam shaping module, wherein the beam shaping module is arranged on a light path of the laser emitted by the light source module, and the beam shaping module is used for shaping the laser into an illumination spot in a one-dimensional direction.
In some embodiments, the light path module further includes a filter disposed on a light path through which the dichroic mirror transmits the fluorescence, the filter for transmitting the fluorescence and filtering out residual excitation light mixed in the fluorescence.
In some embodiments, the optical path module further includes a barrel lens disposed on an optical path of the fluorescence diffracted by the beam splitter, the barrel lens configured to collect the diffracted fluorescence into the imaging module for imaging.
In a third aspect, embodiments of the present invention further provide a genetic sequencer comprising a hyperspectral sequencing system according to any one of the embodiments of the second aspect of the present invention.
The embodiment of the invention at least comprises the following beneficial effects: the embodiment of the invention provides a hyperspectral sequencing method, a hyperspectral sequencing system and a gene sequencer, wherein in the process of realizing gene sequencing by the hyperspectral sequencing method, the embodiment of the invention can emit laser to a sample to be tested so as to enable bases in the sample to be tested to generate fluorescence under the excitation of the laser, then collect fluorescence generated after base excitation, diffract and image the fluorescence, obtain a fluorescence image containing a corresponding fluorescence spectrum after diffraction, determine a target fluorescence spectrum corresponding to a target base in the fluorescence spectrum, identify target diffraction light with a light splitting function in the target fluorescence spectrum in the fluorescence image, judge the category of the target base according to the target emission light, then obtain the wavelength of the target base corresponding to the target fluorescence spectrum according to the target diffraction light, and different bases have different wavelengths, so that the base category of the target base can be determined according to the wavelength, realize gene sequencing.
Drawings
FIG. 1a is a schematic diagram of an imaging optical path of a four-channel sequencing system in the related art;
FIG. 1b is a schematic diagram of an imaging optical path of a dual channel sequencing system in the related art;
FIG. 2 is a schematic diagram of an imaging optical path of a hyperspectral sequencing system provided in one embodiment of the present invention;
FIG. 3 is a schematic diagram of an illumination spot illuminating a sample to be measured according to one embodiment of the present invention;
FIG. 4 is a schematic representation of a fluorescence image provided by one embodiment of the present invention;
FIG. 5 is a flow chart of a method for hyperspectral sequencing according to one embodiment of the present invention;
FIG. 6 is a flow chart of a method for hyperspectral sequencing according to another embodiment of the present invention;
FIG. 7 is a flow chart of a hyperspectral sequencing method according to another embodiment of the present invention
FIG. 8 is a flow chart of a method for hyperspectral sequencing according to another embodiment of the present invention;
FIG. 9 is a graph comparing fluorescence of a single dye and fluorescence of a mixed dye according to one embodiment of the invention;
FIG. 10 is a graph showing sub-spectral intensity after linear spectral unmixing provided by an embodiment of the invention;
FIG. 11 is a flow chart of a method for hyperspectral sequencing according to another embodiment of the present invention.
Detailed Description
The present invention will be described in further detail with reference to the drawings and examples, in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
It should be understood that in the description of the embodiments of the present invention, plural (or multiple) means two or more, and that greater than, less than, exceeding, etc. are understood to not include the present number, and that greater than, less than, within, etc. are understood to include the present number. If any, the terms "first," "second," etc. are used for distinguishing between technical features only, and should not be construed as indicating or implying a relative importance or implicitly indicating the number of technical features indicated or implicitly indicating the precedence of the technical features indicated.
Deoxyribonucleic acid (Deoxyr iboNuc leic Ac id, DNA) is simple in structure, and DNA molecules are composed of two long sugar chain structures that are bonded together by base pairs, just like a ladder. The whole molecule forms a double helix around its own central axis. In the base pairs forming the stable helix structure, 4 different bases are shared, and are called A (ADENINE adenine), T (THYMINE thymine), G (GUANINE guanine) and C (CYTOSINE cytosine) according to the initial letters of their English names. Each base is completely complementary to the chemical nature of the other base, so that A always pairs with T, G always pairs with C, and A pairs with U upon transcription.
In the sequencing process, the gene sequencer needs to perform fluorescence imaging on four bases of ATCG respectively, and then obtains the sequence of the bases in DNA according to the imaging result. Traditional sequencing methods require imaging by means of multiple channels, typically four-channel imaging systems, each channel corresponding to a single base. Another common scheme is a two-channel imaging system, which photographs fluorescence of two bases at a time, completing fluorescence imaging of four bases in two.
The applicant found that in the four-channel imaging system of the related art, as shown in fig. 1a, in order to realize the distinction of four different types of bases, different dichroic mirrors are required to be matched for light splitting, and different filters are matched for filtering the influence of stray light and residual excitation light from other channels on the signal of the channel. Because of the large number of base fluorescence types detected at the same time, crosstalk is inevitably generated between base fluorescence signals. In order to increase the signal quantity of the channel to the maximum extent, improve the accuracy, reduce the influence of other channels on the channel signal at the same time, the bandwidth of the optical filter of each channel needs to be strictly optimized. That is, the filter needs to be customized to meet the use requirement, and the cost of such customization is relatively high, which increases the cost of the system. Secondly, four-channel imaging system needs four the same cameras, and four the same sleeve lenses come formation of image respectively, and quantity is more, and shooting efficiency is low, and the cost is also higher.
Applicants have also found that in the dual channel imaging system of the related art, as shown in fig. 1b, there are fewer imaging channels than four channels, and the cost of the system is significantly reduced. However, there are various disadvantages, for example, since two channels are used to collect four-color base fluorescence, the filters of each channel must be double-bandpass, that is, each filter supports the passage of fluorescence of two bases, which saves cost but increases the risk of fluorescence signal crosstalk, for example, assuming that channel 1 is the fluorescence of detecting a base and G base, when detecting a base, since the absorption spectrum of the selected G base dye has a certain width, it is theoretically possible that the fluorescence is excited simultaneously by excitation light of a base (intensity is not high), which results in the fluorescence signal of G base entering channel 1 when detecting a base, causing interference to the recognition of a base; secondly, the two channels need to be shot twice to collect fluorescence of four bases, so that shooting time is doubled, shooting efficiency is low, and sequencing data flux in unit time is reduced; in addition, although the number of channels is reduced, two repeated imaging devices are still required to complete four-color base fluorescence imaging, increasing the system cost.
