CN111893576B - Construction method of trace cell genome sequencing library - Google Patents

Construction method of trace cell genome sequencing library Download PDF

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CN111893576B
CN111893576B CN202010649228.9A CN202010649228A CN111893576B CN 111893576 B CN111893576 B CN 111893576B CN 202010649228 A CN202010649228 A CN 202010649228A CN 111893576 B CN111893576 B CN 111893576B
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nucleic acid
dna
transposase
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CN111893576A (en
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范小英
苏丹
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Bioisland Laboratory
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    • C40B50/00—Methods of creating libraries, e.g. combinatorial synthesis
    • C40B50/06—Biochemical methods, e.g. using enzymes or whole viable microorganisms
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    • C12Q1/6869—Methods for sequencing

Abstract

The invention relates to the technical field of molecular biology experiments, in particular to a construction method of a trace cell genome sequencing library. The method comprises the following steps: a) obtaining a DNA mixture to be detected; b) fragmenting the DNA mixture into nucleic acid fragments using a transposase, and adding identical transposable linkers to both ends of the nucleic acid fragments; controlling the average length of the nucleic acid fragments to be 5 kb-20 kb by adjusting the working concentration of the transposase; c) and amplifying the nucleic acid fragment by using an amplification primer. The method is prominent in the detection research of chromosome structure variation and extrachromosomal circular DNA, and provides a new prospect for the assembly of genome and the accurate positioning of sequence structure.

Description

Construction method of trace cell genome sequencing library
Technical Field
The invention relates to the technical field of molecular biology experiments, in particular to a construction method of a trace cell genome sequencing library.
Background
The sequencing of micro-cells, especially Single-cell whole-genome sequencing (scWGS), is a leading-edge tool in modern molecular biology research, can well reveal cell heterogeneity in samples such as tumors, discover various mutations, evaluate genomic instability, especially can more accurately excavate hidden information in mixed samples in the process of cancer development, and has a good application prospect. Several single cell genome technologies are currently available including DOP-PCR technology, Multiple Displacement Amplification (MDA) technology, multiple annealing and loop-based amplification cycling (MALBAC) technology, LIANTI technology for linear amplification by transposon insertion, etc. The single cell genome sequencing methods can generate highly accurate short-read long genome sequencing data, and are very suitable for discovering Copy Number Variation (CNV), small fragment insertion or deletion, and Single Nucleotide Variation (SNV), however, due to the defect of second-generation sequencing short-read long, the detection of Structural Variation (SV) based on single cell genome sequencing still has great challenges, and currently, there are few related reports.
The existing single cell genome sequencing method is limited by the short reading length of sequencing by the traditional Next Generation Sequencing (NGS) technology, so that the complex chromosome structure variation of a large fragment or multiple complex sequences is difficult to effectively discover. Structural chromosomal variations include deletions, insertion repeats, inversions, and translocations, which are the major sources of somatic genetic variation and can promote tumor development and metastasis. In addition to SV, extrachromosomal circular dna (ecdna) has recently been found in human cells. The current research shows that the ecDNA is mainly present in tumor tissues and tumor cell lines, and a plurality of research results show that the ecDNA plays a regulating role in cancer, particularly, large molecular ecDNA with hundreds to thousands of kb can generate a plurality of copies in cells so as to promote the expression of protooncogenes, drive the occurrence of tumor heterogeneity and possibly participate in the drug resistance regulation of tumors. These abnormal changes in genome architecture are important for us to uncover unknown true phase and complex regulation of cancer, but there is currently no efficient method to achieve single cell resolution for SV and ecDNA detection.
The single molecule real-time (SMRT) sequencing technology has unique advantages in whole genome SV detection, and high-fidelity sequencing reads of a DNA template are generated by adopting a continuous rolling circle consensus sequence sequencing (CCS) mode, so that high-precision (99.8%) long-read sequencing can be realized, and the reading possibility of directly crossing SV connection points and crossing complex regions containing repeated sequences is improved. In addition, the high fidelity of long read length sequencing improves the sensitivity and reliability of SV detection over other sequencing platform technologies. However, because SMRT DNA sequencing requires a large number of long DNA fragments, genomic DNA from a single cell cannot meet normal library construction requirements, which presents a challenge to successfully implement single cell-scale genomic detection.
