[发明专利]基于Illumina测序的串联条形码、其标记的DNA文库及其构建方法

权利要求书

1.基于Illumina测序的串联条形码，其特征在于，包括IS1串联条形码和IS2串联条形码，其中：所述IS1串联条形码在95℃以0.1℃/秒的速率降温至12℃的过程中IS1序列和与所述IS1序列具有部分互补片段的IS3X’序列结合形成；所述IS2串联条形码在95℃以0.1℃/秒的速率降温至12℃的过程中IS2序列和与所述IS2序列具有部分互补片段的IS3Y’序列结合形成；

所述IS1序列包括IS1_Ind1至IS1_Ind24，所述IS2序列包括IS2_Ind25至IS2_Ind48，所述IS3X’序列包括IS3_Ind1至IS3_Ind24，所述IS3Y’序列包括IS3_Ind25至IS3_Ind48，如下表所示，其中*表示PTO修饰：

	Name	Sequence	Name	Sequence
TCTGCC	IS1_Ind1	A*C*A*C*TCTTTCCCTACACGACGCTCTTCCGATCTtc*t*g*c*c	IS3_Ind1	ggcagaAGATCGGAA*G*A*G*C
GTCTCT	IS1_Ind2	A*C*A*C*TCTTTCCCTACACGACGCTCTTCCGATCTgt*c*t*c*t	IS3_Ind2	agagacAGATCGGAA*G*A*G*C
ATATTG	IS1_Ind3	A*C*A*C*TCTTTCCCTACACGACGCTCTTCCGATCTat*a*t*t*g	IS3_Ind3	caatatAGATCGGAA*G*A*G*C
TGGAAG	IS1_Ind4	A*C*A*C*TCTTTCCCTACACGACGCTCTTCCGATCTtg*g*a*a*	IS3_Ind4	cttccaAGATCGGAA*G*A*G*C
TCTAGT	IS1_Ind5	A*C*A*C*TCTTTCCCTACACGACGCTCTTCCGATCTtc*t*a*g*t	IS3_Ind5	actagaAGATCGGAA*G*A*G*C
AGAGTA	IS1_Ind6	A*C*A*C*TCTTTCCCTACACGACGCTCTTCCGATCTag*a*g*t*a	IS3_Ind6	tactctAGATCGGAA*G*A*G*C
GGCCAA	IS1_Ind7	A*C*A*C*TCTTTCCCTACACGACGCTCTTCCGATCTgg*c*c*a*	IS3_Ind7	ttggccAGATCGGAA*G*A*G*C
TATCTC	IS1_Ind8	A*C*A*C*TCTTTCCCTACACGACGCTCTTCCGATCTta*t*c*t*c	IS3_Ind8	gagataAGATCGGAA*G*A*G*C
TTATGC	IS1_Ind9	A*C*A*C*TCTTTCCCTACACGACGCTCTTCCGATCTtt*a*t*g*c	IS3_Ind9	gcataaAGATCGGAA*G*A*G*C
AGTTGG	IS1_Ind10	A*C*A*C*TCTTTCCCTACACGACGCTCTTCCGATCTag*t*t*g*g	IS3_Ind10	ccaactAGATCGGAA*G*A*G*C
GTCAAG	IS1_Ind11	A*C*A*C*TCTTTCCCTACACGACGCTCTTCCGATCTgt*c*a*a*	IS3_Ind11	cttgacAGATCGGAA*G*A*G*C
CAGCAA	IS1_Ind12	A*C*A*C*TCTTTCCCTACACGACGCTCTTCCGATCTca*g*c*a*	IS3_Ind12	ttgctgAGATCGGAA*G*A*G*C
TCGCCG	IS1_Ind13	A*C*A*C*TCTTTCCCTACACGACGCTCTTCCGATCTtc*g*c*c*g	IS3_Ind13	cggcgaAGATCGGAA*G*A*G*C
CTAAGA	IS1_Ind14	A*C*A*C*TCTTTCCCTACACGACGCTCTTCCGATCTct*a*a*g*a	IS3_Ind14	tcttagAGATCGGAA*G*A*G*C
