Soft tissue sarcoma gene fusion detection panel targeting rna and kits and methods thereof

Abstract

This invention relates to the field of biomedical technology, specifically to a panel for detecting gene fusions in soft tissue sarcoma targeting RNA, along with its kit and method. This invention can simultaneously target 256 core genes closely related to the occurrence, development, pathological classification, and targeted therapy of soft tissue sarcoma, covering key gene loci for common and rare fusion events in soft tissue sarcoma, corresponding to a total of 4500 specific chromosomal transcriptional regions, covering the entire transcriptome regions of the 256 genes. This ensures a high detection rate and reduces the risk of missed detections. The method achieves a sensitivity of 96.15%, a specificity of 100%, and 100% precision both within and between batches.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and more specifically, to a panel for detecting gene fusions in soft tissue sarcomas targeting RNA, as well as its kits and methods. Background Technology

[0002] Soft tissue sarcomas are a group of malignant tumors originating from mesenchymal pluripotent stem cells. Their origin tissues include fibrous, adipose, smooth muscle, striated muscle, mesothelial, vascular, lymphatic, synovial, and neuroectodermal tissues, but exclude bone, cartilage, and lymphohematopoietic tissue. Different pathological types of these tumors exhibit significant heterogeneity in terms of location, transformed cell phenotype, and histopathological characteristics, with over a hundred subtypes identified, posing a serious challenge to clinicopathological diagnosis. Many subtypes present similar histomorphological features, making accurate differentiation difficult using only traditional microscopic observation. Furthermore, different subtypes differ significantly in biological behavior, treatment strategies, and prognosis; therefore, accurate pathological diagnosis is a prerequisite for effective clinical treatment of these tumors.

[0003] Molecular biology studies have confirmed that gene fusion is one of the main oncogenic driver mutations in soft tissue sarcomas. Many specific fusion genes have become the "gold standard" molecular markers for subtype diagnosis, such as EWSR1-FLI1 for Ewing sarcoma and SS18-SSX for synovial sarcoma. Meanwhile, with the development of precision medicine, targeted drugs against gene fusions such as NTRK, ALK, and RET have been applied clinically, making fusion gene detection both diagnostically significant and valuable for medication guidance. Both domestic and international clinical guidelines strongly recommend molecular detection techniques for the auxiliary diagnosis and subtyping of soft tissue sarcomas.

[0004] Currently, commonly used gene fusion detection technologies in clinical practice have significant limitations: while fluorescence in situ hybridization (FISH) is intuitive, it has low throughput and cannot identify unknown fusion partner genes; immunohistochemistry (IHC) is simple to operate, but the interpretation of results is subjective, and the lack of specific antibodies for some fusion gene products can easily lead to false positives or false negatives; polymerase chain reaction (RT-PCR) has high sensitivity, but it requires prior knowledge of the fusion breakpoint location, makes it difficult to detect multiple fusion types simultaneously, and has high requirements for sample RNA quality.

[0005] Next-generation sequencing (NGS) technology has brought breakthroughs to gene fusion detection, but different technical approaches still have drawbacks: DNA-based NGS panels face problems such as low detection sensitivity, high cost, and incomplete coverage due to the need to cover intron regions; while whole transcriptome sequencing can unbiasedly discover fusion genes, data redundancy leads to high detection costs, complex analysis, and long cycles, making it unsuitable for routine clinical diagnosis. RNA-based targeted sequencing technology balances detection breadth, depth, and cost-effectiveness, but existing panels for soft tissue sarcomas often have problems such as limited gene coverage and probes designed only for hotspot exons, making it easy to miss fusion events with unknown partner genes or breakpoints located in rare positions. Summary of the Invention

[0006] This invention aims to overcome the shortcomings of existing soft tissue sarcoma gene fusion detection technologies, such as limited coverage, high risk of missed detection, poor sample adaptability, and imbalance between cost and efficiency.

[0007] This invention discloses a panel for detecting gene fusions in soft tissue sarcoma targeting RNA. The panel includes genes closely related to the occurrence, development, and targeted therapy of soft tissue sarcoma, and comprises the following genes:

[0008] There are hundreds, even thousands, of genes associated with soft tissue sarcoma, and the literature and databases contain a vast amount of even contradictory association information, posing a huge "fog of choice" for those skilled in the art. The technical solution of this invention uses multi-dimensional and inherently conflicting selection criteria, such as clinical diagnostic value, therapeutic guidance significance (whether the gene involves targets of existing targeted drugs such as NTRK, ALK, RET, or has potential future therapeutic value), technical feasibility, and cost controllability, to precisely screen, weigh, and ultimately solidify an "optimal solution" set of 256 genes from a massive pool of candidate genes, covering key gene loci for common and rare fusion events in soft tissue sarcoma.

