CN105603052A - Probes and uses thereof - Google Patents

Probes and uses thereof Download PDF

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Publication number
CN105603052A
CN105603052A CN201410629930.3A CN201410629930A CN105603052A CN 105603052 A CN105603052 A CN 105603052A CN 201410629930 A CN201410629930 A CN 201410629930A CN 105603052 A CN105603052 A CN 105603052A
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probe
sample
optionally
coding region
gene coding
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CN201410629930.3A
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CN105603052B (en
Inventor
魏晓明
杨昀
田甜
孙岩
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Shenzhen Bgi Medicine Co Ltd
Wuhan Bgi Medical Laboratory Co Ltd
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Shenzhen Bgi Medicine Co Ltd
Wuhan Bgi Medical Laboratory Co Ltd
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Abstract

The present invention discloses probes and uses thereof. The group of the probes specifically recognize at least one part of a FLG gene coding region, and meet at least one condition selected from the following conditions that (1) the length of the probe is 75 bp; (2) the probe specifically recognizes the sequence between the upstream 10 bp and the downstream 10 bp of the FLG gene coding region; (3) the multiplier of the probe specifically recognizing the region having the GC content of higher than 0.6 and lower than 0.3 is more than 2; (4) the melting temperatures of the probe and the target sequence is 60-10 DEG C, preferably 80 DEG C; (5) the probe does not contain a hairpin structure; (6) the probe is matched with two sites on a reference genome at the most; and (7) the window sliding size is 10 bp during the probe selection. According to the present invention, the group of the probes provides good capture specificity, high sensitivity and high coverage for at least one part of the specifically-recognized FLG gene coding region, and can be effectively used for the capture detection of the FLG gene coding region.

Description

Probe and uses thereof
Technical field
The present invention relates to biological technical field, especially target gene high throughput sequencing technologies field. Particularly, the present invention relates toProbe and uses thereof, more specifically, the present invention relates to one group of probe, builds the method for high-throughput sequencing library, definite test sample for the treatment ofThe method of this FLG gene coding region nucleotide sequence, device and definite sample to be tested FLG base of structure high-throughput sequencing libraryBecause of the system of code area nucleotide sequence.
Background technology
Ordinary type ichthyosis (Ichthyosisvulgaris) is a kind of autosomal dominant inherited disease, and the incidence of disease of this disease is1:250-1:1000. Main clinic symptoms comprises that skin lesion is " fish scale " or " snakeskin " shape, follicular keratosis papule, poreBe point-like, the lines of the hand and pin line are many, dark, random. With the gene of this disease association be the coding silk polyprotein (Filaggrin) that is positioned at 1q21.3FLG gene, this gene is a part for the gene cluster of epidermal differentiation coding structure in latter stage albumen. FLG full length gene12.7 – 14.7kb, comprise 3 extrons (Exon), and Exon1 (15bp) is non-coding sequence, and Exon2 (159bp) comprises initialCoded sequence, Exon3 (12-14kb) comprises that 10-12 length of N end and coding silk polyprotein reaches the repetitive sequence of 1k.At present, FLG gene tester is mainly long PCR, then long PCR product is carried out to nido amplification, obtains specialProperty aim sequence after, understand FLG sequence in conjunction with sanger order-checking. But this section of sequence not only combines complexity, also hasIndividual specificity, uses the method often to there will be and does not obtain specific sequence or check order unsuccessfully, and experimentation is very unstable, leadsCause and can not thoroughly understand FLG. In addition, the sample number that first generation sequenator checks order is simultaneously few, and need to first pass through PCR process,Workload is large, and flux is little, and recall rate is less than 30%.
Thereby current FLG gene tester still haves much room for improvement.
Summary of the invention
The present invention is intended at least solve one of technical problem existing in prior art. For this reason, one object of the present invention is to carryGoing out one can efficient detection specific sequence, thoroughly understand FLG gene, and experimentation is stable, workload is little, fluxLarge FLG gene tester.
It should be noted that, the following work of the present invention based on inventor completes:
Inventor utilizes target area capture technique, uses special exon trapping chip to carry out high flux to hFL G geneOrder-checking. The general principle of this technology is to catch the target sequence on genome with a set of oligonucleotide probe, then uses logicalWith primer pair, these sequences that capture are carried out pcr amplification, then these amplified productions are carried out to high-flux sequence, thus identificationBase sequence in DNA sample, analyzes order-checking gained sequence information by analysis of biological information method, thereby findsThe variation information of target sequence, comprises single nucleotide variations, and insertion/deletion, repeats etc., and the method can significantly improve FLG baseThe recall rate of cause, is about 83%.
And then, according to an aspect of the present invention, the invention provides one group of probe. According to embodiments of the invention, described oneAt least a portion of group probe specificity identification FLG gene coding region, and described probe meet be selected from following condition at least itOne:
(1) length of described probe is 75bp;
(2) described probe specificity identification FLG gene coding region upstream 10bp is to the sequence between the 10bp of downstream;
(3) specific recognition GC content is higher than 0.6 and lower than the probe in 0.3 region, and multiplier is greater than 2;
(4) melting temperature of described probe and target sequence is 60-10 degree Celsius, preferably 80 degrees Celsius;
(5) described probe does not comprise hairpin structure;
(6) described probe with mate with reference to 2 sites at the most on genome;
(7) window sliding size when described probe is selected is 10bp.
Inventor is surprised to find, one group of probe of the present invention, the FLG gene coding region to its specific recognition at least onePart catch that specificity is good, sensitivity and coverage very high, can be effective to carry out catching of FLG gene coding regionDetect. According to embodiments of the invention, utilize one group of probe of the present invention capture probe specific recognition accurately and effectivelyTarget sequence---at least a portion of FLG gene coding region, thus can effectively build the nucleic acid sequencing literary composition that obtains target sequenceStorehouse, further, for high-flux sequence, can effectively determine at least one of FLG gene coding region by this nucleic acid sequencing libraryThe sequence information of part, and the order-checking degree of depth of target sequence is high, and data user rate is high, and then can realize the gene to FLGDetection. In addition, utilize said method to build high-throughput sequencing library, and and then for FLG genetic test, specificity is good,Sensitivity and coverage are high, favorable repeatability, thus can successfully understand FLG gene, find sudden change, and the method experimentProcess stabilization, cost is low, simple to operate, workload is little, easily promote.
According to a further aspect in the invention, the invention provides a kind of method that builds high-throughput sequencing library. According to of the present inventionEmbodiment, the method comprises the following steps: by genomic DNA fragment, to obtain DNA fragmentation; By described DNAFragment is carried out end reparation, to obtain the DNA fragmentation of repairing through end; At the described DNA sheet of repairing through end3 ' end of section adds base A, to obtain the DNA fragmentation with cohesive end A; Have cohesive end A's by describedDNA fragmentation is connected with joint, connects product to obtain; Utilize foregoing one group of probe to carry out described connection productScreening, to obtain object fragment, described object fragment forms described high-throughput sequencing library.
According to embodiments of the invention, the FLG gene coding region of one group of probe of the present invention to its specific recognitionAt least a portion catch that specificity is good, sensitivity and coverage very high, can be effective to carry out FLG gene coding regionAcquisition Detection. And then, utilize the method for the present invention target sequence of capture probe specific recognition accurately and effectively---At least a portion of FLG gene coding region, the nucleic acid sequencing library of establishing target sequence, and then this nucleic acid sequencing library is used forAfter high-flux sequence, can effectively determine the sequence information of at least a portion of FLG gene coding region, and target sequenceThe order-checking degree of depth is high, and data user rate is high, and then can realize the detection to FLG gene. In addition, utilize method of the present inventionBuild high-throughput sequencing library, and then for FLG genetic test, specificity is good, sensitivity and coverage high, repeatabilityGood, thus can successfully understand FLG gene, find sudden change, and the method experimentation is stable, cost is low, simple to operate,Workload is little, easily popularization.
According to another aspect of the invention, the invention provides a kind of definite sample to be tested FLG gene coding region nucleotide sequenceMethod. According to embodiments of the invention, the method comprises the following steps: according to foregoing structure high-throughput sequencing libraryMethod, the high-throughput sequencing library of structure sample to be tested, described high-throughput sequencing library comprises FLG gene coding region nucleic acid orderRow; High-throughput sequencing library to described sample to be tested checks order, to obtain sequencing result; And based on described order-checking knotReally, determine the nucleotide sequence of described sample to be tested FLG gene coding region.