CCGCTT	IS1_Ind15	A*C*A*C*TCTTTCCCTACACGACGCTCTTCCGATCTcc*g*c*t*t	IS3_Ind15	aagcggAGATCGGAA*G*A*G*C
AAGTTA	IS1_Ind16	A*C*A*C*TCTTTCCCTACACGACGCTCTTCCGATCTaa*g*t*t*a	IS3_Ind16	taacttAGATCGGAA*G*A*G*C
GGTACC	IS1_Ind17	A*C*A*C*TCTTTCCCTACACGACGCTCTTCCGATCTgg*t*a*c*c	IS3_Ind17	ggtaccAGATCGGAA*G*A*G*C
CCAGGT	IS1_Ind18	A*C*A*C*TCTTTCCCTACACGACGCTCTTCCGATCTcc*a*g*g*t	IS3_Ind18	acctggAGATCGGAA*G*A*G*C
AATCGA	IS1_Ind19	A*C*A*C*TCTTTCCCTACACGACGCTCTTCCGATCTaa*t*c*g*a	IS3_Ind19	tcgattAGATCGGAA*G*A*G*C
AACGCA	IS1_Ind20	A*C*A*C*TCTTTCCCTACACGACGCTCTTCCGATCTaa*c*g*c*	IS3_Ind20	tgcgttAGATCGGAA*G*A*G*C
GACGAC	IS1_Ind21	A*C*A*C*TCTTTCCCTACACGACGCTCTTCCGATCTga*c*g*a*	IS3_Ind21	gtcgtcAGATCGGAA*G*A*G*C
CGCGCT	IS1_Ind22	A*C*A*C*TCTTTCCCTACACGACGCTCTTCCGATCTcg*c*g*c*t	IS3_Ind22	agcgcgAGATCGGAA*G*A*G*C
CCGTAG	IS1_Ind23	A*C*A*C*TCTTTCCCTACACGACGCTCTTCCGATCTcc*g*t*a*g	IS3_Ind23	ctacggAGATCGGAA*G*A*G*C
GTAATC	IS1_Ind24	A*C*A*C*TCTTTCCCTACACGACGCTCTTCCGATCTgt*a*a*t*c	IS3_Ind24	gattacAGATCGGAA*G*A*G*C
GACCTT	IS2_Ind25	G*T*G*A*CTGGAGTTCAGACGTGTGCTCTTCCGATCTga*c*c*t*t	IS3_Ind25	aaggtcAGATCGGAA*G*A*G*C
TCATAA	IS2_Ind26	G*T*G*A*CTGGAGTTCAGACGTGTGCTCTTCCGATCTtc*a*t*a*a	IS3_Ind26	ttatgaAGATCGGAA*G*A*G*C
CAAGAG	IS2_Ind27	G*T*G*A*CTGGAGTTCAGACGTGTGCTCTTCCGATCTca*a*g*a*g	IS3_Ind27	ctcttgAGATCGGAA*G*A*G*C
CGATCA	IS2_Ind28	G*T*G*A*CTGGAGTTCAGACGTGTGCTCTTCCGATCTcg*a*t*c*a	IS3_Ind28	tgatcgAGATCGGAA*G*A*G*C
TTGATT	IS2_Ind29	G*T*G*A*CTGGAGTTCAGACGTGTGCTCTTCCGATCTtt*g*a*t*t	IS3_Ind29	aatcaaAGATCGGAA*G*A*G*C
TCCGAG	IS2_Ind30	G*T*G*A*CTGGAGTTCAGACGTGTGCTCTTCCGATCTtc*c*g*a*g	IS3_Ind30	ctcggaAGATCGGAA*G*A*G*C
CCTGAA	IS2_Ind31	G*T*G*A*CTGGAGTTCAGACGTGTGCTCTTCCGATCTcc*t*g*a*a	IS3_Ind31	ttcaggAGATCGGAA*G*A*G*C
ATTCTT	IS2_Ind32	G*T*G*A*CTGGAGTTCAGACGTGTGCTCTTCCGATCTat*t*c*t*t	IS3_Ind32	aagaatAGATCGGAA*G*A*G*C
GCGACT	IS2_Ind33	G*T*G*A*CTGGAGTTCAGACGTGTGCTCTTCCGATCTgc*g*a*c*t	IS3_Ind33	agtcgcAGATCGGAA*G*A*G*C