[0009] Secondly, this invention provides a panel probe composition for detecting soft tissue sarcoma gene fusions targeting RNA. The probe composition comprises probes that hybridize with transcript sequences from one or more chromosomal locus regions, specifically: 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chr5|112116482|112116602,chr5|112128124|112128244,chr5|112136968|112137088,chr5|112145812|112146016,chr5|112151180|112151300,chr5|112154652|112155050,chr5|112157580|112157700,chr5|112158970|112159090,chr5|112162814|112162934,chr5|112163604|112163724chr5|112164550|112164670,chr5|112170638|112170870,chr5|112173240|112181944,chr5|142149931|142150489,chr5|142150781|142150949,chr5|142252951|142253071,chr5|142254645|142254765,chr5|142258913|142259033,chr5|142264853|142264973,chr5|142273797|142273917, chr5|142281491|142281611,chr5|142283109|142283229,chr5|142287259|142287379,chr5|142292751|142292871,chr5|142311591|142311711,chr5|142393603|142393723,chr5|142416733|142416853,chr5|142421357|142421477,chr5|142433987|142434107,chr5|142435561|142435681 chr5|142437199|142437319,chr5|142500543|142500721,chr5|142513541|142513661,chr5|142526785|142526955,chr5|142586753|142587047,chr5|142593547|142593667,chr5|142601863|142604743,chr5|142605013|142605783,chr5|142606141|142607821,chr5|142607915|142608635 chr5|149493391|149495517,chr5|149497171|149497291,chr5|149497301|149497421,chr5|149498301|149498421,chr5|149499019|149499139,chr5|149499569|149499689,chr5|149500451|149500571,chr5|149500765|149500885,chr5|149501433|149501611,chr5|149502595|149502773,chr5|149503807|149503927,chr5|149504281|149504401,chr5|149505013|149505133,chr5|149506069|149506189,chr5|149509309|149509539,chr5|149510103|149510223,chr5|149511539|149511659,chr5|149512303|149512513,chr5|149513139|149513331,chr5|149513447|149513567, chr5|149514269|149514587,chr5|149515107|149515449,chr5|149516533|149516653,chr5|149531891|149533529,chr5|149534949|149535417,chr5|149546331|149546893,chr5|149562321|149562485,chr5|149563027|149564129,chr5|149781191|149781821,chr5|149782097|149782217, chr5|149782673|149782883,chr5|149784225|149784345,chr5|149784635|149784755,chr5|149785793|149785913,chr5|149786423|149786543,chr5|149786705|149786895,chr5|149792177|149792481,chr5|170814119|170814239,chr5|170814803|170815059,chr5|170817011|170817131, chr5|170818301|170818421,chr5|170818695|170818815,chr5|170819731|170820041,chr5|170827125|170827245,chr5|170827859|170827979,chr5|170832259|170832379,chr5|170833381|170834147,chr5|170834681|170834801,chr5|170837549|170837669,chr5|170837843|170837963chr5|176513905|176514087,chr5|176516541|176516703,chr5|176517381|176517835,chr5|176517929|176518113,chr5|176518687|176518807,chr5|176519311|176519521 chr5|176519655|176519775,chr5|176520129|176520561,chr5|176520655|176520775,chr5|176522321|176522733,chr5|176523059|176523179,chr5|176523263|176523383 chr5|176523613|176523733,chr5|176524285|176524405,chr5|176524517|176525153,chr5|179382007|179382727,chr5|179390457|179390577,chr5|179393795|179394019 chr5|179397397|179397517,chr5|179405183|179405303,chr5|179407103|179407223,chr5|179440051|179440319,chr5|179467443|179467655,chr5|179498445|179498767 chr6|31132104|31132652,chr6|31132854|31132974,chr6|31132992|31133112,chr6|31133288|31134174,chr6|31134538|31135006,chr6|31137982|31138468,chr6|33378780|33379000,chr6|33380014|33380374,chr6|33380462|33380582,chr6|33380962|33381082,chr6|33381176|333 81342,chr6|33381500|33381620,chr6|33381792|33381912,chr6|33382020|33382140,chr6|33382226|33382346,chr6|33382494|33382614,chr6|33382722|33383114,chr6|33383346|33383466,chr6|33383576|33384224,chr6|34204640|34205102,chr6|34206558|34206742,chr6|342085 04|34208700,chr6|34210470|34210590,chr6|34211210|34211330,chr6|34212558|34214056,chr6|38136166|38139286,chr6|38139366|38139632,chr6|38139776|38143016,chr6|38160274|38160394,chr6|38224178|38224298,chr6|38256038|38256246,chr6|38312754|38312874,chr6| 38545374|38545494,chr6|38547984|38548222,chr6|38560342|38560624,chr6|38561730|38562160,chr6|38563536|38563852,chr6|38565676|38565906,chr6|38580600|38580818,chr6|38607580|38607700,chr6|41651706|41652824,chr6|41653818|41653984,chr6|41654808|41654928,chr6|41655480|41655774,chr6|41657448|41657568,chr6|41658390|41658982,chr6|41700386|41700506,chr6|41702034|41702154,chr6|41702628|41702806,chr6|41703078|41703354,chr6|41703862|41703982,chr6|43138996|43139916,chr6|43140100|43140220,chr6|43141574|4314 1860,chr6|43143434|43143714,chr6|43144284|43144404,chr6|431460 22|43146232,chr6|43146522|43146642,chr6|43146772|43149302,chr6 |108881017|108881227,chr6|108882109|108883041,chr6|108984647|1 08986101,chr6|109000969|109001915,chr6|109001957|109003597,chr 6|109003641|109004705,chr6|109004971|109006035,chr6|117586711| 117587015,chr6|117589335|117589663,chr6|117591695|117592235,ch r6|117593607|117594737,chr6|117608455|117608815,chr6|117609087 |117610023,chr6|117622127|117622309,chr6|117629963|117630083,c hr6|117631233|117631453,chr6|117632171|117632291,chr6|117638309|117638429,chr6|117639323|117639443,chr6|117641021|117641201,chr6|117642429|117642549,chr6|117645475|117645595,chr6|117647377|117647585,chr6|117650489|117650609,chr6|117658325|117658511,chr6|117662287|117662803,chr6|117663553|117663715,chr6|117665213|117665433,chr6|117674143|117674341,chr6|117677781|117678087,chr6|117678957|117679181,chr6|117680961|117681183,chr6|117681475|117681595,chr6|117683755|117684037,chr6|117686213|117686375, chr6|117686733|117686913,chr6|117687229|117687465,chr6|117700211|117700331,chr6|117704469|117704679,chr6|117706835|117707033,chr6|117708047|117708167,chr6|117708933|117709205,chr6|117710503|117711017,chr6|117714375|117714495,chr6|117715315|117715517, chr6|117715779|117715899,chr6|117717329|117717449,chr6|117718067|117718287,chr6|117724311|117724431,chr6|117725433|117725599,chr6|117730715|117730835,chr6|117735635|117735755,chr6|117737389|117737509,chr6|117739587|117739707,chr6|117746687|117747113, chr6|117881421|117884555,chr6|117888007|117888205,chr6|117890725|117890907,chr6|117892009|117892129,chr6|117894619|117894803,chr6|117896329|117896523,chr6|117898561|117898681,chr6|117900053|117900235,chr6|117923157|117923683,chr6|131160477|131161747,chr6|131179267|131179387,chr6|131184757|131184877,chr6|131186665|131186785,chr6|131188599|131188719,chr6|131190693|131191275,chr6|131191433|131191553,chr6|131197803|131197923,chr6|131199233|131199399,chr6|131201255|131201375,chr6|131206225|131206417, chr6|131211423|131211615,chr6|131215471|131215591,chr6|131216097|131216267,chr6|131220613|131220733,chr6|131222091|131222329,chr6|131225581|131225701,chr6|131229921|131230041,chr6|131247737|131247857,chr6|131276235|131276465,chr6|131277123|131277647, chr6|131291391|131291511,chr6|131321753|131321873,chr6|131334665|131334785,chr6|131384273|131384393,chr6|135502435|135502555,chr6|135502563|135502683,chr6|135507039|135507159,chr6|135508947|135509067,chr6|135510915|135511035,chr6|135511255|135511493, chr6|135513451|135513571,chr6|135513585|135513705,chr6|135514955|135515075,chr6|135515485|135515605,chr6|135516097|135516217,chr6|135516875|135517149,chr6|135517853|135518469,chr6|135520035|135520197,chr6|135520631|135520751,chr6|135521213|135521703,chr6|135522771|135522891,chr6|135524347|135524467,chr6|135538991|135540319,chr6|139692933|139695097,chr6|139695259|139695793,chr6|151815141|151815329,chr6|151857457|151857577,chr6|151859169|151859445,chr6|151865697|151865859,chr6|151869429|151869633, chr6|151894299|151894635,chr6|151907013|151907233,chr6|151914231|151914423,chr6|151917459|151917721,chr6|151936567|151936687,chr6|151936703|151936823,chr6|151939021|151939837,chr6|151940151|151940735,chr6|151940985|151941337,chr6|151941339|151942387, chr6|151977791|151977911,chr6|152011669|152011789,chr6|152023015|152023135,chr6|152126837|152126957,chr6|152128321|152128653,chr6|152128807|152129507,chr6|152130231|152130415,chr6|152163721|152163937,chr6|152201787|152201907,chr6|152265297|152265651, chr6|152332799|152332919,chr6|152382131|152382251,chr6|152415509|152415711,chr6|152419807|152421847,chr6|152421909|152422183,chr6|152422187|152424467,chr6|152446391|152450763,chr6|159186770|159188118,chr6|159188282|159188552,chr6|159190344|159190464,chr6|159190806|159190984,chr6|159191800|159191920,chr6|159192226|159192488,chr6|159197428|159197548,chr6|159204542|159204706,chr6|1592056 56|159205776,chr6|159206330|159206624,chr6|159208126|159208246,chr6|159210300|159210420,chr6|159239104|159239276,chr6|159240314|159240434, chr7|5566718|5567266,chr7|5567352|5567580,chr7|5567614|5568368,chr7|5568742|5569080,chr7|5569170|5569290,chr7|5570132|5570252,chr7|13930794|13933792,chr7|13933880|13935770,chr7|13940352|13940472,chr7|13946044|13946232,chr7|13947388|13947508,chr7|1394 9230|13949350,chr7|13950838|13950958,chr7|13971116|13971382,chr7|13975322|13975530,chr7|13978746|13978866,chr7|14017018|14017138,chr7|14025730|14026046,chr7|14026226|14026346,chr7|14027694|14027814,chr7|14028638|14028758,chr7|14028882|14029300,chr7|1 4030168|14030340,chr7|14030736|14031058,chr7|27870186|27872604,chr7|27880306|27880494,chr7|27934828|27935044,chr7|28031504|28031624,chr7|28220072|28220422,chr7|74072023|74072413,chr7|74103449|74103569,chr7|74105313|74105433,chr7|74113297|74113417,chr 7|74114537|74114799,chr7|74119463|74119583,chr7|74120681|74120801,chr7|74125341|74125461,chr7|74129163|74129283,chr7|74131203|74131395,chr7|74133169|74133289,chr7|74143119|74143239,chr7|74144539|74144659,chr7|74146809|74147011,chr7|74148259|74148379chr7|74149777|74149897,chr7|74150831|74151033,chr7|74152355|74152475,chr7|74157763|74157883,chr7|74159087|74159319,chr7|74160205|74160325,chr7|74160653|74160773,chr7|74162341|74162461,chr7|74163333|74163453,chr7|74163595|74163797,chr7|74165659|741 65779,chr7|74166401|74166521,chr7|74167407|74167527,chr7|74168167|74168369,chr7|74169785|74169905,chr7|74171111|74171231,chr7|74172251|74172371,chr7|74173087|74173207,chr7|74173867|74175031,chr7|94024196|94024422,chr7|94027640|94027760,chr7|940283 18|94028438,chr7|94029494|94029614,chr7|94030844|94030964,chr7|94033858|94034212,chr7|94034476|94034596,chr7|94034950|94035070,chr7|94035528|94035648,chr7|94037120|94037240,chr7|94037484|94037700,chr7|94038048|94038168,chr7|94038622|94038742,chr7| 94038838|94038958,chr7|94039024|94039144,chr7|94039520|94039640,chr7|94039724|94039844,chr7|94040166|94040286,chr7|94040356|94040476,chr7|94041346|94041466,chr7|94041884|94042004,chr7|94042360|94042480,chr7|94042968|94043088,chr7|94043172|94043292chr7|94043500|94043620,chr7|94044500|94044620,chr7|94045706|94045826,chr7|94047028|94047148,chr7|94047776|94047896,chr7|94048776|94048896,chr7|94049510|94049630,chr7|94049692|94049968,chr7|94050286|94050406,chr7|94051176|94051296,chr7|94052258| 94052438,chr7|94053640|94053760,chr7|94054422|94054542,chr7|94054912|94055178,chr7|94055276|94055396,chr7|94055728|94055848,chr7|94056286|94056406,chr7|94056492|94056612,chr7|94056928|94057206,chr7|94057594|94057798,chr7|94058490|94058610,chr7|9 4058630|94058750,chr7|94059548|94060548,chr7|100770375|100770533,chr7|100771663|100771977,chr7|100773691|100773811,chr7|100773823|100773943,chr7|100775145|100775359,chr7|100776965|100777183,chr7|100778765|100778885,chr7|100778985|100779105,chr7| 100780253|100780373,chr7|100780475|100782105,chr7|100782249|100782369,chr7|100782467|100782587,chr7|116312239|116312639,chr7|116339115|116340347,chr7|116371711|116371921,chr7|116380011|116380131,chr7|116380895|116381087,chr7|116395399|116395577chr7|116397481|116397837,chr7|116398503|116398683,chr7|116399381|116399553,chr7|116403093|116403331,chr7|116409689|116409809,chr7|116409823|116409943,chr7|116411541|116411717,chr7|116411893|116412051,chr7|116414925|116415045,chr7|116415053|116415173, chr7|116417423|116417543,chr7|116418819|116419019,chr7|116422035|116422155,chr7|116423347|116423531,chr7|116435649|116436697,chr7|116436953|116438489,chr7|137559665|137559785,chr7|137559831|137561013,chr7|137561101|137565355,chr7|137567147|137567383, chr7|137569743|137569863,chr7|137570137|137570257,chr7|137586073|137586193,chr7|137588673|137588793,chr7|137590437|137590603,chr7|137592987|137593191,chr7|137597559|137597833,chr7|137600573|137600767,chr7|137612885|137613121,chr7|137617631|137617751, 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50052209|50052329,chr20|50071075|50071233,chr20|50090507|50090697,chr20|50091985|50092205,chr20|50133313|50133503,chr20|50139609|50140657,chr20|50158889|50159277,chr20|50179089|50179387,chr20|57414793|57415949,chr20|57416665|57416939,chr20|574277 61|57430397,chr20|57430593|57430713,chr20|57431175|57431295,chr20|57463457|57463881,chr20|57464225|57464417,chr20|57466465|57466929,chr20|57467181|57467301,chr20|57470643|57470763,chr20|57473957|57474077,chr20|57474993|57475563,chr20|57478573|574 78855,chr20|57480425|57480545,chr20|57484183|57484303,chr20|57484395|57484515,chr20|57484523|57484643,chr20|57484739|57484859,chr20|57485011|57485131,chr20|57485361|57485481,chr20|57485727|57486255,chr20|60718825|60718945,chr20|60729327|60729529chr20|60733717|60734007,chr20|60734915|60735035,chr20|60736481|60736645,chr20|60737797|60737995,chr20|60738503|60738687,chr20|60739191|60739311, chr20|60740463|60740583,chr20|60747737|60747857,chr20|60749575|60749695,chr20|60754177|60755595,chr20|60755967|60756087,chr20|60756093|60757629, chr21|39739173|39739579,chr21|39751929|39755863,chr21|39762879|39762999,chr21|39763549|39763669,chr21|39764271|39764391,chr21|39771983|39772575,chr21|39774455|39774575,chr21|39775417|39775639, chr21|39795321|39795491,chr21|39817317|39817553,chr21|39842687|39844011,chr21|39845937|39846057,chr21|39870277|39870437,chr21|39947567|39947687,chr21|39956757|39956877,chr21|40032435|40032599, chr21|40033583|40033703,chr21|42836228|42838088,chr21|42839650|42839822,chr21|42840312|42840474,chr21|42842562|42842682,chr21|42843722|42843916,chr21|42845242|42845432,chr21|42848464|42848584, chr21|42851094|42851214,chr21|42852406|42852526,chr21|42860320|42860440,chr21|42861416|42861536,chr21|42866272|42866392,chr21|42866394|42866514,chr21|42870020|42870140,chr21|42879866|42880094, chr22|24129143|24129457,chr22|24133951|24134071,chr22|24135749|24135869,chr22|24143121|24143331,chr22|24145485|24145605,chr22|24158947|24159131,chr22|24167401|24167611,chr22|24175763|24175883,chr22|24176317|24180205,chr22|28144255|28145673,chr22| 28145675|28147093,chr22|28192691|28194817,chr22|28194917|28197803,chr22|28373941|28375141,chr22|28375469|28377231,chr22|28377257|28379897,chr22|28381229|28381349,chr22|28385869|28385989,chr22|28386727|28386847,chr22|28388553|28388673,chr22|283892 65|28389385,chr22|28389395|28389515,chr22|28392125|28392245,chr22|28394517|28395257,chr22|28397351|28397549,chr22|28410225|28410389,chr22|28426203|28426363,chr22|28490057|28490241,chr22|28492167|28492405,chr22|28494907|28495027,chr22|28497153|284 97273,chr22|28501257|28501799,chr22|28503039|28504399,chr22|28559069|28559595,chr22|28692191|28692311,chr22|28693557|28693849,chr22|28702473|28702639,chr22|28838833|28839685,chr22|29025529|29025827,chr22|29075573|29075887,chr22|29664237|29664357chr22|29668163|29668283,chr22|29668333|29668453,chr22|29669731|29669851,chr22|29670201|29670321,chr22|29674009|29674213,chr22|29678369|29678555,chr22|29682901|29683131,chr22|29684585|29684783,chr22|29686391|29686681,chr22|29687509|29687629,chr22| 29688081|29688201,chr22|29688475|29688595,chr22|29692233|29692353,chr22|29693817|29693937,chr22|29694713|29694893,chr22|29695211|29695331,chr22|29695579|29695849,chr22|29696051|29696481,chr22|29999535|30000109,chr22|30032741|30032861,chr22|300350 79|30035199,chr22|30038171|30038291,chr22|30050619|30050739,chr22|30051563|30051683,chr22|30054155|30054275,chr22|30057201|30057321,chr22|30060955|30061075,chr22|30064317|30064437,chr22|30067815|30067935,chr22|30069247|30069483,chr22|30070817|300 70937,chr22|30074187|30074307,chr22|30077417|30077599,chr22|30078999|30080173,chr22|30090681|30091981,chr22|30092309|30092429,chr22|30092433|30092553,chr22|30092559|30092797,chr22|30093091|30093565,chr22|30093937|30094647,chr22|30752603|30753129chr22|30757939|30758111,chr22|30761873|30762781,chr22|30765411|30765601,chr22|30766305|30766947,chr22|30767617|30767737,chr22|30768053|30768283,chr22|30769575|30769761,chr22|30769901|30770089,chr22|30771457|30771661,chr22|30772231|30774355,chr22| 31721779|31723303,chr22|31724781|31724901,chr22|31731667|31731857,chr22|31736655|31737677,chr22|31738853|31738973,chr22|31740307|31742341,chr22|36134723|36139989,chr22|36140021|36140141,chr22|36140235|36140355,chr22|36141945|36142065,chr22|361425 03|36142623,chr22|36146427|36146547,chr22|36152103|36152223,chr22|36155925|36156087,chr22|36157235|36157355,chr22|36157427|36157547,chr22|36161439|36161559,chr22|36164299|36164419,chr22|36174037|36174157,chr22|36177637|36177801,chr22|36205817|362 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18579,chr22|36722599|36722719,chr22|36723459|36723579,chr22|36737405|36737579,chr22|36744929|36745319,chr22|36783841|36784021,chr22|39619313|39621367,chr22|39621689|39621861,chr22|39626079|39626241,chr22|39627617|39627841,chr22|39629423|39629543chr22|39631771|39631891,chr22|39639845|39641045,chr22|41697519|41697785,chr22|41716627|41716747,chr22|41721523|41721643,chr22|41721715|41721931, chr22|41723199|41723377,chr22|41726007|41726127,chr22|41728143|41728263,chr22|41734277|41734397,chr22|41734995|41735203,chr22|41735809|41736149, chr22|41737061|41737181,chr22|41738521|41738641,chr22|41739409|41739589,chr22|41741997|41742221,chr22|41744061|41744181,chr22|41745113|41745313 chr22|41747547|41747667,chr22|41751479|41751599,chr22|41751751|41752075,chr22|41752353|41752473,chr22|41752639|41752821,chr22|41753171|41756159, chrX|39910488|39911662,chrX|39913128|39913304,chrX|39913486|39913606,chrX|39914610|39914774,chrX|39916398|39916582,chrX|39921382|39921654,chrX|39921988|39922332,chrX|39922840|39923224,chrX|39923578|39923860,chrX|39930216|39930420,chrX|39930856|39930976,chrX|39931592|39933010,chrX|39933024|39934442,chrX|39935668|39935788,chrX|39937098|39937218,chrX|39956458|39957220,chrX|40036250|40036590,chrX|44732424|44732966,chrX|44733140|44733260,chrX|44820522|44820642,chrX|44833874|44833994,chrX|44870174|44870294,chrX|44879854|44879974,chrX|44894142|44894262,chrX|44896856|44896976,chrX|44910940|44911060,chrX|44913076|44913196,chrX|44918240|44918360,chrX|44918482|44918720,chrX|44919274|44919394,chrX|44919844|44920018,chrX|44920556|44920676,chrX|44921882|44922002,chrX|44922656|44923070,chrX|44928814|44929610,chrX|44935946|44936066,chrX|44937636|44937756,chrX|44938380|44938604,chrX|44941810|44942042,chrX|44942694|44942862,chrX|44945106|44945226,chrX|44948978|44949184,chrX|44949958|44950118,chrX|44966658|44966778,chrX|44969314|44969502,chrX|44970596|44972052,chrX|48114786|48114906,chrX|48116640|48116760,chrX|48117178|48117298,chrX|48117958|48118078,chrX|48121178|48121298,chrX|48123224|48123344,chrX|48125714|48125834,chrX|48126228|48126938,chrX|48242932|48243052,chrX|48243458|48243578,chrX|48244000|48244120,chrX|48244780|48244900,chrX|48246732|48246852,chrX|48248794|48248914,chrX|48251298|48251418,chrX|48252188|48252798,chrX|48886226|48887172,chrX|48887174|48888120,chrX|48888902|48889068,chrX|48890956|48891076,chrX|48891208|48891328,chrX|48891646|48891766,chrX|48895524|48895976,chrX|48896622|48896944,chrX|48897978|48898098,chrX|48900626|48900892,chrX|49969355|49969659,chrX|50027515|50027635,chrX|50028133|50028253,chrX|50031773|50031893,chrX|50037867|50037987,chrX|50051495|50054505,chrX|50055523|50055643,chrX|50056841|50056961,chrX|50085209|50085329,chrX|50089641|50089815,chrX|50090615|50090783,chrX|50094245|50094365,chrX|50094595|50094921,chrX|52725886|52726112,chrX|52726126|52726626,chrX|52727030|52727150,chrX|52727830|52727990,chrX|52729500|52729620,chrX|52731574|52731694,chrX|52733534|52733654,chrX|52734174|52734294,chrX|52734714|52734834,chrX|52736158|52736278,chrX|70315690|70316840,chrX|70320524|70321598,chrX|70321916|70323390,chrX|70503468|70503588,chrX|70504214|70504334,chrX|70504830|70504950,chrX|70510468|70510650,chrX|70511618|70511830,chrX|70514066|70514386,chrX|70516402|70516522,chrX|70516690|70516906,chrX|70517208|70517328,chrX|70517676|70517796,chrX|70518278|70518398,chrX|70518550|70518670,chrX|70519732|70520194,chrX|70520436|70521076,chrX|70752944|70753064,chrX|70753074|70753194,chrX|70756018|70756216,chrX|70757668|70757930,chrX|70764390|70764510,chrX|70767754|70767874,chrX|70774342|70774462,chrX|70775030|70775244,chrX|70775794|70775952,chrX|70776520|70776640,chrX|70776782|70777164,chrX|70777332|70777530,chrX|70779106|70779284,chrX|70779422|70779542,chrX|70781626|70781746,chrX|70782686|70782876,chrX|70782970|70783358,chrX|70784440|70784612,chrX|70787340|70787610,chrX|70787844|70787964,chrX |70793420|70794602,chrX|70794608|70795790,chrX|129114935|129115327,chrX|12 9115627|129115747,chrX|129116601|129116875,chrX|129139167|129139287,chrX|1 29146539|129146659,chrX|129146915|129150197,chrX|129154949|129155133,chrX| 129156851|129156971,chrX|129158955|129159363,chrX|129162599|129162719,chr X|129162725|129162845,chrX|129168419|129168659,chrX|129171331|129171517,ch rX|129173101|129173265,chrX|129184669|129184789,chrX|129185825|129185999,c hrX|129189769|129191591,chrX|129191623|129191859,chrX|129191869|129192105. ,