Based on the above, the embodiment of the invention provides a hyperspectral sequencing method, a hyperspectral sequencing system and a gene sequencer, which can overcome the problems in the prior art, abandons a light splitting mode of combining a dichroic mirror and an optical filter, directly obtains the complete spectrum of base fluorescence in a grating light splitting mode, can record the fluorescence of four-color bases by one-time shooting in a single channel, effectively distinguishes the four-color bases in a linear spectrum unmixing mode, and has high shooting efficiency and accuracy and low system cost.
In the embodiment of the invention, a gene sequencing chip is taken as a sample to be detected, four types of ATCG bases on the gene sequencing chip are taken as an example, the four types of bases are respectively dyed through different fluorescent dyes, and the four types of bases respectively excite four types of fluorescence with different wavelengths after being irradiated by laser.
The embodiment of the invention provides a hyperspectral sequencing method, a hyperspectral sequencing system and a gene sequencer, and firstly describes the hyperspectral sequencing system in the embodiment of the invention.
In one embodiment, a hyperspectral sequencing system in an embodiment of the present invention includes:
The light source module is used for emitting laser;
the optical path module comprises a dichroic mirror, an objective lens and a beam splitter;
the dichroic mirror is arranged on a light path of the laser emitted by the light source module, and is used for receiving the laser and reflecting the laser;
The objective lens is arranged on a light path of laser reflected by the dichroic mirror, and is used for converging the laser and forming an illumination light spot on the sample to be detected so that bases in the sample to be detected generate fluorescence under the excitation of the laser, and is also used for receiving the fluorescence generated after the base excitation and transmitting the fluorescence to the dichroic mirror, and the dichroic mirror is also used for transmitting the fluorescence;
the beam splitter is arranged on a light path of the two-way transmission fluorescence and is used for diffracting the fluorescence;
The imaging module is arranged on a light path of fluorescence diffracted by the beam splitter and is used for receiving the diffracted fluorescence and obtaining a fluorescence image containing a corresponding fluorescence spectrum;
The processing module is used for determining a target fluorescence spectrum in the fluorescence spectrum and identifying target diffraction light which plays a role in light splitting in the target fluorescence spectrum in the fluorescence image; the method is used for obtaining the wavelength of the target base corresponding to the target fluorescence spectrum according to the target diffraction light, and determining the base class of the target base according to the wavelength.
It should be noted that, as shown in fig. 2, the hyperspectral sequencing system in the embodiment of the present invention may be an imaging system, and specifically, the embodiment of the present invention may obtain a data cube of information of a sample to be measured, where the data includes not only spatial information but also spectral information of the sample to be measured. The light source module, the light path module, the imaging module and the processing module in the embodiment of the invention can be provided with a plurality of elements to execute functions required by the modules, and in one embodiment, the light source module is a laser, and the imaging module is a camera.
In the optical path system of fig. 2, laser 111 emits laser, the laser passes through an optical path to reach a dichroic mirror 121, the laser reaches an objective lens 122 after being reflected by the dichroic mirror 121, then the laser passes through the objective lens 122 and forms a linear illumination spot on a sample 131 to be measured, one-dimensional scanning imaging is performed on the sample 131 to be measured, after the sample 131 to be measured is excited, fluorescence emitted by a base dye on the sample 131 to be measured firstly passes through the objective lens 122 and is transmitted to a beam splitter 123 through the dichroic mirror 121 to be diffracted by the beam splitter 123, and diffracted fluorescence signals are converged to a camera 141 to be imaged to obtain a fluorescence image.
It should be noted that, the processing module in the embodiment of the present invention may process the fluorescent image so as to determine the base type of each base in the sample to be detected, specifically, the number of bases on the sample to be detected scanned in the embodiment of the present invention is several, taking fig. 3 as an example, the embodiment of the present invention may scan a plurality of bases at one time, the resulting fluorescent image is shown in fig. 4, each spectrum in the resulting fluorescent image is a fluorescent spectrum formed after diffraction of a different base, when the embodiment of the present invention needs to determine the type of a certain base in the scanned bases, i.e., the type of the target base, it is to be understood that, because the fluorescent spectrum of the corresponding position in the fluorescent image needs to be determined as the target fluorescent spectrum in the embodiment of the present invention, the scanning of the sample to be detected not only can scan the spectrum information thereof, because of the different positions of the bases on the sample to be detected, the space information can be scanned to obtain the target fluorescence spectrum corresponding to the target base, the target diffraction light with the light splitting function in the target fluorescence spectrum can be obtained sequentially, the wavelength of the target base corresponding to the target fluorescence spectrum can be obtained according to the target diffraction light, and the base type of the target base can be determined according to the wavelength, wherein the fluorescence excited by the base is diffracted through the light splitter, the diffracted fluorescence signal is divided into two parts, one part is a 0-level light signal without light splitting, and the other part is a high-level light signal with light splitting function, the high-level light signal with light splitting function is taken as the target diffraction light in the embodiment of the invention, the corresponding wavelength can be identified in the fluorescence spectrum through the target diffraction light, and the base type of the target base can be finally identified, so the hyperspectral sequencing system in the embodiment of the invention has high shooting efficiency and accuracy, the fluorescence of four-color bases can be recorded by shooting in a single channel, the gene sequencing can be realized without setting a complex optical structure, and the system cost is low.
In an embodiment, as shown in fig. 3, a sample 131 to be tested in the embodiment of the present invention is provided with a plurality of rows of DNA cluster arrays sequentially arranged, each row of DNA cluster array is provided with a plurality of DNA clusters, as shown in fig. 3, each row of DNA cluster array is provided with a plurality of DNA clusters, each DNA cluster is a circular pattern as shown in fig. 3, each DNA cluster may include a plurality of DNAs, each DNA cluster includes a plurality of bases, wherein the plurality of DNA cluster arrays may be divided into a first DNA cluster array and a second DNA cluster array, the first DNA cluster array is a DNA cluster array of a certain row currently being scanned, or may be a DNA cluster array of a first row in the sample to be tested, the second DNA cluster array is a DNA cluster array of a next row after the first DNA cluster array is scanned, the base includes a base in the first DNA cluster array and a base in the second DNA cluster array, specifically, the base in the first DNA cluster array is a base in a certain DNA cluster in the first DNA cluster array, the base in the second DNA cluster array is a base in a certain DNA cluster in the second DNA cluster array, as shown in fig. 3, when the laser reaches the sample 131 to be measured along the optical path, an illumination spot 1111 is formed on the sample 131 to be measured, it is understood that the illumination spot 1111 in the embodiment of the present invention is a linear illumination spot, and the beam shaping module shapes the illumination spot 1111 in a one-dimensional direction to form a linear spot, which can just irradiate on a certain DNA cluster array in the sample 131 to be measured.