Inventor finds, at least one portion of the FLG gene coding region of one group of probe of the present invention to its specific recognitionPoint catch that specificity is good, sensitivity and coverage very high, can be effective to the Acquisition Detection of FLG gene coding region.And then, utilize the method for the present invention target sequence of capture probe specific recognition accurately and effectively---FLG gene codeAt least a portion in district, the nucleic acid sequencing library of establishing target sequence, and carry out high-flux sequence, thus can be effectively definiteThe sequence information of at least a portion of FLG gene coding region, and the order-checking degree of depth of target sequence is high, and data user rate is high, entersAnd can realize the detection to FLG gene. In addition, utilize method of the present invention to build high-throughput sequencing library, and determine and treatThis FLG of test sample gene coding region nucleotide sequence, specificity is good, sensitivity and coverage high, favorable repeatability, result is accurateReliably, thereby can successfully understand FLG gene, find sudden change, and the method experimentation is stable, cost is low, operation is simpleSingle, workload is little, easily promote.
In accordance with a further aspect of the present invention, the invention provides a kind of device that builds high-throughput sequencing library. According to of the present inventionEmbodiment, this device comprises: fragmentation unit, described fragmentation unit is used for genomic DNA fragment, to obtainDNA fragmentation; End is repaired unit, and described end is repaired unit and is connected with described fragmentation unit, for by described DNA sheetDuan Jinhang end is repaired, to obtain the DNA fragmentation of repairing through end; Base A adding device, described base A addsUnit is repaired unit with described end and is connected, and adds base A for the 3 ' end at the described DNA fragmentation of repairing through end,To obtain the DNA fragmentation with cohesive end A; Joint linkage unit, described joint linkage unit has described in inciting somebody to actionThe DNA fragmentation of cohesive end A is connected with joint, connects product to obtain; And screening unit, described screening unit withDescribed joint linkage unit is connected, and is provided with foregoing one group of probe, for utilizing described one group of probe to described connectionProduct screens, to obtain object fragment, described object fragment forms described high-throughput sequencing library.
According to embodiments of the invention, the FLG gene coding region of one group of probe of the present invention to its specific recognitionAt least a portion catch that specificity is good, sensitivity and coverage very high, can be effective to carry out FLG gene coding regionAcquisition Detection. And then, utilize the device of the present invention target sequence of capture probe specific recognition accurately and effectively---At least a portion of FLG gene coding region, and the nucleic acid sequencing library of establishing target sequence, and then this nucleic acid sequencing library is usedAfter high-flux sequence, can effectively determine the sequence information of at least a portion of FLG gene coding region, and target sequenceThe order-checking degree of depth high, data user rate is high, and then can effectively realize the detection to FLG gene. In addition, utilize the present inventionDevice build high-throughput sequencing library, and then for FLG genetic test, specificity is good, sensitivity and coverage high, canReproducible, thus can successfully understand FLG gene, find sudden change, and this apparatus structure is simple, applicable experimentationStable, production cost is low, simple to operate, easily promote.
According to a further aspect in the invention, the present invention also provides a kind of definite sample to be tested FLG gene coding region nucleotide sequenceSystem. According to embodiments of the invention, this system comprises: library construction device, described library construction device is noted earlierThe device of structure high-throughput sequencing library, for building the high-throughput sequencing library of sample to be tested, described high-throughput sequencing libraryComprise FLG gene coding region nucleotide sequence; Sequencing device, described sequencing device is connected with described library construction device, forHigh-throughput sequencing library to described sample to be tested checks order, to obtain sequencing result; And analytical equipment, described analysisDevice is connected with described sequencing device, for based on described sequencing result, determines described sample to be tested FLG gene coding regionNucleotide sequence.
Inventor finds, at least one portion of the FLG gene coding region of one group of probe of the present invention to its specific recognitionPoint catch that specificity is good, sensitivity and coverage very high, can be effective to the Acquisition Detection of FLG gene coding region.And then, utilize the system of the present invention target sequence of capture probe specific recognition accurately and effectively---FLG gene codeAt least a portion in district, the nucleic acid sequencing library of establishing target sequence, and carry out high-flux sequence, thus can be effectively definiteThe sequence information of at least a portion of FLG gene coding region, and the order-checking degree of depth of target sequence is high, and data user rate is high, entersAnd can realize the detection to FLG gene. In addition, utilize system constructing high-throughput sequencing library of the present invention, and determine and treatThis FLG of test sample gene coding region nucleotide sequence, specificity is good, sensitivity and coverage high, favorable repeatability, result is accurateReliably, thereby can successfully understand FLG gene, find sudden change, and this system architecture is simple, applicable experimentation is stable,Production cost is low, simple to operate, easily popularization.
Additional aspect of the present invention and advantage in the following description part provide, and part will become bright from the following descriptionAobvious, or recognize by practice of the present invention.
Brief description of the drawings
Above-mentioned and/or additional aspect of the present invention and advantage from conjunction with below accompanying drawing to the description of embodiment, will become obviously withEasily understand, wherein:
Fig. 1 has shown according to one embodiment of the invention, the structural representation of the device of structure high-throughput sequencing library of the present inventionFigure;
Fig. 2 has shown according to one embodiment of the invention, definite sample to be tested FLG of the present invention gene coding region nucleotide sequenceThe structural representation of system;
Fig. 3 has shown according to one embodiment of the invention, the size of Insert Fragment and distribution situation;
Fig. 4 has shown according to one embodiment of the invention, the quality distribution diagram of sequencing data;
Fig. 5 has shown according to one embodiment of the invention, the average order-checking error rate assessment result figure of single base;
Fig. 6 has shown according to one embodiment of the invention, the GC content distribution situation of sample;
Fig. 7 has shown according to one embodiment of the invention, GC (AT) the content distribution situation of poor reads;
Fig. 8 has shown according to one embodiment of the invention, information analysis report the test file cut-away view;
Fig. 9 has shown according to one embodiment of the invention, the degree of depth of each CDS of target gene FLG, the statistics of coverageResult;
Figure 10 has shown according to one embodiment of the invention, single base degree of depth Poisson distribution figure of sample;
Figure 11 has shown according to one embodiment of the invention, sample FLG partial data sectional drawing after analysis of biological information;
Figure 12 has shown according to one embodiment of the invention, the schematic flow sheet that data are understood;
Figure 13 has shown according to one embodiment of the invention, the result that sudden change detects;
Figure 14 shown according to one embodiment of the invention, and FLG gene is c.5383G > the normal chain Reads comparison result of T sudden change;
Figure 15 has shown according to one embodiment of the invention, the normal chain Reads comparison result that c.3321delA FLG gene suddenlys change;And
Figure 16 has shown according to one embodiment of the invention, sample to be tested is carried out to the flow process signal of the method for FLG genetic testFigure.
Detailed description of the invention
Describe embodiments of the invention below in detail. Embodiment described below is exemplary, only for explaining the present invention, andCan not be interpreted as limitation of the present invention.
Probe
According to an aspect of the present invention, the invention provides one group of probe. According to embodiments of the invention, described one group of probeAt least a portion of specific recognition FLG gene coding region, and described probe meet be selected from following condition one of at least:
(1) length of described probe is 75bp;
(2) described probe specificity identification FLG gene coding region upstream 10bp is to the sequence between the 10bp of downstream;
(3) specific recognition GC content is higher than 0.6 and lower than the probe in 0.3 region, and multiplier is greater than 2;
(4) melting temperature of described probe and target sequence is 60-10 degree Celsius, preferably 80 degrees Celsius;
(5) described probe does not comprise hairpin structure;
(6) described probe with mate with reference to 2 sites at the most on genome;
(7) window sliding size when described probe is selected is 10bp.