In view of the above, the present invention is particularly proposed.
Disclosure of Invention
The invention relates to a construction method of a trace cell sequencing library, which comprises the following steps:
a) obtaining a DNA mixture to be detected;
b) fragmenting the DNA mixture into nucleic acid fragments using a transposase, and adding identical transposable linkers to both ends of the nucleic acid fragments;
controlling the average length of the nucleic acid fragments to be 5 kb-20 kb by adjusting the working concentration of the transposase;
c) and amplifying the nucleic acid fragment by using an amplification primer.
According to a further aspect, the invention relates to the use of the method as described above in the sequencing of micro-cellular genomes.
The invention also relates to a kit for constructing a trace cell genome sequencing library, which is characterized by comprising Tn5 transposase with the packaging concentration of 0.01 ng/mu L-0.05 ng/mu L and a fragmentation buffer solution.
Compared with the prior art, the invention has the beneficial effects that:
1) the method provided by the invention is outstanding in the detection of chromosome Structural Variation (SV), the possibility of directly capturing the complete variant structure is greatly increased by long-reading sequencing data, and the result after structural variation can be directly read in one complete sequencing data.
2) On the level of trace cells, particularly single cells, the long-read sequencing data enhances the analysis capability of a complex region containing a large number of repeated sequences, and provides a new prospect for the assembly of a genome and the accurate positioning of a sequence structure.
3) The method provided by the present invention provides a method for studying extrachromosomal circular dna (ecdna) at the single cell level.
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In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 is a schematic technical flow chart of the Smile-seq method of the present invention;
FIG. 2 is a graph showing a single-cell gDNA amplification product length distribution according to an embodiment of the present invention, showing that most of the amplification products are around 6kb in length;
FIG. 3 is a graph showing the read length distribution of CCS reads obtained by PB sequencing and analysis in accordance with the amplification products before library construction in one embodiment of the present invention;
FIG. 4 is an example of SV detection results using Smile-seq in one embodiment of the present invention; a, the result statistics of SV mutation types such as deletion, insertion, duplication, inversion and the like detected in the experiment are shown; panel b shows two of the translocation mutations detected with Smile-seq that are common in the k562 cell line: translocation of chromosome 9 and 22 resulted in BCR-ABL1 fusion gene and NUP214-XKR3 fusion gene examples;
FIG. 5 is an example of the result of detection and identification of ecDNA in K562 cells using the Smile-seq technique in an embodiment of the present invention, and a is a schematic diagram showing the procedure for screening the ecDNA in K562 cells; b, statistics of the candidate ecDNA detected in the experiment shown in the graph and the length distribution thereof; panels c and d demonstrate the results of partial ecDNA validation by PCR and Sanger sequencing.
Detailed Description
Reference will now be made in detail to embodiments of the invention, one or more examples of which are described below. Each example is provided by way of explanation, not limitation, of the invention. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the scope or spirit of the invention. For instance, features illustrated or described as part of one embodiment, can be used on another embodiment to yield a still further embodiment.
It is therefore intended that the present invention cover the modifications and variations of this invention provided they come within the scope of the appended claims and their equivalents. Other objects, features and aspects of the present invention are disclosed in or are apparent from the following detailed description. It is to be understood by one of ordinary skill in the art that the present discussion is a description of exemplary embodiments only, and is not intended as limiting the broader aspects of the present invention.
The invention relates to a construction method of a trace cell sequencing library, which comprises the following steps:
a) obtaining a DNA mixture to be detected;
b) fragmenting the DNA mixture into nucleic acid fragments using a transposase, and adding identical transposable linkers to both ends of the nucleic acid fragments;
controlling the average length of the nucleic acid fragments to be 5 kb-20 kb by adjusting the working concentration of the transposase;
c) and amplifying the nucleic acid fragment by using an amplification primer.
