KR102146113B1 - 인간 알파코로나바이러스 전장유전체 증폭을 통한 진단키트 및 전장 유전체 서열 확인 방법 - Google Patents

인간 알파코로나바이러스 전장유전체 증폭을 통한 진단키트 및 전장 유전체 서열 확인 방법 Download PDF

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KR102146113B1
KR102146113B1 KR1020180145560A KR20180145560A KR102146113B1 KR 102146113 B1 KR102146113 B1 KR 102146113B1 KR 1020180145560 A KR1020180145560 A KR 1020180145560A KR 20180145560 A KR20180145560 A KR 20180145560A KR 102146113 B1 KR102146113 B1 KR 102146113B1
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한국표준과학연구원
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Abstract

본 발명은 코로나 바이러스 감염 여부를 판별하기 위한 전장 유전체 서열 획득 방법 및 이의 용도에 관한 것이다. 본 발명의 방법, 키트, 조성물은 코로나 바이러스의 유전체 서열을 증폭할 수 있는 11종의 범용 프라이머 세트를 포함하고, 이에 따라 시료 내 목적 바이러스의 존재 유무 및 관련 질환을 간편하고 빠르게 확인할 수 있다. 또한 전장 유전체 분석을 통해 신종 코로나 바이러스의 변이여부를 판별하여 신규 바이러스 동정에도 효과적으로 사용될 수 있다.

Description

인간 알파코로나바이러스 전장유전체 증폭을 통한 진단키트 및 전장 유전체 서열 확인 방법{Human alphacoronavirus universal primer sets for whole genome amplification method and diagnosis kit}
본 발명은 인간 알파코로나바이러스의 전장유전체를 증폭할 수 있는 프라이머 세트를 활용하여 미량의 시료에서 바이러스 유전체를 증폭하고 염기서열을 분석하여 전장 유전체 정보를 얻어내는 기술에 관한 것이다.
코로나바이러스(Coronavirus)는 30kb의 positive-sense single-stranded RNA genome을 갖고 있고 있으며 이는 RNA virus 중 가장 크다. 알파코로나바이러스는 코로나바이러의 일종으로 spherical morphology를 가지며, 120-160nm의 capsid를 가지고 있다. 알파코로나바이러스는 인간에게 호흡기 감염을 일으키는 인간 감염 바이러스와 돼지, 고양이, 박쥐 등에서는 호흡 계 감염뿐 아니라 장 감염을 유발하는 동물 감염 바이러스 등이 알려져 있다. 알파코로나 중 인체 감염 종으로는 229E와 NL63 두 종이 있고, 현재 229E의 complete genome과 partial genome이 각 22건 및 5건씩 등록되어 있으며, NL63은 총 45건의 complete genome이 NCBI에 등록되어 있다.
2001년 vaccinia virus를 cloning vector로 하여 1973-deposited laboratory-adapted prototype strain of HCoV-229E (VR-740)의 HCoV-229E cDNA clone (Inf-1)과 recombinants를 제작하여 세포에서 증식시킨 뒤 RNA를 추출하고 RT-PCR을 수행하여 complete genome을 추출하였다(Genbank accession: NC_002645). 이후 빠른 분석을 위해 2012년에는 임상샘플에서 HCov-229E total RNA를 추출한 뒤 2001년 시퀀싱 된 genome을 scaffold로 하여 최소 80bp 이상 겹쳐서 합성되는 500bp의 PCR product를 생성하는 bidirectional primer 조합으로 시퀀싱한 complete genome이 보고되었다.
2017년에는 Haiti의 어린이들의 plasma에서 채취한 HCov-229E를 Vero E6 cell에서 추출하여 Sanger와 Miseq으로 시퀀싱을 수행하였다. HCoV-NL63은 2004년 암스테르담에서 분리되어 Second-strand DNA synthesis를 한 후 restriction enzyme으로 digestion 한 뒤, anchor를 붙이고 primer로 amplify를 하는 cDNA-amplified restriction fragment-length polymorphism technique (VIDISCA : Virus-Discovery-cDNA-AFLP)으로 발견되었고, 475개의 genome fragment를 이용한 complete genome이 보고되었다(Genbank accession: NC_005831).
덴마크에서는 paediatric patients의 92개의 respiratory specimens로부터 추출한 NL63 RNA를 역전사하고 modified sequence-independent single-primer amplification (SISPA) approach를 이용하여 amplicon을 생성한 후 Hiseq platform으로 시퀀싱을 하여 assembly를 하였다(Genbank accession: JQ765564).
2013년 베타코로나바이러스를 분석하기 위해 기존에 알려진 15개의 베타코로나바이러스를 바탕으로 2.5kb 정도의 15개 amplicon을 만드는 60개의 프라이머 세트를 구성하여 이를 이용하여 reverse transcription을 진행 한 후 NGS로 분석함으로써 기존 방법보다 짧은 시간에 바이러스 전장유전체를 분석하였다.
2015년 South Korea에서 Complete Genome Sequence of Middle East Respiratory Syndrome Coronavirus를 환자의 객담 샘플에서 바이러스를 추출하여 vero cell로 배양한 후 추출한 RNA로부터 cDNA를 합성하여 평균 300bp 길이의 overlapping PCR primer를 제작하였다. Fragments들은 Illumina MiSeq 50-bp single-end platform으로 시퀀싱되어 complete genome을 얻었다.
2015년 브라질에서 창궐한 Zika virus는 임산부의 amniotic fluid(양수)에서 채취하여 RNA로부터 double strand cDNA를 합성하고 MiSeq으로 시퀀싱하여 Zika virus genome을 얻었다.
2015년 Sierra Leone, Liberia, Guinea에서 10,000명 이상의 사상자를 낸 Ebola virus의 genome은 38개에서 11개의 primer pair를 이용하여 현장에서 수시간 내에 MinION을 이용하여 시퀀싱하였다.
hCoV의 발견은 일찍이 되었지만, 의학적 중요성이 낮고 분리 및 동정의 어려움으로 관심을 받지 못하였다가 2003년 SARS로 진단의 중요성이 커졌다. 그러나 모든 종류의 Human Coronavirus를 검출할 수 있는 sensitive하고 rapid 검출법은 존재하지 않고, monoclonal antibodies를 이용하거나 reverse-transcriptase polymerase chain reaction (RT-PCR)을 이용하여 진단해오고 있다.
알파코로나바이러스에서는 진단을 위해서 2000년대부터 gene 1ab나 gene N을 타켓으로 하는 universal primer를 사용하고자 하였으나, 약 30% 정도만을 정확히 검출하였다. 따라서 각 group별 specific primer를 이용한 diagnosis의 중요성이 대두되었다.
1. 대한민국 공개특허공보 제10-2010-0018993호
1. Alphacoronavirus. The Springer Index of Viruses: 371-383. doi:10.1007/978-0-387-95919-1_56. Retrieved 2. Coronavirus Pathogenesis and the Emerging Pathogen Severe Acute Respiratory Syndrome Coronavirus. Microbiol Mol Biol Rev. 2005 Dec; 69(4): 635-664 3. Infectious RNA transcribed in vitro from a cDNA copy of the human coronavirus genome cloned in vaccinia virus. J Gen Virol. 2001 Jun;82(Pt 6):1273-81 4. The first complete genome sequences of clinical isolates of human coronavirus 229E. Virus Genes. 2012 Dec;45(3):433-9 5. Complete Genome Sequence of Human Coronavirus Strain 229E Isolated from Plasma Collected from a Haitian Child in 2016. Genome Announc. 2017 Nov 22;5(47). pii: e01313-17 6. Identification of a new human coronavirus. Nat Med. 2004 Apr;10(4):368-73 7. Genomic analysis of 16 Colorado human NL63 coronaviruses identifies a new genotype, high sequence diversity in the N-terminal domain of the spike gene and evidence of recombination. J Gen Virol. 2012 Nov;93(Pt 11):2387-98 8. Full genome deep sequencing and phylogenetic analysis of novel human betacoronavirus. Emerg Infect Dis. 2013 May;19(5):736-42B 9. Complete Genome Sequence of Middle East Respiratory Syndrome Coronavirus KOR/KNIH/002_05_2015, Isolated in South Korea. Genome Announc. 2015 Aug 13;3(4). pii: e00787-15 10. Real-time, portable genome sequencing for Ebola surveillance. Nature. 2016 Feb 11;530(7589):228-232 11. Genetic variability of human coronavirus OC43-, 229E-, and NL63-like strains and their association with lower respiratory tract infections of hospitalized infants and immunocompromised patients. J Med Virol. 2006 Jul;78(7):938-49
본 발명자들은 NL63과 229E를 포함한 인간 알파코로나 바이러스 및 유사 알파 코로나 바이러스에서 증폭이 가능한 범용 프라이머를 활용하여 미량의 유전체로부터 PCR등을 통하여 증폭하여 증폭된 유전체를 염기서열분석법을 통하여 빠르고 간편하게 바이러스의 전장 유전체 염기서열을 확보하는 방법 및 이를 이용한 진단방법을 개발하여 본 발명을 완성하게 되었다.
본 발명의 목적은 시료 내 알파 코로나 바이러스의 전장 유전체 서열 확인 방법을 제공한다.
본 발명의 다른 목적은 시료 내 알파 코로나 바이러스의 진단 키트를 제공하는 데 있다.
본 발명의 또 다른 목적은 알파 코로나 바이러스 유발 질환 검출용 조성물을 제공하는 데 있다.
본 발명의 다른 목적 및 이점은 하기의 발명의 상세한 설명, 청구범위 및 도면에 의해 보다 명확하게 된다.
본 발명의 일 양태에 따르면, 본 발명의 다음의 단계를 포함하는 분리된 시료 내 알파 코로나 바이러스의 전장 유전체 서열 확인 방법을 제공한다. (a) 알파 코로나 바이러스 전장 유전체에 상보적인 하나 이상의 정방향 및 역방향 프라이머 세트를 제조하는 단계; (b) 시료에서 RNA를 분리하는 단계; (c) 분리된 RNA를 사용하여 역전사 반응을 수행하는 단계; (d) 역전사 반응이 수행된 시료에 하나 이상의 프라이머 세트를 첨가하여 중합효소 연쇄반응을 실시하는 단계; 및 (e) 차세대 염기서열 분석(Next Generation Seqeuncing, NGS)을 이용한 증폭된 산물의 서열을 분석하는 단계를 포함하는 알파코로나 바이러스의 유전체 서열 확인 방법을 제공한다.
본 발명에서 상기 프라이머 세트는 서열번호 1 내지 서열번호 20으로 이루어진 군에서 선택된 프라이머 세트를 포함하는 것이 바람직하며, 보다 바람직하게는 상기 프라이머 세트는 서열번호 1 내지 서열번호 20으로 이루어진 10개의 프라이머 세트를 포함하는 것이다. 더불어 상기 (e) 단계는 서열번호 31 내지 87의 프라이머를 사용하는 방법을 제공한다.
본 발명에서 상기 시료는 혈액, 혈청, 객담, 소변 또는 생체 조직으로부터 분리된 것일 수 있으나 이에 제한되지는 않는다.
또한 본 발명에서 상기 알파 코로나 바이러스는 229E 또는 NL63을 포함하는 방법을 제공한다.
본 발명의 다른 양태에 따르면, 본 발명은 알파 코로나 바이러스 전장 유전체에 상보적인 하나 이상의 정방향 및 역방향 프라이머 세트를 포함하는 알파 코로나 바이러스 진단 키트를 제공한다.
본 발명의 또 다른 양태에 따르면, 본 발명은 알파 코로나 바이러스 전장 유전체에 상보적인 하나 이상의 정방향 및 역방향 프라이머 세트를 포함하는 알파 코로나 바이러스 유발 질환 진단용 조성물을 제공한다.
본 발명의 특징 및 이점을 요약하면 다음과 같다:
(a) 본 발명은 인간 알파 코로나 바이러스 전장 유전체의 서열 획득 방법 및 이의 용도에 관한 것이다.
(b) 본 발명의 방법, 키트 및 조성물은 인간 알파 코로나 바이러스의 전장 유전체를 증폭시킬 수 있는 정방향 및 역방향 프라이머 세트를 이용하고, 유전체 증폭을 통하기 때문에 미량의 바이러스 시료 및 미배양 임상시료 등에서 인간 알파 코로나 바이러스의 존재 유무 및 변종유무를 간편하게 빠르게 확인할 수 있다.
(c) 따라서, 본 발명의 방법, 키트 및 조성물은 인간 알파 코로나 바이러스의 전장 게놈 서열을 매우 정확하고 신속하게 얻을 수 있게 해주며, 이를 통해 인간 알파 코로나 바이러스로 유발된 질환의 진단에 효과적으로 적용될 수 있다.
도 1은 본 발명을 통한 바이러스 전장 유전체 분석의 개념도로써 프라이머 및 증폭체의 숫자는 도면에 제시된 것으로 제한되는 것은 아니다.
도 2은 본 발명의 실시예를 통한 229E와 NL63을 증폭한 핵산 조각의 전기 영동 분석 결과이다.
도 3은 본 발명의 실시예를 통해 확보된 229E 염기 서열의 계통도로써 확보된 염기서열이 원래 염기서열과 같은 분류에 속함을 보여주는 결과이다.
본 발명의 목적을 달성하기 위하여, 본 발명은
(a) 알파 코로나 바이러스 전장 유전체에 상보적인 하나 이상의 정방향 및 역방향 프라이머 세트를 제조하는 단계; (b) 시료에서 RNA를 분리하는 단계; (c) 분리된 RNA를 사용하여 역전사 반응을 수행하는 단계; (d) 역전사 반응이 수행된 시료에 하나 이상의 프라이머 세트를 첨가하여 중합효소 연쇄반응을 실시하는 단계; 및 (e) 차세대 염기서열 분석(Next Generation Seqeuncing, NGS)을 이용한 증폭된 산물의 서열을 분석하는 단계
를 포함하는 알파코로나 바이러스의 유전체 서열 확인 방법을 제공한다.
본 발명의 실시예에 따른 방법에 있어서, 상기 (c)단계의 중합효소 연쇄반응 (Polymerase Chain Reaction, PCR)은, 재조합효소 중합효소 증폭(Recombinase Polymerase amplification, RPA)도 가능하다.
또한 본 발명의 실시예에 따른 방법에 있어서, 상기 (e) 단계의 차세대 염기서열 분석(NGS)은 롱리드 시퀀싱 (Long Read Sequecing) 방법으로 단일 DNA 분자 시퀀싱이고 전류를 염기신호를 읽는 Oxford Nanopore Technologies의 Minion을 사용하였으나, 동일한 롱리드 시퀀싱 방법이며, 단일 DNA 분자 시퀀싱이지만 형광 펄스(Pulse)를 이용한 Pacific Bioscience (Pacbio)의 SMRT (Single molecule real time)도 가능하다.
본 발명의 방법의 전체 개념도는 도1에 나타난 바와 같다.
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하기로 한다. 이들 실시예는 단지 본 발명을 예시하기 위한 것이므로, 본 발명의 범위가 이들 실시예에 제한되는 것으로 해석되지는 않는다.
<실시예 1> 바이러스 origin
1966년에 분리보고된 이 균주의 참조유전체가 존재하지 않으므로, HCoV-229E는 ATCC에서 VR-740을 구입하였고, NL63 RNA는 국가병원체자원은행(NCCP 43214)에서 구입하여 사용하였다.
<실시예 2> 바이러스 세포배양 및 RNA 추출
바이러스 세포는 생물안전성 2등급 추출 배양 시설 내 이산화탄소 배양기에서 배양하였고, MRC-5(ATCC CCL171) 및 Caco-2(ATCC HTB-37)을 사용하였다. 바이러스 접종 전 37℃ 에서 MEM 배지를 이용하여 배양하였으며 접종후에는 같은 배지에서 33℃에서 5일간 배양하였다.
RNA 추출을 위하여 감염된 세포 배양액에서는 QIAmp viral RNA minikit (Qiagen)를 사용하였고, 감염된 세포에서는 RNeasy minikit (Qiagen)를 제조자의 사용설명서에 따라 추출하였다.
<실시예 3> 프라이머 설계 및 선정 기준
유전체 정렬을 통해 보존된 서열 중심으로 primer 후보군을 표2와 같이 69개 선정하였다. Primer 후보군 가운데 50 ℃ < Tm < 60 ℃이며 degeneracy 가 3개 이하이고 Inosine(N base)가 1개 이하인 primer 가운데 2912-5342bp 사이로 amplicon이 생성되는 총 20개의 primer, 10개 pair를 선정하여 실험에 사용하였다. 선정된 총 20개의 프라이머 세트 (Primer sets)는 표1과 같다.
프라이머 세트
Primer Forward
(5＇-> 3＇)		 Reverse
(5＇-> 3＇)		 length
(bp)
Set 1 43 (서열번호 1)
(TAGACTTTGTGTCTACTYYTCTC)		 5385 (서열번호 2)
(GCATTACCCAACAATTATTT)		 5342
Set 2 4358 (서열번호 3)
(TTTAATGTTGTWGGKCCICG)		 9077 (서열번호 4)
(GCATAACAAGCCACGTA)		 4719
Set 3 8201 (서열번호 5)
(GGTGGTRAYAATGTTTATTGYTA)		 11113 (서열번호 6)
(AGCCAAAATCACTTGTTA)		 2912
Set 4 9077 (서열번호 7)
(TAYCGYTGYGCTTGTTATGC)		 13477 (서열번호 8)
(GGACACCATCATAAAAC)		 4400
Set 5 12255 (서열번호 9)
(CTGGTARYGGTCARGCTAT)		 15343 (서열번호 10)
(CATTAGTCACAACATGG)		 3088
Set 6 13477 (서열번호 11)
(GTKTTTATWGATGGTGTWCC)		 17266 (서열번호 12)
(AACAAACTTGACTCCAGG)		 3789
Set 7 15343 (서열번호 13)
(CCKATGTTGTGYACTAARTG)		 20047 (서열번호 14)
(CTCCATCTACATATCGA)		 4704
Set 8 17266 (서열번호 15)
(CCTGGYAGTCAYAGTTTGTT)		 22457 (서열번호 16)
(TAAGGCTCTTCATGTTTTACA)		 5191
Set 9 19333 (서열번호 17)
(TATGGTGATGTKTCWAAAACTAC)		 24066 (서열번호 18)
(TCTAATAAGGAAGTTTAGTTGA)		 4733
Set 10 22457 (서열번호 19)
(TGTAAAACYATWGAAGAYGCCTTA)		 polyT (서열번호 20)
(TTTTTTTTTTTTT-GTGTATCCATATC)		 5000
<실시예 4> Reverse Transcription
HCoV-229E와 HCoV-NL63 RNA를 Random hexamer와 Superscrip III First-Strand Synthesis SuperMix (Cat No. 18080400) 를 이용하여 Reverse Transcript 반응을 수행하였다.