It can be understood that the DNA cluster arrays on the sample to be tested are arranged at intervals, and the distance between the DNA cluster arrays only needs to meet the requirement that the illumination light spots cannot irradiate on two adjacent DNA cluster arrays, so that the arrangement interval between the base groups can be freely set according to the size of the illumination light spots in the embodiment of the invention, or a certain number of base groups meeting the sequencing requirement can be set according to the size of the sample to be tested, namely the size of the gene sequencing chip in the embodiment of the invention, and the size of the illumination light spots is regulated, so that the embodiment of the invention can improve the shooting efficiency and the efficiency of gene sequencing.
In an embodiment, the imaging module in the embodiment of the present invention is further configured to: imaging fluorescence generated by the bases in the first DNA cluster array to obtain a diffracted fluorescence image, wherein the fluorescence image comprises fluorescence spectra corresponding to the luminous bases in the first DNA cluster array, and imaging fluorescence generated by the bases in the second DNA cluster array in sequence to obtain another diffracted fluorescence image, and the other fluorescence image comprises fluorescence spectra corresponding to the luminous bases in the second DNA cluster array.
It can be understood that in the scanning process of the sample to be detected through the line illumination light spots in the embodiment of the invention, the sample to be detected is an array formed by fluorescent DNA clusters, the illuminated line light spots excite only one line of DNA cluster array at a time, namely, one DNA cluster array in the embodiment, therefore, each time the imaging is performed only to the fluorescence of the base in the line to obtain a corresponding fluorescence image, the base generates fluorescence under the excitation of laser, therefore, the base generating fluorescence at present is called as the luminescence base, the embodiment of the invention can image the DNA cluster array in the sample to be detected row by row, for example, when the first DNA cluster array is scanned, the fluorescence excited by the base in the first DNA cluster array is formed into a fluorescence image, specifically, the fluorescence spectrum corresponding to the luminescence base in each DNA cluster in the first DNA cluster array can be imaged, the fluorescence spectrum corresponding to the luminescence base in the first DNA cluster array, the first DNA cluster array can be corresponding to the fluorescence spectrum array one by one, the base in any one in the first DNA cluster array can be identified by one, the base type of any one can be identified by one in the fluorescence image, after the base in any one line of the first DNA cluster array is completely, the base in the second DNA cluster can be detected, the fluorescence array can be excited by one in the second line, and the fluorescence array can be imaged by one by the second base in the second cluster array, which is corresponding to the fluorescence in the fluorescence array in the first line, which is formed by the fluorescence array, and the fluorescence array can be detected by the one, and the second in the second line, and the fluorescence array can be detected by the one. And (5) completing one round of detection.
Referring to fig. 2, in an embodiment of the present invention, the apparatus further includes a displacement stage 132, where the displacement stage 132 is disposed on an optical path where the objective lens 122 converges laser light, the displacement stage 132 is used for placing the sample 131 to be measured, and the displacement stage 132 is further used for moving the sample 131 to be measured, so that the imaging module sequentially images fluorescence excited by the base in the first DNA cluster array and the base in the second DNA cluster array.
It should be noted that, the displacement table in the embodiment of the invention is an electric displacement table, and can perform one-dimensional scanning imaging on the sample to be detected in cooperation with the electric displacement table, when the first DNA cluster array is scanned, fluorescent imaging is performed on the base excitation in the first DNA cluster array, after the signal is collected, the sample to be detected is moved onto the second DNA cluster array through the electric displacement table, that is, the second DNA cluster array is moved to the position under the illumination light spot, thereby completing the base fluorescent excitation of another row of DNA cluster array, and then the displacement table is sequentially moved to complete the excitation of the subsequent position, thereby completing one round of detection.
In an embodiment, the target diffracted light in the embodiment of the present invention is 1 st order light that plays a role in splitting light in the target fluorescence spectrum, and the processing module is further configured to identify 0 st order light in the target fluorescence spectrum in the fluorescence image, and further configured to calculate a relative distance between the 0 st order light and the 1 st order light in the target fluorescence spectrum, and further configured to obtain a wavelength of a corresponding target base according to the relative distance.
After the fluorescence excited by the base reaches the beam splitter, the diffracted fluorescence signal is divided into two parts by the beam splitter, one part is a 0-level optical signal without beam splitting, the other part is a 1-level optical signal with beam splitting effect, the invention selects 1-level light as target diffraction light, in a fluorescence image, the fluorescence image emitted by bases of a line of DNA cluster array is shown as figure 4, the fluorescence signal of each DNA cluster base corresponds to each line in figure 3 to be imaged, the fluorescence image of each line can be obviously divided into two parts, as in figure 4, a circular spot on the left side is 0-level light after the fluorescence signal is diffracted, the bright spot has no beam splitting effect, the part is a strip part on the right side of each line, the part is 1-level light after the diffraction of a certain base fluorescence signal corresponding to the line, and the spectrum of the base fluorescence is also, and the wavelength corresponding to each pixel in the strip can be calculated from the transverse relative distance between the strip and 0-level, thus the base dye type corresponding to the line can be obtained, and finally the base dye type and the gene sequencing can be completed.
It will be appreciated that, on the premise of meeting the requirements of the embodiments of the present invention, other high-level light such as 2-level light or-1-level light may be selected as the target diffracted light, which is not particularly limited herein.
In an embodiment, as shown in fig. 2, the embodiment of the present invention further includes a beam shaping module 124, where the beam shaping module 124 is disposed on a light path of the laser emitted by the light source module, the beam shaping module 124 is configured to shape the laser into an illumination spot in a one-dimensional direction, and after the laser 111 emits the excitation light, the excitation light enters the beam shaping module 124 first, and the function of the module is to shape the illumination spot in the one-dimensional direction.
In an embodiment, as shown in fig. 2, the optical path module further includes a filter 125, where the filter 125 is disposed on an optical path of the fluorescence transmitted by the dichroic mirror 121, and the filter 125 is used to transmit the fluorescence and filter out residual excitation light mixed in the fluorescence.