To achieve the goal of long DNA fragment library in micro cells, we developed a new micro cell sequencing method and named Smile-seq, which amplifies single cell genomic DNA by transposon insertion for long fragment amplification. Unlike the commonly used design, we embedded two-molecule adapters of the same sequence onto transposase, theoretically allowing 100% recovery of the original DNA fragments by transposable PCR (traditional transposases only 50% efficient with two different adapters). In addition, the present invention also overcomes the problem that adapters of the same sequence are susceptible to circularization by controlling the length of the nucleic acid fragments by adjusting the working concentration of the transposase. The amplified long fragment DNA is used for directly sequencing on a Third Generation Sequencing (TGS) platform such as an SMRT DNA sequencing platform to obtain high-quality single-cell genome sequence information. The detection of SV and/or ecDNA has unique advantages.
The DNA mixture in the present invention may be derived from any sample, wherein the term "sample" is used in its broadest sense. In one sense, it is meant to include cells (e.g., human, bacterial, yeast, and fungi), tissues or living bodies, or samples or cultures obtained from any source, as well as biological samples. Biological samples may be obtained from animals (including humans) and refer to biological materials or compositions found therein, including but not limited to bone marrow, blood, serum, platelets, plasma, interstitial fluid, urine, cerebrospinal fluid, nucleic acids, DNA, tissue, and purified or filtered forms thereof. The sample may be derived from a healthy sample or a diseased sample, such as tumor tissue/cells. However, these examples should not be construed as limiting the type of sample that can be used in the present invention.
The term "amplification" when co-occurring in the context of the term "nucleic acid" or "nucleic acid fragment" refers to the generation of multiple copies of a polynucleotide, or portion of a polynucleotide, typically starting from a small amount of the polynucleotide (e.g., as little as a single polynucleotide molecule), wherein the amplification product or amplicon is typically detectable. Amplification of polynucleotides includes a variety of chemical and enzymatic methods. The generation of multiple copies of DNA from one or a few copies of a target or template DNA molecule during Polymerase Chain Reaction (PCR), Rolling Circle Amplification (RCA) or Ligase Chain Reaction (LCR) is an amplified form. Amplification is not limited to the strict replication of the starting molecule. For example, the use of reverse transcription RT-PCR to generate multiple cDNA molecules from a limited amount of RNA in a sample is an amplified version. In addition, the production of multiple RNA molecules from a single DNA molecule during the transcription process is also an amplified version.
In some embodiments, the nucleic acid fragments are controlled to have an average length of 5kb to 15 kb;
in some embodiments, the nucleic acid fragments are controlled to have an average length of 5kb to 10 kb;
in some embodiments, the nucleic acid fragments are controlled to have an average length of 5.5kb to 7 kb;
in some embodiments, the nucleic acid fragments are controlled to have an average length of 5.9kb to 6.1 kb;
in some embodiments, the nucleic acid fragments are controlled to have an average length of 6 kb.
In some embodiments, the transposase is highly active.
In some embodiments, the transposase is selected from the group consisting of one or a combination of any one of Tn1, Tn2, Tn3, Tn4, Tn5, Tn6, Tn7, Tn9, Tn10, Tn551, Tn971, Tn916, Tn1545, Tn1681, Tgf2, Tol2, Himar1, and HARBI 1.
Tgf2 and Tol2 are from the hAT family, Himar1 from the Tcl/Mariner family, and HARBI1 from the PIF/Harbinger family.
A preferred transposase is Tn 5.
In some embodiments, the transposase is Tn5 at a working concentration of 0.01 ng/. mu.L to 0.05 ng/. mu.L, alternatively 0.015 ng/. mu.L to 0.025 ng/. mu.L, or 0.017 ng/. mu.L, 0.020 ng/. mu.L, or 0.023 ng/. mu.L.
In the present invention, the Tn5 enzyme is controlled within a suitable range to ensure that the genomic DNA of a single cell is cut into suitably long fragments (fragments are enriched in a length of 5kb to 20 kb).
In some embodiments, in step b), the fragmentation buffer used comprises 40mM to 60mM TAPS-NaOH or TAPS-KOH, 20mM to 30mM Mg2+PEG8000 of 35g/100ml to 45g/100ml, pH 8.0 to 8.6.
In some embodiments, in step b), the main active ingredient of the fragmentation buffer used comprises 45mM to 55mM TAPS-NaOH or TAPS-KOH, 22mM to 28mM Mg2+PEG8000 of 37g/100ml to 43g/100ml, pH 8.1 to 8.5.