GGCTTC	IS2_Ind34	G*T*G*A*CTGGAGTTCAGACGTGTGCTCTTCCGATCTgg*c*t*t*c	IS3_Ind34	gaagccAGATCGGAA*G*A*G*C
AATACG	IS2_Ind35	G*T*G*A*CTGGAGTTCAGACGTGTGCTCTTCCGATCTaa*t*a*c*g	IS3_Ind35	cgtattAGATCGGAA*G*A*G*C
TACGGT	IS2_Ind36	G*T*G*A*CTGGAGTTCAGACGTGTGCTCTTCCGATCTta*c*g*g*t	IS3_Ind36	accgtaAGATCGGAA*G*A*G*C
ACCGTC	IS2_Ind37	G*T*G*A*CTGGAGTTCAGACGTGTGCTCTTCCGATCTac*c*g*t*c	IS3_Ind37	gacggtAGATCGGAA*G*A*G*C
AGAAGC	IS2_Ind38	G*T*G*A*CTGGAGTTCAGACGTGTGCTCTTCCGATCTag*a*a*g*c	IS3_Ind38	gcttctAGATCGGAA*G*A*G*C
CATAGC	IS2_Ind39	G*T*G*A*CTGGAGTTCAGACGTGTGCTCTTCCGATCTca*t*a*g*c	IS3_Ind39	gctatgAGATCGGAA*G*A*G*C
AGGCTC	IS2_Ind40	G*T*G*A*CTGGAGTTCAGACGTGTGCTCTTCCGATCTag*g*c*t*c	IS3_Ind40	gagcctAGATCGGAA*G*A*G*C
CTGCGG	IS2_Ind41	G*T*G*A*CTGGAGTTCAGACGTGTGCTCTTCCGATCTct*g*c*g*g	IS3_Ind41	ccgcagAGATCGGAA*G*A*G*C
CTCGGC	IS2_Ind42	G*T*G*A*CTGGAGTTCAGACGTGTGCTCTTCCGATCTct*c*g*g*c	IS3_Ind42	gccgagAGATCGGAA*G*A*G*C
GATTAG	IS2_Ind43	G*T*G*A*CTGGAGTTCAGACGTGTGCTCTTCCGATCTga*t*t*a*g	IS3_Ind43	ctaatcAGATCGGAA*G*A*G*C
AGATAT	IS2_Ind44	G*T*G*A*CTGGAGTTCAGACGTGTGCTCTTCCGATCTag*a*t*a*t	IS3_Ind44	atatctAGATCGGAA*G*A*G*C
TGGTCC	IS2_Ind45	G*T*G*A*CTGGAGTTCAGACGTGTGCTCTTCCGATCTtg*g*t*c*c	IS3_Ind45	ggaccaAGATCGGAA*G*A*G*C
GTTCCG	IS2_Ind46	G*T*G*A*CTGGAGTTCAGACGTGTGCTCTTCCGATCTgt*t*c*c*g	IS3_Ind46	cggaacAGATCGGAA*G*A*G*C
GTACGT	IS2_Ind47	G*T*G*A*CTGGAGTTCAGACGTGTGCTCTTCCGATCTgt*a*c*g*t	IS3_Ind47	acgtacAGATCGGAA*G*A*G*C
AAGAAC	IS2_Ind48	G*T*G*A*CTGGAGTTCAGACGTGTGCTCTTCCGATCTaa*g*a*a*c	IS3_Ind48	gttcttAGATCGGAA*G*A*G*C

2.权利要求1所述基于Illumina测序的串联条形码标记DNA文库的构建方法，其特征在于，包括以下步骤：

(1)生物样本DNA序列破碎，获取DNA片段序列；

(2)修复上述DNA片段序列平末端；

(3)在所述DNA片段序列5’端加上所述IS1串联条形码，所述DNA片段序列3’端加上所述IS2串联条形码，引物IS7的结合位点位于所述IS1串联条形码外侧，引物IS8的结合位点位于所述IS2串联条形码外侧；

(4)修复所述DNA片段序列空缺并延伸；

(5)采用所述引物IS7、IS8对DNA序列进行PCR扩增。