[0010] The core of this technical solution is that the probe composition can target 256 core genes closely related to the occurrence, development, pathological classification, and targeted therapy of soft tissue sarcoma, covering key gene loci of common and rare fusion events in soft tissue sarcoma, corresponding to a total of 4,500 specific chromosomal transcriptional regions, covering the full transcript regions of 256 genes. This is the core design to ensure a high detection rate and reduce the risk of missed detection. At the same time, the invention creatively introduces the exon RNA sequence consensus (ERCC) Spike-in control sequence and incorporates its probe into the panel to monitor the efficiency and quantitative accuracy of the entire experimental process. This is a non-standardized design that reflects the innovative quality control of this invention.

[0011] Thirdly, the present invention provides a soft tissue sarcoma gene fusion detection kit, the kit comprising the probe composition described in the second aspect.

[0012] Preferably, the detection kit further includes sample processing reagents, sequencing library construction reagents, targeted capture reagents, quality control controls, and instructions.

[0013] Preferably, the sample processing reagents include FFPE sample dewaxing solution, RNA extraction lysis buffer, proteinase K, DNase I digestion solution, RNA washing solution, etc., for efficient extraction of high-quality total RNA.

[0014] Preferably, the library construction reagent contains RNA fragmentation buffer, reverse transcriptase, random primers, dNTP mixture, end repair enzyme, adapter sequence, DNA polymerase, etc., and is adapted for FFPE-degraded RNA library construction.

[0015] Preferably, the targeted capture reagent comprises streptavidin-labeled magnetic beads, hybridization buffer, and washing buffer, used to specifically capture target RNA reverse transcription products.

[0016] Preferably, the quality control controls are: ERCC Spike-in RNA standard, known fusion gene positive control RNA, and template-free negative control, used to monitor the quality of the entire process.

[0017] Preferably, the instruction manual details the reagent kit composition, storage conditions, operating procedures, quality control standards, and the basis for result interpretation.

[0018] Fourthly, the present invention provides a method for detecting gene fusions in soft tissue sarcomas for non-disease diagnosis purposes, comprising the following steps: S1: Sample pretreatment and RNA extraction; S2: Library Construction; S3: Sequencing and Bioinformatics Analysis; S4: Result Interpretation Criteria; The probe composition described in the second aspect was used in step S2.

[0019] Fifthly, the present invention provides a soft tissue sarcoma gene fusion detection device, wherein the detection device performs RNA template detection, comprising: Nucleic acid extraction unit: used to extract RNA from biological samples; Preliminary library construction unit: used to fragment, reverse transcribe, add A tails to the extracted RNA, ligate adapters, and perform PCR amplification to construct a preliminary library; Hybridization capture unit: used to capture the target fragment from the pre-library using the probe composition described in the second aspect, and to perform PCR amplification to construct a probe capture library; Sequencing unit: Used for sequencing probe-captured libraries and data processing.

[0020] In a sixth aspect, the present invention provides the use of the probe composition of the second aspect, the kit of the third aspect, or the detection device of the fifth aspect in the preparation of diagnostic products or devices for soft tissue sarcoma.

[0021] Compared with the prior art, the present invention has the following significant advantages: Comprehensive detection range and low risk of missed detection: The panel of 256 genes covers the vast majority of soft tissue sarcoma-related genes, and the probe design covers the entire transcript region. Combined with the ERCC Spike-in control design, it ensures efficient detection of known fusions while reducing the risk of missed detection of unknown partner genes or rare breakpoint fusions, thus solving the problem of diagnosing soft tissue sarcoma subtypes.

[0022] Excellent technical performance and strong sample adaptability: The RNA-based targeted sequencing strategy directly detects mature mRNA and accurately identifies fusion breakpoints; the experimental parameters (starting amount, break conditions, etc.) optimized for FFPE samples and the customized bioinformatics workflow enable the method to achieve a sensitivity of 96.15%, a specificity of 100%, and intra- and inter-batch precision of 100%, and can stably process degraded clinical samples.

[0023] Balancing clinical practicality and cost-effectiveness: Compared to FISH / IHC, it has higher throughput; compared to DNA NGS, it has lower cost and more functionally relevant results; compared to whole transcriptome sequencing, it has smaller data volume and faster analysis. It successfully identified fusion genes in 77.3% of cases with unclear FISH results, providing a reliable basis for the diagnosis of difficult cases and the screening of targeted therapies.

[0024] Outstanding creativity: The screening of 256 genes is based on a trade-off of multi-dimensional conflict criteria. The customized optimization of probe design and experimental procedures does not simply rely on tools or routine operations. The resulting complete technical system has non-obviousness. Attached Figure Description

[0025] Figure 1 This invention provides a flowchart of bioinformatics analysis. The flowchart illustrates the complete analysis process from sequencing data to fusion gene results output, including data splitting and format conversion, raw data quality control, sequence alignment and fusion analysis, result filtering and annotation, and quality control information summarization. Each step is sequentially connected to ensure the reliability of the analysis results. Detailed Implementation the term

[0026] Gene fusion refers to the fusion of partial or complete sequences of two different genes into a new gene due to some mechanism (such as genomic variation). Generally, gene fusion refers to fusion at the genome level. However, fusion can also occur at the transcriptome level, mainly due to the fusion of RNA produced by the transcription of two different genes, forming a new fusion RNA. This RNA may encode a protein or be non-coding. The fusion gene generated at the genome level may or may not be expressed, depending on the fusion process (e.g., due to disruption of the promoter region or other reasons).