According to embodiments of the invention, one group of probe of the present invention has the nucleotide sequence shown in SEQIDNO:1-1095, toolBody is as shown in the table:
Probe sequence SEQ ID NO:
ATTCTGGCCATGGGGAAGTATGTAATTTGTGTTTATGACAAATAAGAATACAAGAGACAAACAGTATTATATGAT 1
TAGTTTATTTTTAATTTAGATGCAGCTTACTATAATATTAATTATGTCCAAGATGATTTTTTGAATACAGAATAC 2
TAGAAGGATAATAGAGAAAGATGTGCTAGCCCTGATGTTGATATAGCCACTTTGGTATACAGAACTGTTTTATAT 3
ATTTTTGGCTCCTTCGATATTTCTGAAAAAGATTAATTTAGAAATTTGGGGAGTGTCTAAAACTTAAACTTTCAA 4
AAAAACATAAAACATTACTTGACCCAGAATCTCCTAAAATACTCCAGCTAGTTTTCTAAAGTTAGCTCTCCATGA 5
TTGATTTCTTCCATTTAATATTTCTGAAATATAGCGTTTAAAGATCATTACACAATAAAAATAAGCTACCACCAA 6
TACCACCAAACTAATGAAATACTATAGCATATTTTAAACAGATTGACAGGAAAAGATAACTTCCCTGAAAGTATT 7
TTTCTTGATTGAAAGTGAACTTGCTTCATTCTTCTATTCTTGGATTAATTCCTTTGCCATTAATTTCTTACTCAT 8
CATAGTAATAGTATCTCTGTGACTGACTAAATCCCAGTTGTTTCGATATATCACTAGAATGGCCACATAAACCTG 9
TAAACCTGGGTCCTTATTAATATACGTTGCATAATACCTTGGATGATCTTTACCAAACGCACTTGCTTTACAGAT 10
TCAGATCTTTCCTTGAAAACAACAGGATTGGAATTGTAACTAACACTTCCGTGCTGAGAGTGTCTAAACCCGGAT 11
ATAATCATAATCTGCACTACCATAGCTGCCATGTCTCCAAACTAAACCTGATTGACCTTTTTGCCTTTCAGTGCC 12
AGATTGATAATGATAAGAACTAGAACTGTGAGGACTGCCACGTGACTGTATTCCTGAGTGATACGCAGAATCTTG 13
ACTAAAGTGACCATGTTCCTTAGCGGTACTAGAGTCTGACTGTACAGGTGAAGACTGTACATGACTGGCTGTATC 14
CATGACTGGCTGTATCGCGGTGAGAGGATCCGGGGTGTCTGGAGCCATCTCTTGACTGCTCCCGAGAAGATCCAT 15
GGTTTCTGGAAGCAGACTCAGATCGCCTCTCAGAGTCCTCTGGGTATGCCTCACTGTCACTGTCCTGGCTAACAC 16
GGTTTCTGGAAGCAGACTCAGATCGCCTCTCAGAGTCCTCTGGGTATGCCTCACTGTCACTGTCCTGGCTAACAC 17
TAACACTGGATCCCTGGCGCCTGCTTCTCCTGGACCCCGCTGATTCACCCTGGCCGGACTGTGAGTGTCTAGAGC 18
TAACACTGGATCCCTGGCGCCTGCTTCTCCTGGACCCCGCTGATTCACCCTGGCCGGACTGTGAGTGTCTAGAGC 19
AGTGGAAGCTTCATGGTGACGCGACCCTGAGTGCCTGGAGCCGTCTCCTGACTGTTCCTCATTACGTGTTTCTCT 20
AGTGGAAGCTTCATGGTGACGCGACCCTGAGTGCCTGGAGCCGTCTCCTGACTGTTCCTCATTACGTGTTTCTCT 21
GTTTCTCTGCTTGCACTTCTGGATCCTGACTGCCCATGGGAGGCATCAGACCTTCCCTGGGATGTGGTGTGGCTG 22
GTTTCTCTGCTTGCACTTCTGGATCCTGACTGCCCATGGGAGGCATCAGACCTTCCCTGGGATGTGGTGTGGCTG 23
GTGTCCAGACCTATCTACCGATTGCTCGTGGTAGGATCCCTGTCTTCCTCCTCTCCTTGACCCCGGGTGTCCACG 24
GTGTCCAGACCTATCTACCGATTGCTCGTGGTAGGATCCCTGTCTTCCTCCTCTCCTTGACCCCGGGTGTCCACG 25
CGGGTGTCCACGAATGGTGTCCTGACCCTCTTGGGACGCTGAGTGCCTGGAGCTGTCTCGTGCCTGCTCGTGGCG 26
CGGGTGTCCACGAATGGTGTCCTGACCCTCTTGGGACGCTGAGTGCCTGGAGCTGTCTCGTGCCTGCTCGTGGCG 27
CCTTGTCTTCCTCCAGTACTGGGCCCAGCCCGTCCATGGGCAGACTCAGACTGTTCATGAGTGCTCACCTGGTAG 28
CCTTGTCTTCCTCCAGTACTGGGCCCAGCCCGTCCATGGGCAGACTCAGACTGTTCATGAGTGCTCACCTGGTAG 29
GAGGAAAGACCCTGAACGTCCAGACCTTCCTGCTGACCGGCCACGTGTGGACTCTTGGTGGCTCTGCTGATGGGG 30
GAGGAAAGACCCTGAACGTCCAGACCTTCCTGCTGACCGGCCACGTGTGGACTCTTGGTGGCTCTGCTGATGGGG 31
GCTGACTGCTGGTGGTGGGATCCATGTCTCTCTCCTGCACTTGATCTTGCCTGTTCATGGGATGATGCAGCCTGT 32
GCTGACTGCTGGTGGTGGGATCCATGTCTCTCTCCTGCACTTGATCTTGCCTGTTCATGGGATGATGCAGCCTGT 33
GATGCAGCCTGTCCACCAGAGGAATTCTCTGCATGATGAGTGCCTGATTGTCTGGAGCTCTCTGCAGAGTGCCCG 34
GATGCAGCCTGTCCACCAGAGGAATTCTCTGCATGATGAGTGCCTGATTGTCTGGAGCTCTCTGCAGAGTGCCCG 35
TGACCGGCTCTGTCTTCGTGATGGGACGTGGGGTGTCTGGAGCCATCTCTTGACTGCTCCTGAGCAGATCCACGA 36
TGACCGGCTCTGTCTTCGTGATGGGACGTGGGGTGTCTGGAGCCATCTCTTGACTGCTCCTGAGCAGATCCACGA 37
CTGGATCCCTGGTTCCTGCTTGTCCTGGGCCCCGCTGATTGTCCCTGGCCGGACTGTGAGTGTCTAGAGCTGTCC 38
CTGGATCCCTGGTTCCTGCTTGTCCTGGGCCCCGCTGATTGTCCCTGGCCGGACTGTGAGTGTCTAGAGCTGTCC 39
CCTGAGTGGAAGCTTCATGGTGATGCGACCATGAGTGCCTGGAGCCATCTCCTGATTGTTCGTCATTACGAGTTT 40
CCCTGAGTGTCCAGACCTATCTACCGATTGCTCGTAGTGGGATCCCTGCCTTCCTCTTCTGCTTGACCCCGGGTG 41
CCCTGAGTGTCCAGACCTATCTACCGATTGCTCGTAGTGGGATCCCTGCCTTCCTCTTCTGCTTGACCCCGGGTG 42
CGGGTGTCCACGAATGGTGTCCTGACCCTCTTGGGACGCTGAGTGCCTGGAGCTGTCTCGTGCCTGCTCGTGGTG 43
CGGGTGTCCACGAATGGTGTCCTGACCCTCTTGGGACGCTGAGTGCCTGGAGCTGTCTCGTGCCTGCTCGTGGTG 44
GTCTTCGTCCAGTGCTGGTCCTGGTCCGCCCATGGGCAGACTCAGACTGTTCATGAGTGCTCACCTGGTAGAGGA 45
GTCTTCGTCCAGTGCTGGTCCTGGTCCGCCCATGGGCAGACTCAGACTGTTCATGAGTGCTCACCTGGTAGAGGA 46
ACCCTGAACGTCCAGACCTTCCCCCTGACCGGTCACGTGCGGACTCTTGGTGGCTCTGCTGATGGGGCCCAGCCT 47
ACCCTGAACGTCCAGACCTTCCCCCTGACCGGTCACGTGCGGACTCTTGGTGGCTCTGCTGATGGGGCCCAGCCT 48
GGCCCAGCCTGTCCATGGCCTGACACTGACTGTGTGTCTGACTCTTCTGAATGTCCCTCACTATCACTGGCCTGA 49
GGCCCAGCCTGTCCATGGCCTGACACTGACTGTGTGTCTGACTCTTCTGAATGTCCCTCACTATCACTGGCCTGA 50
