The quality of the NGS library was judged by measuring the concentration and fragment size of the NGS library using a qubit3.0 fluorescence quantifier and Agilent2100 system. The step 3 can be carried out when the quality of the NGS library is qualified.

Step 3, Capture of targets Using the Children solid tumor Gene Panel of the invention

According to the procedures of IDT Hybridization elution Kit (XGen Hybridization and Washkit), 400-600ng of each of the three patient NGS libraries (Sample01, Sample02, Sample03) and 6 standard NGS libraries (Wt1a, Wt1b, MUT1a, MUT1b, MUT2a, MUT2b) was used for target capture.

After the target capture DNA is mixed together, it is hybridized with the inventive panel probe for a period of 4-16h at 65 ℃ and an overnight hybridization for 16h is recommended.

Dynabeads Using streptavidin magnetic beads^TMM-270 Streptavidin Beads separate target DNA sequences captured by the probe, and the target DNA library is obtained by purification after hot elution and room temperature elution. Finally, the concentration of the target DNA library is amplified by using a PCR method and then purified.

The quality of the NGS library was judged by measuring the concentration and fragment size of the NGS library using a qubit3.0 fluorescence quantifier and Agilent2100 system (fig. 1). And (4) performing machine sequencing after the library quality is qualified.

Step 4, sequencing on computer and data analysis

The library was put into an Illumina NGS sequencer (e.g. Novaseq6000) for sequencing to obtain the original sequence data file.

And (4) performing letter generation analysis on the off-line data file to obtain a sequence QC report and obtain a genetic variation information file of each sample.

And (4) judging a positive mutation site by combining clinical characteristics and annotations of various databases such as HGMD, OMIM, Clinvar and the like on the mutation. Table 1 shows the SNV mutation detection of one sample, and shows that the gene Panel can detect the mutation with the mutation frequency of 1% and can reach the minimum detection limit of tumor detection.

Table 2 shows the variation and clinical significance of clinical information related to 3 samples, wherein the fusion of Sample01 suggests the sensitivity of targeted drug application and the tumor typing and prognosis conditions, the variation of the gene copy number of Sample02 suggests the prognosis conditions, the evaluation is a high-risk group, and the gene fusion of Sample03 clearly suggests the tumor type.

Table 1 shows the detection of SNV (single nucleotide variation) in one example

Gene	Chromosome position a	Genetic variation	Frequency of variation
				SMARCA4	chr19:11172458	NM_003072:intron34:c.4912-2A>C	10.5％
CLCN6	chr1:11888205	NM_001286:exon11:c.883C>T:p.R295C	51.6％
				PBX1	chr1:164529166	NM_002585:exon1:c.107A>G:p.E36G	6.7％
OBSCN	chr1:228456386	NM_052843:exon17:c.5017C>T:p.R1673C	1.1％
				OBSCN	chr1:228506884	NM_052843:exon54:c.14431G>A:p.A4811T	49.8％
NEB	chr2:152534437	NM_004543:exon33:c.3520G>A:p.A1174T	51.7％
				FGFR3	chr4:1807297	NM_000142:exon12:c.1546G>C:p.D516H	48.3％
PDGFRB	chr5:149505089	NM_002609:exon12:c.1726G>T:p.G576C	47.2％
				THBS2	chr6:169634880	NM_003247:exon11:c.1600G>A:p.V534M	47.2％
NUTM2B	chr10:81470422	NM_001278495:exon5:c.1636A>G:p.K546E	1.6％
				GNAS	chr20:57429939	NM_080425:exon1:c.1619C>T:p.P540L	1.3％
RLIM	chrX:73811792	NM_016120:exon4:c.1358G>A:p.S453N	2.9％

TABLE 2-3 samples Positive variants and their clinical significance

In conclusion, the pediatric solid tumor Panel of the invention integrates the hot spot genes of the current pediatric solid tumor, covers the hot spot gene mutation, gene fusion and gene copy number variation, efficiently and specifically captures target DNA and performs sequencing analysis, can well replace the mode of simultaneously detecting WES (gene mutation detection) and RNAseq (gene fusion detection), and has high cost performance. Meanwhile, the kit can prompt the tumor classification, the prognosis curative effect and the targeted medication condition, and provides comprehensive evidence of a gene level for a clinician in the diagnosis and treatment process.

The panel of the invention was then sensitivity-verified using three standards of commercial standard HD780 (horizons) wild type, at concentrations of 1% and 5%, repeated 1 time for each concentration of standard, with the results shown in Table 3;

table 3-standard HD780 wild type (Wt1a, Wt1b), 1% (MUT1a, MUT1b) and 5% (MUT2a, MUT2b) detected.