1μl RNA (conc. 50 ng/ul), 0.5ul random hexamer (5＇ -NNNNNN-3 ＇50ng/ul), 1μl 10mM dNTP mix, 최종 볼륨이 8μl가 되도록 DEPC water를 0.2μl PCR tube에 넣고 가볍게 혼합하였고, 65℃에서 5분간 incubation 한 후 ice에서 최소 1분 이상 식히고 가볍게 centrifugation한 후, 얼음에 tube를 꽂고 10μl 2X first-strand reaction mix, 2μl Superscript III enzyme mix를 넣은 후 가볍게 혼합하였다. Thermal cycler를 이용하여 25℃에서 10분간 incubation 한 후 50℃에서 70분 역전사를 진행한 뒤, 85℃에서 5분간 두어 효소의 불활성화를 진행하였다.
1μl RNase H를 첨가한 후 37℃에서 20분간 incubation하였고, RT 반응을 최적화하기 위하여 다양한 RNA template(50ng, 1ug), random hexamer(1ul, 0.5ul, 0.1ul) 및 RT reaction time(50, 70, 90min) 조건에서 최적화하였다.
<실시예 5> Polymerase Chain Reaction
설계한 primer set를 이용하여 PCR을 수행하였다.
1μl First strand cDNA, 1μl Forward primer (conc. 10 pmol/ul), 1μl Reverse primer (conc. 10 pmol/ul), 25μl KAPA hotstart ready mix (catalog number KR0370), 22μl 멸균증류수를 0.2μl PCR tube에 넣고 혼합하였다.
PCR 조건은 94℃ 5분 후, denaturation 94℃ 30초, annealing 55℃ 1분, extension 72℃ 4분으로 30회를 진행하고 final extension 72℃ 10분 동안 수행하였고, QIAquick gel extraction kit (Qiagen, Germantown, MD) 을 이용하여 PCR products를 purified하였으며 동일한 양의 amplicon으로 pooling하였다.
PCR 최적화를 위하여 Annealing temperature는 55-60℃ temperature gradient PCR (55, 55.3, 56, 56.9, 58.1, 59, 59.6, 60)을 수행하였고, Extension time은 3, 4, 5분동안 수행하였으며, Number of PCR cycle은 30회, 40회 동안 수행하였다.
<실시예 6> Sequencing
PCR amplicon은 Oxford nanopore사가 제시한 표준적인 방법에 따라 PCR에 사용된 프라이머를 기반으로 추가로 229E, NL63 확보된 염기서열정보의 끝부분의 서열의 프라이머를 이용하여 primer walking법으로 sequencing을 진행하여 완료하였다.
Supplement table primer list(229E, NL63)
서열번호 Name Sequence(5＇-> 3＇)
21 Alpha13F TTWTCTATCTAYAGATAGARAA
22 Alpha41F TTTAGACTTTGTGTCTACTYYTCTC
23 Alpha43F TAGACTTTGTGTCTACTYYTCTC
24 Alpha62F TCTCAACTAAACGAAATTTTT
25 Alpha252F AAGCAAMGTTYTGTCKTTGTGG
26 Alpha950F GGTTCICCIKTTATKACIAATGGT
27 Alpha1421F GGTGTYATWGAYATTAGT
28 Alpha2981F GGTGGYAARRTWTCWTTYTCTGA
29 Alpha3940F TRTIGATGGTTTYGGTGT
30 Alpha4358F TTTAATGTTGTWGGKCCICG
31 Alpha4904F TAYATGTGYGTTKTRCCATC
32 Alpha5385F AYAATAATTGTTGGGTKAATGC
33 Alpha5546F AAGGGTGATGCTGARGA
34 Alpha6842F TATGTTAATGCWAATGGTGGT
35 Alpha7517F GCKTGYTGTATGCGYGC
36 Alpha8201F GGTGGTRAYAATGTTTATTGYTA
37 Alpha8432F TGGTWTGTTAAYGATGGAMGTGT
38 Alpha8933F GARAGTGCTGCWGCWGG
39 Alpha8961F TWGAYATKCGTTCTTATGA
40 Alpha9077F TAYCGYTGYGCTTGTTATGC
41 Alpha9203F ATGGCRCARCCRTCTGG
42 Alpha10476F TGGCGYTTTKCTARAGA
43 Alpha10814F AARTAYATGRTWGCTAATGG
44 Alpha11113F TAARCAYAGTGATTTTGGWCT
45 Alpha11189F TCITCWTTTGTTRGTATGCC
46 Alpha11360F AGRATGGCTGAACAAGCTGC
47 Alpha11507F GCACGTRATGGTGTTGT
48 Alpha11624F TAYGCKGGIGTTGTTTGGAC
49 Alpha12179F GCTGTTARACAWGGYGCAAAACC
50 Alpha12255F CTGGTARYGGTCARGCTAT
51 Alpha12532F TCTAGTGCMGCTCGACTAGA
52 Alpha12717F GGAMCACGAGCARTCCATGTA
53 Alpha13477F GTKTTTATWGATGGTGTWCC
54 Alpha13816F GGTGATGCTGCWRTTAAAGATTTTGA
55 Alpha13868F CTTAATAARAGYGCTGGKTGGCC
56 Alpha14036F CARAAIGGTGATGCTGC
57 Alpha14220F CTTAATAARAGYGCTGKTTGGCC
58 Alpha14742F GGTACRACTTCWGGTGAYGC
59 Alpha15343F CCKATGTTGTGYACTAARTG
60 Alpha15532F GCTGGWAATRTITTTGGTTT
61 Alpha16114F ACAATACAGGGTCCICCTGGTAGTGG
62 Alpha16561F CGWATGTGTGCWATAGGMCC
63 Alpha17266F CCTGGYAGTCAYAGTTTGTT
64 Alpha17320F GGTTGGTTRGGWATGGATGT
65 Alpha17989F TTTAAACAYGCWTTGGGITGTGA
66 Alpha18574F TTTGAWKKTTATAATTTGTGGCA
67 Alpha18874F TTRTGGGATTATGAAGCWGA
68 Alpha19333F TATGGTGATGTKTCWAAAACTAC
69 Alpha19735F TGTAATTTRTATAATTAYGGTGC
70 Alpha20047F TCWGATATGTAYGATGGYAG
71 Alpha20829F01 TGYTTATGGTCTGTTTCTGG
72 Alpha20829F02 TGYTTATGGCCTGTACCTGG
73 Alpha20829F03 TGYYTCIGGCCTGTWCCTGG
74 Alpha20846F01 GGTTTTGTYTATTTTAAYGGTACTGG
75 Alpha20846F02 GGTTTTGTYTATTTTAAYGCCACTGG
76 Alpha22436F CTYAAGCAGTAYACTTCTGCTTG
77 Alpha22457F TGTAAAACYATWGAAGAYGCCTTA
78 Alpha22789F TGATGCWGARCGWATGGC
79 Alpha23909F TGGCCITGGTGGGTKTGG
80 Alpha24066F TCAACTAAACTTCCTTATTAYGA
81 Alpha25053F GTTGGAATITWATYCTAAC
82 Alpha25287F01 GTTTCAGACTTTTCCGWCGTG
83 Alpha25287F02 GTTTCAGACTTTGGCGCCGTG
84 Alpha25679F TTGCTTCATTTIWTCTAAACTRAAC
85 Alpha25980F GGTGTYGTYTGGGTTGCT
86 Alpha27065F TAGTCTTAYACACAATGGTARGCC
87 Alpha27168F TGGAGTAMTYAAAGATCGC
<실시예 7> Assembly
Minion을 통해서 얻어진 염기서열은 bowtie2를 이용하여 알려진 유전체 정보를 참조로 하여 assembly하였다. Assembly된 유전체 염기서열은 참조서열 대비 99.88%의 일치도를 나타내었으며 염기서열은 다음과 같다.
>1 229E|NC_002645:1
ACTTAAGTACCTTATCTATCTACAGATAGAAAAGTTGCTTTTTAGACTTTGTGTCTACTT
TTCTCAACTAAACGAAATTTTTGCTATGGCCGGCATCTTTGATGCTGGAGTCGTAGTGTA
ATTGAAATTTCATTTGGGTTGCAACAGTTTGGAAGCAAGTGCTGTGTGTCCTAGTCTAAG
GGTTTCGTGTTCCGTCACGAGATTCCATTCTACAAACGCCTTACTCGAGGTTCCGTCTCG
TGTTTGTGTGGAAGCAAAGTTCTGTCTTTGTGGAAACCAGTAACTGTTCCTAATGGCCTG
CAACTGTGTGACACTTGCCGTAGCAAGTGATTCTGAAATTTCTGCAAATGGCTGTTCTAC
TATTGCGCAAGCCGTCCGCCGTTATAGCGAGGCCGCTAGCAATGGTTTTAGGGCATGCCG
ATTTGTTTCATTAGATTTGCAGGATTGCATCGTTGGCATTGCAGACGATACATATGTTAT
GGGTCTGCATGGCAATCAGACGTTGTTTTGCAACATAATGAAATTTTCTGACCGTCCTTT
TATGCTTCATGGGTGGTTGGTTTTTTCCAATTCAAATTACCTTTTGGAGGAATTTGATGT
TGTCTTCGGTAAGAGAGGTGGTGGTAATGTGACATACACTGACCAGTATCTCTGTGGCGC
CGATGGCAAACCTGTTATGAGTGAAGATTTATGGCAGTTTGTTGACCATTTCGGTGAGAA
CGAAGAAATTATCATCAATGGTCATACTTACGTTTGTGCTTGGCTTACTAAGCGTAAGCC
CTTAGATTACAAACGTCAGAACAACCTTGCCATTGAAGAGATTGAATATGTGCATGGTGA
TGCTTTGCATACACTACGCAATGGTTCTGTTCTTGAAATGGCTAAGGAAGTGAAGACATC
TAGTAAAGTTGTGTTAAGCGATGCTCTTGACAAACTTTACAAAGTCTTTGGTTCTCCTGT
TATGACAAATGGTTCCAACATCCTAGAGGCCTTTACTAAACCTGTGTTTATTAGTGCATT
AGTTCAATGTACTTGTGGTACCAAGTCTTGGTCTGTTGGTGATTGGACCGGTTTTAAATC
CTCTTGTTGCAACGTGATCAGTAATAAACTGTGTGTTGTTCCCGGTAATGTTAAACCTGG
TGATGCTGTGATTACCACTCAGCAAGCTGGTGCTGGTATTAAGTATTTTTGTGGCATGAC
TCTTAAGTTTGTTGCAAATATTGAAGGTGTCTCTGTTTGGAGAGTGATTGCTCTTCAGAG
TGTGGATTGCTTTGTTGCTTCTTCCACTTTTGTAGAAGAGGAACATGTTAATAGAATGGA
TACATTCTGCTTCAATGTACGCAATAGTGTTACTGATGAGTGTCGTCTGGCCATGTTGGG
TGCTGAAATGACTAGTAATGTCAGAAGACAAGTTGCTTCAGGTGTCATAGACATTAGTAC
CGGTTGGTTTGATGTTTATGATGACATCTTTGCTGAAAGCAAACCATGGTTTGTTCGCAA
GGCTGAAGACATTTTTGGCCCTTGTTGGTCCGCTCTTGTTTCTGCACTTAAACAACTTAA
AGTCACTACAGGTGAACTTGTGAGATTTGTTAAGTCTATTTGCAATTCAGCTGTTGCTGT
CGTGGGTGGTACTATACAAATTCTCGCTAGTGTGCCTGAGAAGTTTTTGAATGCGTTTGA
CGTGTTTGTCACAGCTATTCAAACTGTCTTTGACTGTGCTGTTGAAACTTGTACTATTGC
CGGTAAAGCATTTGACAAGGTTTTTGACTATGTCTTGCTTGATAATGCGCTTGTAAAACT
TGTCACCACAAAGCTTAAGGGTGTTCGTGAACGTGGCCTTAATAAAGTTAAGTATGCAAC
AGTTGTTGTTGGTTCCACTGAAGAAGTTAAATCTTCACGTGTTGAACGTAGCACTGCTGT
ACTTACAATCGCCAACAATTATTCCAAACTTTTTGATGAAGGGTATACTGTTGTAATTGG
CGATGTGGCGTACTTTGTTAGTGACGGCTACTTCCGTCTTATGGCCAGTCCAAATAGTGT
GTTGACTACTGCAGTCTATAAACCATTGTTTGCTTTTAATGTGAATGTTATGGGTACTAG
ACCTGAAAAATTTCCAACCACTGTGACTTGTGAAAATTTAGAGTCTGTTGTTTTGTTTGT
TAATGACAAAATTACTGAATTCCAATTGGATTACAGTATTGATGTCATTGATAATGAAAT
AATTGTCAAACCTAATATCAGCCTATGTGTTCCACTTTATGTGAGAGACTATGTTGACAA
ATGGGATGATTTTTGCAGACAATATAGTAACGAGTCTTGGTTTGAGGATGATTACAGGGC
TTTTATCAGTGTTTTGGACATCACTGATGCTGCTGTGAAAGCTGCAGAGTCTAAAGCTTT
CGTTGATACTATTGTTCCACCTTGCCCATCTATTTTGAAAGTTATAGATGGAGGCAAAAT
ATGGAATGGTGTTATTAAAAATGTTAACTCTGTTAGAGACTGGCTTAAGTCTTTGAAGTT
AAATCTCACACAACAGGGTTTGCTTGGAACATGTGCAAAGCGTTTTAAACGTTGGCTTGG
CATTTTGCTAGAGGCCTATAATGCGTTTTTAGACACTGTGGTTTCTACTGTTAAAATTGG
TGGCTTGACCTTTAAAACATATGCTTTTGATAAACCTTACATTGTGATACGTGATATCGT
GTGTAAGGTTGAAAATAAAACAGAAGCAGAATGGATTGAGCTTTTTCCACATAATGACAG
GATTAAGTCTTTTAGTACTTTCGAGAGTGCTTACATGCCAATTGCAGACCCTACACATTT
TGACATTGAAGAAGTTGAACTTTTAGATGCAGAGTTTGTAGAACCAGGCTGTGGTGGTAT
TTTGGCAGTAATAGATGAGCACGTCTTTTATAAGAAGGATGGTGTTTATTATCCATCAAA
TGGTACTAACATTCTACCTGTTGCATTTACAAAAGCCGCTGGTGGTAAAGTTTCATTTTC
TGATGACGTTGAAGTAAAAGACATTGAACCTGTTTACAGAGTCAAGCTTTGCTTTGAGTT
TGAAGATGAAAAACTTGTAGATGTTTGTGAAAAGGCAATTGGCAAGAAAATTAAACATGA
AGGTGACTGGGATAGCTTTTGTAAGACTATTCAATCAGCACTTTCTGTTGTTTCTTGCTA
TGTAAATCTACCTACTTATTACATTTATGATGAAGAAGGCGGTAATGACTTGAGTTTGCC
CGTTATGATTTCTGAATGGCCTCTTTCTGTTCAACAAGCTCAACAAGAAGCTACTTTACC
TGATATTGCTGAGGATGTTGTTGACCAAGTTGAAGAAGTCAATAGCATTTTTGACATTGA
GACAGTGGATGTTAAACATGATGTGAGTCCTTTTGAAATGCCATTTGAAGAGTTAAATGG
TTTAAAGATACTCAAACAATTGGATAACAACTGCTGGGTTAACTCAGTTATGTTACAAAT
ACAATTAACTGGTATACTTGATGGTGACTATGCTATGCAGTTTTTTAAAATGGGCCGAGT
TGCCAAGATGATTGAACGCTGCTACACTGCTGAGCAATGTATACGTGGTGCTATGGGTGA
TGTTGGTTTGTGTATGTATAGACTGCTTAAAGACTTACACACTGGTTTTATGGTTATGGA
TTATAAATGTAGTTGTACCAGTGGTAGGCTTGAAGAATCGGGAGCTGTTTTGTTTTGTAC
GCCCACTAAGAAGGCGTTTCCTTATGGTACTTGTCTAAATTGTAACGCACCTCGCATGTG
TACAATTAGGCAGTTACAAGGTACCATAATATTTGTGCAACAAAAACCAGAACCTGTTAA
TCCTGTTTCTTTTGTTGTTAAACCAGTCTGCTCATCAATTTTTCGTGGTGCTGTGTCTTG
TGGTCATTACCAGACTAACATCTATTCACAAAATTTGTGTGTGGATGGTTTTGGTGTTAA
CAAGATTCAGCCCTGGACAAATGATGCACTTAATACTATTTGTATTAAGGATGCAGATTA
TAATGCAAAAGTTGAAATATCTGTTACACCAATTAAAAATACAGTTGATACAACACCTAA
GGAAGAATTTGTTGTTAAAGAGAAGTTGAACGCCTTCCTCGTTCATGACAATGTAGCTTT
CTACCAAGGTGATGTTGATACTGTTGTTAATGGTGTTGACTTTGACTTTATTGTAAATGC
TGCTAATGAGAACCTTGCTCATGGTGGAGGACTTGCCAAAGCTTTAGATGTGTACACTAA
AGGTAAACTTCAACGTTTATCTAAAGAACACATTGGATTAGCGGGTAAAGTAAAAGTTGG
TACAGGAGTTATGGTTGAGTGTGATAGCCTTAGAATTTTTAATGTTGTTGGTCCACGCAA
GGGTAAACATGAACGTGATTTACTCATAAAAGCTTACAACACTATTAATAATGAACAAGG
CACACCTTTAACACCAATTTTGAGCTGTGGTATTTTTGGTATCAAACTCGAAACTTCATT
AGAAGTTTTGCTTGATGTTTGTAATACAAAAGAAGTTAAAGTTTTTGTTTATACAGACAC
AGAGGTTTGTAAGGTTAAGGATTTTGTGTCTGGTTTAGTGAATGTTCAAAAAGTTGAGCA
ACCTAAAATAGAACCAAAACCAGTGTCCGTAATTAAAGTTGCACCCAAGCCTTACAGGGT
AGATGGTAAATTTAGTTACTTTACAGAAGACTTGTTGTGTGTCGCTGATGACAAACCCAT
TGTTTTGTTTACTGACTCTATGCTTACTTTGGATGACCGTGGTTTAGCTCTAGACAATGC
ACTTAGTGGTGTGCTTAGTGCTGCTATTAAGGATTGTGTTGACATAAATAAAGCTATACC
TTCTGGTAATCTTATTAAGTTTGATATAGGTTCTGTTGTTGTCTACATGTGTGTTGTGCC
ATCCGAAAAGGACAAACATTTAGATAATAATGTTCAACGATGCACACGTAAGTTGAATAG
ACTTATGTGTGATATAGTTTGTACTATACCAGCTGACTACATCTTGCCATTGGTGTTGTC
TAGTTTGACTTGTAATGTTTCTTTTGTAGGTGAACTTAAAGCTGCTGAAGCTAAAGTTAT
AACTATAAAGGTGACAGAGGATGGTGTTAATGTTCATGATGTGACCGTGACAACAGACAA
GTCATTTGAACAACAAGTTGGTGTTATTGCTGATAAGGACAAAGATCTTTCTGGTGCAGT
ACCAAGTGATCTTAACACATCTGAATTGCTTACTAAAGCAATAGATGTTGATTGGGTCGA
ATTTTATGGCTTTAAAGATGCTGTTACTTTTGCAACAGTTGATCATAGTGCTTTTGCCTA
TGAAAGTGCTGTTGTTAATGGTATTAGAGTGTTAAAAACTAGTGATAATAATTGTTGGGT
GAATGCTGTTTGTATTGCACTACAGTATTCGAAACCCCATTTTATTTCACAAGGTCTTGA
TGCTGCGTGGAATAAATTTGTTTTAGGCGATGTTGAAATTTTTGTTGCATTTGTTTACTA
TGTTGCAAGACTAATGAAAGGTGACAAGGGTGATGCTGAAGACACTTTGACTAAGTTGTC
TAAGTATCTTGCTAATGAAGCTCAAGTTCAATTAGAACATTATAGTTCTTGTGTTGAATG
TGATGCTAAATTTAAAAACTCTGTTGCATCTATCAATTCTGCTATAGTTTGTGCTAGTGT
CAAACGTGATGGTGTGCAAGTTGGTTATTGTGTCCATGGTATTAAGTACTATTCACGTGT
TAGAAGTGTTAGAGGTAGAGCTATTATAGTCAGTGTCGAACAGCTTGAACCGTGTGCTCA
GTCTAGACTTTTGAGTGGTGTTGCTTATACTGCTTTTTCTGGACCTGTTGACAAAGGTCA
TTATACTGTTTATGATACTGCAAAGAAATCAATGTATGATGGTGATCGTTTTGTTAAACA
TGATCTTTCTCTGCTGTCTGTCACATCAGTTGTTATGGTTGGTGGTTATGTTGCACCTGT
TAATACAGTGAAACCTAAACCAGTCATTAATCAACTTGATGAAAAGGCACAGAAGTTCTT
TGATTTTGGTGATTTTTTGATTCATAATTTTGTTATTTTTTTCACATGGTTATTGAGTAT
GTTTACTTTGTGTAAAACTGCAGTAACTACAGGTGATGTTAAAATAATGGCCAAAGCACC
ACAAAGGACGGGTGTTGTTTTAAAACGTAGTCTTAAATATAACTTAAAAGCGTCAGCAGC
TGTTCTTAAATCTAAGTGGTGGCTGCTTGCTAAGTTTACGAAACTACTGTTACTCATATA
TACATTGTACTCAGTAGTTTTGCTTTGTGTACGTTTTGGACCGTTTAATTTTTGTAGTGA
GACTGTTAATGGTTATGCTAAGTCAAACTTTGTCAAGGATGATTACTGTGATGGTTCATT
GGGCTGCAAGATGTGTCTTTTTGGTTACCAAGAGTTAAGTCAATTTAGCCATTTGGATGT
TGTGTGGAAGCATATAACAGACCCTTTGTTTAGTAATATGCAACCTTTCATTGTCATGGT
TTTGCTGCTTATATTTGGTGACAATTATTTGAGATGCTTCTTGCTGTATTTTGTTGCTCA
GATGATAAGCACAGTTGGTGTTTTTCTAGGTTACAAGGAAACAAATTGGTTCTTGCACTT
TATTCCATTTGATGTTATTTGTGATGAACTGCTTGTCACTGTTATTGTTATTAAGGTTAT
TTCTTTTGTCAGACATGTGCTTTTTGGTTGTGAAAACCCAGATTGCATTGCGTGTTCTAA
GAGTGCTAGACTTAAGAGATTCCCTGTTAACACAATTGTCAATGGTGTGCAACGTTCATT
