CN112961902B - DNA bar code marking method for marking large number of samples - Google Patents
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- 108090000623 proteins and genes Proteins 0.000 claims description 5
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Abstract
The invention discloses a DNA bar code marking method for marking a large number of samples. The method comprises the following steps: establishing a three-dimensional hole array; (2) Marking the three-dimensional hole array as N along the X, Y and Z directions respectively X1 ～N Xn 、N Y1 ～N Yn And N Z1 ～N Zn (ii) a (3) Labeling the first, second and third sequences linked to the magnetic beads in the X, Y and Z directions, respectively, and corresponding to the wells; (4) And sequentially adding the first sequence, the second sequence and the third sequence which have the same marks with the magnetic bead hole coordinates into corresponding holes, and then placing the tissue sample into the corresponding holes. The application designs a cubic well array, so that the operation of adding the barcode sequence is systematized, and thousands of tissue samples can be simultaneously marked on the premise of known synthetic magnetic bead barcode sequences.
Description
Technical Field
The invention belongs to a high-throughput bar code marking technology, and particularly relates to a DNA bar code marking method for marking a large number of samples.
Background
The high throughput stochastic barcode labeling technology has led to rapid development of single cell sequencing technology. The key driving force is how to realize unique labeling of tens of thousands of single cells simultaneously. The single cell sequencing technology (such as Drop-seq, inDrop-seq, microwell-seq, SPLiT-seq, etc.) widely used at present increases the diversity of barcode sequences by synthesizing random barcodes, so as to achieve the purpose of simultaneously labeling a large number of cells. The principle of the SPLiT-pool stochastic barcode synthesis used by Microwell-seq and SPLiT-seq is exemplified: on the carboxyl-coated magnetic beads, 3 oligonucleotide chain sequences (barcode) were sequentially linked, each of which was 96. The split-pool method needs to disperse magnetic beads into a 96-well plate, one of 96 first-stage barcodes is added into each well, all the magnetic beads are combined after the first-stage barcodes are connected to the magnetic beads, then the magnetic beads are re-dispersed into a new 96-well plate, one of 96 second-stage barcodes is added into each well, after the second-stage barcodes are connected to the magnetic beads, all the magnetic beads are combined and continuously dispersed into the new 96-well plate, one of 96 third-stage barcodes is added into each well, and after the connection reaction, all the magnetic beads are combined. The bead sequences thus obtained are arranged in nearly one million (96X 96) arrays, and the probability that any two beads are identical is only one part per million, and therefore these beads can be considered unique. However, this method results in the loss of "spatial information" from the beads at each pooling (pooling) and the specific barcode combination on a particular bead cannot be determined at the end of the synthesis because the synthesized beads are fully randomized. Thus, the random barcode labeling technique cannot be applied directly to label a large number of designated samples.
Currently emerging technologies for multi-sample sequencing, sequential sample sequencing, and spatial transcriptome sequencing all require simultaneous labeling of large numbers of tissue or cell samples. If a unique barcode sequence is designed for each sample independently, the required barcode sequence is increased along with the increase of the sample flux, which causes waste of manpower, material resources and financial resources. Therefore, there is a need for a customized barcode strategy with multiplexing that can increase the diversity of barcodes under the premise of knowing barcode sequences, thereby greatly increasing the sample throughput and realizing unique labeling of large batches of samples.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides a DNA barcode labeling method for labeling a large number of samples, and the method abandons two steps of split and pool, so that the sequence on the magnetic beads is completely determined; a cubic pore array is then designed so that the addition of barcode sequences is systematic. This allows for labeling of thousands of tissue samples simultaneously, given the known synthetic magnetic bead barcode sequences.