[0027] "Nucleic acid" refers to a sequence of nucleoside or nucleotide monomers composed of naturally occurring bases, sugars, and interglycosylation (backbone) bonds. The term also includes modified or substituted sequences containing non-naturally occurring monomers or portions thereof. The nucleic acid sequence in this application can be a deoxyribonucleic acid (DNA) sequence or a ribonucleic acid (RNA) sequence and can include naturally occurring bases, including adenine, guanine, cytosine, thymine, and uracil, as well as inosine. These sequences can also contain modified bases. Examples of such modified bases include aza- and deazo-adenine, guanine, cytosine, thymine, and uracil; and xanthine and hypoxanthine. Nucleic acids can be double-stranded or single-stranded and represent sense or antisense strands. Furthermore, the term "nucleic acid" includes complementary nucleic acid sequences and equivalents of codon-optimized or synonymous codons. Nucleic acids may, for example, contain one or more locked nucleic acid (LNA) portions.

[0028] RNA-seq, or transcriptome sequencing analysis, is a high-throughput sequencing technology. Initially used to study the expression levels of mRNA, small RNA, and non-coding RNA, and to identify differentially expressed genes, it has rapidly developed over the past decade. Today, RNA-seq has large-scale applications in transcript variation, gene fusion, and alternative splicing detection. Targeted RNA-seq focuses on analyzing specific transcripts. Similar to standard RNA-seq, targeted enrichment methods can be used to assess gene expression, analyze RNA species, and detect gene fusions and mutations. However, compared to standard RNA-seq, it offers advantages such as high sensitivity, wide dynamic range, low cost, and high throughput. Several methods for targeted long-read RNA-seq have been developed to date. Single or multiplex long-range RT-PCR amplification followed by long-read sequencing utilizes primer pairs located at the terminal exons to amplify the target transcript (Clark et al., 2020). However, due to primer cross-reactivity and amplification bias, this method may not be able to enrich transcripts using novel selective first or last exons and may not be able to scale to large gene combinations. Hybridization-based capture enrichment (Mamanova et al., 2010; Karamitros & Magiorkinis, 2018) (using biotinylated capture oligonucleotides, such as RNA capture long seq (CLS) (Lagarde et al., 2017)) is an efficient method for targeting long-read RNA-Seq. Sheynkman et al. recently described an alternative hybridization-based capture method that uses directly synthesized biotinylated capture oligonucleotides from open-reading frame (ORF) clones (Sheynkman et al., 2020). Alternatively, the universal, easy-to-implement, and highly cost-effective method described in CN202280074462.0, utilizing isothermal linear amplification of capture oligonucleotides for targeted sequencing, can also be employed. The sequencing may include Sanger sequencing, synthetic sequencing, including but not limited to Illumina NGS platform sequencing and PacBio long-read sequencing or nanopore sequencing. The sequencing may include long-read sequencing. The sequencing may include short-read sequencing.

[0029] The streptavidin beads may be magnetic. The sample may be a dsDNA library, including but not limited to cDNA libraries and fragmented genomic DNA libraries, for example, wherein the cDNA library is generated by reverse transcription-polymerase chain reaction of an RNA sample. The sequencing may provide a transcriptome profile, for example, wherein the transcriptome profile includes changes in gene expression and RNA splicing.

[0030] The method can be a method for targeted sequencing of full-length transcripts, non-full-length transcripts, or any genomic fragment.

[0031] When used in conjunction with the term "comprising / including" in the claims and / or description, the use of a noun without a quantifier may mean "one / type," but it is also consistent with the meaning of "one / type or more / types," "at least one / type," and "one / type or more than one / type." The word "about" means adding or subtracting 5% of the specified number.

[0032] The term "panel" originates from the development of NGS (next-generation sequencing) technology. It primarily refers to the simultaneous detection of multiple genes and multiple loci, specifically a defined set of enriched or sequenced genes. A panel may contain pathogen-related genes and possible variants of selected genes. In some implementations, the panel contains a set of characteristic genes. As used herein, the term "characteristic gene" refers to a gene whose expression is positively or negatively correlated with disease severity, disease outcome, or other predictive indicators of disease severity or outcome. In some implementations, these panels are used for sequencing assays. Sequencing-based assays are techniques known in the art. Some non-limiting examples of sequencing-based assays include next-generation sequencing (NGS), Sanger sequencing, bisulfite sequencing, direct RNA sequencing, etc. In next-generation sequencing (NGS) panel assays, only clinically important genes are detected to obtain genomic data in a timely and cost-effective manner. In some implementations, these panels are used for next-generation sequencing (NGS). In some implementations, these panels are used for non-sequencing assays. Non-sequencing assays are techniques known in the art. Non-restrictive examples of non-sequencing detection include microarrays, polymerase chain reaction (PCR) (including real-time PCR, reverse transcription PCR (RT-PCR), quantitative reverse transcription PCR (RT-qPCR), quantitative PCR (qPCR), digital droplet PCR (ddPCR), etc.) (as described by Shemer, R. et al. in Current Protocols in Molecular Biology, 127.1 (2019): e90; and Zemmour, Hai et al. in Nature Communications, 9.1 (2018): 1-9, both of which are incorporated herein by reference in their entirety).

[0033] "Biological samples" (also referred to as "samples") include bodily samples from animals or humans, including biological fluids such as serum, plasma, vitreous fluid, lymph, synovial fluid, follicular fluid, semen, amniotic fluid, breast milk, whole blood, urine, cerebrospinal fluid, saliva, sputum, tears, sweat, mucus, and tissue culture media, as well as tissue extracts such as homogenized tissue and cell extracts. In some embodiments, the biological sample is a serum, plasma, or urine sample. In some embodiments, the biological sample is an FFPE sample.

[0034] This invention provides the application of the Panel probe composition described in the second aspect, the kit described in the third aspect, or the detection device described in the fifth aspect in the preparation of diagnostic products for soft tissue sarcoma. Based on the gene coverage characteristics and detection performance of this Panel, its core applications include, but are not limited to, the following clinical and research scenarios: Soft tissue sarcoma auxiliary diagnosis: For tumor samples with unclear morphological diagnosis, the nature of the tumor (differentiation between benign and malignant) can be helped to be determined by detecting the status of fusion genes. Soft tissue sarcoma subtype classification: Based on specific fusion gene characteristics (such as EWSR1-FLI1 corresponding to Ewing sarcoma), more than 100 soft tissue sarcoma subtypes can be accurately distinguished; Targeted therapy target screening: Detecting the gene fusion status of drug-available targets such as NTRK, ALK, and RET to provide a basis for matching patients with appropriate targeted drugs; Treatment efficacy monitoring: Dynamically detect changes in the abundance of fusion genes during treatment to assess the efficacy of targeted therapy or chemotherapy; Research applications: Used for basic and clinical translational research such as soft tissue sarcoma fusion gene profile research, discovery of novel fusion biomarkers, and prognostic correlation analysis.

[0035] The panel described in this invention includes 256 genes closely related to the occurrence, development, and targeted therapy of soft tissue sarcoma. The gene information can be queried, but is not limited to, through the website https: / / www.ncbi.nlm.nih.gov / gene / . This panel is primarily applicable to routine clinical FFPE samples, and has broad clinical applicability.

[0036] The panel probe composition for detecting soft tissue sarcoma gene fusions targeting RNA described in this invention specifically targets the entire transcript region of the aforementioned genes, targeting a total of 4500 specific chromosomal loci (i.e., target regions) to ensure efficient and comprehensive capture of the target gene transcripts and reduce missed detections due to insufficient probe coverage; the sequences of the 4500 specific chromosomal loci can be found, but are not limited to, through the website http: / / genome.ucsc.edu / . I. Experimental Materials

[0037] Unless otherwise specified, all experimental materials and reagents used are commercially available consumables and reagents. II. Panel and Probe 1. Determination of the gene panel for detection

[0038] First, based on domestic and international guidelines and expert consensus on the diagnosis and treatment of soft tissue sarcoma, combined with a list of clinically needed genes provided by pathologists specializing in soft tissue sarcoma at our hospital, extensive literature review, and analysis of the gene coverage of existing testing products on the market both domestically and internationally, an initial candidate gene pool was formed. Subsequently, considering factors such as the clinical diagnostic value of the genes, their research significance, testing costs, and ensuring the final product pricing is within the acceptable range for patients, multiple rounds of communication and discussion with clinical pathologists were conducted to ultimately determine a testing panel containing 256 genes closely related to the occurrence, development, and targeted therapy of soft tissue sarcoma. This panel ensures a balance between comprehensiveness and targeted therapy in the testing. 2. Design and synthesis of targeted capture probe libraries

[0039] Based on the 256 genes identified in step one, a probe library for hybridization capture was designed. This invention provides a detection probe library that specifically targets the entire transcript regions of the aforementioned genes. These probes target a total of 4500 specific chromosomal loci (i.e., target regions) to ensure efficient and comprehensive capture of target gene transcripts, reducing missed detections due to insufficient probe coverage. III. Detailed Implementation of Testing Procedures 1. RNA extraction and quality control

[0040] Depending on the tissue size, cut 15-25 4µm thick paraffin tissue sections. Use the "Jianshi Bio FFPE Sample DNA / RNA Extraction Kit" to extract genomic RNA from the FFPE samples according to the instructions. Use a Qubit 3.0 fluorescence quantitative instrument (purchased from Thermofisher) and an Agilent 2200 system to check the quantity and quality of the extracted genomic RNA. The total amount of RNA extracted should be ≥50ng. 2. Specific steps for building a document library 2.1 cDNA Synthesis

[0041] 50-200 ng of genomic RNA was randomly fragmented to a suitable sequencing length using a commercial fragmentation reagent at 65°C for 1 min. Using the fragmented RNA as a template, the first strand of cDNA was synthesized using a commercial one-strand synthesis reagent (containing reverse transcriptase and random primers) at 20°C for 10 min, 42°C for 15 min, 70°C for 15 min, and 4°C at infinity. Using this strand as a template, the second strand of cDNA was synthesized using a commercial two-strand synthesis and A-tailing reagent to obtain double-stranded cDNA. The ends of the double-stranded cDNA were repaired, and an adenine (A) base was added to the 3' end at 42°C for 5 min, 62°C for 10 min, and 4°C at infinity. 2.2 Pretext Library Construction

[0042] Using commercially available adapter ligation kits, sequencing adapters with thymine (T) overhangs were ligated to both ends of cDNA. The sequencing program was: 20℃ for 15 min, 4℃ ∞. The ligation products were purified using magnetic beads to remove unligated adapters and other impurities. A limited number of cycles of polymerase chain reaction (PCR) were performed using commercially available PCR kits to amplify the cDNA library with adapters. The sequencing program is shown in Table 1 below. The pre-amplified products were purified to obtain the pre-library library. The pre-library library was quantified and quality-controlled to ensure that the library quality was up to standard. The total amount of the pre-library library was required to be ≥300 ng.

[0043]

[0044] 2.3 Determination of Sample Size Two FFPE fusion-positive samples were selected and verified by other methods. The mutation information of the samples is shown in Table 2 below:

[0045] Libraries were constructed by reverse transcription of 50 ng and 100 ng RNA into cDNA. Each starting amount gradient was repeated once, resulting in a total of four libraries. All libraries were sequenced using the same amount of data. The sequencing results of the four libraries constructed with different starting amounts are shown in Table 3 below. The starting amount of RNA for library construction ranges from 50ng to 100ng. There are no significant differences in the QC performance parameters and fusion ratio of the above four libraries. Fusion can be detected stably, meaning that the starting amount of RNA can be as low as 50ng. It is recommended to use 100ng when there are enough samples.

[0046] 2.4 Determination of interruption conditions and the number of Pre-PCR cycles One FFPE fusion-positive sample, verified by other methods, was selected. Different interruption conditions and PCR cycle numbers were set with a fixed input amount. The pre-library library was then quality-tested using a Qubit 3.0 quantitative PCR instrument and a Qsep100 bioanalyzer. The results are shown in Table 4 below. Based on the above results, when the sample input is 100ng, the interruption conditions are set to 65℃ for 1min, and the PCR cycle number is set to 15, the library yield is the highest and the fragment distribution is more in line with the expected requirements.