TTATGTTAATGCAAATGGTGGTAGTAAGTTTTGTAAGAAACATAGATTTTTCTGTGTTGA
TTGTGACTCTTATGGTTATGGCAGCACGTTTATAACACCCGAAGTTTCTAGAGAACTTGG
TAACATTACCAAAACAAATGTGCAACCAACAGGCCCAGCGTATGTCATGATTGACAAAGT
GGAGTTTGAAAATGGTTTTTACAGATTGTATTCCTGTGAAACATTTTGGCGTTACAACTT
TGATATAACTGAAAGCAAGTATTCTTGCAAAGAGGTTTTTAAAAATTGTAATGTTTTGGA
TGATTTCATCGTGTTTAACAATAATGGGACCAATGTAACGCAGGTTAAAAATGCTAGTGT
TTACTTTTCACAGTTGTTGTGTAGGCCCATTAAATTAGTTGACAGTGAACTTTTGTCCAC
TTTGTCAGTTGATTTTAATGGTGTCTTACACAAGGCATACATTGATGTACTACGTAATAG
CTTTGGTAAAGATCTTAATGCTAATATGTCTTTAGCCGAGTGCAAGAGTGCTTTAGGCCT
GTCTATTAGTGATCATGAATTTACTAGTGCTATTTCTAATGCACATCGTTGTGACGTGTT
GTTATCTGATTTGTCATTTAACAACTTTGTCAGTTCGTATGCTAAACCTGAGGAAAAATT
ATCAGCTTATGACTTGGCGTGTTGTATGCGTGCAGGTGCTAAGGTTGTTAATGCCAATGT
TCTGACAAAGGACCAAACTCCTATTGTTTGGCATGCAAAGGATTTTAACAGTCTTTCTGC
TGAAGGTCGCAAGTATATTGTAAAAACTAGCAAAGCTAAGGGTTTGACTTTCTTGTTGAC
AATTAATGAAAACCAAGCTGTCACGCAAATACCTGCAACTAGCATTGTTGCTAAGCAAGG
TGCTGGTGATGCTGGCCATTCATTAACATGGCTGTGGCTACTGTGTGGTCTTGTGTGTTT
GATTCAATTCTACTTGTGCTTTTTCATGCCCTATTTTATGTACGATATCGTGAGTAGTTT
TGAGGGTTATGATTTTAAGTATATAGAAAATGGTCAGTTGAAGAATTTTGAAGCGCCACT
TAAATGCGTCAGAAACGTTTTTGAAAACTTTGAGGACTGGCATTATGCTAAGTTTGGCTT
CACACCTTTAAACAAGCAAAGCTGTCCTATTGTAGTTGGAGTTTCTGAAATTGTTAATAC
TGTCGCTGGCATTCCATCTAATGTGTATCTTGTTGGTAAAACTTTAATTTTTACACTACA
AGCTGCTTTTGGTAATGCTGGTGTTTGTTATGACATTTTTGGAGTCACAACACCTGAAAA
GTGCATTTTTACTTCTGCTTGTACTAGATTAGAAGGTTTGGGTGGTGACAATGTTTATTG
TTATAACACAGCGCTTATGGAAGGTTCTTTGCCTTACAGTTCAATACAAGCTAATGCATA
TTATAAATATGACAATGGCAATTTTATTAAGTTGCCAGAAGTTATTGCACAAGGCTTTGG
TTTTAGAACAGTGCGTACTATTGCCACCAAATACTGCCGCGTAGGTGAATGTGTTGAATC
CAATGCAGGTGTGTGTTTTGGCTTTGACAAGTGGTTTGTTAACGATGGACGTGTTGCCAA
TGGTTACGTTTGTGGTACTGGTTTGTGGAACCTTGTATTTAACATACTTTCCATGTTTTC
ATCTTCATTCTCTGTTGCTGCAATGTCAGGTCAAATTTTACTTAATTGTGCATTAGGTGC
TTTTGCTATTTTTTGTTGTTTTCTTGTGACAAAGTTTAGACGCATGTTTGGTGACCTTTC
TGTAGGTGTTTGCACTGTTGTTGTGGCTGTTTTGCTTAACAATGTCTCTTACATTGTAAC
TCAGAATTTAGTAACAATGATTGCTTATGCCATATTGTATTTCTTTGCTACTAGAAGCTT
ACGCTATGCATGGATTTGGTGTGCTGCATATTTAATTGCGTATATTTCTTTTGCTCCATG
GTGGTTGTGTGCTTGGTACTTTCTTGCTATGTTGACAGGTTTGTTACCTAGTTTGCTGAA
GCTTAAAGTTTCGACAAATCTTTTCGAAGGTGACAAATTTGTAGGTACATTTGAAAGTGC
TGCTGCAGGAACATTTGTCATTGACATGCGTTCTTATGAGAAACTTGCTAATAGCATCTC
TCCAGAAAAGTTGAAAAGTTATGCTGCTAGCTATAATAGATATAAGTACTATAGTGGTAA
TGCAAATGAAGCTGATTACTGTTGCGCTTGTTATGCCTATTTAGCAAAAGCAATGTTGGA
CTTTTCGCGTGATCATAATGACATCTTGTACACACCTCCGACTGTCAGTTATGGTTCTAC
ATTACAGGCTGGTTTGCGCAAAATGGCACAACCATCTGGCTTTGTGGAGAAATGTGTTGT
CCGTGTCTGCTATGGAAACACTGTGTTGAATGGGTTGTGGCTTGGTGATATTGTTTATTG
CCCACGTCATGTTATCGCATCTAACACAACTTCTGCTATAGATTATGATCACGAATATAG
TATTATGCGGTTGCATAATTTTTCTATAATATCTGGTACAGCATTTCTTGGTGTTGTAGG
TGCTACTATGCATGGAGTAACTCTTAAAATTAAGGTTTCACAGACTAACATGCACACACC
TAGACATTCTTTTAGAACACTAAAATCTGGTGAAGGTTTTAACATCTTAGCATGCTATGA
TGGTTGTGCTCAAGGTGTTTTTGGTGTGAACATGAGAACTAATTGGACTATCCGTGGTTC
ATTTATTAATGGTGCGTGTGGTTCCCCTGGCTACAATCTTAAAAATGGCGAGGTGGAATT
TGTTTATATGCATCAAATTGAACTCGGAAGTGGTAGCCATGTAGGTTCTAGCTTTGATGG
TGTTATGTATGGTGGTTTTGAAGACCAACCTAATCTTCAAGTTGAATCTGCAAACCAGAT
GTTAACAGTTAATGTGGTTGCATTTCTTTATGCTGCTATATTGAATGGTTGCACATGGTG
GCTTAAAGGTGAAAAATTGTTTGTGGAGCATTATAATGAGTGGGCACAGGCTAATGGTTT
CACAGCTATGAATGGTGAAGACGCTTTTTCCATTCTTGCTGCTAAAACTGGTGTCTGTGT
GGAAAGATTACTTCATGCTATTCAAGTTTTGAATAATGGCTTTGGTGGTAAACAAATTTT
GGGTTATTCTAGTCTCAATGATGAGTTCAGTATTAATGAAGTTGTCAAACAAATGTTTGG
TGTTAACCTGCAAAGTGGTAAAACCACTAGTATGTTTAAATCCATAAGCTTATTTGCTGG
CTTCTTTGTCATGTTCTGGGCTGAATTATTTGTTTATACCACCACTATTTGGGTTAACCC
TGGTTTTCTTACTCCGTTTATGATTTTGCTTGTTGCTTTGTCACTCTGTCTTACATTTGT
TGTTAAACATAAGGTTTTGTTTTTGCAAGTGTTTTTGTTGCCTTCAATTATTGTGGCTGC
TATTCAAAACTGTGCTTGGGACTACCATGTTACAAAGGTGTTGGCAGAGAAGTTTGATTA
TAATGTTTCTGTTATGCAAATGGACATCCAGGGTTTTGTTAACATTTTTATTTGTCTTTT
TGTTGCACTGTTGCATACTTGGCGCTTTGCTAAAGAGCGTTGTACACATTGGTGCACTTA
TTTGTTCTCACTCATTGCTGTTTTATACACTGCATTGTATAGTTATGACTACGTTAGTTT
GCTGGTTATGCTACTTTGTGCAATTTCTAATGAATGGTATATTGGTGCTATTATTTTTAG
AATTTGTCGTTTTGGTGTTGCATTTTTACCAGTGGAATACGTGTCTTACTTTGATGGTGT
TAAAACTGTGCTGTTGTTTTACATGTTGTTAGGCTTTGTTAGCTGTATGTACTATGGTTT
GTTGTACTGGATTAACAGGTTCTGTAAGTGCACATTAGGTGTTTATGATTTCTGTGTTAG
TCCAGCCGAATTTAAGTATATGGTTGCTAATGGTTTGAATGCACCAAATGGCCCTTTTGA
TGCGCTCTTTCTGTCTTTTAAACTAATGGGTATTGGCGGTCCTAGAACCATTAAAGTTTC
TACTGTACAGTCTAAATTGACTGATCTTAAGTGCACAAACGTCGTTCTAATGGGCATTTT
GTCTAACATGAACATAGCTTCTAATTCAAAGGAGTGGGCATATTGTGTTGAAATGCACAA
TAAAATAAACTTGTGTGACGACCCTGAAACTGCTCAAGAGTTATTGCTGGCGTTGTTGGC
CTTTTTCTTGTCTAAGCATAGTGATTTTGGTCTTGGTGATCTTGTCGATTCTTATTTTGA
GAACGACTCCATTTTGCAAAGTGTTGCATCTTCTTTTGTTGGTATGCCATCTTTTGTTGC
ATATGAAACAGCAAGACAAGAGTATGAAAATGCTGTTGCAAATGGTTCCTCACCACAAAT
AATCAAACAATTGAAGAAGGCTATGAATGTTGCAAAAGCTGAGTTTGACAGGGAATCATC
TGTTCAAAAGAAAATTAACAGAATGGCTGAACAAGCTGCTGCAGCTATGTACAAAGAAGC
ACGTGCTGTTAATAGAAAATCAAAAGTTGTTAGTGCCATGCATAGTTTACTCTTTGGCAT
GCTCCGACGTTTGGACATGTCTAGTGTTGACACTATCCTTAATATGGCACGTAATGGTGT
TGTCCCTCTTTCCGTTATCCCTGCTACTTCTGCAGCCAGGCTCGTCGTCGTAGTACCAGA
TCATGATTCATTTGTGAAAATGATGGTAGATGGTTTTGTGCACTACGCTGGTGTTGTTTG
GACATTACAGGAAGTTAAGGATAATGATGGTAAGAATGTGCATCTTAAAGATGTTACAAA
GGAAAACCAGGAAATACTTGTTTGGCCTCTGATTTTGACTTGTGAACGTGTCGTTAAATT
GCAGAACAATGAAATAATGCCGGGCAAGATGAAGGTCAAGGCCACCAAAGGTGAAGGTGA
TGGAGGCATTACTAGTGAAGGTAATGCTCTATACAACAATGAAGGTGGACGTGCATTCAT
GTATGCATATGTGACTACGAAGCCTGGCATGAAGTATGTTAAATGGGAACATGACTCTGG
TGTGGTTACAGTTGAATTGGAACCACCTTGCAGATTTGTTATAGACACACCTACTGGACC
CCAAATTAAGTATCTTTATTTTGTTAAGAATCTTAACAATTTAAGGAGAGGTGCTGTTTT
GGGTTACATTGGTGCCACTGTGAGATTGCAAGCTGGCAAACAGACTGAGTTTGTTTCAAA
CTCCCATTTATTAACACATTGTTCTTTTGCTGTTGACCCAGCTGCAGCCTATCTTGATGC
TGTTAAACAAGGCGCAAAACCTGTTGGCAATTGTGTAAAGATGTTGACTAATGGTTCTGG
TAGCGGTCAGGCTATTACTTGTACCATTGATTCCAACACTACGCAGGACACATATGGTGG
CGCGTCTGTTTGTATTTATTGCAGAGCACATGTTGCACATCCAACCATGGACGGTTTTTG
TCAGTACAAAGGCAAGTGGGTACAAGTGCCTATAGGTACAAATGACCCTATAAGATTTTG
TCTTGAAAATACTGTTTGTAAAGTTTGTGGTTGTTGGCTTAATCATGGCTGTACATGTGA
CCGGACTGCTATCCAAAGTTTTGATAACAGTTATTTAAACGAGTCCGGGGCTCTAGTGCC
GCTCGACTAGAGCCCTGTAATGGTACAGACATAGATTACTGTGTCCGTGCATTTGACGTT
TACAATAAAGATGCGTCTTTTATCGGGAAAAATCTGAAGTCCAATTGTGTGCGCTTCAAG
AATGTAGATAAGGATGACGCGTTCTATATTGTTAAACGTTGCATTAAGTCAGTTATGGAC
CACGAGCAGTCCATGTATAACTTACTTAAAGGCTGTAATGCTGTTGCTAAGCATGATTTC
TTTACTTGGCATGAGGGCAGAACCATTTATGGTAATGTTAGTAGACAGGATCTTACTAAA
TACACCATGATGGATTTGTGCTTCGCTCTGCGTAACTTTGATGAAAAAGACTGTGAAGTT
TTTAAGGAGATATTGGTTCTTACTGGTTGTTGTAGTACTGATTACTTTGAAATGAAGAAT
TGGTTTGACCCCATAGAAAATGAGGACATACACCGTGTGTATGCTGCTTTAGGTAAGGTA
GTTGCAAATGCAATGCTTAAGTGTGTTGCTTTTTGCGACGAAATGGTGCTCAAAGGAGTT
GTTGGTGTTTTGACCTTAGACAACCAAGATCTTAATGGGAATTTCTATGACTTCGGTGAC
TTTGTATTGTGTCCTCCTGGAATGGGAATACCCTACTGCACGTCATACTATTCTTATATG
ATGCCTGTTATGGGTATGACTAATTGTTTAGCTAGTGAGTGCTTTATGAAAAGTGACATC
TTTGGTCAAGACTTCAAAACTTTTGATTTGTTGAAATATGATTTCACAGAACATAAGGAG
GTTTTGTTTAACAAGTACTTTAAGTATTGGGGACAGGATTATCATCCTGATTGTGTTGAT
TGCCATGACGAGATGTGTATTTTGCATTGTTCAAATTTTAACACACTCTTCGCAACCACA
ATTCCAAACACGGCTTTTGGACCTCTATGCAGAAAAGTGTTTATTGATGGTGTACCCGTA
GTTGCTACTGCTGGTTACCACTTTAAACAATTAGGACTTGTGTGGAACAAAGATGTTAAC
ACTCATTCTACCAGACTTACTATTACTGAACTCTTACAGTTTGTGACAGATCCAACGCTT
ATAGTTGCGTCATCGCCTGCCTTGGTGGATAAACGCACTGTTTGTTTTTCTGTCGCTGCT
TTGAGTACAGGATTAACATCCCAAACAGTAAAACCTGGCCATTTTAATAAGGAGTTTTAT
GACTTCTTACGTTCTCAGGGGTTTTTCGATGAGGGTTCAGAATTAACATTGAAGCATTTC
TTTTTTACACAAAAGGGTGATGCTGCAATTAAAGATTTTGATTATTATCGTTACAACAGA
CCTACTATGCTGGATATTGGACAAGCTCGCGTAGCATATCAAGTGGCAGCTCGCTATTTT
GACTGTTACGAGGGTGGCTGTATTACATCTAGAGAGGTTGTTGTTACAAACCTTAATAAA
AGCGCTGGTTGGCCCCTTAATAAGTTTGGTAAAGCTGGTTTATATTATGAGTCTATTAGT
TATGAGGAACAAGATGCTATTTTTTCATTAACAAAGCGTAATATTCTCCCTACTATGACT
CAGTTAAATCTTAAATACGCCATATCTGGTAAGGAACGCGCACGTACAGTGGGTGGCGTC
TCTTTATTAGCTACTATGACTACAAGACAGTTTCATCAGAAATGTCTGAAATCCATAGTA
GCTACCAGAAATGCCACCGTTGTTATCGGCACTACCAAGTTTTATGGCGGGTGGGATAAT
ATGTTAAAGAACCTGATGGCCGATGTTGATGATCCTAAATTGATGGGATGGGACTATCCT
AAGTGTGATAGAGCTATGCCCTCAATGATTCGTATGTTGTCGGCTATGATCTTAGGTTCT
AAGCATGTCACATGTTGTACGGCTAGTGATAAATTTTATAGACTTAGTAATGAGCTTGCT
CAAGTTTTGACCGAGGTTGTTTATTCAAATGGTGGGTTTTATTTTAAACCTGGTGGTACA
ACTTCTGGTGATGCAACTACAGCCTATGCCAATTCTGTCTTTAATATATTTCAGGCTGTA
AGTTCTAACATTAATTGCGTTTTGAGCGTTAACTCGTCAAATTGCAATAATTTTAATGTT
AAGAAGTTACAGAGACAACTTTATGATAATTGCTATAGAAATAGTAATGTTGATGAATCT
TTTGTGGATGACTTTTATGGTTATTTGCAAAAGCATTTTTCTATGATGATTCTTTCTGAT
GATAGTGTTGTGTGCTATAATAAAACTTATGCTGGACTTGGTTACATTGCTGATATTAGT
GCTTTTAAAGCCACTTTGTATTATCAGAATGGTGTGTTTATGAGTACAGCTAAGTGTTGG
ACTGAGGAAGATCTTTCTATAGGACCTCATGAATTTTGCTCACAGCACACTATGCAGATT
GTAGATGAAAATGGTAAGTATTATCTACCATATCCAGATCCTAGCCGTATTATTTCTGCT
GGTGTTTTTGTGGATGACATCACTAAGACTGATGCTGTCATTCTTTTGGAACGCTATGTT
TCTCTGGCTATAGATGCCTACCCATTGTCTAAGCATCCTAAACCTGAGTACAGGAAGGTG
TTTTACGCATTGTTAGACTGGGTCAAACATCTCAACAAGACTCTTAACGAAGGTGTTTTG
GAGTCTTTTTCTGTTACACTTTTAGATGAACATGAGTCTAAGTTTTGGGATGAAAGCTTT
TATGCTAGTATGTATGAGAAGTCTACAGTATTACAAGCTGCTGGTCTTTGTGTAGTATGT
GGTTCTCAAACAGTTCTAAGATGCGGTGATTGTTTACGCAGACCGATGTTGTGCACTAAG
TGCGCCTATGATCATGTGTTTGGCACTGATCATAAGTTCATTTTAGCTATTACACCATAT
GTGTGTAACACATCTGGCTGCAATGTAAATGACGTTACAAAACTGTATCTTGGAGGTTTG
AATTATTACTGTGTAGACCACAAACCACATCTTTCATTCCCACTGTGTTCAGTTGGTAAT
GTCTTTGGTTTGTACAAAAGTTCTGCTTTGGGTTCCATGGACATTGATGTCTTTAACAAA
CTTTCTACCTCTGATTGGTCTGACATTCGCGACTACAAGCTTGCTAATGATGCAAAAGAG
TCACTAAGGTTGTTTGCAGCTGAAACGGTCAAGGCTAAAGAGGAAAGTGTTAAGTCATCA
TACGCTTATGCTACCCTAAAGGAGATTGTAGGTCCTAAGGAACTTTTGCTCTTATGGGAA
AGTGGAAAAGCCAAACCACCGTTAAACCGTAATTCTGTTTTTACATGCTTCCAAATTACA
AAAGACTCCAAGTTTCAAGTTGGTGAGTTTGTGTTTGAGAAAGTAGATTACGGTTCTGAT
ACGGTTACTTACAAATCCACTGCTACTACTAAGTTAGTACCAGGTATGTTGTTTATTTTG
ACTTCTCATAATGTTGCTCCACTTAGAGCGCCAACAATGGCAAACCAGGAGAAATATTCT
ACCATTTACAAGTTGCACCCATCATTTAATGTTAGTGATGCTTATGCAAATCTTGTACCT
TATTACCAACTTATTGGCAAACAGCGTATCACCACAATACAGGGTCCTCCTGGTAGTGGA
AAATCGCATTGTTCTATTGGTATTGGTGTGTATTACCCTGGAGCGAGGATCGTGTTCACC
GCTTGTTCTCACGCTGCTGTTGATTCGCTCTGTGCAAAAGCTGTCACAGCCTATAGTGTT
GATAAGTGTACACGTATTATTCCTGCACGTGCCAGAGTTGAGTGTTATAGTGGTTTTAAA
CCTAACAATAATAGTGCACAATACGTGTTTAGTACTGTTAATGCGTTACCTGAAGTTAAT
GCAGACATTGTTGTCGTGGATGAGGTGTCTATGTGCACTAACTATGACTTGTCTGTGATT
AACCAGCGTATATCATATAAACACATTGTATATGTTGGTGATCCTCAACAGCTTCCAGCT
CCTAGAGTTCTTATCTCTAAAGGTGTTATGGAACCAATTGACTATAATGTTGTGACACAA
CGTATGTGTGCTATAGGACCCGATGTCTTTTTACACAAGTGTTACAGATGTCCTGCTGAA
ATAGTTAACACTGTTTCAGAGCTTGTTTATGAAAACAAGTTTGTACCTGTCAAAGAAGCT
AGTAAGCAGTGCTTCAAAATCTTTGAACGCGGTAGTGTTCAGGTAGACAATGGCTCCAGT
ATAAATAGGCGTCAACTTGATGTTGTTAAGCGATTTATACATAAAAACTCCACATGGAGC
AAGGCTGTGTTTATCTCACCTTACAATAGTCAAAATTATGTAGCTGCCAGGCTTTTAGGC
TTACAAACTCAGACAGTGGATTCTGCTCAAGGTAGTGAATATGACTATGTTATATTCGCA
CAGACATCAGATACTGCTCATGCCTGTAATGCCAATCGTTTTAACGTTGCCATTACTAGA
GCAAAGAAAGGTATTTTCTGTATTATGTCTGACAGAACTTTGTTTGATGCACTTAAGTTC
TTTGAAATCACTATGACAGATTTACAGTCTGAAAGTAGTTGTGGTTTGTTTAAGGATTGT
GCACGTAACCCTATTGATTTACCACCAAGTCATGCCACTACTTATTTGTCATTGTCTGAT
AGATTTAAGACTAGTGGTGACTTGGCTGTTCAAATAGGTAACAACAATGTTTGTACCTAT
GAACATGTGATTTCATATATGGGTTTCAGGTTTGATGTTAGCATGCCTGGTAGTCATAGT
TTGTTCTGTACTAGAGACTTTGCCATGCGTCATGTCAGAGGTTGGTTAGGAATGGATGTG
GAAGGTGCACATGTCACAGGTGACAATGTTGGCACTAATGTACCTCTACAAGTTGGTTTT
TCCAATGGTGTTGATTTTGTAGCTCAACCTGAAGGTTGTGTTCTAACAAACACTGGCAGT
GTTGTAAAACCTGTTCGTGCTCGTGCACCACCTGGAGAACAATTTACTCACATTGTACCT
CTGTTACGCAAGGGACAACCTTGGAGTGTGTTGAGAAAACGTATTGTTCAAATGATAGCA
GATTTTCTTGCTGGCTCATCTGATGTACTGGTGTTTGTACTTTGGGCTGGCGGTTTAGAG
TTGACCACTATGCGTTATTTTGTTAAGATTGGAGCTGTTAAACATTGCCAATGTGGTACT
GTTGCAACATGCTACAATTCTGTTAGTAATGACTATTGTTGCTTTAAACATGCATTGGGC