AGTGTCTACTGTCTCTGACTGCAGATGAAGCTTGTCCACGCGGAATGCCTGAGTGTCTGGAGCTGTCTGCTGACT 51
AGTGTCTACTGTCTCTGACTGCAGATGAAGCTTGTCCACGCGGAATGCCTGAGTGTCTGGAGCTGTCTGCTGACT 52
CTGTCTGCTGACTGCTGGTGGCGGGATCCGTGTCTCTCTCCTGGACTTGATCTTGCCTGTTCATGGGATGATGCA 53
CTGTCTGCTGACTGCTGGTGGCGGGATCCGTGTCTCTCTCCTGGACTTGATCTTGCCTGTTCATGGGATGATGCA 54
CTGTCCACCAGAGGAAGTCTCTGCGTGACGAGTGCCTGATTGTCTGGAGCGGTCTGCAGAGTGCCCGTGACCGGC 55
CTGTCCACCAGAGGAAGTCTCTGCGTGACGAGTGCCTGATTGTCTGGAGCGGTCTGCAGAGTGCCCGTGACCGGC 56
CGTGATGGGACCTGGGGTGTCTGGAGCCGTGCCTTGACTGCTCCTGAACAGATCCACGATGGTTTCTGGAAGCAG 57
CGTGATGGGACCTGGGGTGTCTGGAGCCGTGCCTTGACTGCTCCTGAACAGATCCACGATGGTTTCTGGAAGCAG 58
CCCAGACCACCTCTCAGAGTCTTCTGAATGTCCCTCACTGTCACTGTCCTGGCTCACACTGGATCCCTGGCGCCT 59
CCCAGACCACCTCTCAGAGTCTTCTGAATGTCCCTCACTGTCACTGTCCTGGCTCACACTGGATCCCTGGCGCCT 60
CGCCTGCTTCTCCTGGACCCCTCTGATTGTCCCTGGACTGCCTGTGAGTGTCTAGAGATGTCGGCATGAGTGGAA 61
CGCCTGCTTCTCCTGGACCCCTCTGATTGTCCCTGGACTGCCTGTGAGTGTCTAGAGATGTCGGCATGAGTGGAA 62
TGAGTGGAAGCTTCATGGTGACGTGACACTGAGTGCCTGGAGCTGTCTCCTGATTGTTCCTCATTACGTGTTGTT 63
TGAGTGGAAGCTTCATGGTGACGTGACACTGAGTGCCTGGAGCTGTCTCCTGATTGTTCCTCATTACGTGTTGTT 64
AGTGTCCAGAGCTATCTACCGAATGCTCGTGGTGGTACCCCTGCCTTCCTCCTCTGCTTGACCCCGGGTGTCCAC 65
AGTGTCCAGAGCTATCTACCGAATGCTCGTGGTGGTACCCCTGCCTTCCTCCTCTGCTTGACCCCGGGTGTCCAC 66
GGTGTCCTGACCGTATTGGGATGCTGAGTGCCTGGAGCTGTCTTGTGCCTGCTCATGGCGGGATCCTTGTCTTCC 67
GGTGTCCTGACCGTATTGGGATGCTGAGTGCCTGGAGCTGTCTTGTGCCTGCTCATGGCGGGATCCTTGTCTTCC 68
CTTGTCTTCCTCTAGTGCTGGGCCCCGTCCATCCATGGGAGGACTCAGACTGTTCATGAGTGCTCACCTGGTAGA 69
CTTGTCTTCCTCTAGTGCTGGGCCCCGTCCATCCATGGGAGGACTCAGACTGTTCATGAGTGCTCACCTGGTAGA 70
TCCAGACGTTTCCCCTGACCGGCCACGTGCGGACTCTTGGTGGCTCTGCTGATGGGACCCAGCCTGTCCGTGGGC 71
TCCAGACGTTTCCCCTGACCGGCCACGTGCGGACTCTTGGTGGCTCTGCTGATGGGACCCAGCCTGTCCGTGGGC 72
AGCTGTCTGCTGACTGCTGGTGGTGGGATCCATGTCTTTCTCCTGCACTTGATCTTGCCTGTTCATGGGATGATG 73
TGGGACCTGGGGTGTCTGGAGCCATCTCTTAGCTGCTCCTGAGCAGATCCATGATGGTTTCTGGAAGCAGACCCA 74
TGGGACCTGGGGTGTCTGGAGCCATCTCTTAGCTGCTCCTGAGCAGATCCATGATGGTTTCTGGAAGCAGACCCA 75
ACCACCTCTCAGAGTCTTCTGAATGTCCCTCACTGTCACTGTCCTGGCTCACACTGGATCCCTGGCGCCTGCTTC 76
ACCACCTCTCAGAGTCTTCTGAATGTCCCTCACTGTCACTGTCCTGGCTCACACTGGATCCCTGGCGCCTGCTTC 77
CTGGCGCCTGCTTCTCCTGGACCCCTCTGATTGTCCCTGGACTGCCTGTGAGTGTCTAGAGATGTCGGCATGAGT 78
GAAGCTTCATGGTGACGCGACCCTGAGTGCCTGGAGCCGTCTCCTGATTGTTCCTCATTACGTGTTGTTCTGCTT 79
GAAGCTTCATGGTGACGCGACCCTGAGTGCCTGGAGCCGTCTCCTGATTGTTCCTCATTACGTGTTGTTCTGCTT 80
CCCTGAGTGTCCAGAGCTATCTACCGAATGCTCGTGGTGGTACCCCTGCCTTCCTCCTCTGCTTGACCCCGGGTG 81
CCCTGAGTGTCCAGAGCTATCTACCGAATGCTCGTGGTGGTACCCCTGCCTTCCTCCTCTGCTTGACCCCGGGTG 82
TGGTGTCCTGACCGTCTTGGGATGCTGAGTGCCTGGAGCTGTCTTGTGCCTGCTCATGGCGGGATCCTTGTCTTC 83
TGGTGTCCTGACCGTCTTGGGATGCTGAGTGCCTGGAGCTGTCTTGTGCCTGCTCATGGCGGGATCCTTGTCTTC 84
TTGTCTTCCTCTAGTGCTGGGCCCCGTCCATCCATGGGAGGACTCAGACTGTTCATGAGTGCTCACCTGGTAGAG 85
GAATGTCCAGACGTTTCCCCTGACCGGCCACGTGCGGACTCTTGGTGGCTCTGCTGATGGGACCCAGCCTGTCCG 86
GAATGTCCAGACGTTTCCCCTGACCGGCCACGTGCGGACTCTTGGTGGCTCTGCTGATGGGACCCAGCCTGTCCG 87
CACTGACTGTGTGTCTGAGTCTTCTGAATGTCCCTCATTGTCACTGGCCTGACTACCACTGTACCCTCGGTGTCC 88
GGACCTGGGGTGTCTGGAGCCATCTCTTAGCTGCTCCTGAGCAGATCCATGATGGTTTCTGGAAGCAGACCCAGA 89
GGACCTGGGGTGTCTGGAGCCATCTCTTAGCTGCTCCTGAGCAGATCCATGATGGTTTCTGGAAGCAGACCCAGA 90
TGTCCTGGGCCCCTCTGATTGTCCCTGGCCCACCTGCGAGTGTCCAGAGCTGTCGGCCCGAGAGGAAGCTTCATG 91
TGTCCTGGGCCCCTCTGATTGTCCCTGGCCCACCTGCGAGTGTCCAGAGCTGTCGGCCCGAGAGGAAGCTTCATG 92
GAAGCTTCATGGTGACGCGACCCTGAGTGCCTGGAGCCGTCTCCTGATTGTTCATCGTTACGAGTTTGTCTGCTT 93
GAAGCTTCATGGTGACGCGACCCTGAGTGCCTGGAGCCGTCTCCTGATTGTTCATCGTTACGAGTTTGTCTGCTT 94
AGATCTATCTACCAATTGCTCGTAGTGGGATCCCTGCCTTCCTCCACTGCTTGACCCCGGGTGTCCATGAATGGT 95
ACCCTCTTGGGACGTTGAGTGCCTGGAGCTGTCTCGTGCCTGCTTGTGGTGGGATCCTTGTCTTCCTCCAGTGCT 96
ACCCTCTTGGGACGTTGAGTGCCTGGAGCTGTCTCGTGCCTGCTTGTGGTGGGATCCTTGTCTTCCTCCAGTGCT 97
TCTTCCTCCAGTGCTGGTCCCGGTCCGTCCATGGGCGGACTCAGACTGTTCATGAGTGCTCACCTGGTAGAGGAA 98
TCTTCCTCCAGTGCTGGTCCCGGTCCGTCCATGGGCGGACTCAGACTGTTCATGAGTGCTCACCTGGTAGAGGAA 99
CCTGAACGTCCAGACCTTCCTGCTGACCGGCCACGTGTGGACTCTTGGTGGCTCTGCTGATGGGGCCCAGCCTGT 100
CCTGAACGTCCAGACCTTCCTGCTGACCGGCCACGTGTGGACTCTTGGTGGCTCTGCTGATGGGGCCCAGCCTGT 101