In some embodiments, in step b), the main active ingredient of the fragmentation buffer used comprises 47mM to 53mM TAPS-NaOH or TAPS-KOH, 22mM to 27mM Mg2+38g/100ml to 42g/100ml of PEG8000, and pH 8.2 to 8.4.
In some embodiments, in step b), the main active ingredient of the fragmentation buffer used comprises 50mM TAPS-NaOH or TAPS-KOH, 25mM Mg2+40g/100ml PEG8000, pH 8.3.
In the present invention, the buffer provided in the commercial Tn5 transposase kit is not suitable for picogram-scale transposition reaction of nucleic acid fragments. 5 × TAPS _ PEG8000 stabilized in single cell reactions after buffer formulation adjustment.
In the present invention, it is necessary to amplify the nucleic acid fragments to obtain a sufficient amount of the initial DNA of the library. The number of cycles of the PCR reaction cannot exceed 22, otherwise it will be amplified too much, resulting in a bias of shorter fragments. In some embodiments, the number of amplification cycles is 15 to 22, and optionally 18 to 20, preferably 20.
In some embodiments, the linker is 9bp to 18bp in length, e.g., 10bp, 11bp, 12bp, 13bp, 14bp, 15bp, 16bp, 17 bp.
In some embodiments, the amplification primers comprise a sequencing adaptor, a cell identification barcode, and an anchor sequence for binding to the transposable adaptor and performing PCR extension amplification.
Wherein the cell identification barcodes are used to identify and distinguish samples from different cells, or from minute amounts of cells within the same group.
In some embodiments, the DNA mixture is genomic DNA.
In some embodiments, the trace amount of cells has a cell number of 10000 cells or less;
in some embodiments, the cell number of the trace amount of cells is 1000 cells or less;
in some embodiments, the cell number of the trace amount of cells is a single cell.
In some embodiments, the trace amount of cells is a monoclonal line.
In some embodiments, the length of the identification tag and the random cell barcode may be independently selected from 10bp to 35bp, such as 11bp, 12bp, 13bp, 14bp, 15bp, 16bp, 17bp, 18bp, 19bp, 20bp, 21bp, 22bp, 23bp, 24bp, 25bp, 26bp, 27bp, 28bp, 29bp, 30bp, 31bp, 32bp, 33bp, 34 bp.
In some embodiments, the resulting amplification product is also subjected to at least one purification. The method can be used for purifying and screening by using XP magnetic beads, the using ratio of the magnetic beads is 0.3-0.6 times of the volume, more preferably 0.4 times of the volume, and the purifying times are 2 times.
According to a further aspect, the invention relates to the use of the method as described above in the sequencing of micro-cellular genomes.
In some embodiments, the minicell genome sequencing is based on a three-generation sequencing platform, such as the Pacific Biosciences sequencing platform.
In some embodiments, the application includes a step of performing mixed library construction on amplified products of different cell barcodes after amplification.
When the number of cells in each group of micro cells is single cells, the total amount of DNA obtained by mixing amplification products of different cell barcodes and purifying the amplification products can reach the microgram level by using the method.
In some embodiments, the micro-cellular genomic sequencing is used to discover DNA comprising one or more of copy number variation, small fragment insertions or deletions, single nucleotide variation, chromosomal structural variation, extrachromosomal circular DNA, preferably comprising at least one of genomic single base mutations, chromosomal structural variation, extrachromosomal circular DNA.
The invention also relates to a kit for constructing the trace cell genome sequencing library, which comprises Tn5 transposase with the packaging concentration of 0.01 ng/mu L-0.05 ng/mu L and a fragmentation buffer solution.
In some embodiments, the fragmentation buffer comprises 40mM to 60mM TAPS-NaOH or TAPS-KOH, 20mM to 30mM Mg2+PEG8000 of 35g/100ml to 45g/100ml, pH 8.0 to 8.6.
In some embodiments, the kit further comprises one or more of a transposable linker, an amplification primer as described above, dntps, a DNA polymerase, an amplification buffer, DNA purification reagents, and water.
In some embodiments, the DNA polymerase is high fidelity.
Embodiments of the present invention will be described in detail with reference to examples.