[0047] 3. Targeted capture and sequencing parameters

[0048] 3.1 Probe Hybridization and Capture: The pre-library was hybridized with the designed targeted capture probe library at 55℃ for 16-20 h in liquid chromatography to allow the target sequence to bind to the probe. Streptavidin-labeled magnetic beads were used to bind the target sequence bound to the biotin-labeled probe, and a series of washing steps were performed to remove unhybridized and non-specifically bound nucleic acids. The target sequence hybridized with the probe was enriched by magnetic separation, and the captured target sequence was eluted from the magnetic beads. The captured and enriched target fragments were amplified by PCR to construct the final sequencing library. The PCR reaction system is shown in Table 5 below, and the PCR program settings are shown below. The final library was purified and quality-tested using a Qubit 3.0 quantitative PCR instrument and a Qsep100 bioanalyzer. Only libraries that passed the quality test were used for sequencing.

[0049] Post-PCR reaction procedure: Step 1: +98°C, 45 s Step 2: +98°C, 15 s Step 3: +60°C, 30 s Step 4: +72°C, 30 s Step 5: Repeat steps 2-4 6 times (7 cycles in total) Step 6: +72°C, 1 min Step 7: Store at +4°C 3.2 Sequencing: The library was sequenced by paired ends (2×150bp) using the Illumina NextSeq 550 sequencing platform.

[0050] 4. Bioinformatics Analysis Parameter Settings The raw data generated from sequencing were processed using the following custom analysis workflow, the flowchart of which is shown below. Figure 1 As shown in the diagram, the analysis steps are as follows: (1) Data splitting and format conversion: The raw data files (bcl format) from the sequencer are split according to the sample index using the bcl2fastq software and converted into fastq format files containing sequence information and their quality scores.

[0051] (2) Raw data quality control: The quality of the fastq files is evaluated, including the distribution of sequencing quality values, base composition, adapter contamination, and the proportion of repetitive sequences, to ensure that the data quality is up to standard.

[0052] (3) Sequence alignment and fusion analysis: The high-quality sequences after quality control are aligned to the human reference genome (GRCh37). This process uses specialized fusion gene detection tools (such as Arriba, STAR-Fusion, etc.) to directly identify and screen high-confidence fusion breakpoints from the alignment results.

[0053] (4) Results filtering and annotation: The fusion events initially identified were filtered to remove false positive signals (such as alignment errors and common noise in the database), and the clinical significance of the finally confirmed fusion genes was annotated.

[0054] (5) Quality control information summary: Generate a comprehensive quality control report containing key indicators such as sequencing depth, coverage uniformity, alignment rate, and fusion detection sensitivity to evaluate the overall quality of this experiment.

[0055] IV. Performance Validation and Clinical Application Data

[0056] To confirm the reliability and effectiveness of this technical solution, rigorous performance verification and retrospective clinical studies were conducted: 1. Performance Verification:

[0057] (1) Accuracy: FFPE clinical samples containing known fusions and validated by fluorescence in situ hybridization (FISH) or next-generation sequencing (NGS) and known negative cell line standards were used for testing. In the validation of 26 positive samples and 1 negative standard, the detection results are shown in Tables 6 and 7. The sensitivity reached 96.15% and the specificity was 100%, which meets the requirements for clinical testing.

[0058] (2) Precision: The intra-batch (the same batch of experiments was repeated 3 times) and inter-batch (experiments were performed on different dates and by different operators) precision of 2 clinical samples (sample information is shown in Table 8) were verified. The results are shown in Tables 9 and 10. All expected fusion variants were detected stably, and the intra-batch and inter-batch precision were both 100%.

[0059] (3) Detection limit: For one positive specimen containing fusion variants near the detection threshold (sample information is shown in Table 11), the experiment was repeated 3 times for each specimen, and the 3 data analysis results were compared in parallel, as shown in Table 12. All expected variant sites were detected stably and repeatedly.

[0060] 2. Clinical application examples:

[0061] (1) This invention can help identify difficult / atypical FISH signals: In 22 difficult cases with unclear FISH test results (e.g., signals at threshold boundaries or atypical patterns), this panel was used for detection. Following the steps outlined above, sample RNA extraction, nucleic acid fragmentation, RNA reverse transcription, library construction, hybridization capture, sequencing, and bioinformatics analysis were completed. Specific fusion genes were successfully identified in 17 cases (77.3%), for example: A spindle cell tumor was detected by FISH, which indicated a suspicious rearrangement of the SS18 gene but with atypical signal (80% of cells showed SS18 as a single red dot signal). Panel analysis confirmed that the tumor was positive for SS18-SSX2 fusion and was diagnosed as synovial sarcoma.

[0062] A case of small round cell malignant tumor was suspected to be negative for EWSR1 gene break signal in FISH testing, but a large number of single red atypical signals were present (accounting for 88%). After accurate detection of EWSR1-ERG fusion by our panel, it was diagnosed as Ewing sarcoma.

[0063] In one case of spindle cell tumor, FISH testing suggested a possible rearrangement of the MAML2 gene, but the result was near the threshold and the separation distance was relatively close. Our Panel accurately detected the YAP1-MAML2 fusion, and the diagnosis was metaplastic thymoma.

[0064] (2) Application of the present invention in pathological diagnosis: In 16 cases of soft tissue sarcomas with extremely difficult histopathological diagnosis, following the steps outlined above—sample RNA extraction, nucleic acid fragmentation, RNA reverse transcription, library construction, hybridization capture, sequencing, and bioinformatics analysis—this panel helped clarify the diagnosis in 11 cases (68.75%). For example, in one case with a highly morphologically suspected "angioma-like fibrous histiocytoma," FISH testing showed negative results for EWSR1 and FUS. However, our panel detected LAMTOR1-PRKCD fusion, leading to a definitive diagnosis of "deep benign fibrous histiocytoma," distinguishing it from hemangioma-like fibrous histiocytoma. Furthermore, in 3 cases (1 case of NTRK fusion and 2 cases of ALK translocation inflammatory myofibroblastic tumors), targeted therapy guidance was directly provided to the patients.

[0065] In summary, this invention, through the above-described complete technical solution, achieves efficient, accurate, and comprehensive detection of gene fusions in soft tissue sarcomas.

[0066] The scope of protection of this invention is not limited to the specific embodiments described above. All equivalent modifications or substitutions based on the technical solutions of this invention should be included within the scope of protection of this invention. The specific gene composition of the gene panel in this kit and method can be adjusted according to clinical needs, or the reagent models in the experimental procedure can be optimized. As long as the core probe design concept and detection method logic are consistent with this invention, they all fall within the scope of protection of this invention.

[0067] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as defined by the appended claims and their equivalents.

