Method for detecting human papilloma virus based on Solexa sequencing method

Abstract

The present invention relates to a method for detecting Human Papilloma Virus (HPV), in particular, to a method for detecting HPV based on Solexa sequencing method.

Claims

1. A method for sequencing, detecting, or typing HPV in human samples, comprising the following steps: 1) for each sample, amplifying DNA in the sample with one index primer group, wherein said index primer group comprises 11 index primers, the sequence of each of said index primers comprises an index sequence and a PCR primer sequence, and the index sequence is linked to 5 end of the PCR primer sequence, optionally via a linker sequence, wherein i) said index sequence is selected from the group consisting of SEQ ID NOS: 1-95, and each of said 11 index primers has the same index sequence, and ii) the PCR primer sequences of said 11 index primers consist of SEQ ID NOS: 96-106, wherein the index primer groups used in different samples are different, and different index primer groups employ different index sequences; 2) mixing the amplification products obtained by conducting amplification with different index primer groups in step 1), so as to obtain one or more PCR product libraries; 3) adding adapters to the one or more PCR product libraries obtained in step 2) by PCR-free methods, thereby constructing one or more sequencing libraries, wherein the adapters used in different sequencing libraries may be the same or different, and different adapters share the same sequencing sequence but have different characteristic sequences, 4) optionally, mixing the sequencing libraries with different adapters obtained in step 3), so as to obtain one or more library mixtures; 5) conducting sequencing of one or more sequencing libraries obtained in step 3) or one or more library mixtures obtained in step 4) by using a high throughput sequencing technology; 6) correlating the sequencing results to the samples, according to the index primer sequences of the index primer group or according to the index primer sequences of the index primer group and the characteristic sequence of the adapter.

2. The method according to claim 1, wherein in step 3), the adapters are added to the amplification products in the PCR product libraries by using DNA ligase.

3. The method according to claim 1, wherein in step 5), the high throughput sequencing technology is a paired-end sequencing technology.

4. The method according to claim 1, wherein in step 5), the high throughput sequencing technology is a sequencing by synthesis method.

5. The method according to claim 1, wherein said samples are exfoliative cells.

6. The method according to claim 1, wherein at least 50 index primer groups are used in step 1).

7. The method according to claim 1, wherein 95 index primer groups are used in step 1).

8. The method according to claim 1, wherein the index sequences used in step 1) comprise at least SEQ ID NOS: 1-10, or SEQ ID NOS: 11-20, or SEQ ID NOS: 21-30, or SEQ ID NOS: 31-40, or SEQ ID NOS: 41-50, or SEQ ID NOS: 51-60, or SEQ ID NOS: 61-70, or SEQ ID NOS: 71-80, or SEQ ID NOS: 81-90, or SEQ ID NOS: 91-95.

9. The method according to claim 1, wherein at least 50 adapters are used in step 3).

10. The method according to claim 1, wherein at least 200 adapters are used in step 3).

11. The method according to claim 1, wherein said adapters used in step 3) comprise a sequence selected from the group consisting of SEQ ID NOS: 121-132.

12. The method according to claim 1, further comprising aligning the sequencing results of said samples with sequence(s) in an HPV database to accurately type HPV in the samples.

Description

DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is an illustration of the PCR products labeled by an index and an adapter having a unique characteristic sequence. In an exemplary method according to the present invention, indexes are added simultaneously to both ends of the PCR products from each sample by PCR, and multiple PCR products carrying different indexes are mixed together to construct a sequencing library. During the construction of the sequencing library, if necessary, multiple sequencing libraries can be constructed, wherein adapters carrying different characteristic sequences are used to label the sequencing libraries. After construction of libraries is finished, multiple libraries labeled is by adapters carrying different characteristic sequences are mixed together and are sequenced simultaneously by Solexa sequencing method (the indexes used in different sequencing libraries may be the same or different). Finally, according to the sequence information of the characteristic sequences of the adapters and indexes in the sequencing results, the sequencing results can be corresponded to the samples one by one.

(2) FIG. 2 is an agarose gel electrophorogram of a part of PCR products. It can be seen from the electrophorogram that the band of the PCR products is of about 170 bp, wherein lane M is a molecular ladder of 50 bp DNA, lanes 1-14 refer to the PCR products from the randomly selected HPV positive samples.