It can be understood that after the sample to be tested is excited, the fluorescence emitted by the base dye passes through the objective lens and then reaches the optical filter through the dichroic mirror, the optical filter has the function of filtering residual excitation light mixed with the fluorescence of the base dye, the signal-to-noise ratio of the fluorescence signal is improved, and then the fluorescence signal reaches the optical splitter and is diffracted by the optical splitter.
In one embodiment, as shown in fig. 2, the optical path module further includes a barrel lens 126, where the barrel lens 126 is disposed on the optical path of the fluorescence diffracted by the beam splitter 123, and the barrel lens 126 is used to collect the diffracted fluorescence into the imaging module for imaging, that is, into the camera 141 for imaging.
In an embodiment, the beam splitter is a grating or a prism, taking the grating as an example, in the embodiment of the invention, the complete spectrum of the base fluorescence is directly obtained by a grating beam splitting mode, the fluorescence signal generated by base excitation reaches the grating and is diffracted by the grating, the diffracted fluorescence signal is divided into two parts, one part is a 0-level signal without beam splitting, and the other part is a 1-level signal with beam splitting function, and it is understood that only one grating capable of generating 1-level diffraction is needed to be arranged in the embodiment of the invention, so that the camera can acquire 1-level light generated after 1-level diffraction of the grating, and detection of base types can be realized by combining 0-level light.
Referring to fig. 5, the embodiment of the present invention further provides a hyperspectral sequencing method, which can be applied to the hyperspectral sequencing system in the above embodiment, and the hyperspectral sequencing system is not described herein again, and the hyperspectral sequencing method in the embodiment of the present invention includes, but is not limited to, the following steps S101 to S104.
Step S101, laser is emitted to the sample to be detected, so that bases in the sample to be detected generate fluorescence under the excitation of the laser.
And S102, collecting fluorescence generated after base excitation, diffracting and imaging the fluorescence, and obtaining a fluorescence image containing a corresponding fluorescence spectrum after diffraction.
Step S103, determining a target fluorescence spectrum in the fluorescence spectrum, and identifying target diffraction light playing a spectroscopic role in the target fluorescence spectrum in the fluorescence image.
Step S104, obtaining the wavelength of the target base corresponding to the target fluorescence spectrum according to the target diffraction light, and determining the base type of the target base according to the wavelength.
It should be noted that, the hyperspectral sequencing method in the embodiment of the present invention can obtain the data cube of the information of the sample to be tested, and the data not only has the spatial information of the sample to be tested, but also has the spectral information.
It should be noted that, in the embodiment of the present invention, laser is first emitted to the sample to be tested, so that the base in the sample to be tested generates fluorescence under the excitation of the laser, fluorescence generated after base excitation is collected, and diffraction and imaging are performed on the fluorescence, so as to obtain a fluorescence image containing a corresponding fluorescence spectrum after diffraction, and the hyperspectral sequencing method can process the fluorescence image, thereby judging the base type of each base in the sample to be tested, specifically, the base on the sample to be tested scanned in the embodiment of the present invention has a plurality of bases, and using fig. 3 as an example, the embodiment of the present invention can scan a plurality of bases at a time, the fluorescent image obtained is shown in fig. 4, each spectrum in the obtained fluorescence image is a fluorescence spectrum formed after diffraction of a different base, when the embodiment of the present invention needs to determine the type of a certain base in the scanned base, that is the target fluorescent spectrum, it needs to be determined, and as the target fluorescent spectrum, since the scanning of the sample to be tested in the embodiment of the present invention can scan not only the base type of the base to obtain the base information, and then can also obtain a fluorescence signal by diffraction signal according to the target spectrum of a part of the target according to the diffraction order, wherein the fluorescence signal can be a part of the fluorescence signal is obtained by the diffraction order of the fluorescence signal of the fluorescence of the target, and the fluorescence signal can be a part of the target has a diffraction order of the fluorescence signal of the fluorescence according to the fluorescence of the two different base, and the target spectrum can be obtained by the diffraction order, and the diffraction signal has a certain part of the target diffraction according to the wavelength of the target has a certain wavelength, and a certain part of the target diffraction signal has a high effect, and a certain spectrum can be obtained, in the embodiment of the invention, the high-grade optical signal with the light splitting effect is taken as the target diffraction light, and the corresponding wavelength can be identified in the fluorescence spectrum through the target diffraction light, so that the base type of the target base is finally identified, therefore, the hyperspectral sequencing method in the embodiment of the invention has high shooting efficiency and accuracy, the fluorescence of four-color bases can be recorded by one shooting in a single channel, the gene sequencing can be realized without setting a complex optical structure, and the system cost is low.
Referring to fig. 6, in an embodiment, a sample to be measured is provided with a first DNA cluster array and a second DNA cluster array arranged in this order, and bases include bases in the first DNA cluster array and bases in the second DNA cluster array; among the above-mentioned step S102, the following steps S201 to S203 may be included but are not limited thereto.
Step S201, sequentially collecting the base in the first DNA cluster array and the fluorescence generated after the base in the second DNA cluster array is excited.
Step S202, diffracting and imaging fluorescence generated by bases in the first DNA cluster array to obtain a diffracted fluorescence image, wherein the fluorescence image comprises a fluorescence spectrum corresponding to the luminescent bases in the first DNA cluster array.
Step S203, sequentially diffracting and imaging fluorescence generated by the bases in the second DNA cluster array to obtain a diffracted fluorescence image, wherein the other fluorescence image comprises a fluorescence spectrum corresponding to the luminescent bases in the second DNA cluster array.