Claims

1. A panel for detecting gene fusions in soft tissue sarcoma targeting RNA, characterized in that, The panel includes genes closely related to the occurrence, development, and targeted therapy of soft tissue sarcomas, including the following genes: ABL1, ACLY, ACTB, ACTG1, ACTG2, ACVR2A, ADAP2, AHRR, ALDH2, ALK, ANTXR2, APC, ARHGAP26, ASPSCR1, ATF1, ATIC, BCOR, BCORL1, BRAF, BRD3, BRD4, BTBD9, C5orf38, CALB1, CAMTA1, CAND1, CARS1, CCDC157, CCDC170, CCDC6, CCNB3, CD74, CDH11, CDH18, and CDKN2A. , CDX1, CIC, CIITA, CITED2, CLTC, CNBP, COL1A1, COL1A2, COL6A3, COP1, COX6C, CREB1, CREB3L1, CREB3L2, CREBBP, CRTC1, CRTC3, CSF1, CTDP1, CTNNB1, C YBRD1, DDIT3, DNAH17, DNMT1, DUX4, DVL2, EED, EMILIN2, EML4, EP400, EPB41L2, EPC1, EPC2, ERG, ESR1, ETV1, ETV4, ETV5, ETV6, EWSR1, EZR, FBXW7, FEV, FGFR1, FGFR2, FGFR3, FGFR4, FLI1, FN1, FOSB, FOXO1, FOXO3, FOXO4, FUS, GAB1, GCGR, GLI1, GLIS3, GLT8D2, GNAS, GOPC, GTF2I, H3-3A, H3-3B, HAS2, HECT D4, HEY1, HMGA1, HMGA2, HRAS, ICA1L, IDH1, IDH2, IGF2, IGFL2, IRF2BP2, IRX2, JAZF1, KDM2A, KDM6A, KIAA1549, KIF5B, KIT, KLF17, KMT2A, KMT2D, KRAS, LEUTX, LHFPL6, LMNA, LPP, LRIG3, LRP1, MALAT1, MAML2, MAML3, MBTD1, MEAF6, MEF2A, MEIS1, MET, MITF, MN1, MRTFB, MTHFD1, MYB, MYBL1, MYH9, MYOD1, NA B2, NCOA1, NCOA2, NCOA4, NF1, NF2, NFATC1, NFATC2, NFIB, NOC4L, NONO, NPEPPS, NPM1, NR4A3, NRAS, NRG1, NTRK1, NTRK2, NTRK3, NUP210L, NUTM1, NUTM2A,NUTM2B、OGA、OGT、OMD、PATZ1、PAX3、PAX7、PAX8、PBX1、PBX3、PDGFB、PDGFD、PDGF RA、PDGFRB、PHF1、PIK3CA、PLAG1、POU5F1、PPARG、PPFIBP1、PRCC、PRDM10、PRKAC A、PRKAR1A、PRKCA、PRKCB、PRKCD、PTBP1、PTCH1、RAD51B、RAF1、RANBP2、RBFOX2、 RELA、RET、RNF130、ROS1、SDC4、SEC31A、SERPINE1、SFPQ、SLC34A2、SLC39A5、SMAD 3、SMARCA4、SMARCA5、SMARCB1、SP3、SRF、SS18、SS18L1、SSX1、SSX2、SSX4、STAT6 、STRN、SUZ12、SYTL1、TAF15、TAFA2、TCF12、TEAD1、TERT、TESPA1、TFE3、TFEB、TF G、TGFBR3、THBS1、THRAP3、TMPRSS2、TP53、TPM3、TPM4、TPR、TRIO、TSC2、TTC28、U CK2、USP6、VGLL2、VMP1、WT1、WWTR1、YAP1、YWHAE、YY1、ZC3H7B、ZNF444、ZNF521。、 2. A panel probe composition for detecting soft tissue sarcoma gene fusions targeting RNA, characterized in that, The probe composition comprises probes that hybridize with transcript sequences from one or more of the following chromosomal locus regions: chr1|6845514|6845634,chr1|6867034|6867154,chr1|6880214|6880334,chr1|6885150|6885270,chr1|6931806|6932084,chr1|6946290|6948270,chr1|7151336|7151456,chr1|7309558|7309678,chr1|7527864|7527984,chr1|7700450|7700622,chr1|7721776|7721934,chr1|7723402|7724334,chr1|7724336|7725268,chr1|7729792|7729912,chr1|7730974|7731094,chr1|7737666|7737786,chr1|7740150|7740270,chr1|7764786|7764962,chr1|7792498|7792668,chr1|7796394|7796608,chr1|7796970|7797090,chr1|7797304|7797638,chr1|7798008|7798550,chr1|7804884|7805090,chr1|7805894|7806160,chr1|7807744|7807864,chr1|7809780|7809900,chr1|7811248|7811460,chr1|7812496|7812616,chr1|7815652|7815772,chr1|7826458|7827042,chr1|7827122|7827822,chr1|7827904|7828140,chr1|7828178|7829824,chr1|18957330|18958190,chr1|18960786|18960906,chr1|18960920|18961040,chr1|18961608|18961728,chr1|18962738|18962858,chr1|19018238|19018456,chr1|19027136|19027320,chr1|19029578|19029798,chr1|19062116|19062640,chr1|19071298|19075368,chr1|27668516|27668636,chr1|27671778|27671898,chr1|27671904|27672024,chr1|27673902|27674068,chr1|27674288|27674512,chr1|27675546|27675666,chr1|27675878|27675998,chr1|27676138|27676258,chr1|27676452|27676632,chr1|27676868|27676988,chr1|27677274|27677452,chr1|27677728|27677848,chr1|27677936|27678056,chr1|27679764|27679988,chr1|27680194|27680432,chr1|35641968|35642088,chr1|35642900|35643020,chr1|35643552|35643672,chr1|35648476|35648832,chr1|35649102|35649768,chr1|35649778|35650252,chr1|35652592|35652860,chr1|35653572|35653692,chr1|35654594|35654992,chr1|35656088|35656604,chr1|35656932|35657138,chr1|35657812|35658754,chr1|36690014|36690134,chr1|36690576|36690696,chr1|36724974|36725094,chr1|36748124|36748310,chr1|36751958|36752880,chr1|36754650|36755374,chr1|36756964|36757156,chr1|36758194|36758314,chr1|36759434|36759554,chr1|36762174|36762380,chr1|36766476|36766694,chr1|36767094|36767356,chr1|36769336|36770016,chr1|36770056|36770618,chr1|36770672|36771016,chr1|37955534|37956350,chr1|37956632|37956752,chr1|37957102|37957222,chr1|37957318|37957438,chr1|37957908|37958144,chr1|37958170|37959766,chr1|37961442|37961562,chr1|37962116|37962236,chr1|37962262|37962382,chr1|37967394|37967606,chr1|37970758|37970878,chr1|37974840|37974960,chr1|37975038|37975158,chr1|37979020|37979140,chr1|37980252|37980372,chr1|44584482|44584668,chr1|44595014|44595876,chr1|44596174|44596436,chr1|44598850|44599946,chr1|44600184|44600760,chr1|92145840|92147612,chr1|92147616|92147972,chr1|92148054|92149472,chr1|92161222|92161342,chr1|92163606|92163726,chr1|92174220|92174340,chr1|92177790|92178108,chr1|92181782|92181960,chr1|92182114|92182274,chr1|92184858|92185030,chr1|92185440|92185796,chr1|92187502|92187710,chr1|92193206|92193372,chr1|92195352|92195538,chr1|92200322|92200524,chr1|92224178|92224298,chr1|92262834|92263036,chr1|92327018|92327210,chr1|92351424|92351716,chr1|92365164|92365284,chr1|92371372|92371568,chr1|110453460|110453692,chr1|110456882|110457002,chr1|110458226|110458346,chr1|110459904|110460094,chr1|110464458|110464624,chr1|110465778|110466820,chr1|110467364|110467484,chr1|110467736|110467856,chr1|110468674|110469374,chr1|110471464|110473622,chr1|115247030|115248212,chr1|115248392|115250730,chr1|115250734|115250854,chr1|115251152|115251272,chr1|115252180|115252358,chr1|115256410|115256608,chr1|115258674|115258794,chr1|115259274|115259394,chr1|153965155|153965573,chr1|153967543|153967663,chr1|153973321|153973545,chr1|153974201|153974469,chr1|153982437|153982605,chr1|153984723|153984843,chr1|153991417|153991537,chr1|153994591|153994827,chr1|153995587|153995781,chr1|153998007|153998201,chr1|153999967|154000087,chr1|154002361|154002539,chr1|154018535|154018853,chr1|154026693|154026891,chr1|154027217|154027337,chr1|154029297|154029417,chr1|154030567|154030687,chr1|154031045|154031165,chr1|154033005|154033125,chr1|154033409|154033529,chr1|154034045|154034165,chr1|154042739|154042949,chr1|154061887|154062079,chr1|154067401|154067641,chr1|154072463|154072627,chr1|154076477|154076695,chr1|154090191|154090365,chr1|154091155|154091275,chr1|154098789|154098947,chr1|154099779|154099899,chr1|154101727|154101847,chr1|154108279|154108457,chr1|154110587|154110707,chr1|154112267|154112437,chr1|154113893|154114013,chr1|154115927|154116047,chr1|154125219|154125339,chr1|154127307|154127601,chr1|154127723|154128443,chr1|154128653|154129253,chr1|154129315|154131583,chr1|154134229|154134585,chr1|154134849|154135323,chr1|154135565|154136039,chr1|154136389|154136745,chr1|154136915|154137593,chr1|154137887|154138007,chr1|154138277|154138751,chr1|154138839|154138959,chr1|154139059|154139179,chr1|154139529|154140475,chr1|154141759|154141879,chr1|154142849|154142969,chr1|154143095|154143215,chr1|154143865|154143985,chr1|154144481|154144601,chr1|154145359|154145479,chr1|154145557|154145677,chr1|154148597|154148717,chr1|154150575|154150695,chr1|154155443|154155713,chr1|154159723|154159927,chr1|154163663|154163783,chr1|154164097|154164217,chr1|154164367|154164585,chr1|156052349|156052469,chr1|156052729|156052849,chr1|156052863|1560529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chr5|112116482|112116602,chr5|112128124|112128244,chr5|112136968|112137088,chr5|112145812|112146016,chr5|112151180|112151300,chr5|112154652|112155050,chr5|112157580|112157700,chr5|112158970|112159090,chr5|112162814|112162934,chr5|112163604|112163724chr5|112164550|112164670,chr5|112170638|112170870,chr5|112173240|112181944,chr5|142149931|142150489,chr5|142150781|142150949,chr5|142252951|142253071,chr5|142254645|142254765,chr5|142258913|142259033,chr5|142264853|142264973,chr5|142273797|142273917, chr5|142281491|142281611,chr5|142283109|142283229,chr5|142287259|142287379,chr5|142292751|142292871,chr5|142311591|142311711,chr5|142393603|142393723,chr5|142416733|142416853,chr5|142421357|142421477,chr5|142433987|142434107,chr5|142435561|142435681 chr5|142437199|142437319,chr5|142500543|142500721,chr5|142513541|142513661,chr5|142526785|142526955,chr5|142586753|142587047,chr5|142593547|142593667,chr5|142601863|142604743,chr5|142605013|142605783,chr5|142606141|142607821,chr5|142607915|142608635 chr5|149493391|149495517,chr5|149497171|149497291,chr5|149497301|149497421,chr5|149498301|149498421,chr5|149499019|149499139,chr5|149499569|149499689,chr5|149500451|149500571,chr5|149500765|149500885,chr5|149501433|149501611,chr5|149502595|149502773,chr5|149503807|149503927,chr5|149504281|149504401,chr5|149505013|149505133,chr5|149506069|149506189,chr5|149509309|149509539,chr5|149510103|149510223,chr5|149511539|149511659,chr5|149512303|149512513,chr5|149513139|149513331,chr5|149513447|149513567, chr5|149514269|149514587,chr5|149515107|149515449,chr5|149516533|149516653,chr5|149531891|149533529,chr5|149534949|149535417,chr5|149546331|149546893,chr5|149562321|149562485,chr5|149563027|149564129,chr5|149781191|149781821,chr5|149782097|149782217, chr5|149782673|149782883,chr5|149784225|149784345,chr5|149784635|149784755,chr5|149785793|149785913,chr5|149786423|149786543,chr5|149786705|149786895,chr5|149792177|149792481,chr5|170814119|170814239,chr5|170814803|170815059,chr5|170817011|170817131, chr5|170818301|170818421,chr5|170818695|170818815,chr5|170819731|170820041,chr5|170827125|170827245,chr5|170827859|170827979,chr5|170832259|170832379,chr5|170833381|170834147,chr5|170834681|170834801,chr5|170837549|170837669,chr5|170837843|170837963chr5|176513905|176514087,chr5|176516541|176516703,chr5|176517381|176517835,chr5|176517929|176518113,chr5|176518687|176518807,chr5|176519311|176519521 chr5|176519655|176519775,chr5|176520129|176520561,chr5|176520655|176520775,chr5|176522321|176522733,chr5|176523059|176523179,chr5|176523263|176523383 chr5|176523613|176523733,chr5|176524285|176524405,chr5|176524517|176525153,chr5|179382007|179382727,chr5|179390457|179390577,chr5|179393795|179394019 chr5|179397397|179397517,chr5|179405183|179405303,chr5|179407103|179407223,chr5|179440051|179440319,chr5|179467443|179467655,chr5|179498445|179498767 chr6|31132104|31132652,chr6|31132854|31132974,chr6|31132992|31133112,chr6|31133288|31134174,chr6|31134538|31135006,chr6|31137982|31138468,chr6|33378780|33379000,chr6|33380014|33380374,chr6|33380462|33380582,chr6|33380962|33381082,chr6|33381176|333 81342,chr6|33381500|33381620,chr6|33381792|33381912,chr6|33382020|33382140,chr6|33382226|33382346,chr6|33382494|33382614,chr6|33382722|33383114,chr6|33383346|33383466,chr6|33383576|33384224,chr6|34204640|34205102,chr6|34206558|34206742,chr6|342085 04|34208700,chr6|34210470|34210590,chr6|34211210|34211330,chr6|34212558|34214056,chr6|38136166|38139286,chr6|38139366|38139632,chr6|38139776|38143016,chr6|38160274|38160394,chr6|38224178|38224298,chr6|38256038|38256246,chr6|38312754|38312874,chr6| 38545374|38545494,chr6|38547984|38548222,chr6|38560342|38560624,chr6|38561730|38562160,chr6|38563536|38563852,chr6|38565676|38565906,chr6|38580600|38580818,chr6|38607580|38607700,chr6|41651706|41652824,chr6|41653818|41653984,chr6|41654808|41654928,chr6|41655480|41655774,chr6|41657448|41657568,chr6|41658390|41658982,chr6|41700386|41700506,chr6|41702034|41702154,chr6|41702628|41702806,chr6|41703078|41703354,chr6|41703862|41703982,chr6|43138996|43139916,chr6|43140100|43140220,chr6|43141574|4314 1860,chr6|43143434|43143714,chr6|43144284|43144404,chr6|431460 22|43146232,chr6|43146522|43146642,chr6|43146772|43149302,chr6 |108881017|108881227,chr6|108882109|108883041,chr6|108984647|1 08986101,chr6|109000969|109001915,chr6|109001957|109003597,chr 6|109003641|109004705,chr6|109004971|109006035,chr6|117586711| 117587015,chr6|117589335|117589663,chr6|117591695|117592235,ch r6|117593607|117594737,chr6|117608455|117608815,chr6|117609087 |117610023,chr6|117622127|117622309,chr6|117629963|117630083,c hr6|117631233|117631453,chr6|117632171|117632291,chr6|117638309|117638429,chr6|117639323|117639443,chr6|117641021|117641201,chr6|117642429|117642549,chr6|117645475|117645595,chr6|117647377|117647585,chr6|117650489|117650609,chr6|117658325|117658511,chr6|117662287|117662803,chr6|117663553|117663715,chr6|117665213|117665433,chr6|117674143|117674341,chr6|117677781|117678087,chr6|117678957|117679181,chr6|117680961|117681183,chr6|117681475|117681595,chr6|117683755|117684037,chr6|117686213|117686375, chr6|117686733|117686913,chr6|117687229|117687465,chr6|117700211|117700331,chr6|117704469|117704679,chr6|117706835|117707033,chr6|117708047|117708167,chr6|117708933|117709205,chr6|117710503|117711017,chr6|117714375|117714495,chr6|117715315|117715517, chr6|117715779|117715899,chr6|117717329|117717449,chr6|117718067|117718287,chr6|117724311|117724431,chr6|117725433|117725599,chr6|117730715|117730835,chr6|117735635|117735755,chr6|117737389|117737509,chr6|117739587|117739707,chr6|117746687|117747113, chr6|117881421|117884555,chr6|117888007|117888205,chr6|117890725|117890907,chr6|117892009|117892129,chr6|117894619|117894803,chr6|117896329|117896523,chr6|117898561|117898681,chr6|117900053|117900235,chr6|117923157|117923683,chr6|131160477|131161747,chr6|131179267|131179387,chr6|131184757|131184877,chr6|131186665|131186785,chr6|131188599|131188719,chr6|131190693|131191275,chr6|131191433|131191553,chr6|131197803|131197923,chr6|131199233|131199399,chr6|131201255|131201375,chr6|131206225|131206417, chr6|131211423|131211615,chr6|131215471|131215591,chr6|131216097|131216267,chr6|131220613|131220733,chr6|131222091|131222329,chr6|131225581|131225701,chr6|131229921|131230041,chr6|131247737|131247857,chr6|131276235|131276465,chr6|131277123|131277647, chr6|131291391|131291511,chr6|131321753|131321873,chr6|131334665|131334785,chr6|131384273|131384393,chr6|135502435|135502555,chr6|135502563|135502683,chr6|135507039|135507159,chr6|135508947|135509067,chr6|135510915|135511035,chr6|135511255|135511493, chr6|135513451|135513571,chr6|135513585|135513705,chr6|135514955|135515075,chr6|135515485|135515605,chr6|135516097|135516217,chr6|135516875|135517149,chr6|135517853|135518469,chr6|135520035|135520197,chr6|135520631|135520751,chr6|135521213|135521703,chr6|135522771|135522891,chr6|135524347|135524467,chr6|135538991|135540319,chr6|139692933|139695097,chr6|139695259|139695793,chr6|151815141|151815329,chr6|151857457|151857577,chr6|151859169|151859445,chr6|151865697|151865859,chr6|151869429|151869633, chr6|151894299|151894635,chr6|151907013|151907233,chr6|151914231|151914423,chr6|151917459|151917721,chr6|151936567|151936687,chr6|151936703|151936823,chr6|151939021|151939837,chr6|151940151|151940735,chr6|151940985|151941337,chr6|151941339|151942387, chr6|151977791|151977911,chr6|152011669|152011789,chr6|152023015|152023135,chr6|152126837|152126957,chr6|152128321|152128653,chr6|152128807|152129507,chr6|152130231|152130415,chr6|152163721|152163937,chr6|152201787|152201907,chr6|152265297|152265651, chr6|152332799|152332919,chr6|152382131|152382251,chr6|152415509|152415711,chr6|152419807|152421847,chr6|152421909|152422183,chr6|152422187|152424467,chr6|152446391|152450763,chr6|159186770|159188118,chr6|159188282|159188552,chr6|159190344|159190464,chr6|159190806|159190984,chr6|159191800|159191920,chr6|159192226|159192488,chr6|159197428|159197548,chr6|159204542|159204706,chr6|1592056 56|159205776,chr6|159206330|159206624,chr6|159208126|159208246,chr6|159210300|159210420,chr6|159239104|159239276,chr6|159240314|159240434, chr7|5566718|5567266,chr7|5567352|5567580,chr7|5567614|5568368,chr7|5568742|5569080,chr7|5569170|5569290,chr7|5570132|5570252,chr7|13930794|13933792,chr7|13933880|13935770,chr7|13940352|13940472,chr7|13946044|13946232,chr7|13947388|13947508,chr7|1394 