[go: up one dir, main page]

WO2013086964A1 - Procédé permettant un enrichissement, une construction de génothèque et une analyse du polymorphisme mononucléotidique des régions géniques dans un génome complexe d'une plante supérieure - Google Patents

Procédé permettant un enrichissement, une construction de génothèque et une analyse du polymorphisme mononucléotidique des régions géniques dans un génome complexe d'une plante supérieure Download PDF

Info

Publication number
WO2013086964A1
WO2013086964A1 PCT/CN2012/086355 CN2012086355W WO2013086964A1 WO 2013086964 A1 WO2013086964 A1 WO 2013086964A1 CN 2012086355 W CN2012086355 W CN 2012086355W WO 2013086964 A1 WO2013086964 A1 WO 2013086964A1
Authority
WO
WIPO (PCT)
Prior art keywords
sequence
genome
linker
methylation
sequencing
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/CN2012/086355
Other languages
English (en)
Chinese (zh)
Inventor
郭钰
邵迪
韩长磊
陶晔
杨焕明
张秀清
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
BGI Shenzhen Co Ltd
Original Assignee
BGI Shenzhen Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by BGI Shenzhen Co Ltd filed Critical BGI Shenzhen Co Ltd
Publication of WO2013086964A1 publication Critical patent/WO2013086964A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1093—General methods of preparing gene libraries, not provided for in other subgroups