Examples
Taking the human K562 cell line as an example, the control group adopted standard large-scale cell genome DNA extraction and NGS library construction and sequencing: genomic DNA was extracted using DNeasy Blood and Tissue Kit (QIAGEN,69504), quantified using the Qubit dsDNA HS Assay Kit (Invitrogen, Q32851), and about 500ng of genomic DNA was fragmented using Covaris S220 and then library-constructed using the KAPA Hyper Prep Kit (Roche, KR 0961). The resulting sequencing library was sequenced using the NGS platform XTEN.
This example was repeated to verify the optimal reaction conditions, including control of the amount of enzyme in the transposition reaction (i.e., fragment length control), transposition buffer formulation and screening of DNA polymerase, to achieve single-cell genomic long fragment capture and amplification.
The specific implementation process of Smile-seq is as follows
Single cell lysis
The cell lysate formulation was as follows:
Figure GDA0002965070140000081
separating to obtain single cells, transferring each cell into an independent reaction tube, adding 2.5 mu L of cell lysate into each tube, shaking and mixing, incubating at 50 ℃ for 3 hours, then incubating at 70 ℃ for 30 minutes to fully inactivate protease, and directly performing subsequent treatment on the sample or freezing and storing at-80 ℃.
2. Transposase linker treatment (TTE Mix)
Add Adapter primers and then dilute to 100. mu.M, 1: 1 annealing to obtain a linker mixture (Adapter Mix), gradient annealing in a PCR instrument: 75 ℃, 15 minutes, 60 ℃,10 minutes, 50 ℃,10 minutes, 40 ℃,10 minutes, 25 ℃, 30 minutes.
Tn5 and linker Encapsulated
Composition (I) Final concentration Volume (μ L)
Tn5(2μg/μL) 400ng/μL 10
AdapterMix(50μM) 7μM 7
Coupling Buffer ---- 33
TTE Mix 400ng/μL 50
The mixture was thoroughly pipetted and incubated at 30 ℃ for 1 hour to obtain a Tn5 transposase complex (TTE Mix) at a stock concentration of 400 ng/. mu.L and stored at-20 ℃.
The Adapter primer sequences are as follows:
Adapter primer15’-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-3’
Adapter primer25’-phos-CTGTCTCTTATACACATCT-NH3-3’
the Coupling Buffer is contained in the Vazyme commercial kit S111-01.
3. Transposon-mediated DNA fragmentation
The fragmentation reaction solution has the following formula:
composition (I) Final concentration Volume (μ L)
5×TAPS_PEG 8000 1× 2
TTE Mix(0.2ng/μL) 0.02ng/μL 1
H2O ---- 4.5
General System ---- 7.5
To 2.5. mu.L of the cell lysate, 7.5. mu.L of the fragmentation reaction solution was added, and the mixture was gently shaken several times to mix them. The following reactions were carried out on a temperature controlled cyclic PCR instrument: 55 ℃ for 10 minutes, then held at 4 ℃.
The reaction was stopped by adding 2.5. mu.L of 0.2% SDS, and after mixing, DNA fragmentation was completed by leaving at room temperature for 5 minutes.
5 × TAPS _ PEG8000 ingredients as follows: 50mM TAPS-NaOH (or KOH), pH 8.3(RT), 25mM MgCl2，40％PEG 8000。
PCR amplification reaction
The PCR amplification reaction solution has the following formula:
Figure GDA0002965070140000091
adding 37.5. mu.L of PCR amplification reaction solution into the reaction product of the previous step to make the final reaction system be 50. mu.L, and carrying out the following PCR amplification procedure:
Figure GDA0002965070140000101
PCR primer I5-PB contained a random barcode sequence of 16bp and an anchor sequence of 14bp complementary to the template DNA end-linker: 5 'AATGATACGGCGACCACCGAGATCTNNNNNNNNNNNNNNNNTCGTCGGCAGCGTC 3'. Wherein N represents any one of ATCGs.