In an embodiment, as shown in fig. 3, a sample 131 to be tested in the embodiment of the present invention is provided with a plurality of rows of DNA cluster arrays sequentially arranged, each row of DNA cluster array is provided with a plurality of DNA clusters, as shown in fig. 3, each row of DNA cluster array is provided with a plurality of DNA clusters, each DNA cluster is a circular pattern as shown in fig. 3, each DNA cluster may include a plurality of DNAs, each DNA cluster includes a plurality of bases, wherein the plurality of DNA cluster arrays may be divided into a first DNA cluster array and a second DNA cluster array, the first DNA cluster array is a DNA cluster array of a certain row currently being scanned, or may be a DNA cluster array of a first row in the sample to be tested, the second DNA cluster array is a DNA cluster array of a next row after the first DNA cluster array is scanned, the base includes a base in the first DNA cluster array and a base in the second DNA cluster array, specifically, the base in the first DNA cluster array is a base in a certain DNA cluster in the first DNA cluster array, the base in the second DNA cluster array is a base in a certain DNA cluster in the second DNA cluster array, as shown in fig. 3, when the laser reaches the sample 131 to be measured along the optical path, an illumination spot 1111 is formed on the sample 131 to be measured, it is understood that the illumination spot 1111 in the embodiment of the present invention is a linear illumination spot, and the beam shaping module shapes the illumination spot 1111 in a one-dimensional direction to form a linear spot, which can just irradiate on a certain DNA cluster array in the sample 131 to be measured.
It can be understood that the DNA cluster arrays on the sample to be tested are arranged at intervals, and the distance between the DNA cluster arrays only needs to meet the requirement that the illumination light spots cannot irradiate on two adjacent DNA cluster arrays, so that the arrangement interval between the base groups can be freely set according to the size of the illumination light spots in the embodiment of the invention, or a certain number of base groups meeting the sequencing requirement can be set according to the size of the sample to be tested, namely the size of the gene sequencing chip in the embodiment of the invention, and the size of the illumination light spots is regulated, so that the embodiment of the invention can improve the shooting efficiency and the efficiency of gene sequencing.
In an embodiment, in the embodiment of the present invention, fluorescence generated by a base in a first DNA cluster array is diffracted and imaged to obtain a diffracted fluorescence image, where the fluorescence image includes a fluorescence spectrum corresponding to a luminescent base in the first DNA cluster array, and fluorescence generated by a base in a second DNA cluster array is sequentially diffracted and imaged to obtain another fluorescence image after diffraction, where the other fluorescence image includes a fluorescence spectrum corresponding to a luminescent base in the second DNA cluster array.
It can be understood that in the scanning process of the sample to be detected through the line illumination light spots in the embodiment of the invention, the sample to be detected is an array formed by fluorescent DNA clusters, the illuminated line light spots excite only one line of DNA cluster array at a time, namely, one DNA cluster array in the embodiment, therefore, each time the imaging is performed only to the fluorescence of the base in the line to obtain a corresponding fluorescence image, the base generates fluorescence under the excitation of laser, therefore, the base generating fluorescence at present is called as the luminescence base, the embodiment of the invention can image the DNA cluster array in the sample to be detected row by row, for example, when the first DNA cluster array is scanned, the fluorescence excited by the base in the first DNA cluster array is formed into a fluorescence image, specifically, the fluorescence spectrum corresponding to the luminescence base in each DNA cluster in the first DNA cluster array can be imaged, the fluorescence spectrum corresponding to the luminescence base in the first DNA cluster array, the first DNA cluster array can be corresponding to the fluorescence spectrum array one by one, the base in any one in the first DNA cluster array can be identified by one, the base type of any one can be identified by one in the fluorescence image, after the base in any one line of the first DNA cluster array is completely, the base in the second DNA cluster can be detected, the fluorescence array can be excited by one in the second line, and the fluorescence array can be imaged by one by the second base in the second cluster array, which is corresponding to the fluorescence in the fluorescence array in the first line, which is formed by the fluorescence array, and the fluorescence array can be detected by the one, and the second in the second line, and the fluorescence array can be detected by the one. And (5) completing one round of detection.
Referring to fig. 7, in an embodiment, the DNA cluster array includes a target DNA cluster, where the target DNA cluster includes a plurality of bases, including a first target base and a second target base sequentially arranged on the target DNA cluster, and the hyperspectral sequencing method in an embodiment of the present invention may further include, but is not limited to, the following steps S301 to S302.
Step S301, before a round of base class detection is carried out on the first target base, the first target base is dyed through fluorescent dye to obtain a corresponding first fluorescent group, and the corresponding first fluorescent group is synthesized into a target DNA cluster.
Step S302, after the detection of the base type of the first target base is completed, another round of base type detection is carried out on the second target base, and before the detection of the base type of the second target base, the first fluorescent group is cut off, and the second target base is dyed through fluorescent dye to obtain a corresponding second fluorescent group, and the corresponding second fluorescent group is synthesized into the target DNA cluster.
It should be noted that, in the embodiment of the present invention, sequencing is performed by a hyperspectral sequencing method, the sequencing process is a process of sequencing while synthesizing, the target DNA cluster is any DNA cluster in any row of DNA cluster array, that is, any DNA cluster in a sample to be detected, and it can be understood that each DNA cluster includes a DNA strand formed by a plurality of bases, by the hyperspectral sequencing method in the embodiment of the present invention, the DNA strand in the DNA cluster can be sequenced, and each DNA cluster can include a first target base and a second target base, where the first target base is a base in the target DNA cluster currently being scanned, or may be the first base in the target DNA cluster, and the second target base is the next base after the first target base on the base strand in the target DNA cluster.
It should be noted that, in the embodiment of the present invention, a plurality of bases are in a DNA cluster (in order to sequence them), and in order to detect the sequence, it is necessary to detect the bases one by one through a cycle, and by the steps S201 to S203 of the above embodiment, the first target base in the DNA cluster is imaged in sequence, the base class is detected, after scanning of each row of DNA cluster array is completed, the base class detection of all the first target bases in the sample to be detected is completed, a round of detection is completed, and then the next round of detection is performed, so as to detect the base class of the next second target base in each DNA cluster.
Before the base type detection is carried out on the first target base, the first target base is dyed through fluorescent dye to obtain a corresponding first fluorescent group, the corresponding first fluorescent group is synthesized into a target DNA cluster, before the base type of the second target base is detected, the first fluorescent group is cut off, the subsequent spectrum detection of the two fluorescent groups is avoided, the second target base is dyed through the fluorescent dye to obtain a corresponding second fluorescent group, the corresponding second fluorescent group is synthesized into the target DNA cluster, it can be understood that before each round of sequencing, the fluorescent base is synthesized onto a DNA chain through biochemical reaction, then shooting is carried out, the fluorescent group of the synthesized base is cut off after shooting imaging is completed, then the next round of fluorescent base is synthesized, and then a shooting flow is executed, so that the second round of sequencing is completed.
Referring to fig. 8, in an embodiment, in determining the target fluorescence spectrum corresponding to the second target base, the following steps S401 to S403 may be included in the above step S103.