In order to achieve the purpose, the technical scheme adopted by the invention for solving the technical problems is as follows:
a DNA barcode labeling method for labeling a plurality of samples, comprising the steps of:
(1) Establishing an NXNXN stereoscopic hole array, wherein magnetic beads are added in each hole;
(2) Marking the three-dimensional hole array as N along the X, Y and Z directions respectively X1 ～N Xn 、N Y1 ～N Yn And N Z1 ～N Zn Obtaining a corresponding X, Y, Z coordinate for each well;
(3) Marking a plurality of first segment sequences connected to the magnetic beads as Barcode X1-Xn, barcode Y1-Yn or Barcode Z1-Zn corresponding to the coordinates of the hole; then, a plurality of second-stage sequences connected with the first-stage sequences are marked as Barcode X1-Xn, barcode Y1-Yn or Barcode Z1-Zn, and are different from the first-stage sequences; finally, marking the third section of sequence connected with the second section of sequence as a mark different from the first section and the second section in Barcode X1-Xn, barcode Y1-Yn or Barcode Z1-Zn;
(4) And sequentially adding a first sequence, a second sequence and a third sequence which have the same marks with the coordinates of the magnetic bead hole into the corresponding holes, connecting the first sequence with the magnetic bead through EDC reaction, connecting the first sequence, the second sequence and the third sequence through PCR, and then placing the tissue sample into the corresponding holes.
Furthermore, the first sections of sequences are marked as Barcode X1-Xn; a plurality of second-stage sequences are marked as Barcode Y1-Yn; and a plurality of third segment sequences are marked as Barcode Z1-Zn.
Further, the first sequence is a 5' end amino modified sequence, and two ends of the first sequence are respectively connected with a PCR handle and a first connecting sequence; the first sequence was linked to the carboxyl-coated magnetic beads by EDC reaction.
Furthermore, the reverse complement sequence and the second connecting sequence connected to the first connecting sequence are connected to both ends of the second sequence.
Further, one end of the third segment sequence is connected with a reverse complementary sequence connected with the second connecting sequence, and the other end is sequentially connected with UMI used for gene quantification and Poly T used for capturing a transcript with a Poly A tail at the 3' end.
The invention has the beneficial effects that:
compared with the prior art, the invention has obvious advantages, firstly, compared with the random bar code technology, the invention solves the problem that a large number of samples are marked, which cannot be realized by the random bar code technology. Secondly, compared with the traditional method of simply using a section of synthetic sequence as the barcode, the spatial barcode labeling technology also has obvious advantages, along with the continuous increase of sample flux, the cost of the traditional method on the synthesis of the barcode rises linearly, and meanwhile, when different barcodes are prepared into solutions, the separate treatment is needed, which wastes time and labor. And the space bar code marking technology can effectively improve the experimental efficiency and reduce the experimental cost, and only dozens of bars need to be used for marking the independent marks of thousands of samples.
Drawings
FIG. 1 is a flow chart of the present application; wherein A is a constructed three-dimensional hole array; and B is a technical process.
Detailed Description
The following description of the embodiments of the present invention is provided to facilitate the understanding of the present invention by those skilled in the art, but it should be understood that the present invention is not limited to the scope of the embodiments, and it will be apparent to those skilled in the art that various changes may be made without departing from the spirit and scope of the invention as defined and defined in the appended claims, and all matters produced by the invention using the inventive concept are protected.
Example 1 construction of a volumetric pore array
1. In the three mutually perpendicular spatial directions of X, Y and Z, a 48X 48 three-dimensional hole array is constructed, each layer has 48X 48 holes, and 48 layers are formed (figure 1A). We chose a 384 well plate (24X 16) as the basic component of a planar array (2X 3 plates per layer) and label the three-dimensional array of wells as X1 to X48, Y1 to Y48 and Y1 to Y48 in the X, Y and Z directions, respectively, thereby giving the array specific X, Y and Z coordinates for each well.
2. Marking the first segment of sequence as Barcode X1-X48, and respectively corresponding to 48 holes in the X direction in the three-dimensional hole array; the second segment of sequence is marked as Barcode Y1-Y48 and respectively corresponds to 48 holes in the Y direction in the three-dimensional hole array; and the third segment of sequence is marked as Barcode Z1-Z48 and respectively corresponds to 48 holes in the Z direction in the three-dimensional hole array.
As shown in FIG. 1B, the beads contained a PCR primer for subsequent amplification and library construction, three barcode combinations, a UMI (Unique molecular index) for gene quantification, and a Poly T tail for capture of transcripts with a Poly A tail at the 3' end.