GCTGACACTGACTGTGTGTCTGAGTCTTCTGAATGTCCCTCACTGTCACTGGCCTGACTACCACTGGACCCTCGG 102
GCTGACACTGACTGTGTGTCTGAGTCTTCTGAATGTCCCTCACTGTCACTGGCCTGACTACCACTGGACCCTCGG 103
TGACTGCTGGTGGTGGGATCCGTGTCTCTCTCCTGCACTTGATCTTGCCTGTTCATGGGATGATGCAGCCTGTCC 104
TGACTGCTGGTGGTGGGATCCGTGTCTCTCTCCTGCACTTGATCTTGCCTGTTCATGGGATGATGCAGCCTGTCC 105
AGAGGAATTCTCTGCATGATGAGTGCCTGATTGTCTGGAGCTCTCTGCAGAGTGCCCATGACCGGCTCTGTCTTC 106
AGAGGAATTCTCTGCATGATGAGTGCCTGATTGTCTGGAGCTCTCTGCAGAGTGCCCATGACCGGCTCTGTCTTC 107
TTCGTGATGGGACCTGGGGTGTCTGGAGCCATCTCTTGACTGCTCCTGAGCAGATCCATGATGGTTTCTGGACGC 108
TTCGTGATGGGACCTGGGGTGTCTGGAGCCATCTCTTGACTGCTCCTGAGCAGATCCATGATGGTTTCTGGACGC 109
GACCCAGACCGCCTCTCAGAATCTTCTGAGTGTCCCTCACTGTCACTGTCCTGGCTAACACTGGATCCCCGGGGC 110
GACCCAGACCGCCTCTCAGAATCTTCTGAGTGTCCCTCACTGTCACTGTCCTGGCTAACACTGGATCCCCGGGGC 111
TGCTTGTCCTGGGCCCTGATGATTGTCCCTGGCCCACCAGTGAGTGTCTAGAGCTGTCGGCCCAAGAGGAAGCTT 112
TGCTTGTCCTGGGCCCTGATGATTGTCCCTGGCCCACCAGTGAGTGTCTAGAGCTGTCGGCCCAAGAGGAAGCTT 113
GATGATGCGACCCTGAGTGCCTAGAGCCATCTCCTGATTGTTCCTTGTCATATGTTTTTCTGCTTGCACTTCTGG 114
ATCTACCGATTGCTCTTGGTGGGACCCCTGTCTTCCTCCTCTGCTTGGCCCCGGGTGTCCACGAATGGTGTCCTG 115
ATCTACCGATTGCTCTTGGTGGGACCCCTGTCTTCCTCCTCTGCTTGGCCCCGGGTGTCCACGAATGGTGTCCTG 116
TCTTGGGATGCTGAGTGCCTGGAGCTGTCTTGTGCCTGATCATAATGGGATCCTTGTCTTCCTCCAGTGCTGGGC 117
TCTTGGGATGCTGAGTGCCTGGAGCTGTCTTGTGCCTGATCATAATGGGATCCTTGTCTTCCTCCAGTGCTGGGC 118
CCTCCAGTGCTGGGCGCAGACTGTCCATGGGTGGACTCAGACTGTTCATGAGTGCTCACCTGGTAGAGGAAAGAC 119
CCTCCAGTGCTGGGCGCAGACTGTCCATGGGTGGACTCAGACTGTTCATGAGTGCTCACCTGGTAGAGGAAAGAC 120
AGCTTTCCCCTGACTGGCCACGTGCGGACTCTTTGTGGCTCTGCTGATGGGGCCCAGCTTTTCCCTGTGCTGACA 121
AGCTTTCCCCTGACTGGCCACGTGCGGACTCTTTGTGGCTCTGCTGATGGGGCCCAGCTTTTCCCTGTGCTGACA 122
CCCTGTGCTGACACTGACTGTGTGTCTGAGTCTTCTGAATGTCCCTCACTGTCACTGGCCTGACTACCACTGTAC 123
GGTGTCCACTGTCTCTGACTGCAGATGAAGCTTGTCCATGCCCAATGCCTGAGTGTCTGGAGCTGTCTGCTGACT 124
GGTGTCCACTGTCTCTGACTGCAGATGAAGCTTGTCCATGCCCAATGCCTGAGTGTCTGGAGCTGTCTGCTGACT 125
GGGATCCATGTCTTTCTCCTGCACTTGATCTTGCCTGTTCATGGGATGACGCAGCCTGTCCACGAGAGGAAGACT 126
GGGATCCATGTCTTTCTCCTGCACTTGATCTTGCCTGTTCATGGGATGACGCAGCCTGTCCACGAGAGGAAGACT 127
GTGATGGGACCCAGGGTGTCTGGAGCCATCTCTTGACTGCTCCCAAGCAGATCCAAGATGGTTTCTGGAAGCAGA 128
GTGATGGGACCCAGGGTGTCTGGAGCCATCTCTTGACTGCTCCCAAGCAGATCCAAGATGGTTTCTGGAAGCAGA 129
AGACCACCTCTCAGAGTCTTCTGAGTGTCCCTGACTGTCACTGTCCTGGCTAACACTGGATCCCTGGTTCCTGCT 130
AGACCACCTCTCAGAGTCTTCTGAGTGTCCCTGACTGTCACTGTCCTGGCTAACACTGGATCCCTGGTTCCTGCT 131
CCTGCTTGTCCTGGGCCCTGATGATTGTCCCTGGCCCACCTGCGAGTGTCTAGAGCTGTCGGCCCGAGAGGAAGC 132
CCTGCTTGTCCTGGGCCCTGATGATTGTCCCTGGCCCACCTGCGAGTGTCTAGAGCTGTCGGCCCGAGAGGAAGC 133
CTTCATGGTGACGCGACCCTGAGTGCCTGGAGCCGTCTCCTGATTGTTTCTCATTACGTGTTTGTCTGCTGACAC 134
CTTCATGGTGACGCGACCCTGAGTGCCTGGAGCCGTCTCCTGATTGTTTCTCATTACGTGTTTGTCTGCTGACAC 135
TCCTGACTGCCCACGGGAGACATCAGACCTTTCCTGGGACGTGGTGTGGCTGTGATGAGACCCTGAGTGTCCAGA 136
TCCTGACTGCCCACGGGAGACATCAGACCTTTCCTGGGACGTGGTGTGGCTGTGATGAGACCCTGAGTGTCCAGA 137
AGAACTATCTACCGATTGCTCATAGTGGGATCCCTGCCTTCCTCCTCTGCTTGACCCTGGGTGTCCACGAATGGT 138
AGAACTATCTACCGATTGCTCATAGTGGGATCCCTGCCTTCCTCCTCTGCTTGACCCTGGGTGTCCACGAATGGT 139
TCCTGACCCTCTTGGGACGCTGAGTGCCTGGAGCTGTCTCGTGCCTGCTCGTGGCGGGATCTTTGTCTTCCTCCA 140
TCCTGACCCTCTTGGGACGCTGAGTGCCTGGAGCTGTCTCGTGCCTGCTCGTGGCGGGATCTTTGTCTTCCTCCA 141
CCCTGTGCGTCCATGGGCGGACTCAGACTGTTCATGAGTGCTCACCTGGTAGAGGAAAGACCCTGAACGTCCAGA 142
CCCTGTGCGTCCATGGGCGGACTCAGACTGTTCATGAGTGCTCACCTGGTAGAGGAAAGACCCTGAACGTCCAGA 143
TTTCCCCTGACTGGCCACGTGTGGACTCTTGGTGGCTCTGCTGATGGGGCCCAGCCTGTCCGTGGGCTGACACTG 144
TTTCCCCTGACTGGCCACGTGTGGACTCTTGGTGGCTCTGCTGATGGGGCCCAGCCTGTCCGTGGGCTGACACTG 145
GCTGACACTGACTGTGTGTCTGACTCTTCTGAGTGTCCCTCGCTGTCACTGGCCTGGCTACCACTGGACCCTCGG 146
GCTGACACTGACTGTGTGTCTGACTCTTCTGAGTGTCCCTCGCTGTCACTGGCCTGGCTACCACTGGACCCTCGG 147
TCGGTTTCCACTGTCTCCGACTACAGATGAATCTTGTCTGCGCCCAGTGCCTGAGTCTGTGGAGCTGTCTGCTGA 148
TCGGTTTCCACTGTCTCCGACTACAGATGAATCTTGTCTGCGCCCAGTGCCTGAGTCTGTGGAGCTGTCTGCTGA 149
ACTGCTGGTGGCGGGATCCATGTCTTTCTCCTGGACTTGACCTTGCCTGTTCCTGGGATGATGCAGCCTGTCCAC 150
ACTGCTGGTGGCGGGATCCATGTCTTTCTCCTGGACTTGACCTTGCCTGTTCCTGGGATGATGCAGCCTGTCCAC 151
GTCTCTGCATGACGAGTGCCTGATTGTCTGGAGCTCTCTGCAGAGTGCCCATGACTGGCTCTATCTTCTTGATGG 152
GTCTCTGCATGACGAGTGCCTGATTGTCTGGAGCTCTCTGCAGAGTGCCCATGACTGGCTCTATCTTCTTGATGG 153