Step S401, obtaining the synthesized spectrum characteristic information of the fluorescence spectrum corresponding to the second target base and the sub-spectrum morphological characteristic information of the composition fluorescence spectrum.
And step S402, carrying out linear spectrum unmixing on the fluorescence spectrum according to the synthesized spectrum characteristic information and the sub-spectrum morphological characteristic information, and analyzing to obtain a plurality of sub-spectrums, wherein the sub-spectrums comprise a first sub-spectrum and a plurality of second sub-spectrums, and the brightness of the first sub-spectrum is higher than that of the second sub-spectrum.
In step S403, the first sub-spectrum is determined as the target fluorescence spectrum.
It should be noted that, there is more than one DNA strand in the DNA cluster, and if each DNA strand maintains the same number of reaction rounds, the fluorescent signal emitted by the DNA cluster is purer, and is the fluorescent signal of a single dye. However, the situation that each DNA cluster emits the same fluorescence is more complicated than the above, and the situation may occur when the first round of reaction is performed during the sequencing, and as the sequencing is performed, the actual reaction rounds of the different DNA strands in one DNA cluster are distinguished due to incomplete excision or no synthesis of the base with fluorescence in the previous round, so that fluorescence emitted by one DNA cluster after one round of reaction is superposition of fluorescence of multiple bases, for example, when detecting the base class of the second target base, the fluorescence spectrum in the fluorescence image may be generated after superposition of fluorescence of the first target base in the previous round. As shown in FIG. 9, assuming that a certain DNA cluster is A base in the present round, its fluorescence signal is the spectrum pattern at the upper part of FIG. 9, namely the original spectrum, and the synthesis of some DNA chains is delayed due to insufficient biochemical reaction, the fluorescence signal of the DNA cluster is the spectrum pattern at the lower part of FIG. 9, namely the synthesized spectrum, which is the result of mixing A base fluorescence with other base fluorescence in different proportions, and in the sequencing report, the percentage of the abnormal reaction DNA chains to the normal reaction DNA chains is also an important parameter, so that the embodiment of the invention needs to use the algorithm of linear spectrum unmixing to analyze the proportion of different dyes in the mixed fluorescence spectrum.
In the optical system, different spectrum signals are linearly overlapped, so long as the final synthesized spectrum, namely the synthesized spectrum characteristic information of the fluorescence spectrum of the second target base, and the sub-spectrum form, namely the sub-spectrum form characteristic information, of the spectrum are known, the sub-spectrum can be the spectrum of the base in the previous detection of the rounds, the proportion of the sub-spectrum can be calculated through a linear spectrum unmixing algorithm, and the sub-spectrum form characteristic information comprises the sub-spectrum vector, the percentage of the sub-spectrum, the noise spectrum vector and the like which form the synthesized spectrum.
The linear spectral overlap satisfies the following condition:
Wherein x is a synthetic spectrum vector in the actually measured synthetic spectrum, S k is a sub spectrum vector constituting the synthetic spectrum, a k is a percentage of the sub spectrum, called abundance, all sub spectrum vectors constitute a matrix S, a is an abundance vector, and w is a noise spectrum vector.
The formula of the abundance vector estimation value is:
The mixed spectrum below the graph in fig. 9 can be decomposed into four independent spectrums as shown in fig. 10, namely sub-spectrums, in fig. 10, the top four-color base mixed spectrum consistent with the graph below the graph in fig. 9 is the four-color base mixed spectrum, and the decomposed four-color spectrum is shown in a spectrum chart of 2 to 5 rows. As can be seen from fig. 10, the ratio of the spectrum in the long wavelength band is low, and the brightness is very dark, but in the mixed spectrum, due to the tailing of the dye in the short wavelength band and the superposition of the dye peaks in the long wavelength band, the mixed spectrum also has considerable brightness in the long wavelength band, for example, a linear spectrum unmixing algorithm is not used, and only the ratio of the peak brightness of different dyes is calculated to determine the ratio of different bases, and in the case that the intensity of a certain dye is weak (the ratio of a certain base is low), serious distortion of the calculated ratio is caused, so that the report accuracy is affected.
In the conventional filter system, a tailing signal of a short wavelength spectrum enters a long wavelength channel to cause crosstalk of the signal, and the filter system only detects brightness, so that the signal cannot be distinguished from the peak value of the channel or the tailing of an adjacent channel, if the signal crosstalk is overlapped with multi-dye spectrum mixing caused by biochemical reaction lag, a more complex algorithm is needed to identify and analyze the signal, the required calculation resource is larger, and the algorithm is difficult.
Therefore, in the embodiment of the present invention, the fluorescence spectrum corresponding to the second target base is processed through linear spectral unmixing, so as to obtain multiple sub-spectrums, as shown in fig. 10, the brightness of the sub-spectrum of the second row is higher than that of the spectrums of other rows, that is, the intensity of the sub-spectrum of the second row is higher than that of the spectrums of other rows, so that the sub-spectrum of the second row is a first sub-spectrum, and the spectrums of other 3 to 5 rows are second sub-spectrums, wherein the first sub-spectrum of the second row is the spectrum of the target base corresponding to the channel, that is, the spectrum of the second target base.
It is understood that the processing in the hyperspectral sequencing system in the above embodiment may perform the steps S301 to S302 in the above embodiment, or may perform the steps S401 to S403 in the above embodiment, which will not be described herein.
According to the embodiment of the invention, the hyperspectral method is utilized for gene sequencing, the system cost is greatly reduced, meanwhile, four types of ATGC base fluorescent signals can be effectively identified, and the applied hyperspectral sequencing system considers that the fluorescent signals of a single DNA cluster are not pure due to biochemical reaction lag, so that linear spectral unmixing is introduced to accurately calculate the ratio values of different bases in a round of reaction, and the sequencing accuracy is increased.
Referring to fig. 11, in an embodiment, the target diffracted light is the 1 st order light with a spectroscopic effect in the target fluorescence spectrum, and the above step S104 may further include, but is not limited to, the following steps S501 to S503.
Step S501, the 0-order light in the target fluorescence spectrum is identified in the fluorescence image.
In step S502, the relative distance between the level 0 light and the level 1 light of the target fluorescence spectrum is calculated.
Step S503, obtaining the wavelength of the corresponding target base according to the relative distance.