During synthesis, a first segment of sequence (containing PCR handle, barcode X and linker 1) modified by 5' terminal amino is added, and is connected with magnetic beads coated by carboxyl through EDC reaction, then a second segment of sequence (containing reverse complementary sequences of linker1, barcode Y and linker 2) is added, the sequence on the magnetic beads is extended through PCR, finally a third segment of sequence (containing reverse complementary sequences of linker2, barcode Z, UMI and Poly A) is added, and the final three-segment Barcode combination is obtained through PCR.
Example 2
1. First round of ligation reaction
Before the reaction, the Barcode X sequence primer was dissolved in nuclease-free water to a concentration of 400. Mu.M. 3 1.5mL centrifuge tubes were added to each tube at approximately 3.33X 10 6 The carboxyl group magnetic beads (about 464. Mu.L) were washed 1 to 2 times with 0.1M MES. The centrifuge tubes containing the magnetic beads were placed in a magnetic rack, the supernatant was discarded, 716. Mu.L of 0.1M MES solution and 68.8. Mu.L EDC solution were added to each tube, and after mixing well, the contents of each centrifuge tube were evenly distributed to 8 PCR tubes. 63 μ L of 0.2M MES solution and 63 μ L of the corresponding Barcode X1 to X24 were added to each tube, mixed well, and reacted at room temperature for 20min. 25.65. Mu.L of EDC solution was added to each tube and reacted at room temperature for 10min. The above step was repeated and the reaction was carried out again at room temperature for 80min. The 24 PCR tubes were placed on a magnetic frame, the supernatant was aspirated after the beads had settled completely, the beads in each well were washed once with 0.1M PBS containing 0.02% Tween-20, nuclease-free water and TE buffer (pH 8.0) in that order, and finally the beads were resuspended in 500. Mu.L TE buffer at 4 ℃ for use.
2. Second round of ligation reaction
PCR reaction solutions were prepared in 1.5mL EP tubes: 280. Mu.L of 2 XPphanta Master Mix and 56. Mu.L of the corresponding second sequence of 50. Mu.M (Barcode Y), are vortexed by pipetting. And (3) standing the magnetic beads stored in the TE buffer solution on a magnetic frame for 2min, sucking and removing the supernatant, washing the supernatant with nuclease-free water once, adding 220 mu L of nuclease-free water to resuspend the magnetic beads, and uniformly mixing. Taking a 384-hole PCR plate, adding PCR reaction solution (6 mu L/hole) containing different Barcode Y and magnetic beads (4 mu L/hole) correspondingly connected with different Barcode X into each hole, uniformly mixing, centrifuging, placing in a PCR instrument for reaction, adsorbing the magnetic beads by using a high-flux magnetic bead transfer device after the PCR reaction is finished, discarding the unreacted solution, and washing the magnetic beads once by using nuclease-free water. Adding 20 mu L of nuclease-free water into each well, covering a sealing plate film, placing the 384-well plate in a water bath kettle at 95 ℃ for reacting for 6min, quickly taking out, adsorbing magnetic beads by using a high-pass magnetic bead transfer device, discarding unreacted solution, washing the magnetic beads twice by using nuclease-free water preheated at 95 ℃, finally suspending the magnetic beads in TE-TW solution for later use at 4 ℃.
3. Third round of ligation reaction
Taking 18 reacted 384-well plates, adsorbing the inner magnetic beads (connected with Barcode X and Barcode Y) by using a high-throughput magnetic bead transfer device, discarding the supernatant, washing twice by using nuclease-free water, and adding the nuclease-free water for resuspension (4 mu L/well). Preparing a PCR reaction solution in a 1.5mL centrifuge tube, adding a corresponding third section of primer (Barcode Z1-Z18, 5 mu M), adding a corresponding PCR reaction solution (6 mu L/hole) into each 384-pore plate after uniform mixing, uniformly mixing, centrifuging, placing in a PCR instrument for reaction, adsorbing inner magnetic beads of the 384-pore plates by using a high-flux magnetic bead transfer device after the PCR reaction is finished, discarding the supernatant, washing once by using nuclease-free water, adding 15 mu L of exonuclease mixture (6.125 mL of nuclease-free water, 700 mu L of exonuclease I buffer solution and 175 mu L of exonuclease I) into each pore, and incubating for 60min at 37 ℃. The beads in each well were washed once with 15. Mu.L of TE-SDS solution and once with TE-TW solution, and finally resuspended in 20. Mu.L of nuclease-free water. Covering a sealing plate membrane, placing a 384-pore plate in a water bath kettle at 95 ℃ for reacting for 6min, adsorbing magnetic beads in the 384-pore plate by using a high-flux magnetic bead transfer device, discarding unreacted solution, and washing the magnetic beads twice by using nuclease-free water preheated at 95 ℃. Add 15. Mu.L of TE-TW solution to each well for resuspension and store at 4 ℃.