EXAMPLES

(3) 190 samples, of which the HC-II results are known, were genotyped for HPV by the method according to the present invention. The results showed that the results obtained by the method according to the present invention are not only consistent with the known HC-II results, but also accomplish the accurate typing of HPV.

Example 1

Exaction of DNA Samples

(4) According to the instruction of the manufacturer, KingFisher automatic extractor (US Thermo Scientific Kingfisher Flex full automatic bead extraction and purification system) was used to extract DNA from 190 samples of exfoliative cells, of which the HC-II results are known. The nucleic acids were extracted by using the program Bioeasy_200 l Blood DNA_KF.msz. After the program was finished, about 100 l eluting product (extracted DNA) was obtained, which was used as the template for PCR amplification in next step.

Example 2

PCR Amplification

(5) The 190 DNA samples obtained in Example 1 were designated as No. 1-190, and were divided averagely into two groups (HPV-1 group: Nos. 1-95; HPV-2 group: Nos. 96-190). According to the sequences (Table 2, SEQ ID NOS: 96-106) of the primers in the primer group for amplification of HPV DNA (including 6 forward primers and 5 reverse primers), a set of indexes was designed, including 95 indexes (Table 1, SEQ ID NOS: 1-95). Each of the designed indexes was added to the 5 end of the sequence of each primer of the primer set, respectively, thereby obtaining 95 index primer groups, wherein each index primer group included the corresponding 6 forward index primers and 5 reverse index primers, and different index primer groups employed different indexes (i.e., 95 index primer groups were corresponded to 95 indexes one by one).

(6) PCR reactions were conducted for all the samples in the 96-well plates, and two plates were used (one for HPV-1 group and the other for HPV-2 group). DNA obtained in Example 1 was used as template, and a different index primer group was used to carry out PCR amplification as directed to each of the samples in HPV-1 group and HPV-2 group (each containing 95 samples), that is, 95 samples were corresponded to 95 index primer groups one by one. The number of the sample corresponding to each index primer group (each index) was recorded. In each plate, a negative control was provided without adding template. The primers used in the negative controls in the two plates were the same as the primers used in sample No. 1 and No. 96, respectively.