Definitions

  • the restriction-site associated DNA (RAD) labeling technique is a technique for sequence analysis of a region of a cleavage site after digestion of a genomic DNA with a restriction enzyme.
  • the RAD labeling application initially used gene chip technology to separate RAD markers. For example, Lewis, etc.
  • Baird et al. (2008) developed a library-building method for sequencing nearby regions of RAD markers using Illumina sequencing technology, which has achieved good results in finding SNP and RAD marker mapping.
  • Hohenlohe et al. (2010) used a second-generation sequencing technique to sequence RAD-tagged regions and found 45,000 SNPs in a total of 100 individuals in five S. japonicus populations.
  • Chutimanitsakun et al. (201 1 ) used barley as a model organism to evaluate the application of RAD sequencing technology in QTL. Pfender et al.
  • the linkage map constructed using RAD sequencing technology can quickly distinguish the QTL locus of rust resistance genes in ryegrass stems.
  • RAD technology has been widely used in the production of genetic linkage maps, genotyping, QTL mapping, etc., especially in the absence of In the case of a reference sequence, finding a SNP in the vicinity of the cleavage site exhibits a unique advantage.
  • RAD sequencing technology mainly uses NotI, EcoRI and other enzymes in the establishment of libraries.
  • the genomic library obtained by the RAD database method contains a large number of repetitive sequences and cannot enrich the genome region.
  • the present invention provides a new library. method.
  • the genome is subjected to enzymatic cleavage using a methylation-sensitive enzyme instead of a conventional non-methylation-sensitive enzyme, followed by colonization.
  • the invention provides a method of enriching a genomic gene region, the method comprising the step of enzymatically digesting a genome using a methylation sensitive enzyme in place of a conventional non-methylation sensitive enzyme to obtain enrichment Genomic gene region sequence.
  • the methylation sensitive enzyme is selected from at least one of H/ ⁇ II, c/I and HpvCH4IV.
  • the genome may be derived from higher plants, particularly nucleic acid samples containing a repeat sequence of 80% or greater.
  • the present invention provides a method for constructing a genomic enriched gene region sequencing library, which is obtained by enriching the sequence of the gene region by using the method for enriching the genomic gene region, and specifically includes the following steps. : The genome was digested with a methylation-sensitive enzyme instead of a conventional non-methylation-sensitive enzyme, and then the library was constructed. Similarly, the methylation sensitive enzyme is selected from at least one of HpaT c/I and H/n CH4IV. The genome may be derived from a higher plant, in particular a nucleic acid sample containing a repeat sequence of 80% or greater. In still another aspect, the present invention provides a method of constructing a higher flowering plant suitable for containing a complex genome, comprising the steps of:
  • the present invention also provides a method for finding SNPs suitable for higher flowering plants having a complex genome, including
  • the present invention is an improvement of the conventional RAD method, which can enrich the gene to a large extent, and the enrichment efficiency is higher than that of the MF, HC, and HMPR methods used by the prior art. Therefore, the technology provided by the present invention is a rapid and effective method for building genomes, especially for complex genomes of certain higher flowering plants, and gene enrichment.
  • the method of the present invention is particularly applicable to bioinformatic analysis of higher flowering plants containing complex genomes, for example, SNPs that can be used to quickly find SNPs with reference genome sequences and regions near the restriction regions of gene regions without reference genome sequences. . BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 DNA test results.
  • Ml is ⁇ - ⁇ III digest ( Takara); M2 is D2000 (Tiangen).
  • Figure 2 The enzymatic cleavage effect of four varieties of maize, HpaU and Mspl.
  • M 1 is ⁇ -Hind III digest ( Takara );
  • M 2 is D2000 ( Tiangen) .
  • Figure 3 Glue recovery of the interrupted product.
  • M is a l OObp DNA ladder (Tiangen).
  • Figure 4 is a cut of the PCR product.
  • M is a lOObp DNA ladder (Tiangen).
  • DETAILED DESCRIPTION OF THE EMBODIMENTS In order to make the objects, technical solutions and advantages of the present invention more comprehensible, the present invention will be further described in detail below with reference to the accompanying drawings. It is understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
  • the genome is subjected to enzymatic cleavage using a methylation-sensitive enzyme instead of a conventional non-methylation-sensitive enzyme, and then the library is constructed.
  • the method of building the library can be referred to, for example, the method developed by Baird.
  • the sequencing sequence can be aligned with the reference genomic sequence using, for example, soap (version 2.21, available from http: ⁇ soap. genomics.org.cn/).
  • the corn genome is taken as an example, but the method of the present invention is not limited to the corn genome, but is applicable to any higher flowering plant complex genome and other genomes, especially some higher flowering plants containing complex genomes. For example, more than 80% of the genome is a higher flowering plant with a repeat sequence.
  • the reference sequence is the genetic sequence of the species studied, either from a public database or from a commercial database.
  • the reference sequence can also be derived from the short fragment sequence (reading) obtained by sequencing, and the genome sequencing technology has been marketed.
  • the total amount of DNA initially constructed is preferably higher than the amount of DNA required by the conventional RAD database. If the library is built according to the amount of DNA required by conventional RAD, the final amount of DNA may be insufficient.