9230|13949350,chr7|13950838|13950958,chr7|13971116|13971382,chr7|13975322|13975530,chr7|13978746|13978866,chr7|14017018|14017138,chr7|14025730|14026046,chr7|14026226|14026346,chr7|14027694|14027814,chr7|14028638|14028758,chr7|14028882|14029300,chr7|1 4030168|14030340,chr7|14030736|14031058,chr7|27870186|27872604,chr7|27880306|27880494,chr7|27934828|27935044,chr7|28031504|28031624,chr7|28220072|28220422,chr7|74072023|74072413,chr7|74103449|74103569,chr7|74105313|74105433,chr7|74113297|74113417,chr 7|74114537|74114799,chr7|74119463|74119583,chr7|74120681|74120801,chr7|74125341|74125461,chr7|74129163|74129283,chr7|74131203|74131395,chr7|74133169|74133289,chr7|74143119|74143239,chr7|74144539|74144659,chr7|74146809|74147011,chr7|74148259|74148379chr7|74149777|74149897,chr7|74150831|74151033,chr7|74152355|74152475,chr7|74157763|74157883,chr7|74159087|74159319,chr7|74160205|74160325,chr7|74160653|74160773,chr7|74162341|74162461,chr7|74163333|74163453,chr7|74163595|74163797,chr7|74165659|741 65779,chr7|74166401|74166521,chr7|74167407|74167527,chr7|74168167|74168369,chr7|74169785|74169905,chr7|74171111|74171231,chr7|74172251|74172371,chr7|74173087|74173207,chr7|74173867|74175031,chr7|94024196|94024422,chr7|94027640|94027760,chr7|940283 18|94028438,chr7|94029494|94029614,chr7|94030844|94030964,chr7|94033858|94034212,chr7|94034476|94034596,chr7|94034950|94035070,chr7|94035528|94035648,chr7|94037120|94037240,chr7|94037484|94037700,chr7|94038048|94038168,chr7|94038622|94038742,chr7| 94038838|94038958,chr7|94039024|94039144,chr7|94039520|94039640,chr7|94039724|94039844,chr7|94040166|94040286,chr7|94040356|94040476,chr7|94041346|94041466,chr7|94041884|94042004,chr7|94042360|94042480,chr7|94042968|94043088,chr7|94043172|94043292chr7|94043500|94043620,chr7|94044500|94044620,chr7|94045706|94045826,chr7|94047028|94047148,chr7|94047776|94047896,chr7|94048776|94048896,chr7|94049510|94049630,chr7|94049692|94049968,chr7|94050286|94050406,chr7|94051176|94051296,chr7|94052258| 94052438,chr7|94053640|94053760,chr7|94054422|94054542,chr7|94054912|94055178,chr7|94055276|94055396,chr7|94055728|94055848,chr7|94056286|94056406,chr7|94056492|94056612,chr7|94056928|94057206,chr7|94057594|94057798,chr7|94058490|94058610,chr7|9 4058630|94058750,chr7|94059548|94060548,chr7|100770375|100770533,chr7|100771663|100771977,chr7|100773691|100773811,chr7|100773823|100773943,chr7|100775145|100775359,chr7|100776965|100777183,chr7|100778765|100778885,chr7|100778985|100779105,chr7| 100780253|100780373,chr7|100780475|100782105,chr7|100782249|100782369,chr7|100782467|100782587,chr7|116312239|116312639,chr7|116339115|116340347,chr7|116371711|116371921,chr7|116380011|116380131,chr7|116380895|116381087,chr7|116395399|116395577chr7|116397481|116397837,chr7|116398503|116398683,chr7|116399381|116399553,chr7|116403093|116403331,chr7|116409689|116409809,chr7|116409823|116409943,chr7|116411541|116411717,chr7|116411893|116412051,chr7|116414925|116415045,chr7|116415053|116415173, chr7|116417423|116417543,chr7|116418819|116419019,chr7|116422035|116422155,chr7|116423347|116423531,chr7|116435649|116436697,chr7|116436953|116438489,chr7|137559665|137559785,chr7|137559831|137561013,chr7|137561101|137565355,chr7|137567147|137567383, chr7|137569743|137569863,chr7|137570137|137570257,chr7|137586073|137586193,chr7|137588673|137588793,chr7|137590437|137590603,chr7|137592987|137593191,chr7|137597559|137597833,chr7|137600573|137600767,chr7|137612885|137613121,chr7|137617631|137617751, 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67082,chr19|15367764|15367992,chr19|15374234|15374354,chr19|15375204|15375586,chr19|15376154|15376462,chr19|15378234|15378354,chr19|15379724|15379844,chr19|15383616|15383952,chr19|15391064|15391270,chr19|15443090|15443358,chr19|16178338|16178780chr19|16186858|16186978,chr19|16187244|16187566,chr19|16187822|16187942,chr19|16191714|16191894,chr19|16192728|16192848,chr19|16197230|16197350,chr19|16198812|16198932,chr19|16199832|16199952,chr19|16204336|16204572,chr19|16206014|16206582,chr19| 16207156|16207612,chr19|16212014|16212914,chr19|16213180|16213870,chr19|18794480|18794646,chr19|18853718|18853838,chr19|18854880|18855000,chr19|18856640|18856760,chr19|18857832|18857952,chr19|18860578|18860698,chr19|18864292|18864412,chr19|188703 20|18870440,chr19|18870808|18871046,chr19|18876216|18876336,chr19|18879244|18879652,chr19|18882244|18882364,chr19|18885692|18885812,chr19|18886440|18886640,chr19|18887920|18890164,chr19|18890270|18891550,chr19|18891666|18893202,chr19|40267082|402 67202,chr19|40267204|40267324,chr19|40269470|40269590,chr19|40275156|40275326,chr19|40276328|40276940,chr19|42772654|42772774,chr19|42773408|42773528,chr19|42775916|42778738,chr19|42788610|42788932,chr19|42790862|42792186,chr19|42792990|42793606chr19|42793990|42794110,chr19|42794374|42795906,chr19|42796228|42797442,chr19|42797734|42798464,chr19|42798696|42799706,chr19|42799766|42800006,chr19|45971244|45971978,chr19|45973876|45974216,chr19|45974444|45974564,chr19|45975798|45978446,chr19|466510 42|46651162,chr19|46651464|46651584,chr19|46652072|46652302,chr19|46663638|46663758,chr19|46663830|46664176,chr19|46664316|46664554,chr19|56652630|56652750,chr19|56657566|56657838,chr19|56658248|56658586,chr19|56669856|56669976,chr19|56670982|56672270 chr20|43953867|43955037,chr20|43955167|43956113,chr20|43958995|43959213,chr20|43961625|43961745,chr20|43964431|43964551,chr20|43976953|43977073,chr20|50003459|50008027,chr20|50015163|50015283,chr20|50048593|50049301,chr20|50051727|50051847,chr20| 50052209|50052329,chr20|50071075|50071233,chr20|50090507|50090697,chr20|50091985|50092205,chr20|50133313|50133503,chr20|50139609|50140657,chr20|50158889|50159277,chr20|50179089|50179387,chr20|57414793|57415949,chr20|57416665|57416939,chr20|574277 61|57430397,chr20|57430593|57430713,chr20|57431175|57431295,chr20|57463457|57463881,chr20|57464225|57464417,chr20|57466465|57466929,chr20|57467181|57467301,chr20|57470643|57470763,chr20|57473957|57474077,chr20|57474993|57475563,chr20|57478573|574 78855,chr20|57480425|57480545,chr20|57484183|57484303,chr20|57484395|57484515,chr20|57484523|57484643,chr20|57484739|57484859,chr20|57485011|57485131,chr20|57485361|57485481,chr20|57485727|57486255,chr20|60718825|60718945,chr20|60729327|60729529chr20|60733717|60734007,chr20|60734915|60735035,chr20|60736481|60736645,chr20|60737797|60737995,chr20|60738503|60738687,chr20|60739191|60739311, chr20|60740463|60740583,chr20|60747737|60747857,chr20|60749575|60749695,chr20|60754177|60755595,chr20|60755967|60756087,chr20|60756093|60757629, chr21|39739173|39739579,chr21|39751929|39755863,chr21|39762879|39762999,chr21|39763549|39763669, chr21|39764271|39764391,chr21|39771983|39772575,chr21|39774455|39774575,chr21|39775417|39775639, chr21|39795321|39795491,chr21|39817317|39817553,chr21|39842687|39844011,chr21|39845937|39846057, chr21|39870277|39870437,chr21|39947567|39947687,chr21|39956757|39956877,chr21|40032435|40032599, chr21|40033583|40033703,chr21|42836228|42838088,chr21|42839650|42839822,chr21|42840312|42840474, chr21|42842562|42842682,chr21|42843722|42843916,chr21|42845242|42845432,chr21|42848464|42848584, chr21|42851094|42851214,chr21|42852406|42852526,chr21|42860320|42860440,chr21|42861416|42861536, chr21|42866272|42866392,chr21|42866394|42866514,chr21|42870020|42870140,chr21|42879866|42880094, chr22|24129143|24129457,chr22|24133951|24134071,chr22|24135749|24135869,chr22|24143121|24143331,chr22|24145485|24145605,chr22|24158947|24159131,chr22|24167401|24167611,chr22|24175763|24175883,chr22|24176317|24180205,chr22|28144255|28145673,chr22| 28145675|28147093,chr22|28192691|28194817,chr22|28194917|28197803,chr22|28373941|28375141,chr22|28375469|28377231,chr22|28377257|28379897,chr22|28381229|28381349,chr22|28385869|28385989,chr22|28386727|28386847,chr22|28388553|28388673,chr22|283892 65|28389385,chr22|28389395|28389515,chr22|28392125|28392245,chr22|28394517|28395257,chr22|28397351|28397549,chr22|28410225|28410389,chr22|28426203|28426363,chr22|28490057|28490241,chr22|28492167|28492405,chr22|28494907|28495027,chr22|28497153|284 97273,chr22|28501257|28501799,chr22|28503039|28504399,chr22|28559069|28559595,chr22|28692191|28692311,chr22|28693557|28693849,chr22|28702473|28702639,chr22|28838833|28839685,chr22|29025529|29025827,chr22|29075573|29075887,chr22|29664237|29664357chr22|29668163|29668283,chr22|29668333|29668453,chr22|29669731|29669851,chr22|29670201|29670321,chr22|29674009|29674213,chr22|29678369|29678555,chr22|29682901|29683131,chr22|29684585|29684783,chr22|29686391|29686681,chr22|29687509|29687629,chr22| 29688081|29688201,chr22|29688475|29688595,chr22|29692233|29692353,chr22|29693817|29693937,chr22|29694713|29694893,chr22|29695211|29695331,chr22|29695579|29695849,chr22|29696051|29696481,chr22|29999535|30000109,chr22|30032741|30032861,chr22|300350 79|30035199,chr22|30038171|30038291,chr22|30050619|30050739,chr22|30051563|30051683,chr22|30054155|30054275,chr22|30057201|30057321,chr22|30060955|30061075,chr22|30064317|30064437,chr22|30067815|30067935,chr22|30069247|30069483,chr22|30070817|300 70937,chr22|30074187|30074307,chr22|30077417|30077599,chr22|30078999|30080173,chr22|30090681|30091981,chr22|30092309|30092429,chr22|30092433|30092553,chr22|30092559|30092797,chr22|30093091|30093565,chr22|30093937|30094647,chr22|30752603|30753129chr22|30757939|30758111,chr22|30761873|30762781,chr22|30765411|30765601,chr22|30766305|30766947,chr22|30767617|30767737,chr22|30768053|30768283,chr22|30769575|30769761,chr22|30769901|30770089,chr22|30771457|30771661,chr22|30772231|30774355,chr22| 31721779|31723303,chr22|31724781|31724901,chr22|31731667|31731857,chr22|31736655|31737677,chr22|31738853|31738973,chr22|31740307|31742341,chr22|36134723|36139989,chr22|36140021|36140141,chr22|36140235|36140355,chr22|36141945|36142065,chr22|361425 03|36142623,chr22|36146427|36146547,chr22|36152103|36152223,chr22|36155925|36156087,chr22|36157235|36157355,chr22|36157427|36157547,chr22|36161439|36161559,chr22|36164299|36164419,chr22|36174037|36174157,chr22|36177637|36177801,chr22|36205817|362 05937,chr22|36205939|36206059,chr22|36236229|36236519,chr22|36334859|36334979,chr22|36357583|36357703,chr22|36424277|36424881,chr22|36677265|36678889,chr22|36680129|36680319,chr22|36680443|36680563,chr22|36681157|36681383,chr22|36681693|36682007chr22|36682767|36682887,chr22|36684287|36684467,chr22|36684763|36684993,chr22|36685121|36685351,chr22|36688011|36688299,chr22|36689365|36689535,chr22|36689797|36689917,chr22|36690127|36690353,chr22|36690967|36691131,chr22|36691541|36691771,chr22| 36692879|36693069,chr22|36694965|36695085,chr22|36696181|36696301,chr22|36696887|36697111,chr22|36697585|36697705,chr22|36698607|36698727,chr22|36700031|36700209,chr22|36701053|36701173,chr22|36701975|36702095,chr22|36702449|36702661,chr22|367053 23|36705443,chr22|36708083|36708275,chr22|36710149|36710269,chr22|36710281|36710401,chr22|36712551|36712723,chr22|36714251|36714371,chr22|36715571|36715691,chr22|36716255|36716417,chr22|36716831|36716951,chr22|36717773|36717893,chr22|36718459|367 18579,chr22|36722599|36722719,chr22|36723459|36723579,chr22|36737405|36737579,chr22|36744929|36745319,chr22|36783841|36784021,chr22|39619313|39621367,chr22|39621689|39621861,chr22|39626079|39626241,chr22|39627617|39627841,chr22|39629423|39629543chr22|39631771|39631891,chr22|39639845|39641045,chr22|41697519|41697785,chr22|41716627|41716747,chr22|41721523|41721643,chr22|41721715|41721931, chr22|41723199|41723377,chr22|41726007|41726127,chr22|41728143|41728263,chr22|41734277|41734397,chr22|41734995|41735203,chr22|41735809|41736149, chr22|41737061|41737181,chr22|41738521|41738641,chr22|41739409|41739589,chr22|41741997|41742221,chr22|41744061|41744181,chr22|41745113|41745313 chr22|41747547|41747667,chr22|41751479|41751599,chr22|41751751|41752075,chr22|41752353|41752473,chr22|41752639|41752821,chr22|41753171|41756159, chrX|39910488|39911662,chrX|39913128|39913304,chrX|39913486|39913606,chrX|39914610|39914774,chrX|39916398|39916582,chrX|39921382|39921654,chrX|39921988|39922332,chrX|39922840|39923224,chrX|39923578|39923860,chrX|39930216|39930420,chrX|39930856|39930976,chrX|39931592|39933010,chrX|39933024|39934442,chrX|39935668|39935788,chrX|39937098|39937218,chrX|39956458|39957220,chrX|40036250|40036590,chrX|44732424|44732966,chrX|44733140|44733260,chrX|44820522|44820642,chrX|44833874|44833994,chrX|44870174|44870294,chrX|44879854|44879974,chrX|44894142|44894262,chrX|44896856|44896976,chrX|44910940|44911060,chrX|44913076|44913196,chrX|44918240|44918360,chrX|44918482|44918720,chrX|44919274|44919394,chrX|44919844|44920018,chrX|44920556|44920676,chrX|44921882|44922002,chrX|44922656|44923070,chrX|44928814|44929610,chrX|44935946|44936066,chrX|44937636|44937756,chrX|44938380|44938604,chrX|44941810|44942042,chrX|44942694|44942862,chrX|44945106|44945226,chrX|44948978|44949184,chrX|44949958|44950118,chrX|44966658|44966778,chrX|44969314|44969502,chrX|44970596|44972052,chrX|48114786|48114906,chrX|48116640|48116760,chrX|48117178|48117298,chrX|48117958|48118078,chrX|48121178|48121298,chrX|48123224|48123344,chrX|48125714|48125834,chrX|48126228|48126938,chrX|48242932|48243052,chrX|48243458|48243578,chrX|48244000|48244120,chrX|48244780|48244900,chrX|48246732|48246852,chrX|48248794|48248914,chrX|48251298|48251418,chrX|48252188|48252798,chrX|48886226|48887172,chrX|48887174|48888120,chrX|48888902|48889068,chrX|48890956|48891076,chrX|48891208|48891328,chrX|48891646|48891766,chrX|48895524|48895976,chrX|48896622|48896944,chrX|48897978|48898098,chrX|48900626|48900892,chrX|49969355|49969659,chrX|50027515|50027635,chrX|50028133|50028253,chrX|50031773|50031893,chrX|50037867|50037987,chrX|50051495|50054505,chrX|50055523|50055643,chrX|50056841|50056961,chrX|50085209|50085329,chrX|50089641|50089815,chrX|50090615|50090783,chrX|50094245|50094365,chrX|50094595|50094921,chrX|52725886|52726112,chrX|52726126|52726626,chrX|52727030|52727150,chrX|52727830|52727990,chrX|52729500|52729620,chrX|52731574|52731694,chrX|52733534|52733654,chrX|52734174|52734294,chrX|52734714|52734834,chrX|52736158|52736278,chrX|70315690|70316840,chrX|70320524|70321598,chrX|70321916|70323390,chrX|70503468|70503588,chrX|70504214|70504334,chrX|70504830|70504950,chrX|70510468|70510650,chrX|70511618|70511830,chrX|70514066|70514386,chrX|70516402|70516522,chrX|70516690|70516906,chrX|70517208|70517328,chrX|70517676|70517796,chrX|70518278|70518398,chrX|70518550|70518670,chrX|70519732|70520194,chrX|70520436|70521076,chrX|70752944|70753064,chrX|70753074|70753194,chrX|70756018|70756216,chrX|70757668|70757930,chrX|70764390|70764510,chrX|70767754|70767874,chrX|70774342|70774462,chrX|70775030|70775244,chrX|70775794|70775952,chrX|70776520|70776640,chrX|70776782|70777164,chrX|70777332|70777530,chrX|70779106|70779284,chrX|70779422|70779542,chrX|70781626|70781746,chrX|70782686|70782876,chrX|70782970|70783358,chrX|70784440|70784612,chrX|70787340|70787610,chrX|70787844|70787964,chrX|70793420|70794602,chrX|70794608|70795790,chrX|129114935|129115327,chrX|129115627|129115747,chrX|129116601|129116875,chrX|129139167|129139287,chrX|129146539|129146659,chrX|129146915|129150197,chrX|129154949|129155133,chrX|129156851|129156971,chrX|129158955|129159363,chrX|129162599|129162719,chrX|129162725|129162845,chrX|129168419|129168659,chrX|129171331|129171517,chrX|129173101|129173265,chrX|129184669|129184789,chrX|129185825|129185999,chrX|129189769|129191591,chrX|129191623|129191859,chrX|129191869|129192105。, 3. The probe composition according to claim 2, characterized in that, The probe has biotin attached to its 5' or 3' end.