Example 3
1. Tissue sectioning and staining
The brain of a mouse is taken and quickly frozen, the brain tissue of the mouse is sliced along the sagittal axis and the coronal axis respectively to obtain 25 coronal slices and 11 sagittal slices, and the brain slices are dyed by a tar violet staining method.
2. Micro-dissection
Each brain region (13 different brain regions) in the section was cut using a Laser Capture Microdissection (LCM) system. The cut tissue pieces were collected using 96-well plates, each tissue piece collected from a designated well (total 214 tissue samples, collected from 3 96-well plates). Cell lysate and magnetic beads with different barcodes (magnetic beads prepared by the method of the present application) were added to a 96-well plate, one for each well.
3. RNA-seq sequencing
Combining the micro-cut tissue block with magnetic beads in a 96-well plate, and performing tissue lysis, mRNA capture, reverse transcription, amplification, cDNA library construction and other steps to obtain a sample
Paired-end sequencing was performed on the Xten platform.
The mrnas in the 214 brain area samples are captured and subjected to sequencing analysis, and the sequencing data is subjected to Drop-seq standard analysis to obtain a digital gene expression matrix (DGE) of the tissue sample, wherein the basic statistics are shown in table 1:
TABLE 1 basic statistical List of the number of samples in each brain region and the gene expression matrix
The numbers of reads, transitions and genes in Table 1 are the average of multiple samples of this brain region. It can be seen that the total distribution of the number of genes obtained from each brain region is uniform after data combination.
Claims (2)
1. A DNA barcode labeling method for labeling a plurality of samples, comprising the steps of:
(1) Establishing an NXNXN stereoscopic hole array, wherein magnetic beads are added in each hole;
(2) Marking the three-dimensional hole array as N along the X, Y and Z directions respectively X1 ～N Xn 、N Y1 ～N Yn And N Z1 ～N Zn Obtaining a corresponding X, Y, Z coordinate for each well;
(3) Marking a plurality of first segment sequences connected to the magnetic beads as Barcode X1-Xn, barcode Y1-Yn or Barcode Z1-Zn corresponding to the coordinates of the hole; then, a plurality of second segment sequences connected with the first segment sequences are marked as Barcode X1-Xn, barcode Y1-Yn or Barcode Z1-Zn, and are different from the first segment sequences; finally, marking the third section of sequence connected with the second section of sequence as a mark different from the first section and the second section in Barcode X1-Xn, barcode Y1-Yn or Barcode Z1-Zn;
(4) Sequentially adding a first sequence, a second sequence and a third sequence which have the same marks with the coordinates of the magnetic bead hole into the corresponding holes, wherein the first sequence is connected with the magnetic bead through EDC reaction, the first sequence, the second sequence and the third sequence are connected through PCR, and then placing the tissue sample into the corresponding holes;
the first sequence is a sequence modified by 5' end amino, and two ends of the first sequence are respectively connected with a PCR handle and a first connecting sequence; the first section of sequence is connected with magnetic beads coated by carboxyl through EDC reaction;
the two ends of the second segment of sequence are respectively connected with a reverse complementary sequence connected with the first connecting sequence and a second connecting sequence;
one end of the third sequence is connected with a reverse complementary sequence connected with the second connecting sequence, and the other end of the third sequence is sequentially connected with UMI used for gene quantification and Poly T used for capturing transcripts with a Poly A tail at the 3' end.
2. The method of claim 1, wherein the first segment sequences are Barcode X1-Xn; the plurality of second-segment sequences are marked as Barcode Y1-Yn; the third segment sequences are marked as Barcode Z1-Zn.
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