  • the initial DNA of this method was tested to be 1.5 ⁇ ⁇ .
  • the methylation-sensitive enzyme may be Hpan, Acl HpvC, V, or the like, but a CG sequence must be present in the recognition sequence of the endonuclease.
  • the digestion time is preferably 1.5 hours. More preferably, the following enzymatic cleavage effect is achieved, that is, a diffuse band appears.
  • the disrupted fragment is preferably about 500 bp, for example, 400-600 bp.
  • the size of the segment refers to the position of the main band of the electrophoresis after the interruption, and it is not required that all sequences are the size of the segment.
  • a large portion of the DN A fragment can be subjected to, for example, gel extraction.
  • the P1 linker and the P2 linker are added for amplification and for subsequent sequencing.
  • P1 and P2 linkers are generally available from sequencing platform providers and used in accordance with their instructions, such as from illumina solexa, ABI SOLiD, Roche 454, and others.
  • the label sequence is for distinguishing different samples in the case of multiple samples, depending on the number of samples.
  • the less tag sequence can be 2-10 bp in length or longer.
  • the label portion of the joint can be designed according to commons by those skilled in the art.
  • each P1 linker can be connected to a different tag sequence, different samples can be mixed together and can be distinguished at the time of sequencing.
  • sequencing can be performed on a high throughput sequencing platform such as Illumina Solexa, ABI SOLiD or Roche 454.
  • the sequencing may be Solexa sequencing, unidirectional 50 cycle sequencing with a depth of preferably 0.4 ⁇ .
  • the sequencing sequence can be compared with the reference genome sequence using soap 2.21, and the search for inter-species SNPs.
  • gene enrichment efficiency and gene coverage efficiency can be counted.
  • gene enrichment efficiency and gene coverage efficiency are calculated according to the following formula:
  • Gene enrichment rate number of genes read / total number of reads
  • the gene enrichment rate indicates the effect of gene enrichment.
  • Gene coverage indicates the number of genes that can be covered.
  • corn has 32,000 genes, and in the examples of the present invention, 261 19 genes were obtained from the database construction results, and the gene coverage was 261 19/32000, and the result was 80.27%. In the case of a high gene enrichment rate, the gene coverage should also be considered. The coverage of 80.27% of the genes is sufficient to meet the requirements of subsequent analysis.
  • Quantitative DNA concentration was measured using a Quant-iT ds kit, and then 100 ng was subjected to 1% agarose electrophoresis to detect DNA quality. The main band is clear, free of degradation or a small amount of degraded DNA ( Figure 1). The DNA concentration was then diluted to 50 ng ⁇ L.
  • the corn DNA was digested with two enzymes, HpaU and Mspl. Wherein, by taking DNA HpaU 1.5 ⁇ ⁇ digested with Mspl taken 1 ⁇ ⁇ . See Table 5 for details of the two enzymes and Table 6 for the digestion reaction system. Hpall and M3 ⁇ 49l are homologous enzymes, but 3 ⁇ 49l is a non-methylation sensitive enzyme. This design was designed to compare the enzymatic cleavage effects of methylation-sensitive and non-sensitive enzymes at the same cleavage site.
  • TGGTCGCCGTATCATT-3* (SEQ ID NO. 2)
  • the enzyme-cut fragment was ligated to the PI linker, and the ligation reaction system is shown in Table 7.
  • the cleavage product of Hpall uses buffer 3, 3 ⁇ 49l using buffer 4 . Incubate with Thermomixer at 20 °C for 30 min, then incubate at 65 °C for 25 min to inactivate T4 DNAligase.
  • xxxxx is the label sequence used to distinguish the varieties.
  • the label sequence information of the four varieties is as follows:
  • the DNA Mixture was interrupted with a Covaris SI into a 500 bp fragment.
  • the Covaris SI parameter is set to:
  • the Duty cycle is adjusted to 10%, the Intensity is adjusted to 10, the Cycles/Burst is adjusted to 1000, the Time is set to 45S, and the cycle is adjusted to 2.
  • the 3' end of the fragment obtained in the previous step was subjected to a twisting operation, and the reaction system is shown in Table 10.
  • the Thermomixer was incubated at 37 °C for 30 min, purified by QIAquick PCR Purification Kit, and 23.5 ⁇ EB dissolved.
  • the P2 joint is annealed.
  • the annealing procedure settings are the same as PI.
  • the two single-strand sequences of the P2 linker are: top, 5*-Phos-CTCAGGCATCACTCGATTCCTCCGAGAACAA-3* (SEQ ID N0.3); bottom,
  • the reaction system is shown in Table 11.
  • the Thermomixer was incubated at 20 °C for 30 min, purified by QlAquick PCR Purification Kit, and 27 LEB dissolved.
  • the reaction system is shown in Table 12.
  • the reaction conditions were set to: 1 cycle, 98 ° C for 30 sec; 12 cycles including 98 ° C for 10 sec, 65 ° C for 30 sec, 72 ° C for 30 sec ; 1 cycle of 72 ° C for 5 min; 4 ° C storage.
  • the reaction primer sequence is:
  • P1-forward primer 5'-AATGATACGGCGACCACCGA-3' (SEQ ID NO. 5);
  • P2-inverted primer 5'-CAAGCAGAAGACGGCATACGA-3' (SEQ ID NO. 6).
  • Table 12 PCR amplification system
  • the amplified product was electrophoresed on a 2% agarose gel, and the gel was cut to obtain a 350-550 bp fragment (Fig. 4).
  • the sol was recovered using a QlAquick Gel Extraction Kit, and the recovered product was dissolved in 27 ⁇ EB solution. 8.
  • the recovered product was subjected to Solexa sequencing at a depth of 0.4 ⁇ and sequenced in 50 cycles.
  • Gene enrichment rate number of genes read / total number of reads
  • the gene enrichment rate obtained by Hpall is also higher than that obtained by the previous MF, HC, and HMPR methods. Therefore, the use of Hpall enzyme digestion in the present invention is a good method for gene enrichment.
  • Vanyushin BF Ashapkin VA. DNA methylation in higher plants: Past, present and future. Biochim Biophys Acta, 201 1 , 1809: 360-368.
  • n is a, c, g, or t