TGTGACTATGTTTATAATCCATATGTCATAGATATTCAACAATGGGGTTATGTTGGTTCA
CTCTCCACTAATCACCATGCAATTTGTAATGTTCATAGAAATGAGCATGTTGCTTCTGGT
GATGCTATTATGACTAGATGTTTGGCTGTGTATGACTGCTTTGTTAAGAATGTGGATTGG
TCAATTACCTACCCTATGATAGCTAATGAAAATGCCATAAACAAGGGCGGTCGCACTGTG
CAGAGTCATATTATGCGTGCTGCTATTAAATTGTACAACCCTAAAGCAATCCATGACATT
GGTAATCCTAAGGGTATTCGTTGTGCTGTAACTGATGCCAAGTGGTATTGTTATGACAAG
AACCCTATTAATTCTAATGTGAAAACATTGGAGTATGATTACATGACACATGGCCAAATG
GATGGCTTGTGTTTGTTTTGGAATTGTAATGTGGATATGTACCCTGAATTCTCAATTGTT
TGCAGGTTTGACACACGTACACGATCTACATTGAACCTTGAAGGTGTAAATGGTGGGTCA
TTGTATGTCAATAATCATGCATTTCACACTCCTGCTTATGATAAACGTGCTATGGCTAAA
TTGAAACCAGCACCGTTTTTCTACTATGACGACGGTTCATGTGAGGTTGTTCACGATCAA
GTTAACTATGTTCCTTTGAGAGCCACTAATTGCATTACCAAGTGTAATATTGGTGGTGCT
GTATGTTCTAAGCACGCTAATCTCTATAGAGCATATGTTGAGTCATATAACATTTTTACT
CAAGCTGGTTTTAATATTTGGGTTCCTACCACGTTTGATTGTTATAATTTGTGGCAGACA
TTCACAGAGGTCAATTTACAAGGTTTAGAGAACATTGCTTTTAACGTTGTTAATAAAGGT
TCATTTGTTGGTGCTGATGGTGAATTACCAGTAGCCATTAGTGGTGATAAAGTGTTCGTA
CGTGATGGTAACACTGATAATTTAGTCTTTGTTAACAAAACATCACTGCCTACAAACATA
GCATTTGAACTTTTTGCTAAGAGGAAGGTTGGTTTAACACCACCTCTCAGTATTCTCAAA
AACCTTGGTGTTGTCGCCACATATAAGTTTGTCTTGTGGGATTATGAAGCTGAGCGTCCC
TTGACAAGCTTTACTAAGTCTGTTTGTGGTTATACAGACTTTGCAGAGGATGTTTGTACT
TGTTACGATAATAGTATACAAGGTTCATACGAACGTTTTACTCTGTCAACTAATGCTGTG
TTATTCTCTGCTACTGCTGTGAAAACAGGTGGTAAGAGTTTGCCGGCTATTAAATTGAAT
TTTGGAATGCTTAATGGTAATGCAATTGCTACTGTCAAATCAGAAGATGGTAACATAAAA
AATATTAACTGGTTTGTTTACGTACGCAAAGATGGCAAACCTGTTGATCATTATGATGGT
TTTTATACCCAAGGTCGTAATTTACAAGACTTTTTGCCTCGCAGCACAATGGAAGAAGAC
TTTTTGAACATGGATATAGGCGTGTTTATTCAAAAGTATGGTCTAGAGGATTTCAACTTC
GAGCACGTTGTGTATGGTGATGTTTCAAAAACTACTCTAGGCGGTTTACACTTGTTGATT
TCACAAGTACGTCTGAGTAAAATGGGCATCTTAAAGGCAGAGGAGTTTGTGGCAGCATCT
GACATAACACTCAAATGTTGTACTGTGACTTATCTTAATGATCCTAGTTCTAAGACTGTT
TGTACTTACATGGATTTGTTGTTGGATGATTTTGTTTCTGTATTGAAGTCTTTGGATTTG
ACTGTTGTATCCAAGGTTCATGAGGTCATAATTGACAACAAACCATGGAGATGGATGCTA
TGGTGTAAAGATAATGCCGTTGCTACATTCTATCCTCAGTTGCAGAGTGCAGAATGGAAA
TGCGGGTATTCTATGCCTGGTATTTATAAGACACAACGTATGTGCTTAGAACCATGTAAT
TTGTATAATTATGGTGCAGGTTTGAAGTTGCCCAGTGGCATTATGTTCAATGTTGTTAAA
TACACTCAATTGTGTCAATATTTTAACAGTACCACGTTATGTGTTCCTCATAATATGAGA
GTGTTACACTTGGGTGCTGGCTCTGATTATGGTGTTGCACCAGGAACTGCTGTTCTTAAA
AGGTGGTTGCCGCACGACGCAATTGTTGTTGACAACGATGTTGTTGACTATGTGAGTGAC
GCTGATTTTAGTGTTACTGGTGATTGTGCAACCGTTTATTTGGAAGACAAGTTTGACTTG
TTAATCTCTGATATGTACGATGGCAGGACAAAGGCAATTGATGGTGAAAATGTTTCGAAA
GAAGGATTTTTCACTTACATCTATGGTTTCATTTGTGAAAAACTTGCCATCGGAGGTTCG
ATTGCTATTAAAGTAACAGAGTATAGCTGGAATAAGAAATTGTATGAACTTGTACAAAGA
TTTTCTTTTTGGACTATGTTTTGCACTTCTGTTAATACGTCATCATCAGAAGCCTTTGTT
GTCGGAATTAACTATCTTGGTGATTTCGCACAAGGACCTTTTATAGATGGTAACATAATA
CACGCAAATTATGTATTTTGGCGTAACTCCACTGTTATGAGTTTGTCCTACAACTCTGTT
TTAGACCTGAGTAAATTTAATTGCAAACACAAAGCGACTGTTGTTGTGCAATTAAAGGAT
AGTGATATTAATGAAATGGTGCTTAGTCTTGTTAGGAGTGGTAAGTTGCTTGTAAGGGGT
AATGGCAAGTGTTTGAGTTTTAGTAATCATTTAGTCTCAACTAAATAAAATGTTTGTTTT
GCTTGTTGCATATGCCTTGTTGCATATTGCTGGTTGTCAAACTACAAATGGGCTGAACAC
TAGTTACTCTGTTTGCAACGGCTGTGTTGGTTATTCAGAAAATGTATTTGCTGTTGAGAG
TGGTGGTTATATACCCTCCGACTTTGCATTCAATAATTGGTTCCTTCTAACTAATACCTC
ATCTGTTGTAGATGGTGTTGTGAGGAGTTTTCAGCCTTTGTTGCTTAATTGCTTATGGTC
TGTTTCTGGCTTGCGGTTTACTACTGGTTTTGTCTATTTTAATGGTACTGGGAGAGGTGA
TTGTAAAGGTTTTTCCTCAGATGTTTTGTCTGATGTCATACGTTACAACCTCAATTTTGA
AGAAAACCTTAGACGTGGAACCATTTTGTTTAAAACATCTTATGGTGTTGTTGTGTTTTA
TTGTACCAACAACACTTTAGTTTCAGGTGATGCTCACATACCATTTGGTACAGTTTTGGG
CAATTTTTATTGCTTTGTAAATACTACTATTGGCAATGAAACTACGTCTGCTTTTGTGGG
TGCACTACCTAAGACAGTTCGTGAGTTTGTTATTTCACGCACAGGACATTTTTATATTAA
TGGCTATCGCTATTTCACTTTAGGTAATGTAGAAGCCGTTAATTTCAATGTCACTACTGC
AGAAACCACTGATTTTTGCACTGTTGCGTTAGCTTCTTATGCTGACGTTTTGGTTAATGT
GTCACAAACCTCTATTGCTAATATAATTTATTGCAACTCTGTTATTAACAGACTGAGATG
TGACCAGTTGTCCTTTGATGTACCAGATGGTTTTTATTCTACAAGCCCTATTCAATCCGT
TGAGCTACCTGTGTCTATTGTGTCGCTACCTGTTTATCATAAACATACGTTTATTGTGTT
GTACGTTGACTTCAAACCTCAGAGTGGCGGTGGCAAGTGCTTTAACTGTTATCCTGCTGG
TGTTAATATTACACTGGCCAATTTTAATGAAACTAAAGGGCCTTTGTGTGTTGACACATC
ACACTTCACTACCAAATACGTTGCTGTTTATGCCAATGTTGGTAGGTGGAGTGCTAGTAT
TAACACGGGAAATTGCCCTTTTTCTTTTGGCAAAGTTAATAACTTTGTTAAATTTGGCAG
TGTATGTTTTTCGCTAAAGGATATACCCGGTGGTTGCGCAATGCCTATAGTGGCTAATTG
GGCTTATAGTAAGTACTATACTATAGGCTCATTGTATGTTTCTTGGAGTGATGGTGATGG
AATTACTGGCGTCCCACAACCTGTTGAGGGTGTTAGTTCCTTTATGAATGTTACATTGGA
CAAATGTACTAAATATAATATTTATGATGTATCTGGTGTGGGTGTTATTCGCGTTAGCAA
TGACACCTTTCTTAATGGAATTACGTACACATCAACTTCAGGTAACCTTCTGGGTTTTAA
AGATGTTACTAAGGGCACCATCTACTCTATCACTCCTTGTAACCCACCAGATCAGCTTGT
TGTTTATCAGCAAGCTGTTGTTGGTGCTATGTTGTCTGAAAATTTTACTAGTTACGGCTT
TTCTAATGTTGTAGAACTGCCGAAATTTTTCTATGCGTCCAATGGCACTTATAATTGCAC
AGACGCTGTTTTAACTTATTCTAGTTTTGGCGTTTGTGCAGATGGTTCTATAATTGCTGT
TCAACCACGTAATGTTTCATATGATAGTGTTTCAGCTATCGTCACAGCTAATTTGTCTAT
ACCTTCCAATTGGACCACTTCGGTCCAGGTTGAGTATTTACAAATTACAAGTACACCTAT
CGTAGTTGATTGCTCCACTTATGTTTGCAATGGTAATGTGCGCTGTGTTGAATTGCTTAA
GCAGTATACTTCTGCTTGTAAAACCATAGAAGACGCCTTAAGAAATAGCGCCAGGCTGGA
GTCTGCAGATGTTAGTGAGATGCTCACTTTTGACAAGAAAGCGTTTACACTTGCTAATGT
TAGTAGTTTTGGTGACTACAACCTTAGCAGCGTCATACCTAGCTTGCCCACAAGTGGTAG
TAGAGTGGCTGGTCGCAGTGCCATAGAAGACATACTTTTTAGCAAACTTGTTACTTCTGG
ACTTGGCACTGTGGACGCAGACTACAAAAATTGCACTAAGGGTCTTTCCATTGCTGACTT
GGCTTGTGCTCAATATTATAATGGCATTATGGTTTTGCCTGGCGTCGCTGATGCTGAACG
AATGGCCATGTATACAGGTTCTTTAATTGGTGGAATTGCTTTAGGAGGTCTAACATCAGC
CGTTTCAATACCATTTTCATTAGCAATTCAGGCACGTTTAAATTATGTTGCATTGCAGAC
TGATGTTTTACAAGAAAATCAGAAAATTCTTGCTGCATCTTTTAACAAAGCAATGACCAA
CATAGTAGATGCCTTTACTGGTGTTAATGATGCTATTACACAAACTTCACAAGCCCTACA
AACAGTTGCTACTGCACTTAACAAGATCCAGGATGTTGTTAATCAACAAGGCAACTTATT
GAACCATTTAACTTCTCAGTTGAGGCAGAATTTTCAAGCTATCTCTAGCTCTATTCAGGC
TATCTATGACAGACTTGACACTATTCAGGCTGATCAACAAGTAGATAGGCTGATTACTGG
TAGATTGGCTGCTTTGAATGTATTCGTTTCTCATACATTGACTAAGTACACTGAAGTTCG
TGCTTCCAGACAGCTTGCACAACAAAAAGTGAATGAGTGTGTCAAATCCCAGTCTAAGCG
TTATGGCTTCTGTGGAAATGGCACTCACATTTTCTCAATTGTTAATGCTGCTCCTGAGGG
GCTTGTTTTTCTCCACACTGTCTTGTTGCCGACACAATATAAGGATGTTGAAGCGTGGTC
TGGGTTGTGCGTTGATGGTACAAACGGTTATGTGTTGCGACAACCTAATCTTGCTCTTTA
CAAAGAAGGCAATTATTATAGAATCACATCTCGCATAATGTTTGAACCACGTATTCCTAC
CATGGCAGATTTTGTTCAAATTGAAAATTGCAATGTCACATTTGTTAACATTTCTCGCTC
TGAGTTGCAAACCATTGTGCCAGAGTATATTGATGTTAATAAGACGCTGCAAGAATTAAG
TTACAAATTGCCAAATTACACTGTTCCAGACCTAGTTGTCGAACAGTACAACCAGACTAT
TTTGAATTTGACCAGTGAAATTAGCACCCTTGAAAATAAATCTGCGGAGCTTAATTACAC
TGTTCAAAAATTGCAAACTCTGATTGACAACATAAATAGCACATTAGTCGACTTAAAGTG
GCTCAACCGGGTTGAGACTTACATCAAGTGGCCGTGGTGGGTGTGGTTGTGCATTTCAGT
CGTGCTCATCTTTGTGGTGAGTATGTTGCTATTATGTTGTTGTTCTACTGGTTGCTGTGG
CTTCTTTAGTTGTTTTGCATCTTCTATTAGAGGTTGTTGTGAATCAACTAAACTTCCTTA
TTACGACGTTGAAAAGATCCACATACAGTAATGGCTCTAGGTTTGTTCACATTGCAACTT
GTGTCTGCTGTTAATCAATCGCTTAGCAATGCGAAAGTTAGTGCTGAAGTTTCACGACAG
GTTATCCAAGACGTGAAAGATGGCACTGTTACCTTCAACTTGCTAGCGTATACACTAATG
AGCCTCTTTGTTGTGTATTTTGCTTTATTTAAAGCAAGATCACACCGTGGCAGAGCTGCT
CTTATAGTGTTTAAAATTCTAATCCTTTTCGTTTATGTGCCATTGCTGTATTGGTCTCAA
GCATATATTGACGCAACTTTGATTGCTGTAATTTTGCTTGGAAGATTTTTCCATACAGCT
TGGCACTGCTGGCTCTACAAGACATGGGATTTCATTGTCTTCAATGTAACCACACTTTGC
TATGCAAGGTAAGTGTTGGTTTCTTGAAAATAAGGCTCTGAAACCATTCGTTTGTTTTTA
CGGAGGGGATCAATTCCTTTACATAGGCGACAGAATTGTTTCTTATCTCTCAACTAACGA
CTTGTACGTTGCTCTTAGAGGACGTATTGATAAAGACCTCAGCCTTTCTAGAAAGGTTGA
GTTATATAACAGTGAATGTGTATACTTGTTTTGTGAACACCCAGCTGTTGGAATAGTCAA
CACAGATTTCAAATTAGAAATCCACTAAGATGTTCCTTAAGCTAGTGGATGATCATGCTT
TGGTTGTTAATGTACTACTCTGGTGTGTGGTGCTTATAGTGATACTACTAGTGTGTATTA
CAATAATTAAACTAACTAAGCTTTGTTTCACTTGCCATATGTTTTGTAATAGAACAGTTT
ATGGCCCCATTAAAAATGTGTACCACATTTACCAATCATATATGCACATAGACCCTTTCC
CTAAACGAGTTATTGATTTCTAAACTAAACGACAATGTCAAATGACAATTGTACGGGTGA
CATTGTCACCCATTTGAAGAAGTGGAATTTTGGTTGGAATGTTATTCTAACCATATTCAT
TGTTATTCTTCAGTTTGGACACTATAAATACTCCAGATTGTTTTATGGTTTGAAGATGCT
TGTACTGTGGCTTCTTTGGCCACTCGTACTTGCTTTGTCAATCTTTGACACCTGGGCTAA
TTGGGATTCTAATTGGGCCTTTGTTGCATTTAGCTTTCTTATGGCCGTATCAACACTCGT
TATGTGGGTGATGTACTTCGCAAACAGTTTCAGACTTTTCCGACGTGCTCGAACTTTTTG
GGCATGGAATCCTGAGGTCAATGCAATCACTGTCACAACCGTGTTGGGACAGACATACTA
TCAACCCATTCAACAAGCTCCAACAGGCATTACTGTGACCTTGCTGAGCGGCGTGCTTTA
CGTTGACGGACATAGATTGGCTTCAGGTGTTCAGGTTCATAACCTACCTGAATACATGAC
AGTTGCCGTGCCGAGCACTACTATAATTTATAGTAGAGTCGGAAGGTCCGTAAATTCACA
AAATAGCACAGGCTGGGTTTTCTACGTACGAGTAAAACACGGTGATTTTTCTGCAGTGAG
CTCTCCCATGAGCAACATGACAGAAAACGAAAGATTGCTTCATTTTTCCTAAACTGAACG
AAAAGATGGCTACAGTCAAATGGGCTGATGCATCTGAACCACAACGTGGTCGTCAGGGTA
GAATACCTTATTCTCTTTATAGCCCTTTGCTTGTTGATAGTGAACAACCTTGGAAGGTGA
TACCTCGTAATTTGGTACCCATCAACAAGAAAGACAAAAATAAGCTTATAGGCTATTGGA
ATGTTCAAAAACGTTTCAGAACTAGAAAGGGCAAACGGGTGGATTTGTCACCCAAGCTGC
ATTTTTATTATCTTGGCACAGGACCCCATAAAGATGCAAAATTTAGAGAGCGTGTTGAAG
GTGTCGTCTGGGTTGCTGTTGATGGTGCTAAAACTGAACCTACAGGTTACGGTGTTAGGC
GCAAGAATTCAGAACCAGAGATACCACACTTCAATCAAAAGCTCCCAAATGGTGTTACTG
TTGTTGAAGAACCTGACTCCCGTGCTCCTTCCCGGTCTCAGTCGAGGTCGCAGAGTCGCG
GTCGTGGTGAATCCAAACCTCAATCTCGGAATCCTTCAAGTGACAGAAACCATAACAGTC
AGGATGACATCATGAAGGCAGTTGCTGCGGCTCTTAAATCTTTAGGTTTTGACAAGCCTC
AGGAAAAAGATAAAAAGTCAGCGAAAACGGGTACTCCTAAGCCTTCTCGTAATCAGAGTC
CTGCTTCTTCTCAAACTTCTGCCAAGAGTCTTGCTCGTTCTCAGAGTTCTGAAACAAAAG
AACAAAAGCATGAAATGCAAAAGCCACGGTGGAAAAGACAGCCTAATGATGATGTGACAT
CTAATGTCACACAATGTTTTGGCCCCAGAGACCTTGACCACAACTTTGGAAGTGCAGGTG
TTGTGGCCAATGGTGTTAAAGCTAAAGGCTATCCACAATTTGCTGAGCTTGTGCCGTCAA
CAGCTGCTATGCTGTTTGATAGTCACATTGTTTCCAAAGAGTCAGGCAACACTGTGGTCT
TGACTTTCACTACTAGAGTGACTGTGCCCAAAGACCATCCACACTTGGGTAAGTTTCTTG
AGGAGTTAAATGCATTCACTAGAGAAATGCAACAACATCCTCTTCTTAACCCTAGTGCAC
TAGAATTCAACCCATCTCAAACTTCACCTGCAACTGCTGAACCAGTGCGTGATGAAGTTT
CTATTGAAACTGACATAATTGATGAAGTAAACTAAACATGCCACTGTGTTGTTTGAAATT
CAGGCTTTAGTTGGAATTTTGCTTTTGTTCTTGCTTTTATTATCTTTCTTTTGCCTGTTT
TTAGAGAGATTTGGCGCCTTGGTGCCGTAGATGAATACATTGCTTTTCTCTGATCTATGT
ATGATGGTACGATCAGAGCTGCTTTTAATTAACATGATCCCTTGCTTTGGCTTGACAAGG
ATCTAGTCTTATACACAATGGTAAGCCAGTGGTAGTAAAGGTATAAGAAATTTGCTACTA
TGTTACTGAACCTAGGTGAACGCTAGTATAACTCATTACAAATGTGCTGGAGTAATCAAA
GATCGCATTGACGAGCCAACAATGGAAGAGCCAGTCATTTGTCTTGAGACCTATCTAGTT
AGTAACTGCTAATGGAACGGTTTCGATATGGATACACAAAAAAAAAAAAAAAAAAAAAAA
AAAAAAAAAAAAAAAAA