After the fluorescence excited by the base reaches the beam splitter, the diffracted fluorescence signal is divided into two parts by the beam splitter, one part is a 0-level optical signal without beam splitting, the other part is a 1-level optical signal with beam splitting effect, the invention selects 1-level light as target diffraction light, in a fluorescence image, the fluorescence image sent by a line of DNA cluster array in fig. 3 is shown in fig. 4, wherein the fluorescence signal of each DNA cluster corresponds to each line in fig. 4 for imaging, the fluorescence image of each line can be obviously divided into two parts, for example, in fig. 4, a circular spot on the left side is 0-level light after the fluorescence signal is diffracted, the bright spot has no beam splitting effect, the part is a strip part on the right side of each line, the part is 1-level light after the diffraction of a certain base fluorescence signal corresponding to the line, and the spectrum of the base fluorescence is also, and the wavelength corresponding to each pixel in the strip can be calculated from the transverse relative distance between the strip and 0-level, thus the base dye type corresponding to the line can be obtained, and finally the base dye type and the gene sequencing can be completed.
It will be appreciated that, on the premise of meeting the requirements of the embodiments of the present invention, other high-level light such as 2-level light or-1-level light may be selected as the target diffracted light, which is not particularly limited herein.
The embodiment of the invention also provides a gene sequencer, which comprises the hyperspectral sequencing system described in any one of the embodiments, and can execute a hyperspectral sequencing method, and emit laser to a sample to be tested, so that bases in the sample to be tested generate fluorescence under the excitation of the laser, then collect fluorescence generated after base excitation, diffract and image the fluorescence, obtain a fluorescence image containing a corresponding fluorescence spectrum after diffraction, determine a target fluorescence spectrum corresponding to a target base in the fluorescence spectrum, identify target diffraction light with a light splitting function in the target fluorescence spectrum in the fluorescence image, judge the category of the target base according to the target emission light, then obtain the wavelength of the target base corresponding to the target fluorescence spectrum according to the target diffraction light, and different bases have different wavelengths, so that the base category of the target base can be determined according to the wavelength, and gene sequencing is realized.
The gene sequencer according to the embodiment of the present invention may further include other devices or systems to achieve the sequencing function of the gene sequencer, which is not particularly limited herein.
The apparatus embodiments described above are merely illustrative, in which the units described as separate components may or may not be physically separate, i.e., may be located in one place, or may be distributed over a plurality of units. Some or all of the modules may be selected according to actual needs to achieve the purpose of the solution of this embodiment.
It should also be appreciated that the various embodiments provided by the embodiments of the present invention may be arbitrarily combined to achieve different technical effects. While the preferred embodiments of the present invention have been described in detail, the present invention is not limited to the above embodiments, and various equivalent modifications and substitutions can be made by those skilled in the art without departing from the spirit of the present invention.

Claims (12)

1. A method of hyperspectral sequencing comprising:
emitting laser to a sample to be detected so that bases in the sample to be detected generate fluorescence under the excitation of the laser;
collecting fluorescence generated after base excitation, diffracting and imaging the fluorescence to obtain a fluorescence image containing a corresponding fluorescence spectrum after diffraction;
determining a target fluorescence spectrum in the fluorescence spectrum, and identifying target diffraction light which plays a role in light splitting in the target fluorescence spectrum in the fluorescence image;
Obtaining the wavelength of a target base corresponding to the target fluorescence spectrum according to the target diffraction light, and determining the base class of the target base according to the wavelength;
The sample to be tested is provided with a plurality of rows of DNA cluster arrays which are sequentially arranged, the DNA cluster arrays comprise target DNA clusters, the target DNA clusters comprise a plurality of bases, and the target DNA clusters comprise a first target base and a second target base which are sequentially arranged on the target DNA clusters;
In determining the target fluorescence spectrum corresponding to the second target base, the determining a target fluorescence spectrum in the fluorescence spectra includes:
The method comprises the steps of obtaining synthetic spectrum characteristic information of a fluorescence spectrum corresponding to a second target base and sub-spectrum morphological characteristic information of the fluorescence spectrum, wherein the synthetic spectrum characteristic information comprises a synthetic spectrum vector, and the sub-spectrum morphological characteristic information comprises a sub-spectrum vector, a sub-spectrum percentage and a noise spectrum vector of the synthetic spectrum;
The linear spectral overlap satisfies the following condition:
Wherein x is the synthesized spectrum vector in the synthesized spectrum obtained by actual measurement, S k is the sub spectrum vector forming the synthesized spectrum, a k is the percentage of the sub spectrum, called abundance, all the sub spectrum vectors form a matrix S, a is an abundance vector, and w is the noise spectrum vector;
the formula of the abundance vector estimation value is:
Linear spectrum unmixing is carried out on the fluorescence spectrum according to the synthesized spectrum characteristic information and the sub-spectrum morphological characteristic information, and a plurality of sub-spectrums are obtained through analysis, wherein the sub-spectrums comprise a first sub-spectrum and a plurality of second sub-spectrums, and the brightness of the first sub-spectrum is higher than that of the second sub-spectrum;
And determining the first sub-spectrum as the target fluorescence spectrum.
2. The hyperspectral sequencing method of claim 1, wherein the DNA cluster array comprises a first DNA cluster array and a second DNA cluster array, the bases comprising bases in the first DNA cluster array and bases in the second DNA cluster array;
Collecting fluorescence generated after base excitation, diffracting and imaging the fluorescence to obtain a fluorescence image containing a corresponding fluorescence spectrum after diffraction, wherein the method comprises the following steps:
sequentially collecting fluorescence generated after the base in the first DNA cluster array and the base in the second DNA cluster array are excited;
Diffracting and imaging the fluorescence generated by the base in the first DNA cluster array to obtain a diffracted fluorescence image, wherein the fluorescence image comprises a fluorescence spectrum corresponding to the luminous base in the first DNA cluster array;
And sequentially diffracting and imaging the fluorescence generated by the bases in the second DNA cluster array to obtain another fluorescence image after diffraction, wherein the other fluorescence image comprises a fluorescence spectrum corresponding to the luminous bases in the second DNA cluster array.
3. The hyperspectral sequencing method of claim 2, wherein the method further comprises:
Before the first target base is subjected to one-round base class detection, the first target base is dyed through fluorescent dye to obtain a corresponding first fluorescent group, and the corresponding first fluorescent group is synthesized into the target DNA cluster;
And after the detection of the base class of the first target base is completed, carrying out another round of base class detection on the second target base, cutting off the first fluorescent group before the detection of the base class of the second target base, dyeing the second target base through fluorescent dye to obtain a corresponding second fluorescent group, and synthesizing the second fluorescent group into the target DNA cluster.