Landscapes

  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Zoology (AREA)
  • Biomedical Technology (AREA)
  • Organic Chemistry (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Microbiology (AREA)
  • Plant Pathology (AREA)
  • Molecular Biology (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biophysics (AREA)
  • Bioinformatics & Computational Biology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention se rapporte à un procédé permettant une construction de génothèque qui convient pour des régions géniques d'une plante supérieure à floraison qui contient un génome complexe. Dans le procédé selon l'invention, les régions géniques du génome sont enrichies par une enzyme sensible à la méthylation à la place d'une enzyme classique non sensible à la méthylation, puis une génothèque est élaborée. L'invention se rapporte également à un procédé qui convient pour une plante supérieure à floraison qui contient un génome complexe, et qui permet une recherche du polymorphisme mononucléotidique.
PCT/CN2012/086355 2011-12-15 2012-12-11 Procédé permettant un enrichissement, une construction de génothèque et une analyse du polymorphisme mononucléotidique des régions géniques dans un génome complexe d'une plante supérieure Ceased WO2013086964A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201110420042.7 2011-12-15
CN201110420042.7A CN103160937B (zh) 2011-12-15 2011-12-15 对高等植物复杂基因组基因进行富集建库和snp分析的方法

Publications (1)

Publication Number Publication Date
WO2013086964A1 true WO2013086964A1 (fr) 2013-06-20

Family

ID=48584457

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2012/086355 Ceased WO2013086964A1 (fr) 2011-12-15 2012-12-11 Procédé permettant un enrichissement, une construction de génothèque et une analyse du polymorphisme mononucléotidique des régions géniques dans un génome complexe d'une plante supérieure

Country Status (2)

Country Link
CN (1) CN103160937B (fr)
WO (1) WO2013086964A1 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104573409A (zh) * 2015-01-04 2015-04-29 杭州和壹基因科技有限公司 基因定位的多重检验方法
CN106192021A (zh) * 2016-08-02 2016-12-07 中国海洋大学 一种串联rad标签测序文库的构建方法
CN112575104A (zh) * 2020-12-11 2021-03-30 黑龙江省科学院大庆分院 一种工业大麻性状相关基因快速定位方法

Families Citing this family (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105849282A (zh) * 2014-01-03 2016-08-10 深圳华大基因研究院 一种对癌症患者进行术后监控的微创方法
CN103882147B (zh) * 2014-04-17 2016-02-03 中国热带农业科学院热带生物技术研究所 基因组随机扩增序列snp多态性及甲基化多态性的方法
CN107075565B (zh) * 2014-11-21 2021-07-27 深圳华大生命科学研究院 个体单核苷酸多态性位点分型方法及装置
EP3294877A1 (fr) * 2015-05-15 2018-03-21 Pioneer Hi-Bred International, Inc. Caractérisation rapide de systèmes d'endonucléases cas, séquences pam et éléments d'arn guide
CN104946765A (zh) * 2015-06-25 2015-09-30 华中农业大学 基于基因组测序的体细胞突变位点挖掘方法
CN106755534B (zh) * 2017-03-01 2020-06-16 北京林业大学 一种植物dna甲基化组织特异性获取方法
CN108034705A (zh) * 2018-01-15 2018-05-15 武汉爱基百客生物科技有限公司 一种全基因组甲基化高通量测序方法

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030190644A1 (en) * 1999-10-13 2003-10-09 Andreas Braun Methods for generating databases and databases for identifying polymorphic genetic markers
JP5206059B2 (ja) * 2008-03-25 2013-06-12 住友化学株式会社 メチル化されたｄｎａの含量を測定する方法
CN101532014B (zh) * 2008-12-12 2010-12-22 深圳华大基因研究院 对目标基因组区域边扩增边连接的公用接头及连接方法
CN102061526B (zh) * 2010-11-23 2014-04-30 深圳华大基因科技服务有限公司 一种DNA文库及其制备方法、以及一种检测SNPs的方法和装置
CN102206704B (zh) * 2011-03-02 2013-11-20 深圳华大基因科技服务有限公司 组装基因组序列的方法和装置