(7) TABLE-US-00002 TABLE1 Informationonindexesandsamples Thecorresponding The The Index Index positioninthe corresponding corresponding SEQ No. sequences 96-wellplate sample(Group1) sample(Group2) IDNO: PI-1 GCTGCGACTC A1 1 96 1 PI-2 GTGTAGATAC A2 2 97 2 PI-3 CTGATATCTA A3 3 98 3 PI-4 ACGATGCTAT A4 4 99 4 PI-5 TAGACTAGAC A5 5 100 5 PI-6 CTGTCTGTGT A6 6 101 6 PI-7 GCATACTGAC A7 7 102 7 PI-8 CTGCTCGCAT A8 8 103 8 PI-9 CATGAGTAGA A9 9 104 9 PI-10 TCTCACTATG A10 10 105 10 PI-11 TGTACTACTA A11 11 106 11 PI-12 GTAGACTAGT A12 12 107 12 PI-13 ATATGCTACT B1 13 108 13 PI-14 CACTCGCTGT B2 14 109 14 PI-15 CATCACGCAC B3 15 110 15 PI-16 AGCATGTGAT B4 16 111 16 PI-17 AGCTAGTAGA B5 17 112 17 PI-18 GCTATGTAGT B6 18 113 18 PI-19 TACGATGATG B7 19 114 19 PI-20 TACGCTGTAC B8 20 115 20 PI-21 TATGTGTACT B9 21 116 21 PI-22 TGACTCAGAC B10 22 117 22 PI-23 TCGTAGCTCA B11 23 118 23 PI-24 GAGACTCGTA B12 24 119 24 PI-25 CTAGATGTCA C1 25 120 25 PI-26 GATGACTCTC C2 26 121 26 PI-27 TCAGTCGCAC C3 27 122 27 PI-28 TGTAGTGAGT C4 28 123 28 PI-29 TCATCGTAGA C5 29 124 29 PI-30 TAGCATCTGT C6 30 125 30 PI-31 TAGTAGTCGT C7 31 126 31 PI-32 CTATACGTGC C8 32 127 32 PI-33 CGACTGTAGA C9 33 128 33 PI-34 ATGTCATGT C10 34 129 34 PI-35 GTCTCGACTG C11 35 130 35 PI-36 AGCTGACGAT C12 36 131 36 PI-37 ATGATATAGT D1 37 132 37 PI-38 ATGTGCTCTA D2 38 133 38 PI-39 CTCACTCGAT D3 39 134 39 PI-40 GCTGCGACTC D4 40 135 40 PI-41 GAGTCATGTC D5 41 136 41 PI-42 CATACGCTCA D6 42 137 42 PI-43 CACTCTCGTC D7 43 138 43 PI-44 GCACTAGATG D8 44 139 44 PI-45 AGTACGCATG D9 45 140 45 PI-46 TCTGTGACGT D10 46 141 46 PI-47 TAGCTCATCT D11 47 142 47 PI-48 AGCATACACT D12 48 143 48 PI-49 GCTATAGTCA E1 49 144 49 PI-50 CGTCTCATGC E2 50 145 50 PI-51 GCTACTACGT E3 51 146 51 PI-52 GAGTGTACTA E4 52 147 52 PI-53 GTCATACGTG E5 53 148 53 PI-54 TATGAGAGAT E6 54 149 54 PI-55 ATCTGAGTAC E7 55 150 55 PI-56 CGATAGCATC E8 56 151 56 PI-57 ACTGATCTCA E9 57 152 57 PI-58 CTCGATACTA E10 58 153 58 PI-59 CATGTGACTG E11 59 154 59 PI-60 CGCATCACTA E12 60 155 60 PI-61 GCATATATCT F1 61 156 61 PI-62 CTGATGCGAC F2 62 157 62 PI-63 TCTCAGAGTC F3 63 158 63 PI-64 CAGTGCGAGT F4 64 159 64 PI-65 ATCTCTGATG F5 65 160 65 PI-66 GCTAGTAGTC F6 66 161 66 PI-67 ATGAGTCGTC F7 67 162 67 PI-68 ATCACTCAGA F8 68 163 68 PI-69 TCTCTCTGAT F9 69 164 69 PI-70 CTCTAGTGCT F10 70 165 70 PI-71 CGTCGTGCTA F11 71 166 71 PI-72 CGACTACTAT F12 72 167 72 PI-73 GCACGTCGAT G1 73 168 73 PI-74 GTAGTGCTCT G2 74 169 74 PI-75 CTGACGAGCT G3 75 170 75 PI-76 CTATAGTCTA G4 76 171 76 PI-77 ACACGCACTA G5 77 172 77 PI-78 CTCGCACTAC G6 78 173 78 PI-79 AGATCTCACT G7 79 174 79 PI-80 ATACTAGTGT G8 80 175 80 PI-81 ATATCTCGTA G9 81 176 81 PI-82 TGACTGCGTA G10 82 177 82 PI-83 TGTAGACGTA G11 83 178 83 PI-84 AGAGACTATG G12 84 179 84 PI-85 GTCGAGTCAC H1 85 180 85 PI-86 TGACAGCTAC H2 86 181 86 PI-87 CGCTAGACAT H3 87 182 87 PI-88 CGTAGATATG H4 88 183 88 PI-89 TGAGTCTGCT H5 89 184 89 PI-90 TAGTCGTATG H6 90 185 90 PI-91 CATACACGAC H7 91 186 91 PI-92 CGCTCAGAGA H8 92 187 92 PI-93 GTGAGTCTCA H9 93 188 93 PI-94 GACAGATGAT H10 94 189 94 PI-95 GCTGTGCGAC H11 95 190 95

(8) TABLE-US-00003 TABLE2 Thesequenceinformationoftheprimersof theprimergroupforamplificationofHPVDNA withoutaddingindexes. Primer SEQ No. Primersequence IDNO: F1 TTTGTTACTGTGGTAGATACTAC 96 F2 TTTGTTACTGTGGTGGATACTAC 97 F3 TTTGTTACCGTTGTTGATACTAC 98 F4 TTTGTTACTAAGGTAGATACCACTC 99 F5 TTTGTTACTGTTGTGGATACAAC 100 F6 TTTGTTACTATGGTAGATACCACAC 101 R1 GAAAAATAAACTGTAAATCATATTCCT 102 R2 GAAAAATAAATTGTAAATCATACTC 103 R3 GAAATATAAATTGTAAATCAAATTC 104 R4 GAAAAATAAACTGTAAATCATATTC 105 R5 GAAAAATAAACTGCAAATCATATTC 106 Note: F represents a forward primer, R represents a reverse primer.