CTGGGGTTCCTGGAGCCATGTCTTGACTGCTCCCGAGCAGATCCATAATGGTTTCTGGAAGCCGACTCAGACCGC 154
CTGGGGTTCCTGGAGCCATGTCTTGACTGCTCCCGAGCAGATCCATAATGGTTTCTGGAAGCCGACTCAGACCGC 155
AGAGTCTTCTGAGTGTCCCTCACTGTCCCTGTCCTGACTAACACTGGATCCCTGGCGCCTGCTTGTCTTGGACCC 156
AGAGTCTTCTGAGTGTCCCTCACTGTCCCTGTCCTGACTAACACTGGATCCCTGGCGCCTGCTTGTCTTGGACCC 157
TGGACCCCGCTGATTCTCCCTGGCCCACCTGTGAGTGTCTAGAGCTGCCGGCCCGAGTGGAAGGTTCATGGTGAC 158
TGGACCCCGCTGATTCTCCCTGGCCCACCTGTGAGTGTCTAGAGCTGCCGGCCCGAGTGGAAGGTTCATGGTGAC 159
GTTCATGGTGACGTGACCCTGAGTGCCTGGAGCCGTCTCCTGATTGTTCCTCATTTCTTGTTTGCCTGCTTGCAC 160
GTTCATGGTGACGTGACCCTGAGTGCCTGGAGCCGTCTCCTGATTGTTCCTCATTTCTTGTTTGCCTGCTTGCAC 161
CCCATGGGAGGCATCAGACCTTCCCTGGGGTGTGGTGTGGCTGTGATGGTACCCTGAGTGTCCAGACCTATCTAC 162
CCCATGGGAGGCATCAGACCTTCCCTGGGGTGTGGTGTGGCTGTGATGGTACCCTGAGTGTCCAGACCTATCTAC 163
GTCCAGACCTATCTACTGATTGCTCGTGGTAGGATCCCTGCCTTCCTCCTCTGCTTGACCCCGGGTGTCCACGAA 164
GTCCAGACCTATCTACTGATTGCTCGTGGTAGGATCCCTGCCTTCCTCCTCTGCTTGACCCCGGGTGTCCACGAA 165
GGATGCTGAGTGCCTAGAGCTGTTTCGTGCCTGCTCATGGCGGGATCCTTGTCTTCCTCCAGTGCTGGGTGCAGT 166
GGATGCTGAGTGCCTAGAGCTGTTTCGTGCCTGCTCATGGCGGGATCCTTGTCTTCCTCCAGTGCTGGGTGCAGT 167
GTGCAGTCTGTCCGTGTGTGGACTCAGACTGTTCATGAGAGCTCACCTGGTAGAGGAAAGACCTTGAACGTCCAG 168
GTGCAGTCTGTCCGTGTGTGGACTCAGACTGTTCATGAGAGCTCACCTGGTAGAGGAAAGACCTTGAACGTCCAG 169
GTCCAGAGCTTTCCCCTGACTGGCCACGTGCGGACTCTTTGTGGCTCTGCTGATGGGGCCCAGCTTGTCCGTGGG 170
GTCCAGAGCTTTCCCCTGACTGGCCACGTGCGGACTCTTTGTGGCTCTGCTGATGGGGCCCAGCTTGTCCGTGGG 171
GTGTGTCTGAGTCTTCTGAATGTCCCTCACTGTTAGTGACCTGACTACCACTGGACCCTCGGTGTCCACTGTCTC 172
GTGTGTCTGAGTCTTCTGAATGTCCCTCACTGTTAGTGACCTGACTACCACTGGACCCTCGGTGTCCACTGTCTC 173
CGGTGTCCACTGTCTCTGACTGCAGATGAAGCTTGTCTGTGCCCAATGCCTGAGTGTCTGGAGCTGTCTGCTGAC 174
CGGTGTCCACTGTCTCTGACTGCAGATGAAGCTTGTCTGTGCCCAATGCCTGAGTGTCTGGAGCTGTCTGCTGAC 175
CCATGTCTTTCTCCTGGACTTGATCTTGCCTGTTCATGGGATGACACAGCCTGTCCATGAGAGGAAGACTCTGTG 176
CCATGTCTTTCTCCTGGACTTGATCTTGCCTGTTCATGGGATGACACAGCCTGTCCATGAGAGGAAGACTCTGTG 177
TGTGATGAGTGCCTGATTGTCTGGAGCTGTCTGCAGAGTGCCCGTGACTGGCTCTGTCTTCTTGATGGAACCCAG 178
TGTGATGAGTGCCTGATTGTCTGGAGCTGTCTGCAGAGTGCCCGTGACTGGCTCTGTCTTCTTGATGGAACCCAG 179
CCCAGGGTGTCTGGAGCCATCTCTTGACTGCTCCCGAGAAGATCCATGATGGTTTCTGGAAGCAGACCCAGACAA 180
CCCAGGGTGTCTGGAGCCATCTCTTGACTGCTCCCGAGAAGATCCATGATGGTTTCTGGAAGCAGACCCAGACAA 181
TCGGAGTCGTCTGAGTGTCTCTCACTGTCACTGTCCTGGCTAACACTGGATCCCTGGTGCCTGCTTGTCCTGGAC 182
TCGGAGTCGTCTGAGTGTCTCTCACTGTCACTGTCCTGGCTAACACTGGATCCCTGGTGCCTGCTTGTCCTGGAC 183
ATGATTGTTCCTGTCCCACCTGTGAGTGTCTAGAGCTGTCAGCCCAAGAGGCAGCTTCATGGTGACGTGACCCTG 184
ATGATTGTTCCTGTCCCACCTGTGAGTGTCTAGAGCTGTCAGCCCAAGAGGCAGCTTCATGGTGACGTGACCCTG 185
ACGTGACCCTGAGTGCCTGGAGCCGTCTCCTGATTGTTTGTCCTTACGAGTTTGTCTGCTTGCACTTCTGGATCC 186
ACGTGACCCTGAGTGCCTGGAGCCGTCTCCTGATTGTTTGTCCTTACGAGTTTGTCTGCTTGCACTTCTGGATCC 187
CCATGGGAGGCATCAGACCTTCCCTGGGATGTGGTGTGGCTGTGATGGGACCCTGAGTGTCCAGATCTATCTACC 188
CCATGGGAGGCATCAGACCTTCCCTGGGATGTGGTGTGGCTGTGATGGGACCCTGAGTGTCCAGATCTATCTACC 189
TGCTCATGGTGGGATCCCTGCCTTCCTCCTCTCCTTGACCCCGGGTGTGCACGAATGGTGTCCTGACCCTCTTGG 190
TGCTCATGGTGGGATCCCTGCCTTCCTCCTCTCCTTGACCCCGGGTGTGCACGAATGGTGTCCTGACCCTCTTGG 191
CGCTGAGTGCCTGGAGCTGTCTCGTGCCTGCTCGTGGTGGGATCCTTGTCTTCGTCCAGTGCTGGTCCTGGTCCG 192
CGCTGAGTGCCTGGAGCTGTCTCGTGCCTGCTCGTGGTGGGATCCTTGTCTTCGTCCAGTGCTGGTCCTGGTCCG 193
CGCCCATGGGCAGACTCAGACTGTTCATGAGTGCTCACCTGGTAGATGAAAGACCCTGAACGTCCAGACCTTCCC 194
CGCCCATGGGCAGACTCAGACTGTTCATGAGTGCTCACCTGGTAGATGAAAGACCCTGAACGTCCAGACCTTCCC 195
TTCCCCCTGACCAGTCACGTGCGGACTCTTGGTGGCTCTGCTGATGGGGCCCATCCTGTCCATGGCCTGACACTG 196
TTCCCCCTGACCAGTCACGTGCGGACTCTTGGTGGCTCTGCTGATGGGGCCCATCCTGTCCATGGCCTGACACTG 197
ACTGTGTGTCTGACTCCTCTGAATGTCCCTCACTATCACTGGCCTGACTACCACTGGACCCCCAGTGTCCACTGT 198
GACTGCAGATGAAGCTTGTCTGCGCGGAATGCCTGAGTGTCTGGAGCTGTCTGCTGACTGCTGGTGGCGGGATCC 199
GACTGCAGATGAAGCTTGTCTGCGCGGAATGCCTGAGTGTCTGGAGCTGTCTGCTGACTGCTGGTGGCGGGATCC 200
GGAAGTCTCTGCGTGAGGAGTTCCTGATTGTCTGGAGCTGTCTGCAGAGTGCCCGTGACCGGCTCTGTCTTCGTG 201
GGAAGTCTCTGCGTGAGGAGTTCCTGATTGTCTGGAGCTGTCTGCAGAGTGCCCGTGACCGGCTCTGTCTTCGTG 202
GATGGGACCTGGGGTGTCTGGAGCCATGTCTTGACTGCTCCTGAGCAGATCCACGATGGTTTCTGGAAGCAGACC 203
GATGGGACCTGGGGTGTCTGGAGCCATGTCTTGACTGCTCCTGAGCAGATCCACGATGGTTTCTGGAAGCAGACC 204