<110> KRIBB <120> Human alphacoronavirus universal primer sets for whole genome amplification method and diagnosis kit <130> M18-5701 <160> 88 <170> KoPatentIn 3.0 <210> 1 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> 43 Forward primer <400> 1 tagactttgt gtctactyyt ctc 23 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 5385 Reverse primer <400> 2 gcattaccca acaattattt 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 4358 Forward primer <220> <221> modified_base <222> (18) <223> i <400> 3 tttaatgttg twggkccncg 20 <210> 4 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> 9077 Reverse primer <400> 4 gcataacaag ccacgta 17 <210> 5 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> 8201 Forward primer <400> 5 ggtggtraya atgtttattg yta 23 <210> 6 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> 11113 Reverse primer <400> 6 agccaaaatc acttgtta 18 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 9077 Forward primer <400> 7 taycgytgyg cttgttatgc 20 <210> 8 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> 13477 Reverse primer <400> 8 ggacaccatc ataaaac 17 <210> 9 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> 12255 Forward primer <400> 9 ctggtarygg tcargctat 19 <210> 10 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> 15343 Reverse primer <400> 10 cattagtcac aacatgg 17 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 13477 Forward primer <400> 11 gtktttatwg atggtgtwcc 20 <210> 12 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> 17266 Reverse primer <400> 12 aacaaacttg actccagg 18 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 15343 Forward primer <400> 13 cckatgttgt gyactaartg 20 <210> 14 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> 20047 Reverse primer <400> 14 ctccatctac atatcga 17 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 17266 Forward primer <400> 15 cctggyagtc ayagtttgtt 20 <210> 16 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> 22457 Reverse primer <400> 16 taaggctctt catgttttac a 21 <210> 17 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> 19333 Forward primer <400> 17 tatggtgatg tktcwaaaac tac 23 <210> 18 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> 24066 Reverse primer <400> 18 tctaataagg aagtttagtt ga 22 <210> 19 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> 22457 Forward primer <400> 19 tgtaaaacya twgaagaygc ctta 24 <210> 20 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> polyT Reverse primer <400> 20 tttttttttt tttgtgtatc catatc 26 <210> 21 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Alpha13F primer <400> 21 ttwtctatct ayagatagar aa 22 <210> 22 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Alpha41F primer <400> 22 tttagacttt gtgtctacty ytctc 25 <210> 23 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Alpha43F primer <400> 23 tagactttgt gtctactyyt ctc 23 <210> 24 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Alpha62F primer <400> 24 tctcaactaa acgaaatttt t 21 <210> 25 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Alpha252F primer <400> 25 aagcaamgtt ytgtckttgt gg 22 <210> 26 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Alpha950F primer <220> <221> modified_base <222> (6) <223> i <220> <221> modified_base <222> (9) <223> i <220> <221> modified_base <222> (18) <223> i <400> 26 ggttcnccnk ttatkacnaa tggt 24 <210> 27 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Alpha1421F primer <400> 27 ggtgtyatwg ayattagt 18 <210> 28 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Alpha2981F primer <400> 28 ggtggyaarr twtcwttytc tga 23 <210> 29 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Alpha3940F primer <220> <221> modified_base <222> (4) <223> i <400> 29 trtngatggt ttyggtgt 18 <210> 30 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Alpha4358F primer <220> <221> modified_base <222> (18) <223> i <400> 30 tttaatgttg twggkccncg 20 <210> 31 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Alpha4904F primer <400> 31 tayatgtgyg ttktrccatc 20 <210> 32 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Alpha5385F primer <400> 32 ayaataattg ttgggtkaat gc 22 <210> 33 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Alpha5546F primer <400> 33 aagggtgatg ctgarga 17 <210> 34 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Alpha6842F primer <400> 34 tatgttaatg cwaatggtgg t 21 <210> 35 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Alpha7517F primer <400> 35 gcktgytgta tgcgygc 17 <210> 36 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Alpha8201F primer <400> 36 ggtggtraya atgtttattg yta 23 <210> 37 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Alpha8432F primer <400> 37 tggtwtgtta aygatggamg tgt 23 <210> 38 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Alpha8933F primer <400> 38 garagtgctg cwgcwgg 17 <210> 39 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Alpha8961F primer <400> 39 twgayatkcg ttcttatga 19 <210> 40 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Alpha9077F primer <400> 40 taycgytgyg cttgttatgc 20 <210> 41 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Alpha9203F primer <400> 41 atggcrcarc crtctgg 17 <210> 42 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Alpha10476F primer <400> 42 tggcgytttk ctaraga 17 <210> 43 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Alpha10814F primer <400> 43 aartayatgr twgctaatgg 20 <210> 44 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Alpha11113F primer <400> 44 taarcayagt gattttggwc t 21 <210> 45 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Alpha11189F primer <220> <221> modified_base <222> (3) <223> i <400> 45 tcntcwtttg ttrgtatgcc 20 <210> 46 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Alpha11360F primer <400> 46 agratggctg aacaagctgc 20 <210> 47 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Alpha11507F primer <400> 47 gcacgtratg gtgttgt 17 <210> 48 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Alpha11624F primer <220> <221> modified_base <222> (9) <223> i <400> 48 taygckggng ttgtttggac 20 <210> 49 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Alpha12179F primer <400> 49 gctgttarac awggygcaaa acc 23 <210> 50 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Alpha12255F primer <400> 50 ctggtarygg tcargctat 19 <210> 51 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Alpha12532F primer <400> 51 tctagtgcmg ctcgactaga 20 <210> 52 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Alpha12717F primer <400> 52 ggamcacgag cartccatgt a 21 <210> 53 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Alpha13477F primer <400> 53 gtktttatwg atggtgtwcc 20 <210> 54 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Alpha13816F primer <400> 54 ggtgatgctg cwrttaaaga ttttga 26 <210> 55 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Alpha13868F primer <400> 55 cttaataara gygctggktg gcc 23 <210> 56 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Alpha14036F primer <220> <221> modified_base <222> (6) <223> i <400> 56 caraanggtg atgctgc 17 <210> 57 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Alpha14220F primer <400> 57 cttaataara gygctgkttg gcc 23 <210> 58 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Alpha14742F primer <400> 58 ggtacractt cwggtgaygc 20 <210> 59 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Alpha15343F primer <400> 59 cckatgttgt gyactaartg 20 <210> 60 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Alpha15532F primer <220> <221> modified_base <222> (12) <223> i <400> 60 gctggwaatr tntttggttt 20 <210> 61 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Alpha16114F primer <220> <221> modified_base <222> (15) <223> i <400> 61 acaatacagg gtccncctgg tagtgg 26 <210> 62 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Alpha16561F primer <400> 62 cgwatgtgtg cwataggmcc 20 <210> 63 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Alpha17266F primer <400> 63 cctggyagtc ayagtttgtt 20 <210> 64 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Alpha17320F primer <400> 64 ggttggttrg gwatggatgt 20 <210> 65 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Alpha17989F primer <220> <221> modified_base <222> (18) <223> i <400> 65 tttaaacayg cwttgggntg tga 23 <210> 66 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Alpha18574F primer <400> 66 tttgawkktt ataatttgtg gca 23 <210> 67 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Alpha18874F primer <400> 67 ttrtgggatt atgaagcwga 20 <210> 68 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Alpha19333F primer <400> 68 tatggtgatg tktcwaaaac tac 23 <210> 69 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Alpha19735F primer <400> 69 tgtaatttrt ataattaygg tgc 23 <210> 70 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Alpha20047F primer <400> 70 tcwgatatgt aygatggyag 20 <210> 71 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Alpha20829F01 primer <400> 71 tgyttatggt ctgtttctgg 20 <210> 72 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Alpha20829F02 primer <400> 72 tgyttatggc ctgtacctgg 20 <210> 73 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Alpha20829F03 primer <220> <221> modified_base <222> (7) <223> i <400> 73 tgyytcnggc ctgtwcctgg 20 <210> 74 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Alpha20846F01 primer <400> 74 ggttttgtyt attttaaygg tactgg 26 <210> 75 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Alpha20846F02 primer <400> 75 ggttttgtyt attttaaygc cactgg 26 <210> 76 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Alpha22436F primer <400> 76 ctyaagcagt ayacttctgc ttg 23 <210> 77 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Alpha22457F primer <400> 77 tgtaaaacya twgaagaygc ctta 24 <210> 78 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Alpha22789F primer <400> 78 tgatgcwgar cgwatggc 18 <210> 79 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Alpha23909F primer <220> <221> modified_base <222> (6) <223> i <400> 79 tggccntggt gggtktgg 18 <210> 80 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Alpha24066F primer <400> 80 tcaactaaac ttccttatta yga 23 <210> 81 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Alpha25053F primer <220> <221> modified_base <222> (9) <223> i <400> 81 gttggaatnt watyctaac 19 <210> 82 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Alpha25287F01 primer <400> 82 gtttcagact tttccgwcgt g 21 <210> 83 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Alpha25287F02 primer <400> 83 gtttcagact ttggcgccgt g 21 <210> 84 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Alpha25679F primer <220> <221> modified_base <222> (12) <223> i <400> 84 ttgcttcatt tnwtctaaac traac 25 <210> 85 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Alpha25980F primer <400> 85 ggtgtygtyt gggttgct 18 <210> 86 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Alpha27065F primer <400> 86 tagtcttaya cacaatggta rgcc 24 <210> 87 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Alpha27168F primer <400> 87 tggagtamty aaagatcgc 19 <210> 88 <211> 27317 <212> DNA <213> Human alphacoronavirus 229E|NC_002645:1 <400> 88 acttaagtac cttatctatc tacagataga aaagttgctt tttagacttt gtgtctactt 60 ttctcaacta aacgaaattt ttgctatggc cggcatcttt gatgctggag tcgtagtgta 120 attgaaattt catttgggtt gcaacagttt ggaagcaagt gctgtgtgtc ctagtctaag 180 ggtttcgtgt tccgtcacga gattccattc tacaaacgcc ttactcgagg ttccgtctcg 240 tgtttgtgtg gaagcaaagt tctgtctttg tggaaaccag taactgttcc taatggcctg 300 caactgtgtg acacttgccg tagcaagtga ttctgaaatt tctgcaaatg gctgttctac 360 tattgcgcaa gccgtccgcc gttatagcga ggccgctagc aatggtttta gggcatgccg 420 atttgtttca ttagatttgc aggattgcat cgttggcatt gcagacgata catatgttat 480 gggtctgcat ggcaatcaga cgttgttttg caacataatg aaattttctg accgtccttt 540 tatgcttcat gggtggttgg ttttttccaa ttcaaattac cttttggagg aatttgatgt 600 tgtcttcggt aagagaggtg gtggtaatgt gacatacact gaccagtatc tctgtggcgc 660 cgatggcaaa cctgttatga gtgaagattt atggcagttt gttgaccatt tcggtgagaa 720 cgaagaaatt atcatcaatg gtcatactta cgtttgtgct tggcttacta agcgtaagcc 780 cttagattac aaacgtcaga acaaccttgc cattgaagag attgaatatg tgcatggtga 840 tgctttgcat acactacgca atggttctgt tcttgaaatg gctaaggaag tgaagacatc 900 tagtaaagtt gtgttaagcg atgctcttga caaactttac aaagtctttg gttctcctgt 960 tatgacaaat ggttccaaca tcctagaggc ctttactaaa cctgtgttta ttagtgcatt 1020 agttcaatgt acttgtggta ccaagtcttg gtctgttggt gattggaccg gttttaaatc 1080 ctcttgttgc aacgtgatca gtaataaact gtgtgttgtt cccggtaatg ttaaacctgg 1140 tgatgctgtg attaccactc agcaagctgg tgctggtatt aagtattttt