(9) The following PCR parameters were used in amplification:

(10) 95 C. 30 s.fwdarw.48 C. 30 s.fwdarw.72 C. 30 s (40 cycles)

(11) 72 C. 10 min.fwdarw.12 C.

(12) PCR reaction system was of 25 l, and its composition was as follows (all the agents were purchased from Enzymatics Company):

(13) TABLE-US-00004 agents volume/reaction H.sub.2O (HPLC grade) 14.375 l 10x Ex Taq Buffer (Mg.sup.2+ plus) 2.5 l dNTP mix (each 2.5 mM) 2 l mixture of F1/F2/F3/F4/F5/F6 carrying an index 0.5 l (each 7.5 pmol) mixture of R1/R2/R3/R4/R5 carrying an index 0.5 l (each 7.5 pmol) Ex Taq HS (5 U/l) 0.125 l Template DNA 5 l Total volume 25 l

(14) PCR reaction was carried out in PTC-200 PCR Instrument from Bio-Rad Company. After finishing PCR, 3 l PCR product was taken to perform electrophoresis on 2.5% agarose gel (FIG. 2).

Example 3

Mixing and Purification of PCR Products

(15) The rest PCR products in HPV-1 group and HPV-2 group each were mixed in a 3 ml EP tube (also marked as HPV-1 group and HPV-2 group), and were mixed homogenously under shaking. 500 l DNA was taken from each of the two tubes, and was purified by using the Qiagen DNA Purification kit according to the instruction of the manufacturer, to provide 200 l purified DNA. The DNA concentrations of the purified mixtures were determined as 98 ng/l (HPV-1 group) and 102 ng/l (HPV-2 group) by using Nanodrop 8000 (Thermo Fisher Scientific Co.), respectively.

Example 4

Construction of Solexa Sequencing Library

(16) 4.1: End-Repair Reaction

(17) DNA end-repair reaction was conducted to the purified DNA mixtures in the two tubes obtained in Example 3 by using Thermomixer (Eppendorf Company), respectively. The reaction system for the repair reaction was of 1004 and its composition was as follow (all the agents were purchased from Enzymatics Company):

(18) TABLE-US-00005 Agents Volume/reaction DNA obtained in last step 75 l 20x Polynucleotide Kinase Buffer(B904) 10 L dNTP mix (20 mM each) 4 L T4 DNA Polymerase 5 L Klenow Fragment 1 L T4 Polynucleotide Kinase 5 L Total volume 100 L

(19) Reaction condition: 20 C., 30 mins.

(20) According to the instruction of the manufacturer, the products of the DNA end-repair reaction were purified and recovered by using QIAquick PCR Purification kit. The recovered product was dissolved in 34 l EB (QIAGEN Elution Buffer).

(21) 4.2: Addition of A at the 3 End

(22) The base A was added to the 3 end of the recovered DNA by using Thermomixer (Eppendorf Company). The reaction system was of 50 ul, and its composition was as follow (all the agents were purchased from Enzymatics Company):

(23) TABLE-US-00006 Agent Volume/reaction DNA obtained in last step 32 l dATP (1 mM, GE Company) 10 l 10x Blue Buffer 5 l Klenow (3-5exo) 3 l Total volume 50 l

(24) Reaction conditions: 37 C., 30 mins.

(25) According to the instruction of the manufacturer, the product having the base A added at 3 end was purified and recovered by using MiniElute PCR Purification Kit (QIAGEN Company). The recovered product was dissolved in 20 l EB.

(26) 4.3: Addition of Solexa Adapter

(27) Different adapters were added to the two products obtained in last step by using Thermomixer (Eppendorf Company), so as to construct 2 sequencing libraries. The corresponding correlation between the adapters and libraries was recorded.