5. Sample mixing and purification
Mixing gDNA amplification samples connected with different cell bar codes together, purifying by using 0.4 multiplied by AMPure PB magnetic bead, mixing the magnetic bead with 0.4 times volume of the product, incubating for 5 minutes at room temperature, then placing on a magnetic frame, and removing the supernatant after the magnetic bead is adsorbed to the side wall. The magnetic beads were washed 2 times with 80% ethanol, air dried, resuspended in an appropriate volume of aqueous solution, allowed to stand at room temperature for 2 minutes, placed on a magnetic rack, and the supernatant product was transferred to a new sample tube. The purification step is repeated once, and the purified product is dissolved in an appropriate amount of PB buffer solution to enable the final concentration to be more than 50 ng/. mu.L so as to meet the initial requirement of the subsequent third-generation sequencing library construction.
PB library construction and sequencing
Library construction and sequencing of the amplified products was performed according to the Pacific Biosciences sequencing platform requirements, and PB sequencing library construction was performed according to the instructions using the SMRTbell Template Prep Kit v.1.0-SPv3(Pacific Biosciences, Ref.No. 100-991-900).
The primer sequences used in the examples of the present invention:
Adapter primer1 5'-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-3'；Adapter primer2 5'-phos-CTGTCTCTTATACACATCT-NH3-3'. Namely the Adapter primer, and a primer dimer formed after annealing treatment contains 19bp double-stranded transposase binding sites and a single-stranded Adapter sequence.
I5-PB primer 5 'AATGATACGGCGACCACCGAGATCTNNNNNNNNNNNNNNNNTCGTCGGCAGCGTC 3' is gDNA template PCR amplification primer, and comprises a bar code positioning label, a random bar code sequence of 16bp and an anchoring sequence of 14bp which is complementary with a template DNA terminal joint.
Other reagent information Tris-EDTA (Santa Cruz, sc-296654); 10% Triton X-100(Sigma-Aldrich, T9284); qiagen protease (Qiagen, 1020952); 1M KCL (Sigma-Aldrich, 58221-; TAPS (Sigma-Aldrich, T51301000); KOH (Sigma-Aldrich, P1767-250G); MgCl2(Sigma-Aldrich,20-303)；PEG 8000(Sigma-Aldrich,V3011)；Tn5(Vazyme,S111-01)；0.2％SDS(Psaitong,PS0062-100mL)；2×Gflex PCR Buffer&Tks Gflex DNA Polymerase(Takara,R060B)；Ampure PBbeads(Pacific Biosciences,100-265-900)；SMRTbell Template Prep Kit(Pacific Biosciences,100-991-900)。
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims (8)

1. The construction method of the micro cell sequencing library is characterized by comprising the following steps:
a) obtaining a DNA mixture to be detected; the DNA mixture is genomic DNA;
b) fragmenting the DNA mixture into nucleic acid fragments using a transposase, and adding identical transposable linkers to both ends of the nucleic acid fragments;
controlling the average length of the nucleic acid fragments to be 5 kb-20 kb by adjusting the working concentration of the transposase;
c) amplifying the nucleic acid fragment by using an amplification primer;
the transposase is Tn5, which is Tn5 transposase with the product number of S111-01 in a Vazyme commercial kit, and the working concentration is 0.02 ng/mu L.
2. The method according to claim 1, wherein in step b), the fragmentation buffer used comprises 40 mM-60 mM TAPS-NaOH or TAPS-KOH and 20 mM-30 mM Mg2+PEG8000 of 35g/100ml to 45g/100ml, and pH =8.0 to 8.6.
3. The method of claim 1, wherein the amplification primers comprise a sequencing adaptor, a cell identification barcode, and an anchor sequence for binding to the transposable adaptor and performing PCR extension amplification.
4. The method according to any one of claims 1 to 3, wherein the number of cycles of amplification in step c) is 15 to 22.
5. The method according to any one of claims 1 to 3, wherein the number of cells in the micro-scale cells is not less than 1 and not more than 10000.
6. Use of the method of any one of claims 1 to 5 for non-diagnostic purposes in sequencing of micro-cellular genomes.
7. The use of claim 6, wherein the micro-cellular genome sequencing is based on a three generation sequencing platform.
8. The use of claim 6 or 7, wherein the sequencing of the genome of the minicell is used to discover at least one of single base mutations in the genome, structural variations in the chromosome, and extrachromosomal circular DNA.
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