gtggcatgac 1200 tcttaagttt gttgcaaata ttgaaggtgt ctctgtttgg agagtgattg ctcttcagag 1260 tgtggattgc tttgttgctt cttccacttt tgtagaagag gaacatgtta atagaatgga 1320 tacattctgc ttcaatgtac gcaatagtgt tactgatgag tgtcgtctgg ccatgttggg 1380 tgctgaaatg actagtaatg tcagaagaca agttgcttca ggtgtcatag acattagtac 1440 cggttggttt gatgtttatg atgacatctt tgctgaaagc aaaccatggt ttgttcgcaa 1500 ggctgaagac atttttggcc cttgttggtc cgctcttgtt tctgcactta aacaacttaa 1560 agtcactaca ggtgaacttg tgagatttgt taagtctatt tgcaattcag ctgttgctgt 1620 cgtgggtggt actatacaaa ttctcgctag tgtgcctgag aagtttttga atgcgtttga 1680 cgtgtttgtc acagctattc aaactgtctt tgactgtgct gttgaaactt gtactattgc 1740 cggtaaagca tttgacaagg tttttgacta tgtcttgctt gataatgcgc ttgtaaaact 1800 tgtcaccaca aagcttaagg gtgttcgtga acgtggcctt aataaagtta agtatgcaac 1860 agttgttgtt ggttccactg aagaagttaa atcttcacgt gttgaacgta gcactgctgt 1920 acttacaatc gccaacaatt attccaaact ttttgatgaa gggtatactg ttgtaattgg 1980 cgatgtggcg tactttgtta gtgacggcta cttccgtctt atggccagtc caaatagtgt 2040 gttgactact gcagtctata aaccattgtt tgcttttaat gtgaatgtta tgggtactag 2100 acctgaaaaa tttccaacca ctgtgacttg tgaaaattta gagtctgttg ttttgtttgt 2160 taatgacaaa attactgaat tccaattgga ttacagtatt gatgtcattg ataatgaaat 2220 aattgtcaaa cctaatatca gcctatgtgt tccactttat gtgagagact atgttgacaa 2280 atgggatgat ttttgcagac aatatagtaa cgagtcttgg tttgaggatg attacagggc 2340 ttttatcagt gttttggaca tcactgatgc tgctgtgaaa gctgcagagt ctaaagcttt 2400 cgttgatact attgttccac cttgcccatc tattttgaaa gttatagatg gaggcaaaat 2460 atggaatggt gttattaaaa atgttaactc tgttagagac tggcttaagt ctttgaagtt 2520 aaatctcaca caacagggtt tgcttggaac atgtgcaaag cgttttaaac gttggcttgg 2580 cattttgcta gaggcctata atgcgttttt agacactgtg gtttctactg ttaaaattgg 2640 tggcttgacc tttaaaacat atgcttttga taaaccttac attgtgatac gtgatatcgt 2700 gtgtaaggtt gaaaataaaa cagaagcaga atggattgag ctttttccac ataatgacag 2760 gattaagtct tttagtactt tcgagagtgc ttacatgcca attgcagacc ctacacattt 2820 tgacattgaa gaagttgaac ttttagatgc agagtttgta gaaccaggct gtggtggtat 2880 tttggcagta atagatgagc acgtctttta taagaaggat ggtgtttatt atccatcaaa 2940 tggtactaac attctacctg ttgcatttac aaaagccgct ggtggtaaag tttcattttc 3000 tgatgacgtt gaagtaaaag acattgaacc tgtttacaga gtcaagcttt gctttgagtt 3060 tgaagatgaa aaacttgtag atgtttgtga aaaggcaatt ggcaagaaaa ttaaacatga 3120 aggtgactgg gatagctttt gtaagactat tcaatcagca ctttctgttg tttcttgcta 3180 tgtaaatcta cctacttatt acatttatga tgaagaaggc ggtaatgact tgagtttgcc 3240 cgttatgatt tctgaatggc ctctttctgt tcaacaagct caacaagaag ctactttacc 3300 tgatattgct gaggatgttg ttgaccaagt tgaagaagtc aatagcattt ttgacattga 3360 gacagtggat gttaaacatg atgtgagtcc ttttgaaatg ccatttgaag agttaaatgg 3420 tttaaagata ctcaaacaat tggataacaa ctgctgggtt aactcagtta tgttacaaat 3480 acaattaact ggtatacttg atggtgacta tgctatgcag ttttttaaaa tgggccgagt 3540 tgccaagatg attgaacgct gctacactgc tgagcaatgt atacgtggtg ctatgggtga 3600 tgttggtttg tgtatgtata gactgcttaa agacttacac actggtttta tggttatgga 3660 ttataaatgt agttgtacca gtggtaggct tgaagaatcg ggagctgttt tgttttgtac 3720 gcccactaag aaggcgtttc cttatggtac ttgtctaaat tgtaacgcac ctcgcatgtg 3780 tacaattagg cagttacaag gtaccataat atttgtgcaa caaaaaccag aacctgttaa 3840 tcctgtttct tttgttgtta aaccagtctg ctcatcaatt tttcgtggtg ctgtgtcttg 3900 tggtcattac cagactaaca tctattcaca aaatttgtgt gtggatggtt ttggtgttaa 3960 caagattcag ccctggacaa atgatgcact taatactatt tgtattaagg atgcagatta 4020 taatgcaaaa gttgaaatat ctgttacacc aattaaaaat acagttgata caacacctaa 4080 ggaagaattt gttgttaaag agaagttgaa cgccttcctc gttcatgaca atgtagcttt 4140 ctaccaaggt gatgttgata ctgttgttaa tggtgttgac tttgacttta ttgtaaatgc 4200 tgctaatgag aaccttgctc atggtggagg acttgccaaa gctttagatg tgtacactaa 4260 aggtaaactt caacgtttat ctaaagaaca cattggatta gcgggtaaag taaaagttgg 4320 tacaggagtt atggttgagt gtgatagcct tagaattttt aatgttgttg gtccacgcaa 4380 gggtaaacat gaacgtgatt tactcataaa agcttacaac actattaata atgaacaagg 4440 cacaccttta acaccaattt tgagctgtgg tatttttggt atcaaactcg aaacttcatt 4500 agaagttttg cttgatgttt gtaatacaaa agaagttaaa gtttttgttt atacagacac 4560 agaggtttgt aaggttaagg attttgtgtc tggtttagtg aatgttcaaa aagttgagca 4620 acctaaaata gaaccaaaac cagtgtccgt aattaaagtt gcacccaagc cttacagggt 4680 agatggtaaa tttagttact ttacagaaga cttgttgtgt gtcgctgatg acaaacccat 4740 tgttttgttt actgactcta tgcttacttt ggatgaccgt ggtttagctc tagacaatgc 4800 acttagtggt gtgcttagtg ctgctattaa ggattgtgtt gacataaata aagctatacc 4860 ttctggtaat cttattaagt ttgatatagg ttctgttgtt gtctacatgt gtgttgtgcc 4920 atccgaaaag gacaaacatt tagataataa tgttcaacga tgcacacgta agttgaatag 4980 acttatgtgt gatatagttt gtactatacc agctgactac atcttgccat tggtgttgtc 5040 tagtttgact tgtaatgttt cttttgtagg tgaacttaaa gctgctgaag ctaaagttat 5100 aactataaag gtgacagagg atggtgttaa tgttcatgat gtgaccgtga caacagacaa 5160 gtcatttgaa caacaagttg gtgttattgc tgataaggac aaagatcttt ctggtgcagt 5220 accaagtgat cttaacacat ctgaattgct tactaaagca atagatgttg attgggtcga 5280 attttatggc tttaaagatg ctgttacttt tgcaacagtt gatcatagtg cttttgccta 5340 tgaaagtgct gttgttaatg gtattagagt gttaaaaact agtgataata attgttgggt 5400 gaatgctgtt tgtattgcac tacagtattc gaaaccccat tttatttcac aaggtcttga 5460 tgctgcgtgg aataaatttg ttttaggcga tgttgaaatt tttgttgcat ttgtttacta 5520 tgttgcaaga ctaatgaaag gtgacaaggg tgatgctgaa gacactttga ctaagttgtc 5580 taagtatctt gctaatgaag ctcaagttca attagaacat tatagttctt gtgttgaatg 5640 tgatgctaaa tttaaaaact ctgttgcatc tatcaattct gctatagttt gtgctagtgt 5700 caaacgtgat ggtgtgcaag ttggttattg tgtccatggt attaagtact attcacgtgt 5760 tagaagtgtt agaggtagag ctattatagt cagtgtcgaa cagcttgaac cgtgtgctca 5820 gtctagactt ttgagtggtg ttgcttatac tgctttttct ggacctgttg acaaaggtca 5880 ttatactgtt tatgatactg caaagaaatc aatgtatgat ggtgatcgtt ttgttaaaca 5940 tgatctttct ctgctgtctg tcacatcagt tgttatggtt ggtggttatg ttgcacctgt 6000 taatacagtg aaacctaaac cagtcattaa tcaacttgat gaaaaggcac agaagttctt 6060 tgattttggt gattttttga ttcataattt tgttattttt ttcacatggt tattgagtat 6120 gtttactttg tgtaaaactg cagtaactac aggtgatgtt aaaataatgg ccaaagcacc 6180 acaaaggacg ggtgttgttt taaaacgtag tcttaaatat aacttaaaag cgtcagcagc 6240 tgttcttaaa tctaagtggt ggctgcttgc taagtttacg aaactactgt tactcatata 6300 tacattgtac tcagtagttt tgctttgtgt acgttttgga ccgtttaatt tttgtagtga 6360 gactgttaat ggttatgcta agtcaaactt tgtcaaggat gattactgtg atggttcatt 6420 gggctgcaag atgtgtcttt ttggttacca agagttaagt caatttagcc atttggatgt 6480 tgtgtggaag catataacag accctttgtt tagtaatatg caacctttca ttgtcatggt 6540 tttgctgctt atatttggtg acaattattt gagatgcttc ttgctgtatt ttgttgctca 6600 gatgataagc acagttggtg tttttctagg ttacaaggaa acaaattggt tcttgcactt 6660 tattccattt gatgttattt gtgatgaact gcttgtcact gttattgtta ttaaggttat 6720 ttcttttgtc agacatgtgc tttttggttg tgaaaaccca gattgcattg cgtgttctaa 6780 gagtgctaga cttaagagat tccctgttaa cacaattgtc aatggtgtgc aacgttcatt 6840 ttatgttaat gcaaatggtg gtagtaagtt ttgtaagaaa catagatttt tctgtgttga 6900 ttgtgactct tatggttatg gcagcacgtt tataacaccc gaagtttcta gagaacttgg 6960 taacattacc aaaacaaatg tgcaaccaac aggcccagcg tatgtcatga ttgacaaagt 7020 ggagtttgaa aatggttttt acagattgta ttcctgtgaa acattttggc gttacaactt 7080 tgatataact gaaagcaagt attcttgcaa agaggttttt aaaaattgta atgttttgga 7140 tgatttcatc gtgtttaaca ataatgggac caatgtaacg caggttaaaa atgctagtgt 7200 ttacttttca cagttgttgt gtaggcccat taaattagtt gacagtgaac ttttgtccac 7260 tttgtcagtt gattttaatg gtgtcttaca caaggcatac attgatgtac tacgtaatag 7320 ctttggtaaa gatcttaatg ctaatatgtc tttagccgag tgcaagagtg ctttaggcct 7380 gtctattagt gatcatgaat ttactagtgc tatttctaat gcacatcgtt gtgacgtgtt 7440 gttatctgat ttgtcattta acaactttgt cagttcgtat gctaaacctg aggaaaaatt 7500 atcagcttat gacttggcgt gttgtatgcg tgcaggtgct aaggttgtta atgccaatgt 7560 tctgacaaag gaccaaactc ctattgtttg gcatgcaaag gattttaaca gtctttctgc 7620 tgaaggtcgc aagtatattg taaaaactag caaagctaag ggtttgactt tcttgttgac 7680 aattaatgaa aaccaagctg tcacgcaaat acctgcaact agcattgttg ctaagcaagg 7740 tgctggtgat gctggccatt cattaacatg gctgtggcta ctgtgtggtc ttgtgtgttt 7800 gattcaattc tacttgtgct ttttcatgcc ctattttatg tacgatatcg tgagtagttt 7860 tgagggttat gattttaagt atatagaaaa tggtcagttg aagaattttg aagcgccact 7920 taaatgcgtc agaaacgttt ttgaaaactt tgaggactgg cattatgcta agtttggctt 7980 cacaccttta aacaagcaaa gctgtcctat tgtagttgga gtttctgaaa ttgttaatac 8040 tgtcgctggc attccatcta atgtgtatct tgttggtaaa actttaattt ttacactaca 8100 agctgctttt ggtaatgctg gtgtttgtta tgacattttt ggagtcacaa cacctgaaaa 8160 gtgcattttt acttctgctt gtactagatt agaaggtttg ggtggtgaca atgtttattg 8220 ttataacaca gcgcttatgg aaggttcttt gccttacagt tcaatacaag ctaatgcata 8280 ttataaatat gacaatggca attttattaa gttgccagaa gttattgcac aaggctttgg 8340 ttttagaaca gtgcgtacta ttgccaccaa atactgccgc gtaggtgaat gtgttgaatc 8400 caatgcaggt gtgtgttttg gctttgacaa gtggtttgtt aacgatggac gtgttgccaa 8460 tggttacgtt tgtggtactg gtttgtggaa ccttgtattt aacatacttt ccatgttttc 8520 atcttcattc tctgttgctg caatgtcagg tcaaatttta cttaattgtg cattaggtgc 8580 ttttgctatt ttttgttgtt ttcttgtgac aaagtttaga cgcatgtttg gtgacctttc 8640 tgtaggtgtt tgcactgttg ttgtggctgt tttgcttaac aatgtctctt acattgtaac 8700 tcagaattta gtaacaatga ttgcttatgc catattgtat ttctttgcta ctagaagctt 8760 acgctatgca tggatttggt gtgctgcata tttaattgcg tatatttctt ttgctccatg 8820 gtggttgtgt gcttggtact ttcttgctat gttgacaggt ttgttaccta gtttgctgaa 8880 gcttaaagtt tcgacaaatc ttttcgaagg tgacaaattt gtaggtacat ttgaaagtgc 8940 tgctgcagga acatttgtca ttgacatgcg ttcttatgag aaacttgcta atagcatctc 9000 tccagaaaag ttgaaaagtt atgctgctag ctataataga tataagtact atagtggtaa 9060 tgcaaatgaa gctgattact gttgcgcttg ttatgcctat ttagcaaaag caatgttgga 9120 cttttcgcgt gatcataatg acatcttgta cacacctccg actgtcagtt atggttctac 9180 attacaggct ggtttgcgca aaatggcaca accatctggc tttgtggaga aatgtgttgt 9240 ccgtgtctgc tatggaaaca ctgtgttgaa tgggttgtgg cttggtgata ttgtttattg 9300 cccacgtcat gttatcgcat ctaacacaac ttctgctata gattatgatc acgaatatag 9360 tattatgcgg ttgcataatt tttctataat atctggtaca gcatttcttg gtgttgtagg 9420 tgctactatg catggagtaa ctcttaaaat taaggtttca cagactaaca tgcacacacc 9480 tagacattct tttagaacac taaaatctgg tgaaggtttt aacatcttag catgctatga 9540 tggttgtgct caaggtgttt ttggtgtgaa catgagaact aattggacta tccgtggttc 9600 atttattaat ggtgcgtgtg gttcccctgg ctacaatctt aaaaatggcg aggtggaatt 9660 tgtttatatg catcaaattg aactcggaag tggtagccat gtaggttcta gctttgatgg 9720 tgttatgtat ggtggttttg aagaccaacc taatcttcaa gttgaatctg caaaccagat 9780 gttaacagtt aatgtggttg catttcttta tgctgctata ttgaatggtt gcacatggtg 9840 gcttaaaggt gaaaaattgt ttgtggagca ttataatgag tgggcacagg ctaatggttt 9900 cacagctatg aatggtgaag acgctttttc cattcttgct gctaaaactg gtgtctgtgt 9960 ggaaagatta cttcatgcta ttcaagtttt gaataatggc tttggtggta aacaaatttt 10020 gggttattct agtctcaatg atgagttcag tattaatgaa gttgtcaaac aaatgtttgg 10080 tgttaacctg caaagtggta aaaccactag tatgtttaaa tccataagct tatttgctgg 10140 cttctttgtc atgttctggg ctgaattatt tgtttatacc accactattt gggttaaccc 10200 tggttttctt actccgttta tgattttgct tgttgctttg tcactctgtc ttacatttgt 10260 tgttaaacat aaggttttgt ttttgcaagt gtttttgttg ccttcaatta ttgtggctgc 10320 tattcaaaac tgtgcttggg actaccatgt tacaaaggtg ttggcagaga agtttgatta 10380 taatgtttct gttatgcaaa tggacatcca gggttttgtt aacattttta tttgtctttt 10440 tgttgcactg ttgcatactt ggcgctttgc taaagagcgt tgtacacatt ggtgcactta 10500 tttgttctca ctcattgctg ttttatacac tgcattgtat agttatgact