(28) The reaction system for the addition of the Solexa adapters was of 50 ul, and its composition was as follow (all the agents were purchased from Illumina Company):

(29) TABLE-US-00007 Agent Volume/reaction DNA obtained in last step 11 L 2x Rapid ligation Buffer 15 L PCR-free Index Adapter oligo mix (25 mM) 1 L T4 DNA Ligase (Rapid, L603-HC-L) 3 L Total volume 30 L

(30) Reaction condition: 20 C., 15 mins.

(31) According to the instruction of the manufacturer, the reaction products were purified by using Ampure Beads (Beckman Coulter Genomics), and the purified products were dissolved in 17 l deionized water. Agilent Bioanalyzer 2100 (Agilent Company) and Fluorescence quantitative PCR (QPCR) were used to detect the DNA concentration of the products, the results were as follow.

(32) TABLE-US-00008 2100 (nM) qPCR (nM) HPV-1 Group 20.4 24.2 HPV-2 Group 21.6 25.8

Example 5

Solexa Sequencing

(33) The concentrations measured by Agilent Bioanalyzer 2100 were used as the standard, and the two products obtained in last step were mixed equimolarly (10 pmol DNA for each). According to the instruction of the manufacturer, Solexa sequencer (Illumina Genome Analyzer IIx sequencer) was used in sequencing under Solexa PE-75 program.

Example 6

Result Analysis

(34) According to the sequence information of the characteristic sequences of the adapters and the index primers (index portion and primer portion) in the sequencing result, the sequencing results were corresponded to the samples one by one. Then, the sequencing result of each sample was aligned with HPV database by using alignment programs known in the art, such as BLAST and SOAP, thereby accomplishing HPV detection and accurately typing HPV.

(35) The detection results obtained were completely identical to the known results (see Table 3), indicating that the method according to the present invention can be applied to accurately detect HPV in samples.