GTCTTCTGAATGTCCCTCACTGTCACTGTCCTGGCTAACACTGGATCCCTGGCGCCTGCTTGTCCTGGACCCCTC 205
GTCTTCTGAATGTCCCTCACTGTCACTGTCCTGGCTAACACTGGATCCCTGGCGCCTGCTTGTCCTGGACCCCTC 206
TGTCCTGGACCCCTCTGATTGTCCCTGGCCTGCCTGTGAGTGTCTAGAGATGTCGGCATGAGAGGAAGCTTCATG 207
TGTCCTGGACCCCTCTGATTGTCCCTGGCCTGCCTGTGAGTGTCTAGAGATGTCGGCATGAGAGGAAGCTTCATG 208
CGTGACCCTGAGTGCCTGGAGCCGTCTCTTGATTGTTCCTCATTACGTGTTGTTCTGCTTGCACTTCTGGATCCT 209
CCGATTGCTCGTGGTGGGACCCCTGCCTTCCTCTTCTGCTTGACCCCGGGTGTCCACGAATGGTGTCCTGACCAT 210
CCGATTGCTCGTGGTGGGACCCCTGCCTTCCTCTTCTGCTTGACCCCGGGTGTCCACGAATGGTGTCCTGACCAT 211
ACCATCTTGGGATGCTGAGTGCCTGGAGTTGTCTCGTGCCTGCTCATGGTGGGATCCTTGTCTTACTCCAGTGCT 212
ACCATCTTGGGATGCTGAGTGCCTGGAGTTGTCTCGTGCCTGCTCATGGTGGGATCCTTGTCTTACTCCAGTGCT 213
CTGGGCCCTGTCCATCCATGGGAGGACTCAGACTGTTTATGAGTGCTCACCTGGTAGAGGAAAGACCCTGAACGT 214
CTGGGCCCTGTCCATCCATGGGAGGACTCAGACTGTTTATGAGTGCTCACCTGGTAGAGGAAAGACCCTGAACGT 215
GTCGAGACCTTTCCCCTGACCGGTCACGTGCGGACTCTTGGTGGCTCTGCTGATGGTGACCAGCCTGTCCATGGC 216
GTCGAGACCTTTCCCCTGACCGGTCACGTGCGGACTCTTGGTGGCTCTGCTGATGGTGACCAGCCTGTCCATGGC 217
TGAGTCTTCTGAATGTCCCTCACTGTCAGTGGCCTGACTACCACTGGACCCTCGGTGTCCACTGTCTCTGACTGC 218
TGAGTCTTCTGAATGTCCCTCACTGTCAGTGGCCTGACTACCACTGGACCCTCGGTGTCCACTGTCTCTGACTGC 219
AGATGAAGCTTGTCCGTGCCCAGTGCCTGAGTGTCTGGAGCTGTCTGCTGACTGGAGCTGGTGGCGGGATCCATG 220
AGATGAAGCTTGTCCGTGCCCAGTGCCTGAGTGTCTGGAGCTGTCTGCTGACTGGAGCTGGTGGCGGGATCCATG 221
TGTCTTTCTCCTGCACTTGATCTTGCCTGTTCATGGGATGACGCAGCCTGTCCACTAGAGGAATTCTGTGTGTGA 222
CGAGTGCCTGATTTTCTGGAGCTGTCTGCAGAGTGCCCATGACCAGCTCTGTCTTCGTGATGGGACCTGGGGTGT 223
CGAGTGCCTGATTTTCTGGAGCTGTCTGCAGAGTGCCCATGACCAGCTCTGTCTTCGTGATGGGACCTGGGGTGT 224
TCAGAGTCTTCTGAGTGTCCCTGACTGTCACTGTCCTGGCTAACACTGGATCCCTGGTTCCTACTTGTCCTGGGC 225
TCAGAGTCTTCTGAGTGTCCCTGACTGTCACTGTCCTGGCTAACACTGGATCCCTGGTTCCTACTTGTCCTGGGC 226
TGTCCTGGGCCCCGATGATTGTCCCTGGCCCACCTGTGAGTGTCTAGAGCTGTCAGCCTGAGAGGAAGCTTCATG 227
TGTCCTGGGCCCCGATGATTGTCCCTGGCCCACCTGTGAGTGTCTAGAGCTGTCAGCCTGAGAGGAAGCTTCATG 228
AGTGCCTGGTGCCGTCTCCTGATTGTTCCTCATTTCGTGTTTGTCTGCTTGCACTTCTGGATCCTGACTGCCCAT 229
AGTGCCTGGTGCCGTCTCCTGATTGTTCCTCATTTCGTGTTTGTCTGCTTGCACTTCTGGATCCTGACTGCCCAT 230
GCCCATGGGAGGCATCAGACCTTCCCTGGGATGTGGTGTGGCTGTGATGGGAACCTGAGTGTCCAGACCTATTTA 231
GCCCATGGGAGGCATCAGACCTTCCCTGGGATGTGGTGTGGCTGTGATGGGAACCTGAGTGTCCAGACCTATTTA 232
TACCGATTGCTCGTGGTGGGATCCCTGCCTTCCTCCTCTGCTTGACCCCGGGTGTCCACGAATGGTGTCCTGACC 233
TACCGATTGCTCGTGGTGGGATCCCTGCCTTCCTCCTCTGCTTGACCCCGGGTGTCCACGAATGGTGTCCTGACC 234
CTCTTGGGACGCTGAATGCCTGGAGCTGTCTCGTGCCTGCTCGTGGTGCGATCCTTGTCTTCCTCCAGTGCTGGT 235
CTCTTGGGACGCTGAATGCCTGGAGCTGTCTCGTGCCTGCTCGTGGTGCGATCCTTGTCTTCCTCCAGTGCTGGT 236
TCCATGGGCAGAGTCAGGCTGTTCATGAGTGCTCACCTGGTAGAGGGAAGACCCTGAACGTCCAGACCGTTCCCC 237
TCCATGGGCAGAGTCAGGCTGTTCATGAGTGCTCACCTGGTAGAGGGAAGACCCTGAACGTCCAGACCGTTCCCC 238
GACCGGCCACGTGTGGACTCTTGGTGGCTCTGCTGTCTCAGCCCAGCCTTTCCGTGGCCTGACACTGATTGTGTG 239
GACCGGCCACGTGTGGACTCTTGGTGGCTCTGCTGTCTCAGCCCAGCCTTTCCGTGGCCTGACACTGATTGTGTG 240
TGTGTCTGAGTTTTCTGAATGTCCCTCACTGTCACTGGCCTGACTACCGCTAGACCCCCGGTGTCCACGATCGCT 241
TGTGTCTGAGTTTTCTGAATGTCCCTCACTGTCACTGGCCTGACTACCGCTAGACCCCCGGTGTCCACGATCGCT 242
GATCGCTGACTGCAGATGAAGCTTGCCCGCGCCCAGTGGCTGAGTGTCTGGAGCTGTCTGCTGACTGCTGGTGGC 243
GATCGCTGACTGCAGATGAAGCTTGCCCGCGCCCAGTGGCTGAGTGTCTGGAGCTGTCTGCTGACTGCTGGTGGC 244
CCGGATCCATGTCTTTCTCCTGGACTTGATCTTGCCTGTTCATGGGATGATGCAGTCTGTCCACGAGAGGAAGTC 245
CTGCGTGACGAGTGCCTGATTGTCTGGAGCTGTCTGCAGAGTGCCCATGACTGGCTCTGTCTTCATCATGGGACC 246
ACCTGGGGTGTCTGGAGCCATCTCTTGACTGCTCCCACGCAGATCCATGATGGTTTCTGGAAGCCGACCCAGAGT 247
ACCTGGGGTGTCTGGAGCCATCTCTTGACTGCTCCCACGCAGATCCATGATGGTTTCTGGAAGCCGACCCAGAGT 248
TGAGTGTCCCTCACTGTCCCTGTCCTGGCTAACTCTGGATCCCCTACGCTTTCTTGTCCTGGACTCCTGCAATGG 249
TGAGTGTCCCTCACTGTCCCTGTCCTGGCTAACTCTGGATCCCCTACGCTTTCTTGTCCTGGACTCCTGCAATGG 250
GGACTCCTGCAATGGTACCTGGCTTGTATTTTCATGTCTTGACCTGTTCACTTGAGATGATGATTTGCCATCAGA 251
CTTGATCTTTCATATATTTTGTTTTCTTCTAATAGACTATCAGTGGTGTCATAGGCTTCATCCTGGATTGTGTAA 252
GTGGCAATATGGCCTGATTGTATCCATTTTTGAGTCATTCTTCCTGTATTTTCATAATCATATACTCCTTCTTCA 253
TGTCTTCTTTCTCTTCAAGTCTTTCACTTAGCCTCTTCCTATTGTCTCCTAATCTAGTATTTTCAGTCTTGTTTT 254
TTTTCTCTTTTTTACTTGAGTTATGATGGTTTTTTCCATATTCTTCTTCTCTATGAGTAGGTGAATATCCTTTTC 255