acgttagttt 10560 gctggttatg ctactttgtg caatttctaa tgaatggtat attggtgcta ttatttttag 10620 aatttgtcgt tttggtgttg catttttacc agtggaatac gtgtcttact ttgatggtgt 10680 taaaactgtg ctgttgtttt acatgttgtt aggctttgtt agctgtatgt actatggttt 10740 gttgtactgg attaacaggt tctgtaagtg cacattaggt gtttatgatt tctgtgttag 10800 tccagccgaa tttaagtata tggttgctaa tggtttgaat gcaccaaatg gcccttttga 10860 tgcgctcttt ctgtctttta aactaatggg tattggcggt cctagaacca ttaaagtttc 10920 tactgtacag tctaaattga ctgatcttaa gtgcacaaac gtcgttctaa tgggcatttt 10980 gtctaacatg aacatagctt ctaattcaaa ggagtgggca tattgtgttg aaatgcacaa 11040 taaaataaac ttgtgtgacg accctgaaac tgctcaagag ttattgctgg cgttgttggc 11100 ctttttcttg tctaagcata gtgattttgg tcttggtgat cttgtcgatt cttattttga 11160 gaacgactcc attttgcaaa gtgttgcatc ttcttttgtt ggtatgccat cttttgttgc 11220 atatgaaaca gcaagacaag agtatgaaaa tgctgttgca aatggttcct caccacaaat 11280 aatcaaacaa ttgaagaagg ctatgaatgt tgcaaaagct gagtttgaca gggaatcatc 11340 tgttcaaaag aaaattaaca gaatggctga acaagctgct gcagctatgt acaaagaagc 11400 acgtgctgtt aatagaaaat caaaagttgt tagtgccatg catagtttac tctttggcat 11460 gctccgacgt ttggacatgt ctagtgttga cactatcctt aatatggcac gtaatggtgt 11520 tgtccctctt tccgttatcc ctgctacttc tgcagccagg ctcgtcgtcg tagtaccaga 11580 tcatgattca tttgtgaaaa tgatggtaga tggttttgtg cactacgctg gtgttgtttg 11640 gacattacag gaagttaagg ataatgatgg taagaatgtg catcttaaag atgttacaaa 11700 ggaaaaccag gaaatacttg tttggcctct gattttgact tgtgaacgtg tcgttaaatt 11760 gcagaacaat gaaataatgc cgggcaagat gaaggtcaag gccaccaaag gtgaaggtga 11820 tggaggcatt actagtgaag gtaatgctct atacaacaat gaaggtggac gtgcattcat 11880 gtatgcatat gtgactacga agcctggcat gaagtatgtt aaatgggaac atgactctgg 11940 tgtggttaca gttgaattgg aaccaccttg cagatttgtt atagacacac ctactggacc 12000 ccaaattaag tatctttatt ttgttaagaa tcttaacaat ttaaggagag gtgctgtttt 12060 gggttacatt ggtgccactg tgagattgca agctggcaaa cagactgagt ttgtttcaaa 12120 ctcccattta ttaacacatt gttcttttgc tgttgaccca gctgcagcct atcttgatgc 12180 tgttaaacaa ggcgcaaaac ctgttggcaa ttgtgtaaag atgttgacta atggttctgg 12240 tagcggtcag gctattactt gtaccattga ttccaacact acgcaggaca catatggtgg 12300 cgcgtctgtt tgtatttatt gcagagcaca tgttgcacat ccaaccatgg acggtttttg 12360 tcagtacaaa ggcaagtggg tacaagtgcc tataggtaca aatgacccta taagattttg 12420 tcttgaaaat actgtttgta aagtttgtgg ttgttggctt aatcatggct gtacatgtga 12480 ccggactgct atccaaagtt ttgataacag ttatttaaac gagtccgggg ctctagtgcc 12540 gctcgactag agccctgtaa tggtacagac atagattact gtgtccgtgc atttgacgtt 12600 tacaataaag atgcgtcttt tatcgggaaa aatctgaagt ccaattgtgt gcgcttcaag 12660 aatgtagata aggatgacgc gttctatatt gttaaacgtt gcattaagtc agttatggac 12720 cacgagcagt ccatgtataa cttacttaaa ggctgtaatg ctgttgctaa gcatgatttc 12780 tttacttggc atgagggcag aaccatttat ggtaatgtta gtagacagga tcttactaaa 12840 tacaccatga tggatttgtg cttcgctctg cgtaactttg atgaaaaaga ctgtgaagtt 12900 tttaaggaga tattggttct tactggttgt tgtagtactg attactttga aatgaagaat 12960 tggtttgacc ccatagaaaa tgaggacata caccgtgtgt atgctgcttt aggtaaggta 13020 gttgcaaatg caatgcttaa gtgtgttgct ttttgcgacg aaatggtgct caaaggagtt 13080 gttggtgttt tgaccttaga caaccaagat cttaatggga atttctatga cttcggtgac 13140 tttgtattgt gtcctcctgg aatgggaata ccctactgca cgtcatacta ttcttatatg 13200 atgcctgtta tgggtatgac taattgttta gctagtgagt gctttatgaa aagtgacatc 13260 tttggtcaag acttcaaaac ttttgatttg ttgaaatatg atttcacaga acataaggag 13320 gttttgttta acaagtactt taagtattgg ggacaggatt atcatcctga ttgtgttgat 13380 tgccatgacg agatgtgtat tttgcattgt tcaaatttta acacactctt cgcaaccaca 13440 attccaaaca cggcttttgg acctctatgc agaaaagtgt ttattgatgg tgtacccgta 13500 gttgctactg ctggttacca ctttaaacaa ttaggacttg tgtggaacaa agatgttaac 13560 actcattcta ccagacttac tattactgaa ctcttacagt ttgtgacaga tccaacgctt 13620 atagttgcgt catcgcctgc cttggtggat aaacgcactg tttgtttttc tgtcgctgct 13680 ttgagtacag gattaacatc ccaaacagta aaacctggcc attttaataa ggagttttat 13740 gacttcttac gttctcaggg gtttttcgat gagggttcag aattaacatt gaagcatttc 13800 ttttttacac aaaagggtga tgctgcaatt aaagattttg attattatcg ttacaacaga 13860 cctactatgc tggatattgg acaagctcgc gtagcatatc aagtggcagc tcgctatttt 13920 gactgttacg agggtggctg tattacatct agagaggttg ttgttacaaa ccttaataaa 13980 agcgctggtt ggccccttaa taagtttggt aaagctggtt tatattatga gtctattagt 14040 tatgaggaac aagatgctat tttttcatta acaaagcgta atattctccc tactatgact 14100 cagttaaatc ttaaatacgc catatctggt aaggaacgcg cacgtacagt gggtggcgtc 14160 tctttattag ctactatgac tacaagacag tttcatcaga aatgtctgaa atccatagta 14220 gctaccagaa atgccaccgt tgttatcggc actaccaagt tttatggcgg gtgggataat 14280 atgttaaaga acctgatggc cgatgttgat gatcctaaat tgatgggatg ggactatcct 14340 aagtgtgata gagctatgcc ctcaatgatt cgtatgttgt cggctatgat cttaggttct 14400 aagcatgtca catgttgtac ggctagtgat aaattttata gacttagtaa tgagcttgct 14460 caagttttga ccgaggttgt ttattcaaat ggtgggtttt attttaaacc tggtggtaca 14520 acttctggtg atgcaactac agcctatgcc aattctgtct ttaatatatt tcaggctgta 14580 agttctaaca ttaattgcgt tttgagcgtt aactcgtcaa attgcaataa ttttaatgtt 14640 aagaagttac agagacaact ttatgataat tgctatagaa atagtaatgt tgatgaatct 14700 tttgtggatg acttttatgg ttatttgcaa aagcattttt ctatgatgat tctttctgat 14760 gatagtgttg tgtgctataa taaaacttat gctggacttg gttacattgc tgatattagt 14820 gcttttaaag ccactttgta ttatcagaat ggtgtgttta tgagtacagc taagtgttgg 14880 actgaggaag atctttctat aggacctcat gaattttgct cacagcacac tatgcagatt 14940 gtagatgaaa atggtaagta ttatctacca tatccagatc ctagccgtat tatttctgct 15000 ggtgtttttg tggatgacat cactaagact gatgctgtca ttcttttgga acgctatgtt 15060 tctctggcta tagatgccta cccattgtct aagcatccta aacctgagta caggaaggtg 15120 ttttacgcat tgttagactg ggtcaaacat ctcaacaaga ctcttaacga aggtgttttg 15180 gagtcttttt ctgttacact tttagatgaa catgagtcta agttttggga tgaaagcttt 15240 tatgctagta tgtatgagaa gtctacagta ttacaagctg ctggtctttg tgtagtatgt 15300 ggttctcaaa cagttctaag atgcggtgat tgtttacgca gaccgatgtt gtgcactaag 15360 tgcgcctatg atcatgtgtt tggcactgat cataagttca ttttagctat tacaccatat 15420 gtgtgtaaca catctggctg caatgtaaat gacgttacaa aactgtatct tggaggtttg 15480 aattattact gtgtagacca caaaccacat ctttcattcc cactgtgttc agttggtaat 15540 gtctttggtt tgtacaaaag ttctgctttg ggttccatgg acattgatgt ctttaacaaa 15600 ctttctacct ctgattggtc tgacattcgc gactacaagc ttgctaatga tgcaaaagag 15660 tcactaaggt tgtttgcagc tgaaacggtc aaggctaaag aggaaagtgt taagtcatca 15720 tacgcttatg ctaccctaaa ggagattgta ggtcctaagg aacttttgct cttatgggaa 15780 agtggaaaag ccaaaccacc gttaaaccgt aattctgttt ttacatgctt ccaaattaca 15840 aaagactcca agtttcaagt tggtgagttt gtgtttgaga aagtagatta cggttctgat 15900 acggttactt acaaatccac tgctactact aagttagtac caggtatgtt gtttattttg 15960 acttctcata atgttgctcc acttagagcg ccaacaatgg caaaccagga gaaatattct 16020 accatttaca agttgcaccc atcatttaat gttagtgatg cttatgcaaa tcttgtacct 16080 tattaccaac ttattggcaa acagcgtatc accacaatac agggtcctcc tggtagtgga 16140 aaatcgcatt gttctattgg tattggtgtg tattaccctg gagcgaggat cgtgttcacc 16200 gcttgttctc acgctgctgt tgattcgctc tgtgcaaaag ctgtcacagc ctatagtgtt 16260 gataagtgta cacgtattat tcctgcacgt gccagagttg agtgttatag tggttttaaa 16320 cctaacaata atagtgcaca atacgtgttt agtactgtta atgcgttacc tgaagttaat 16380 gcagacattg ttgtcgtgga tgaggtgtct atgtgcacta actatgactt gtctgtgatt 16440 aaccagcgta tatcatataa acacattgta tatgttggtg atcctcaaca gcttccagct 16500 cctagagttc ttatctctaa aggtgttatg gaaccaattg actataatgt tgtgacacaa 16560 cgtatgtgtg ctataggacc cgatgtcttt ttacacaagt gttacagatg tcctgctgaa 16620 atagttaaca ctgtttcaga gcttgtttat gaaaacaagt ttgtacctgt caaagaagct 16680 agtaagcagt gcttcaaaat ctttgaacgc ggtagtgttc aggtagacaa tggctccagt 16740 ataaataggc gtcaacttga tgttgttaag cgatttatac ataaaaactc cacatggagc 16800 aaggctgtgt ttatctcacc ttacaatagt caaaattatg tagctgccag gcttttaggc 16860 ttacaaactc agacagtgga ttctgctcaa ggtagtgaat atgactatgt tatattcgca 16920 cagacatcag atactgctca tgcctgtaat gccaatcgtt ttaacgttgc cattactaga 16980 gcaaagaaag gtattttctg tattatgtct gacagaactt tgtttgatgc acttaagttc 17040 tttgaaatca ctatgacaga tttacagtct gaaagtagtt gtggtttgtt taaggattgt 17100 gcacgtaacc ctattgattt accaccaagt catgccacta cttatttgtc attgtctgat 17160 agatttaaga ctagtggtga cttggctgtt caaataggta acaacaatgt ttgtacctat 17220 gaacatgtga tttcatatat gggtttcagg tttgatgtta gcatgcctgg tagtcatagt 17280 ttgttctgta ctagagactt tgccatgcgt catgtcagag gttggttagg aatggatgtg 17340 gaaggtgcac atgtcacagg tgacaatgtt ggcactaatg tacctctaca agttggtttt 17400 tccaatggtg ttgattttgt agctcaacct gaaggttgtg ttctaacaaa cactggcagt 17460 gttgtaaaac ctgttcgtgc tcgtgcacca cctggagaac aatttactca cattgtacct 17520 ctgttacgca agggacaacc ttggagtgtg ttgagaaaac gtattgttca aatgatagca 17580 gattttcttg ctggctcatc tgatgtactg gtgtttgtac tttgggctgg cggtttagag 17640 ttgaccacta tgcgttattt tgttaagatt ggagctgtta aacattgcca atgtggtact 17700 gttgcaacat gctacaattc tgttagtaat gactattgtt gctttaaaca tgcattgggc 17760 tgtgactatg tttataatcc atatgtcata gatattcaac aatggggtta tgttggttca 17820 ctctccacta atcaccatgc aatttgtaat gttcatagaa atgagcatgt tgcttctggt 17880 gatgctatta tgactagatg tttggctgtg tatgactgct ttgttaagaa tgtggattgg 17940 tcaattacct accctatgat agctaatgaa aatgccataa acaagggcgg tcgcactgtg 18000 cagagtcata ttatgcgtgc tgctattaaa ttgtacaacc ctaaagcaat ccatgacatt 18060 ggtaatccta agggtattcg ttgtgctgta actgatgcca agtggtattg ttatgacaag 18120 aaccctatta attctaatgt gaaaacattg gagtatgatt acatgacaca tggccaaatg 18180 gatggcttgt gtttgttttg gaattgtaat gtggatatgt accctgaatt ctcaattgtt 18240 tgcaggtttg acacacgtac acgatctaca ttgaaccttg aaggtgtaaa tggtgggtca 18300 ttgtatgtca ataatcatgc atttcacact cctgcttatg ataaacgtgc tatggctaaa 18360 ttgaaaccag caccgttttt ctactatgac gacggttcat gtgaggttgt tcacgatcaa 18420 gttaactatg ttcctttgag agccactaat tgcattacca agtgtaatat tggtggtgct 18480 gtatgttcta agcacgctaa tctctataga gcatatgttg agtcatataa catttttact 18540 caagctggtt ttaatatttg ggttcctacc acgtttgatt gttataattt gtggcagaca 18600 ttcacagagg tcaatttaca aggtttagag aacattgctt ttaacgttgt taataaaggt 18660 tcatttgttg gtgctgatgg tgaattacca gtagccatta gtggtgataa agtgttcgta 18720 cgtgatggta acactgataa tttagtcttt gttaacaaaa catcactgcc tacaaacata 18780 gcatttgaac tttttgctaa gaggaaggtt ggtttaacac cacctctcag tattctcaaa 18840 aaccttggtg ttgtcgccac atataagttt gtcttgtggg attatgaagc tgagcgtccc 18900 ttgacaagct ttactaagtc tgtttgtggt tatacagact ttgcagagga tgtttgtact 18960 tgttacgata atagtataca aggttcatac gaacgtttta ctctgtcaac taatgctgtg 19020 ttattctctg ctactgctgt gaaaacaggt ggtaagagtt tgccggctat taaattgaat 19080 tttggaatgc ttaatggtaa tgcaattgct actgtcaaat cagaagatgg taacataaaa 19140 aatattaact ggtttgttta cgtacgcaaa gatggcaaac ctgttgatca ttatgatggt 19200 ttttataccc aaggtcgtaa tttacaagac tttttgcctc gcagcacaat ggaagaagac 19260 tttttgaaca tggatatagg cgtgtttatt caaaagtatg gtctagagga tttcaacttc 19320 gagcacgttg tgtatggtga tgtttcaaaa actactctag gcggtttaca cttgttgatt 19380 tcacaagtac gtctgagtaa aatgggcatc ttaaaggcag aggagtttgt ggcagcatct 19440 gacataacac tcaaatgttg tactgtgact tatcttaatg atcctagttc taagactgtt 19500 tgtacttaca tggatttgtt gttggatgat tttgtttctg tattgaagtc tttggatttg 19560 actgttgtat ccaaggttca tgaggtcata attgacaaca aaccatggag atggatgcta 19620 tggtgtaaag ataatgccgt tgctacattc tatcctcagt tgcagagtgc agaatggaaa 19680 tgcgggtatt ctatgcctgg tatttataag acacaacgta tgtgcttaga accatgtaat 19740 ttgtataatt atggtgcagg tttgaagttg cccagtggca ttatgttcaa