(36) TABLE-US-00009 TABLE 3 Detection results of 190 samples. The known Sample HC-II result HPV result of the No. (RLU/CO value) present detection 1 14.2 HPV56 2 0.31 negative 3 196.41 HPV16 4 5.76 HPV18 5 0.35 negative 6 99.86 HPV18, HPV11, HPV16 7 128.86 HPV39 8 35.12 HPV18, HPV6 9 498.69 HPV16, HPV56 10 603.57 HPV18, HPV31, HPV39 11 0.27 negative 12 3420.57 HPV18 13 0.38 negative 14 0.41 negative 15 455.06 HPV16 16 8.93 HPV18 17 0.6 negative 18 0.41 negative 19 0.29 negative 20 27.64 HPV31 21 1985.41 HPV56, HPV68 22 20.71 HPV42 23 1795.83 HPV11, HPV16, HPV52 24 9.55 HPV43 25 237.62 HPV39 26 1.5 HPV6 27 1478.98 HPV68, HPV16 28 115.31 HPV44 29 419.31 HPV16 30 1.81 candHPV89 31 2013.61 HPV52, HPV39 32 1379.09 HPV54, HPV33 33 12.74 HPV42 34 1695.31 HPV16, candHPV89 35 1410.85 HPV35 36 1149.25 HPV18 37 0.24 negative 38 1.55 HPV11 39 2.03 HPV11, HPV6 40 8.45 HPV42 41 0.2 negative 42 0.22 negative 43 0.53 negative 44 10.38 HPV6 45 78.21 HPV16 46 0.23 negative 47 45.42 HPV16, HPV18 48 0.35 negative 49 148.66 HPV18, candHPV89 50 60.27 HPV56 51 0.28 negative 52 360.26 HPV56, HPV68 53 50.31 HPV18 54 0.18 negative 55 0.31 negative 56 196.41 HPV16 57 5.76 HPV51 58 0.23 negative 59 0.88 negative 60 0.16 negative 61 870.63 HPV52, HPV16 62 10.18 HPV42 63 0.15 negative 64 1.36 HPV11 65 68.2 HPV59 66 0.68 negative 67 130.41 HPV45 68 0.26 negative 69 5.25 HPV6 70 0.46 negative 71 8.23 HPV40 72 0.28 negative 73 100.16 HPV43, HPV44 74 450.13 HPV41 75 127.08 HPV39, HPV6 76 602.79 HPV45 77 276 HPV16 78 243.6 HPV6, HPV70, HPV39 79 229.44 HPV35 80 1384.92 HPV52, HPV56, HPV11 81 172.64 HPV26, HPV42 82 855.24 HPV35, HPV6 83 620.69 HPV52 84 128.02 HPV11 85 514.84 HPV33 86 68.3 HPV58 87 402.15 HPV59, HPV16 88 51.72 HPV33 89 1.78 HPV6 90 56.7 HPV11, HPV31 91 186.06 HPV16 92 0.02 negative 93 386.06 HPV18, HPV16 94 28.09 HPV6, HPV44 95 186.06 HPV68, 96 0.28 negative 97 0.33 negative 98 181.29 HPV35, HPV6 99 77.32 HPV16 100 91.22 HPV39 101 188.92 HPV52 102 1352.83 HPV35, HPV11, HPV39 103 1.39 HPV43 104 119.5 HPV45, HPV11 105 292.43 HPV56, HPV31 106 2.91 HPV68 107 193.13 HPV45 108 2.62 HPV6 109 94.12 HPV16 110 792.72 HPV18, HPV31 111 31.76 HPV11 112 0.25 negative 113 0.23 negative 114 750.82 HPV56, HPV16 115 0.4 negative 116 2.75 HPV31 117 396.04 HPV45 118 354.76 HPV18, HPV16 119 6.26 HPV11 120 1719.67 HPV16, HPV45 121 76.92 HPV51 122 1318.02 HPV56, HPV16, HPV42 123 0.28 negative 124 0.33 negative 125 181.29 HPV59 126 77.32 HPV68 127 110.8 HPV52 128 147.25 HPV16 129 0.24 HPV26 130 1.55 HPV11, HPV53 131 2.03 HPV6, HPV66 132 8.45 HPV43 133 0.2 negative 134 0.24 negative 135 10.53 HPV11 136 1410.85 HPV16, HPV53, HPV70 137 1149.25 HPV56, HPV81, HPV73 138 0.24 negative 139 413.9 HPV45 140 17.05 HPV11 141 23.6 HPV52 142 3379.09 HPV16, HPV35, HPV56 143 0.18 negative 144 1.46 HPV18 145 1.25 HPV11, HPV26 146 2.13 HPV6, HPV81 147 872.52 HPV16, HPV45, HPV52 148 1.5 HPV18 149 4.33 HPV16 150 0.82 negative 151 60.35 HPV59 152 0.24 negative 153 0.23 negative 154 0.18 negative 155 1.46 HPV51 156 11.25 HPV16 157 2.13 HPV11 158 0.13 negative 159 90.18 HPV58 160 0.15 negative 161 602.79 HPV68, HPV16 162 132.68 HPV56, HPV11 163 127.08 HPV39, HPV54 164 602.79 HPV33 165 276 HPV18 166 243.6 HPV45 167 229.44 HPV51 168 1384.92 HPV16, HPV58, HPV72 169 172.64 HPV58 170 855.24 HPV16, candHPV89 171 126.47 HPV51 172 86.62 HPV44, HPV11 173 879.37 HPV18, HPV58 174 119.39 HPV56 175 0.61 negative 176 18.02 HPV16 177 16.06 HPV18 178 60.69 HPV56, HPV11 179 2.45 HPV11 180 94.93 HPV39 181 1635.3 HPV16, HPV35, HPV51 182 754.64 HPV33, candHPV89 183 0.23 HPV11 184 20.28 HPV18 185 0.16 negative 186 0.13 negative 187 60.18 HPV59 188 0.15 negative 189 1.36 HPV43 190 0.28 negative

(37) In addition, accurate typing of HPV in samples was also accomplished by the method according to the present invention. Table 4 provides the sequences and typing results of the samples corresponding to lane Nos. 1-14 as shown in FIG. 2.