As is clear from Table 3, the gene Panel of the present invention can detect a mutation at a mutation frequency of 1% and can achieve the minimum detection limit for tumor detection.

Example 2 detection System for solid tumor in Children

An embodiment of the present invention provides a system for detecting a solid tumor of a child, including:

The bioinformatics processing includes: and (4) carrying out biological information analysis on the sequencing result to obtain a sample mutation result. And the sequencing result is a fastq file, and the sequencing result is analyzed by using an algorithm for acquiring the gene mutation issued by Broad to obtain and annotate the gene mutation result. The method mainly comprises the steps of performing annotation by quality control of a fastq file, genome association, analysis of somatic cell mutation and embryonic cell mutation, wherein the used software has the fastq file for quality control; bwa, and GATK and mutect2 obtain somatic mutation; obtaining embryo cell mutation by a HaplotpypeCaller method; ANNOVAR is annotated.

And combining the mutation information with clinical information to obtain final diagnosis.

Example 3 detection System for solid tumor in Children

The embodiment of the invention provides a cervical cancer prognosis system, which comprises:

a processor and a memory coupled to the processor, the memory storing instructions,

the instructions when executed by the processor use the steps of:

Embodiment 4 computer-readable storage Medium

Embodiments of the present invention provide a computer-readable storage medium, on which a computer program is stored, which, when executed by a processor, implements the steps of the embodiment 2 method and/or the embodiment 3 method.

Of course, the storage medium provided by the embodiment of the present invention contains computer-executable instructions, and the computer-executable instructions are not limited to the method operations described above, and may also perform related operations in the method provided by any embodiment of the present invention.

From the above description of the embodiments, it is obvious for those skilled in the art that the present invention can be implemented by software and necessary general hardware, and certainly, can also be implemented by hardware, but the former is a better embodiment in many cases. Based on such understanding, the technical solutions of the present invention may be embodied in the form of a software product, which may be stored in a computer-readable storage medium, such as a floppy disk, a Read-Only Memory (ROM), a Random Access Memory (RAM), a FLASH Memory (FLASH), a hard disk or an optical disk of a computer, and includes instructions for enabling an electronic device (which may be a personal computer, a server, or a network device) to execute the methods according to the embodiments of the present invention.

It should be noted that, in the foregoing embodiment, each included unit and each included module are only divided according to functional logic, but are not limited to the above division as long as the corresponding functions can be implemented; in addition, specific names of the functional units are only for convenience of distinguishing from each other, and are not used for limiting the protection scope of the present invention.

Finally, it should also be noted that the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.

While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. Therefore, it is intended that the appended claims be interpreted as including preferred embodiments and all such alterations and modifications as fall within the scope of the embodiments of the invention.

It will be apparent to those skilled in the art that various modifications and variations can be made in the embodiments of the present invention without departing from the spirit or scope of the embodiments of the invention. Thus, if such modifications and variations of the embodiments of the present invention fall within the scope of the claims of the embodiments of the present invention and their equivalents, the embodiments of the present invention are also intended to encompass such modifications and variations.

Claims

1. Use of a gene marker combination for the preparation of a probe combination and/or a kit for the detection of solid tumors in children, wherein the gene marker combination comprises:

2. The use according to claim 1, wherein the pediatric solid tumor comprises: a brain tumor, one of neuroblastoma, kidney tumor, soft tissue tumor, langerhans cell histopathy, osteogenic sarcoma, germ cell tumor, ewing tumor, retinoblastoma, and germ cell tumor.

3. A probe combination for detecting solid tumors of children, which is characterized by comprising:

targeting DNA probe: for capturing a first gene marker combination targeted to any one of claims 1-2;

fusion DNA Probe: for capturing a gene fusion comprising a DNA fragment produced by the gene fusion of any one of the second gene marker combinations of claims 1-2.

4. The probe combination for detecting solid tumors in children as claimed in claim 3, wherein the targeting DNA probe and the fusion DNA probe are nucleotide oligomers and are respectively complementary to the targeting DNA or the fusion of the targeting DNA.

5. The probe combination for detecting the solid tumors of children as claimed in claim 3, wherein the probe size of the targeting DNA probe and the fusion DNA probe is 75-200 nucleotides.

6. A kit for detecting solid tumors in children, comprising: the probe combination for detecting solid tumors in children according to any one of claims 3 to 5.

7. A system for detecting solid tumors in children, comprising:

a sequencing module for obtaining sequencing results of the gene marker combination of any one of claims 1-2;

8. The system for detecting solid tumors in children according to claim 7, wherein the bioinformatics processing comprises the specific steps of:

9. A system for detecting solid tumors in children, the system comprising:

10. A computer-readable storage medium, on which a computer program is stored, which, when being executed by a processor, uses the steps of claim 9.