4. A soft tissue sarcoma gene fusion detection kit, characterized in that, The kit includes the probe composition of claim 2.

5. The soft tissue sarcoma gene fusion detection kit as described in claim 4, characterized in that, The test kit also includes sample processing reagents, sequencing library construction reagents, targeted capture reagents, quality control controls, and instructions.

6. The soft tissue sarcoma gene fusion detection kit as described in claim 5, characterized in that, The sample processing reagents include FFPE sample dewaxing solution, RNA extraction lysis buffer, proteinase K, DNase I digestion solution, and RNA washing solution; the library construction reagents include RNA fragmentation buffer, reverse transcriptase, random primers, dNTP mixture, end repair enzyme, adapter sequence, and DNA polymerase; the targeted capture reagents include streptavidin-labeled magnetic beads, hybridization buffer, and washing buffer; the quality control controls are: ERCC Spike-in RNA standard, known fusion gene positive control RNA, and template-free negative control.

7. A method for detecting gene fusions in soft tissue sarcomas for non-disease diagnosis purposes, characterized in that, Includes the following steps: S1: Sample pretreatment and RNA extraction; S2: Library Construction; S3: Sequencing and Bioinformatics Analysis; S4: Result Interpretation Criteria; The probe composition of claim 2 was used in step S2.

8. A soft tissue sarcoma gene fusion detection device, characterized in that, The detection device performs RNA template detection, including: Nucleic acid extraction unit: used to extract RNA from biological samples; Preliminary library construction unit: used to fragment, reverse transcribe, add A tails to the extracted RNA, ligate adapters, and perform PCR amplification to construct a preliminary library; Hybridization capture unit: used to capture the target fragment from the pre-library using the probe composition of claim 2, and to perform PCR amplification to construct a probe capture library; Sequencing unit: Used for sequencing probe-captured libraries and data processing.

9. The use of the probe composition of claim 2, the kit of claim 4, or the detection device of claim 8 in the preparation of diagnostic products or devices for soft tissue sarcoma.

Images