4. The hyperspectral sequencing method of claim 1 wherein the target diffracted light is a1 st order light in the target fluorescence spectrum that acts as a light splitting;
The obtaining the wavelength of the target base corresponding to the target fluorescence spectrum according to the target diffraction light comprises the following steps:
Identifying level 0 light in a target fluorescence spectrum in the fluorescence image;
Calculating a relative distance between the level 0 light and the level 1 light of the target fluorescence spectrum;
and obtaining the wavelength of the corresponding target base according to the relative distance.
5. A hyperspectral sequencing system, comprising:
The light source module is used for emitting laser;
The optical path module comprises a dichroic mirror, an objective lens and a beam splitter;
The dichroic mirror is arranged on a light path of the laser emitted by the light source module, and is used for receiving the laser and reflecting the laser;
The objective lens is arranged on a light path of the laser reflected by the dichroic mirror, the objective lens is used for converging the laser and forming an illumination light spot on a sample to be tested so that bases in the sample to be tested generate fluorescence under the excitation of the laser, the objective lens is also used for receiving the fluorescence generated after the base is excited and transmitting the fluorescence to the dichroic mirror, and the dichroic mirror is also used for transmitting the fluorescence;
the beam splitter is arranged on the light path of the fluorescence transmitted by the two directions, and the beam splitter is used for diffracting the fluorescence;
The imaging module is arranged on a light path of the fluorescence diffracted by the beam splitter, and is used for receiving the diffracted fluorescence and obtaining a fluorescence image containing a corresponding fluorescence spectrum;
The processing module is used for determining a target fluorescence spectrum in the fluorescence spectrum and identifying target diffraction light which plays a role in splitting in the target fluorescence spectrum in the fluorescence image; the method comprises the steps of obtaining the wavelength of a target base corresponding to the target fluorescence spectrum according to the target diffraction light, and determining the base class of the target base according to the wavelength;
The sample to be tested is provided with a plurality of rows of DNA cluster arrays which are sequentially arranged, the DNA cluster arrays comprise target DNA clusters, the target DNA clusters comprise a plurality of bases, and the target DNA clusters comprise a first target base and a second target base which are sequentially arranged on the target DNA clusters;
In determining the target fluorescence spectrum corresponding to the second target base, the determining a target fluorescence spectrum in the fluorescence spectra includes:
The method comprises the steps of obtaining synthetic spectrum characteristic information of a fluorescence spectrum corresponding to a second target base and sub-spectrum morphological characteristic information of the fluorescence spectrum, wherein the synthetic spectrum characteristic information comprises a synthetic spectrum vector, and the sub-spectrum morphological characteristic information comprises a sub-spectrum vector, a sub-spectrum percentage and a noise spectrum vector of the synthetic spectrum;
The linear spectral overlap satisfies the following condition:
Wherein x is the synthesized spectrum vector in the synthesized spectrum obtained by actual measurement, S k is the sub spectrum vector forming the synthesized spectrum, a k is the percentage of the sub spectrum, called abundance, all the sub spectrum vectors form a matrix S, a is an abundance vector, and w is the noise spectrum vector;
the formula of the abundance vector estimation value is:
Linear spectrum unmixing is carried out on the fluorescence spectrum according to the synthesized spectrum characteristic information and the sub-spectrum morphological characteristic information, and a plurality of sub-spectrums are obtained through analysis, wherein the sub-spectrums comprise a first sub-spectrum and a plurality of second sub-spectrums, and the brightness of the first sub-spectrum is higher than that of the second sub-spectrum;
And determining the first sub-spectrum as the target fluorescence spectrum.
6. The hyperspectral sequencing system of claim 5, wherein the DNA cluster array comprises a first DNA cluster array and a second DNA cluster array, the bases comprising bases in the first DNA cluster array and bases in the second DNA cluster array;
the imaging module is further configured to sequentially image fluorescence excited by the bases in the first DNA cluster array and the bases in the second DNA cluster array, and includes:
imaging the fluorescence generated by the base in the first DNA cluster array to obtain a diffracted fluorescence image, wherein the fluorescence image comprises a fluorescence spectrum corresponding to the luminous base in the first DNA cluster array;
imaging the fluorescence generated by the bases in the second DNA cluster array in sequence to obtain another diffracted fluorescence image, wherein the other fluorescence image comprises a fluorescence spectrum corresponding to the luminous bases in the second DNA cluster array.
7. The hyperspectral sequencing system of claim 6 further comprising a displacement stage disposed on the optical path of the objective lens converging the laser light, the displacement stage for placing the sample to be tested;
the displacement table is also used for moving the sample to be detected, so that the imaging module images fluorescence excited by the base in the first DNA cluster array and the base in the second DNA cluster array in sequence.
8. The hyperspectral sequencing system of claim 5 wherein the target diffracted light is a1 st order light in the target fluorescence spectrum that acts as a light splitting effect;
The processing module is further used for identifying 0-level light in a target fluorescence spectrum in the fluorescence image; and further for calculating a relative distance between the level 0 light and the level 1 light of the target fluorescence spectrum; and is also used for obtaining the wavelength of the corresponding target base according to the relative distance.
9. The hyperspectral sequencing system of claim 5 further comprising a beam shaping module disposed on an optical path of the laser light emitted by the light source module, the beam shaping module configured to shape the laser light into an illumination spot in one dimension.
10. The hyperspectral sequencing system of claim 5, wherein the light path module further comprises a filter disposed on a light path through which the dichroic mirror transmits the fluorescence, the filter for transmitting the fluorescence and filtering out residual excitation light mixed in the fluorescence.
11. The hyperspectral sequencing system of claim 5, wherein the light path module further comprises a barrel mirror disposed on a light path of the fluorescence diffracted by the beam splitter, the barrel mirror configured to focus the diffracted fluorescence into the imaging module for imaging.
12. A genetic sequencer comprising a hyperspectral sequencing system as claimed in any one of claims 5 to 11.
CN202210660961.XA 2022-06-13 2022-06-13 Hyperspectral sequencing method, hyperspectral sequencing system and gene sequencer Active CN115266662B (en)

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