tgttgttaaa 19800 tacactcaat tgtgtcaata ttttaacagt accacgttat gtgttcctca taatatgaga 19860 gtgttacact tgggtgctgg ctctgattat ggtgttgcac caggaactgc tgttcttaaa 19920 aggtggttgc cgcacgacgc aattgttgtt gacaacgatg ttgttgacta tgtgagtgac 19980 gctgatttta gtgttactgg tgattgtgca accgtttatt tggaagacaa gtttgacttg 20040 ttaatctctg atatgtacga tggcaggaca aaggcaattg atggtgaaaa tgtttcgaaa 20100 gaaggatttt tcacttacat ctatggtttc atttgtgaaa aacttgccat cggaggttcg 20160 attgctatta aagtaacaga gtatagctgg aataagaaat tgtatgaact tgtacaaaga 20220 ttttcttttt ggactatgtt ttgcacttct gttaatacgt catcatcaga agcctttgtt 20280 gtcggaatta actatcttgg tgatttcgca caaggacctt ttatagatgg taacataata 20340 cacgcaaatt atgtattttg gcgtaactcc actgttatga gtttgtccta caactctgtt 20400 ttagacctga gtaaatttaa ttgcaaacac aaagcgactg ttgttgtgca attaaaggat 20460 agtgatatta atgaaatggt gcttagtctt gttaggagtg gtaagttgct tgtaaggggt 20520 aatggcaagt gtttgagttt tagtaatcat ttagtctcaa ctaaataaaa tgtttgtttt 20580 gcttgttgca tatgccttgt tgcatattgc tggttgtcaa actacaaatg ggctgaacac 20640 tagttactct gtttgcaacg gctgtgttgg ttattcagaa aatgtatttg ctgttgagag 20700 tggtggttat ataccctccg actttgcatt caataattgg ttccttctaa ctaatacctc 20760 atctgttgta gatggtgttg tgaggagttt tcagcctttg ttgcttaatt gcttatggtc 20820 tgtttctggc ttgcggttta ctactggttt tgtctatttt aatggtactg ggagaggtga 20880 ttgtaaaggt ttttcctcag atgttttgtc tgatgtcata cgttacaacc tcaattttga 20940 agaaaacctt agacgtggaa ccattttgtt taaaacatct tatggtgttg ttgtgtttta 21000 ttgtaccaac aacactttag tttcaggtga tgctcacata ccatttggta cagttttggg 21060 caatttttat tgctttgtaa atactactat tggcaatgaa actacgtctg cttttgtggg 21120 tgcactacct aagacagttc gtgagtttgt tatttcacgc acaggacatt tttatattaa 21180 tggctatcgc tatttcactt taggtaatgt agaagccgtt aatttcaatg tcactactgc 21240 agaaaccact gatttttgca ctgttgcgtt agcttcttat gctgacgttt tggttaatgt 21300 gtcacaaacc tctattgcta atataattta ttgcaactct gttattaaca gactgagatg 21360 tgaccagttg tcctttgatg taccagatgg tttttattct acaagcccta ttcaatccgt 21420 tgagctacct gtgtctattg tgtcgctacc tgtttatcat aaacatacgt ttattgtgtt 21480 gtacgttgac ttcaaacctc agagtggcgg tggcaagtgc tttaactgtt atcctgctgg 21540 tgttaatatt acactggcca attttaatga aactaaaggg cctttgtgtg ttgacacatc 21600 acacttcact accaaatacg ttgctgttta tgccaatgtt ggtaggtgga gtgctagtat 21660 taacacggga aattgccctt tttcttttgg caaagttaat aactttgtta aatttggcag 21720 tgtatgtttt tcgctaaagg atatacccgg tggttgcgca atgcctatag tggctaattg 21780 ggcttatagt aagtactata ctataggctc attgtatgtt tcttggagtg atggtgatgg 21840 aattactggc gtcccacaac ctgttgaggg tgttagttcc tttatgaatg ttacattgga 21900 caaatgtact aaatataata tttatgatgt atctggtgtg ggtgttattc gcgttagcaa 21960 tgacaccttt cttaatggaa ttacgtacac atcaacttca ggtaaccttc tgggttttaa 22020 agatgttact aagggcacca tctactctat cactccttgt aacccaccag atcagcttgt 22080 tgtttatcag caagctgttg ttggtgctat gttgtctgaa aattttacta gttacggctt 22140 ttctaatgtt gtagaactgc cgaaattttt ctatgcgtcc aatggcactt ataattgcac 22200 agacgctgtt ttaacttatt ctagttttgg cgtttgtgca gatggttcta taattgctgt 22260 tcaaccacgt aatgtttcat atgatagtgt ttcagctatc gtcacagcta atttgtctat 22320 accttccaat tggaccactt cggtccaggt tgagtattta caaattacaa gtacacctat 22380 cgtagttgat tgctccactt atgtttgcaa tggtaatgtg cgctgtgttg aattgcttaa 22440 gcagtatact tctgcttgta aaaccataga agacgcctta agaaatagcg ccaggctgga 22500 gtctgcagat gttagtgaga tgctcacttt tgacaagaaa gcgtttacac ttgctaatgt 22560 tagtagtttt ggtgactaca accttagcag cgtcatacct agcttgccca caagtggtag 22620 tagagtggct ggtcgcagtg ccatagaaga catacttttt agcaaacttg ttacttctgg 22680 acttggcact gtggacgcag actacaaaaa ttgcactaag ggtctttcca ttgctgactt 22740 ggcttgtgct caatattata atggcattat ggttttgcct ggcgtcgctg atgctgaacg 22800 aatggccatg tatacaggtt ctttaattgg tggaattgct ttaggaggtc taacatcagc 22860 cgtttcaata ccattttcat tagcaattca ggcacgttta aattatgttg cattgcagac 22920 tgatgtttta caagaaaatc agaaaattct tgctgcatct tttaacaaag caatgaccaa 22980 catagtagat gcctttactg gtgttaatga tgctattaca caaacttcac aagccctaca 23040 aacagttgct actgcactta acaagatcca ggatgttgtt aatcaacaag gcaacttatt 23100 gaaccattta acttctcagt tgaggcagaa ttttcaagct atctctagct ctattcaggc 23160 tatctatgac agacttgaca ctattcaggc tgatcaacaa gtagataggc tgattactgg 23220 tagattggct gctttgaatg tattcgtttc tcatacattg actaagtaca ctgaagttcg 23280 tgcttccaga cagcttgcac aacaaaaagt gaatgagtgt gtcaaatccc agtctaagcg 23340 ttatggcttc tgtggaaatg gcactcacat tttctcaatt gttaatgctg ctcctgaggg 23400 gcttgttttt ctccacactg tcttgttgcc gacacaatat aaggatgttg aagcgtggtc 23460 tgggttgtgc gttgatggta caaacggtta tgtgttgcga caacctaatc ttgctcttta 23520 caaagaaggc aattattata gaatcacatc tcgcataatg tttgaaccac gtattcctac 23580 catggcagat tttgttcaaa ttgaaaattg caatgtcaca tttgttaaca tttctcgctc 23640 tgagttgcaa accattgtgc cagagtatat tgatgttaat aagacgctgc aagaattaag 23700 ttacaaattg ccaaattaca ctgttccaga cctagttgtc gaacagtaca accagactat 23760 tttgaatttg accagtgaaa ttagcaccct tgaaaataaa tctgcggagc ttaattacac 23820 tgttcaaaaa ttgcaaactc tgattgacaa cataaatagc acattagtcg acttaaagtg 23880 gctcaaccgg gttgagactt acatcaagtg gccgtggtgg gtgtggttgt gcatttcagt 23940 cgtgctcatc tttgtggtga gtatgttgct attatgttgt tgttctactg gttgctgtgg 24000 cttctttagt tgttttgcat cttctattag aggttgttgt gaatcaacta aacttcctta 24060 ttacgacgtt gaaaagatcc acatacagta atggctctag gtttgttcac attgcaactt 24120 gtgtctgctg ttaatcaatc gcttagcaat gcgaaagtta gtgctgaagt ttcacgacag 24180 gttatccaag acgtgaaaga tggcactgtt accttcaact tgctagcgta tacactaatg 24240 agcctctttg ttgtgtattt tgctttattt aaagcaagat cacaccgtgg cagagctgct 24300 cttatagtgt ttaaaattct aatccttttc gtttatgtgc cattgctgta ttggtctcaa 24360 gcatatattg acgcaacttt gattgctgta attttgcttg gaagattttt ccatacagct 24420 tggcactgct ggctctacaa gacatgggat ttcattgtct tcaatgtaac cacactttgc 24480 tatgcaaggt aagtgttggt ttcttgaaaa taaggctctg aaaccattcg tttgttttta 24540 cggaggggat caattccttt acataggcga cagaattgtt tcttatctct caactaacga 24600 cttgtacgtt gctcttagag gacgtattga taaagacctc agcctttcta gaaaggttga 24660 gttatataac agtgaatgtg tatacttgtt ttgtgaacac ccagctgttg gaatagtcaa 24720 cacagatttc aaattagaaa tccactaaga tgttccttaa gctagtggat gatcatgctt 24780 tggttgttaa tgtactactc tggtgtgtgg tgcttatagt gatactacta gtgtgtatta 24840 caataattaa actaactaag ctttgtttca cttgccatat gttttgtaat agaacagttt 24900 atggccccat taaaaatgtg taccacattt accaatcata tatgcacata gaccctttcc 24960 ctaaacgagt tattgatttc taaactaaac gacaatgtca aatgacaatt gtacgggtga 25020 cattgtcacc catttgaaga agtggaattt tggttggaat gttattctaa ccatattcat 25080 tgttattctt cagtttggac actataaata ctccagattg ttttatggtt tgaagatgct 25140 tgtactgtgg cttctttggc cactcgtact tgctttgtca atctttgaca cctgggctaa 25200 ttgggattct aattgggcct ttgttgcatt tagctttctt atggccgtat caacactcgt 25260 tatgtgggtg atgtacttcg caaacagttt cagacttttc cgacgtgctc gaactttttg 25320 ggcatggaat cctgaggtca atgcaatcac tgtcacaacc gtgttgggac agacatacta 25380 tcaacccatt caacaagctc caacaggcat tactgtgacc ttgctgagcg gcgtgcttta 25440 cgttgacgga catagattgg cttcaggtgt tcaggttcat aacctacctg aatacatgac 25500 agttgccgtg ccgagcacta ctataattta tagtagagtc ggaaggtccg taaattcaca 25560 aaatagcaca ggctgggttt tctacgtacg agtaaaacac ggtgattttt ctgcagtgag 25620 ctctcccatg agcaacatga cagaaaacga aagattgctt catttttcct aaactgaacg 25680 aaaagatggc tacagtcaaa tgggctgatg catctgaacc acaacgtggt cgtcagggta 25740 gaatacctta ttctctttat agccctttgc ttgttgatag tgaacaacct tggaaggtga 25800 tacctcgtaa tttggtaccc atcaacaaga aagacaaaaa taagcttata ggctattgga 25860 atgttcaaaa acgtttcaga actagaaagg gcaaacgggt ggatttgtca cccaagctgc 25920 atttttatta tcttggcaca ggaccccata aagatgcaaa atttagagag cgtgttgaag 25980 gtgtcgtctg ggttgctgtt gatggtgcta aaactgaacc tacaggttac ggtgttaggc 26040 gcaagaattc agaaccagag ataccacact tcaatcaaaa gctcccaaat ggtgttactg 26100 ttgttgaaga acctgactcc cgtgctcctt cccggtctca gtcgaggtcg cagagtcgcg 26160 gtcgtggtga atccaaacct caatctcgga atccttcaag tgacagaaac cataacagtc 26220 aggatgacat catgaaggca gttgctgcgg ctcttaaatc tttaggtttt gacaagcctc 26280 aggaaaaaga taaaaagtca gcgaaaacgg gtactcctaa gccttctcgt aatcagagtc 26340 ctgcttcttc tcaaacttct gccaagagtc ttgctcgttc tcagagttct gaaacaaaag 26400 aacaaaagca tgaaatgcaa aagccacggt ggaaaagaca gcctaatgat gatgtgacat 26460 ctaatgtcac acaatgtttt ggccccagag accttgacca caactttgga agtgcaggtg 26520 ttgtggccaa tggtgttaaa gctaaaggct atccacaatt tgctgagctt gtgccgtcaa 26580 cagctgctat gctgtttgat agtcacattg tttccaaaga gtcaggcaac actgtggtct 26640 tgactttcac tactagagtg actgtgccca aagaccatcc acacttgggt aagtttcttg 26700 aggagttaaa tgcattcact agagaaatgc aacaacatcc tcttcttaac cctagtgcac 26760 tagaattcaa cccatctcaa acttcacctg caactgctga accagtgcgt gatgaagttt 26820 ctattgaaac tgacataatt gatgaagtaa actaaacatg ccactgtgtt gtttgaaatt 26880 caggctttag ttggaatttt gcttttgttc ttgcttttat tatctttctt ttgcctgttt 26940 ttagagagat ttggcgcctt ggtgccgtag atgaatacat tgcttttctc tgatctatgt 27000 atgatggtac gatcagagct gcttttaatt aacatgatcc cttgctttgg cttgacaagg 27060 atctagtctt atacacaatg gtaagccagt ggtagtaaag gtataagaaa tttgctacta 27120 tgttactgaa cctaggtgaa cgctagtata actcattaca aatgtgctgg agtaatcaaa 27180 gatcgcattg acgagccaac aatggaagag ccagtcattt gtcttgagac ctatctagtt 27240 agtaactgct aatggaacgg tttcgatatg gatacacaaa aaaaaaaaaa aaaaaaaaaa 27300 aaaaaaaaaa aaaaaaa 27317

Claims (19)

  1. (a) 알파 코로나 바이러스 전장 유전체에 상보적인 하나 이상의 정방향 및 역방향 프라이머 세트를 제조하는 단계;
    (b) 시료에서 RNA를 분리하는 단계;
    (c) 분리된 RNA를 사용하여 역전사 반응을 수행하는 단계;
    (d) 역전사 반응이 수행된 시료에 프라이머 세트를 첨가하여 중합효소 연쇄반응을 실시하는 단계; 및
    (e) 증폭된 산물의 서열을 분석하는 단계;를 포함하고,
    상기 프라이머 세트는 서열번호 1 및 서열번호 2; 서열번호 3 및 서열번호 4; 서열번호 5 및 서열번호 6; 서열번호 7 및 서열번호 8; 서열번호 9 및 서열번호 10; 서열번호 11 및 서열번호 12; 서열번호 13 및 서열번호 14; 서열번호 15 및 서열번호 16; 서열번호 17 및 서열번호 18; 및 서열번호 19 및 서열번호 20;로 이루어진 군에서 선택되는 어느 하나인 것을 특징으로 하는,
    분리된 시료 내 알파 코로나 바이러스의 전장 유전체 서열 확인 방법.
  2. 삭제
  3. 삭제
  4. 제1항에 있어서, 상기 시료는 혈액, 혈청, 객담, 소변 또는 생체 조직으로부터 분리된 것인, 방법
  5. 제1항에 있어서, 상기 알파 코로나 바이러스는 229E 또는 NL63을 포함하는 것인, 방법
  6. 제1항에 있어서, 상기 (e) 단계는 서열번호 31 내지 87의 프라이머를 모두 사용하는 것인, 방법
  7. 알파 코로나 바이러스 전장 유전체에 상보적인 하나 이상의 정방향 및 역방향 프라이머 세트를 포함하는 알파 코로나 바이러스 진단 키트로서,
    상기 프라이머 세트는 서열번호 1 및 서열번호 2; 서열번호 3 및 서열번호 4; 서열번호 5 및 서열번호 6; 서열번호 7 및 서열번호 8; 서열번호 9 및 서열번호 10; 서열번호 11 및 서열번호 12; 서열번호 13 및 서열번호 14; 서열번호 15 및 서열번호 16; 서열번호 17 및 서열번호 18; 및 서열번호 19 및 서열번호 20;로 이루어진 군에서 선택되는 어느 하나의 프라이머 세트를 포함하는 알파 코로나 바이러스 진단 키트.
  8. 삭제
  9. 삭제
  10. 제7항에 있어서, 진단 시료는 혈액, 혈청, 객담, 소변 또는 생체 조직으로부터 분리된 것인, 키트
  11. 제7항에 있어서, 상기 알파 코로나 바이러스는 229E 또는 NL63을 포함하는 것인, 키트
  12. 제7항에 있어서, 알파 코로나 바이러스의 서열 분석은 서열번호 31 내지 87의 프라이머를 모두 사용하는 것인, 키트
  13. 알파 코로나 바이러스 전장 유전체에 상보적인 하나 이상의 정방향 및 역방향 프라이머 세트를 포함하는 알파 코로나 바이러스 유발 질환 진단용 조성물로서,
    상기 프라이머 세트는 서열번호 1 및 서열번호 2; 서열번호 3 및 서열번호 4; 서열번호 5 및 서열번호 6; 서열번호 7 및 서열번호 8; 서열번호 9 및 서열번호 10; 서열번호 11 및 서열번호 12; 서열번호 13 및 서열번호 14; 서열번호 15 및 서열번호 16; 서열번호 17 및 서열번호 18; 및 서열번호 19 및 서열번호 20;로 이루어진 군에서 선택되는 어느 하나의 프라이머 세트를 포함하는 것인 알파 코로나 바이러스 유발 질환 진단용 조성물
  14. 제13항에 있어서, 상기 코로나 바이러스 유발 질환은 호흡기 질환인 것인, 조성물
  15. 삭제
  16. 삭제
  17. 제13항에 있어서, 진단 시료는 혈액, 혈청, 객담, 소변 또는 생체 조직으로부터 분리된 것인, 조성물
  18. 제13항에 있어서, 상기 알파 코로나 바이러스는 229E 또는 NL63을 포함하는 것인, 조성물
  19. 제13항에 있어서, 알파 코로나 바이러스의 서열 분석은 서열번호 31 내지 87의 프라이머를 모두 사용하는 것인, 조성물

KR1020180145560A 2018-11-22 2018-11-22 인간 알파코로나바이러스 전장유전체 증폭을 통한 진단키트 및 전장 유전체 서열 확인 방법 KR102146113B1 (ko)

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