(38) TABLE-US-00010 TABLE4 ThesequencesandtypingresultsofthesamplescorrespondingtolaneNos1-14 asshowninFIG.2. Lane HPV Parameterofalignment SEQID No. type Sequencingresults identity score evalue NO: 1 HPV11 GAAAAATAAACTGTAAATCATATTCCTCCACATGGCGCATGTATTCCT 96.77 107 6.00E27 107 TATAATCTGAATTA 2 HPV16 TTTGTTACTGTGGTAGATACTACCCGCAGTACAAATATGTCATTATGT 96.77 107 6.00E27 108 GCTGCCATATCTAC 3 HPV18 TTTGTTACTAAGGTAGATACCACTCGCAGTACCAATTTAACAATATGT 96.77 107 6.00E27 109 GCTTCTACACAGTC 4 HPV31 GAAAAATAAACTGTAAATCATATTCCTCACCATGTCTTAAATACTCTT 95.16 99.6 2.00E24 110 TAAAATTACTACTT 5 HPV33 GAAAAATAAACTGTAAATCATATTCCTCAACATGTCTTATATATTCTT 96.77 107 6.00E27 111 TAAAATTTTCATTT 6 HPV35 GAAAAATAAACTGTAAATCATATTCTTCACCATGCCTTAAATATTCCT 98.39 115 3.00E29 112 TATAATTGTCATTT 7 HPV39 TTTGTTACTGTGGTAGATACTACCCGTAGTACCAACTTTACATTATCT 94.83 91.7 4.00E22 113 ACCTCTATAGAGTC 8 HPV43 TTTGTTACTAAGGTAGATACCACTCGTAGTACAAACTTAACGTTATGT 100 101 4.00E25 114 GCCTCTACTGACCC 9 HPV45 GAAAAATAAACTGTAAATCATATTCCTCCACATGTCTACTATAGTGCT 100 123 1.00E31 115 TAAACTTAGTAGGA 10 HPV51 GAAATATAAATTGTAAATCAAATTCTTCCCCATGCCTAATATATTGCT 91.94 83.8 9.00E20 116 TAAAGTTACTTGGA 11 HPV52 TTTGTTACTGTGGTGGATACTACTCGTAGCACTAACATGACTTTATGT 98 91.7 4.00E22 117 GCTGAGGTTAAAAA 12 HPV56 TTTGTTACTGTTGTGGATACAACTAGAAGTACTAACATGACTATTAGT 95.16 99.6 2.00E24 118 ACTGCTACAGAACA 13 HPV58 GAAAAATAAACTGTAAATCATATTCCTCAACATGACGTACATATTCCT 96.77 107 6.00E27 119 TAAAATTATCATTT 14 HPV61 TTTGTTACTGTGGTGGATACTACCCGCAGTACTAATTTAGCCATTTGC 94 75.8 2.00E17 120 ACTGCTACATCCCC

REFERENCES

(39) The patents, publications and other materials, which are used to illustrate the present invention or provide further detailed contents about the practice of the present invention, are incorporated by reference herein, and are provided in the following list for the convenience. [1]. Pectasides D, Kanposioras K, Papaxoinis G et al. Chemotherapy for recurrent cervical cancer. Cancer Treatment Reviews, 2008, 34(7): 603-613. [2]. Brink, A. A., P. J. Snijders, and C. J. Meijer. HPV detection methods. Dis. Markers 2007, 23: 273-281. [3]. IARC. Handbooks of cancer prevention. Cervix cancer screening [R]. Lyon: IARC Press, 2005. [4]. Doorbar, J. Molecular biology of human papillomavirus infection and cervical cancer. Clin. Sci. 2006, 110: 525-541. [5]. Cox T, Cuzick J. HPV DNA testing in cervical cancer screening: From evidence to policies. Gyneeol Oncol, 2006, 103: 8-11. [6]. Kulmala S, syIjhen. Human papillomavirus testing with the hybrid capture assay and PCR as screening tools. Clin Microbiol, 2004, 42(6): 2470-2475. [7]. Quail, M. et al., A large genome center's improvements to the Illumina sequencing system. Nat. Methods, 2008, 5, 1005-1010. [8]. Brown, C. G. et al., Solexa/Illumina GAPipeline product and product documentation, Illumina Inc, 2006. [9]. Lozano, R. Successfully integrating human papillomavirus testing into your practice. Arch. Pathol. Lab Med, 2003, 127: 991-994.