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
BAIRD NA ET AL.: "Rapid SNP Discovery and genetic mapping using sequenced RAD Markers", PLOS ONE, vol. 3, no. 10, 13 October 2008 (2008-10-13), pages 1 - 7 *
DAVEY JW ET AL.: "Genome-wide genetic marker discovery and genotyping using next-generation sequencing", NATURE REVIEWS GENETICS, vol. 12, no. 7, July 2011 (2011-07-01), pages 499 - 510 *
ELSHIRE RJ ET AL.: "A robust, simple genotyping-by-sequencing (GBS) approach for high diversity species", PLOS ONE, vol. 6, no. 5, 4 May 2011 (2011-05-04), pages 1 - 10 *
TEER JK ET AL.: "Systematic comparison of three genomic enrichment methods for massively parallel DNA sequencing", GENOME RESEARCH, vol. 20, no. 10, October 2010 (2010-10-01), pages 1420 - 1431 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104573409A (zh) * 2015-01-04 2015-04-29 杭州和壹基因科技有限公司 基因定位的多重检验方法
CN106192021A (zh) * 2016-08-02 2016-12-07 中国海洋大学 一种串联rad标签测序文库的构建方法
CN106192021B (zh) * 2016-08-02 2017-04-26 中国海洋大学 一种串联rad标签测序文库的构建方法
CN112575104A (zh) * 2020-12-11 2021-03-30 黑龙江省科学院大庆分院 一种工业大麻性状相关基因快速定位方法

Also Published As

Publication number Publication date
HK1182148A1 (en) 2014-01-03
CN103160937A (zh) 2013-06-19
CN103160937B (zh) 2015-02-18

Similar Documents

Publication Publication Date Title
WO2013086964A1 (fr) Procédé permettant un enrichissement, une construction de génothèque et une analyse du polymorphisme mononucléotidique des régions géniques dans un génome complexe d'une plante supérieure
US20250154496A1 (en) Methods for targeted genomic analysis
US8980551B2 (en) Use of class IIB restriction endonucleases in 2nd generation sequencing applications
US20170226582A1 (en) Method for constructing a sequencing library based on a single-stranded DNA molecule and application thereof
AU2021204166B2 (en) Reagents, kits and methods for molecular barcoding
CN110139931B (zh) 用于定相测序的方法和组合物
US20110015084A1 (en) Methods for Identifying Genetic Linkage
CN102409042B (zh) 一种高通量基因组甲基化dna富集方法及其所使用标签和标签接头
WO2018149091A1 (fr) Procédé de construction d'une bibliothèque de séquençage à haut débit d'arn circulaire et kit associé
CA2824431A1 (fr) Procedes et systemes pour determination d'haplotype
CN101313078A (zh) 突变群体的高通量筛选
CN109468409B (zh) 一种小麦抗白粉病基因Pm2b高通量检测标记及其应用
CN102839168A (zh) 核酸探针及其制备方法和应用
EP3615683B1 (fr) Procédés de liaison de polynucléotides
CN106566872B (zh) 基于测序基因分型技术的猪snp标记位点的分析方法
CN117802205B (zh) 一种单细胞Hi-C文库构建方法
CN105734130B (zh) 全基因组范围的植物基因功能鉴定的方法
Zhao et al. A prolific and robust whole‐genome genotyping method using PCR amplification via primer‐template mismatched annealing
KR101967609B1 (ko) ChiVMV 저항성 고추 품종을 판별하기 위한 SNP 마커 및 이의 용도
WO2014086037A1 (fr) Procédé de construction d'une banque de séquençage d'acides nucléiques et sa mise en oeuvre
EP4143334A1 (fr) Enrichissement et détection d'acide nucléique
HK1182148B (en) Method for enrichment, library construction and snp analysis of gene of gene regions in complex genome of higher plant
HK40103977A (en) Methods for targeted genomic analysis
CN118215744A (zh) 利用等温线性扩增探针的靶标富集和定量
ALLOPOLYPLOIDS RICHARD JA BUGGS 2, SIMON RENNY-BYFIELD 2, MICHAEL CHESTER 3, 4, INGRID E. JORDON-THADEN 3, 4, LYDERSON FACIO VICCINI 5, SRIKAR CHAMALA 3, ANDREW R. LEITCH 2, PATRICK S. SCHNABLE 6, W. BRADLEY BARBAZUK 3, PAMELA S. SOLTIS 4, 7, AND DOUGLAS E. SOLTIS 3, 7

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 12856958

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

32PN Ep: public notification in the ep bulletin as address of the adressee cannot be established

Free format text: NOTING OF LOSS OF RIGHTS PURSUANT TO RULE 112(1) EPC (EPO FORM 1205A DATED 07/11/2014)

122 Ep: pct application non-entry in european phase

Ref document number: 12856958

Country of ref document: EP

Kind code of ref document: A1