CTTTTCTTTCTTTTTTTTCAGAACTAGATTCATGCCTTTTCCCCCCTGTTTCTCTTGGGCTCTTGGATCTTCCCT 256
GTTTCTTCTTTCCAGACTTGAGGGTCTTTTTCTGTTTTCTTTGTTTTCTTCCTGTTTATTATCTTCATGTTTATC 257
ATGTTTATCATGATGACTGTGCTTTCTGTGCTTGTGTCCTGATATCGGTAAATTCTCTTTTCTGGTAGACTCATA 258
TAATATGCTTGAGCCAACTTGAATACCATCAGAAGAAACTCAGTGAAGTCAATTTTCTTGTTGTGGTCTATATCC 259
AAGTGATCCATGAAGACATCAACCATATCTGGGTCATCTGGATTCTGTACAGAGGGAAGTCACAGAGGGAGACTG 260
TAATTTGTGTTTATGACAAATAAGAATACAAGAGACAAACAGTATTATATGATGAATAGTTTATTTTTAATTTAG 261
TTATTTTTAATTTAGATGCAGCTTACTATAATATTAATTATGTCCAAGATGATTTTTTGAATACAGAATACTAGA 262
GAAGGATAATAGAGAAAGATGTGCTAGCCCTGATGTTGATATAGCCACTTTGGTATACAGAACTGTTTTATATTT 263
CTTCGATATTTCTGAAAAAGATTAATTTAGAAATTTGGGGAGTGTCTAAAACTTAAACTTTCAAAAACATAAAAC 264
AAAAACATAAAACATTACTTGACCCAGAATCTCCTAAAATACTCCAGCTAGTTTTCTAAAGTTAGCTCTCCATGA 265
TTTAATATTTCTGAAATATAGCGTTTAAAGATCATTACACAATAAAAATAAGCTACCACCAAACTAATGAAATAC 266
ACTAATGAAATACTATAGCATATTTTAAACAGATTGACAGGAAAAGATAACTTCCCTGAAAGTATTATGAAGTTT 267
TTGATTGAAAGTGAACTTGCTTCATTCTTCTATTCTTGGATTAATTCCTTTGCCATTAATTTCTTACTCATAGTA 268
ATCTCTGTGACTGACTAAATCCCAGTTGTTTCGATATATCACTAGAATGGCCACATAAACCTGGGTCCTTATTAA 269
TCCTTATTAATATACGTTGCATAATACCTTGGATGATCTTTACCAAACGCACTTGCTTTACAGATATCAGATCTT 270
AGGATTGGAATTGTAACTAACACTTCCGTGCTGAGAGTGTCTAAACCCGGATTCACCATAATCATAATCTGCACT 271
TAGCTGCCATGTCTCCAAACTAAACCTGATTGACCTTTTTGCCTTTCAGTGCCCTCAGATTGATAATGATAAGAA 272
AATGATAAGAACTAGAACTGTGAGGACTGCCACGTGACTGTATTCCTGAGTGATACGCAGAATCTTGTGAAAGAC 273
AGACTACTAAAGTGACCATGTTCCTTAGCGGTACTAGAGTCTGACTGTACAGGTGAAGACTGTACATGACTGGCT 274
CGCGGTGAGAGGATCCGGGGTGTCTGGAGCCATCTCTTGACTGCTCCCGAGAAGATCCATGATGGTTTCTGGAAG 275
CTGGAAGCAGACTCAGATCGCCTCTCAGAGTCCTCTGGGTATGCCTCACTGTCACTGTCCTGGCTAACACTGGAT 276
CTGGAAGCAGACTCAGATCGCCTCTCAGAGTCCTCTGGGTATGCCTCACTGTCACTGTCCTGGCTAACACTGGAT 277
CTGCTTCTCCTGGACCCCGCTGATTCACCCTGGCCGGACTGTGAGTGTCTAGAGCTGTCAGCCTGAGTGGAAGCT 278
CTGCTTCTCCTGGACCCCGCTGATTCACCCTGGCCGGACTGTGAGTGTCTAGAGCTGTCAGCCTGAGTGGAAGCT 279
GTGGAAGCTTCATGGTGACGCGACCCTGAGTGCCTGGAGCCGTCTCCTGACTGTTCCTCATTACGTGTTTCTCTG 280
GTGGAAGCTTCATGGTGACGCGACCCTGAGTGCCTGGAGCCGTCTCCTGACTGTTCCTCATTACGTGTTTCTCTG 281
CCCTGAGTGTCCAGACCTATCTACCGATTGCTCGTGGTAGGATCCCTGTCTTCCTCCTCTCCTTGACCCCGGGTG 282
CCCTGAGTGTCCAGACCTATCTACCGATTGCTCGTGGTAGGATCCCTGTCTTCCTCCTCTCCTTGACCCCGGGTG 283
CCTGACCCTCTTGGGACGCTGAGTGCCTGGAGCTGTCTCGTGCCTGCTCGTGGCGGGATCCTTGTCTTCCTCCAG 284
CCTGACCCTCTTGGGACGCTGAGTGCCTGGAGCTGTCTCGTGCCTGCTCGTGGCGGGATCCTTGTCTTCCTCCAG 285
CTTCCTCCAGTACTGGGCCCAGCCCGTCCATGGGCAGACTCAGACTGTTCATGAGTGCTCACCTGGTAGAGGAAA 286
CTTCCTCCAGTACTGGGCCCAGCCCGTCCATGGGCAGACTCAGACTGTTCATGAGTGCTCACCTGGTAGAGGAAA 287
GACCCTGAACGTCCAGACCTTCCTGCTGACCGGCCACGTGTGGACTCTTGGTGGCTCTGCTGATGGGGCCCAGCC 288
GACCCTGAACGTCCAGACCTTCCTGCTGACCGGCCACGTGTGGACTCTTGGTGGCTCTGCTGATGGGGCCCAGCC 289
GACACTGACTGTGTGTCTGAGTCTTCTGAATGTCCCTCACTGTCACTGGCCTGACTACCACTGGACCCTCGGTGT 290
GACACTGACTGTGTGTCTGAGTCTTCTGAATGTCCCTCACTGTCACTGGCCTGACTACCACTGGACCCTCGGTGT 291
GACTGCTGGTGGTGGGATCCATGTCTCTCTCCTGCACTTGATCTTGCCTGTTCATGGGATGATGCAGCCTGTCCA 292
GACTGCTGGTGGTGGGATCCATGTCTCTCTCCTGCACTTGATCTTGCCTGTTCATGGGATGATGCAGCCTGTCCA 293
AGGAATTCTCTGCATGATGAGTGCCTGATTGTCTGGAGCTCTCTGCAGAGTGCCCGTGACCGGCTCTGTCTTCGT 294
AGGAATTCTCTGCATGATGAGTGCCTGATTGTCTGGAGCTCTCTGCAGAGTGCCCGTGACCGGCTCTGTCTTCGT 295
CTGTCTTCGTGATGGGACGTGGGGTGTCTGGAGCCATCTCTTGACTGCTCCTGAGCAGATCCACGATGGTTTCTG 296
CTGTCTTCGTGATGGGACGTGGGGTGTCTGGAGCCATCTCTTGACTGCTCCTGAGCAGATCCACGATGGTTTCTG 297
CAGACCACCTCTCAGAGTCTTCTGAGTGTCCCTGACTGTCACTGTCCTGGCTAACACTGGATCCCTGGTTCCTGC 298
CAGACCACCTCTCAGAGTCTTCTGAGTGTCCCTGACTGTCACTGTCCTGGCTAACACTGGATCCCTGGTTCCTGC 299
CTGCTTGTCCTGGGCCCCGCTGATTGTCCCTGGCCGGACTGTGAGTGTCTAGAGCTGTCCGCCTGAGTGGAAGCT 300
CTGCTTGTCCTGGGCCCCGCTGATTGTCCCTGGCCGGACTGTGAGTGTCTAGAGCTGTCCGCCTGAGTGGAAGCT 301
CTTCATGGTGATGCGACCATGAGTGCCTGGAGCCATCTCCTGATTGTTCGTCATTACGAGTTTGTCTGCTGGCAC 302
CTTCATGGTGATGCGACCATGAGTGCCTGGAGCCATCTCCTGATTGTTCGTCATTACGAGTTTGTCTGCTGGCAC 303
ACCGATTGCTCGTAGTGGGATCCCTGCCTTCCTCTTCTGCTTGACCCCGGGTGTCCACGAATGGTGTCCTGACCC 304
ACCGATTGCTCGTAGTGGGATCCCTGCCTTCCTCTTCTGCTTGACCCCGGGTGTCCACGAATGGTGTCCTGACCC 305
GTCCTGGTCCGCCCATGGGCAGACTCAGACTGTTCATGAGTGCTCACCTGGTAGAGGAAAGACCCTGAACGTCCA 306
GTCCTGGTCCGCCCATGGGCAGACTCAGACTGTTCATGAGTGCTCACCTGGTAGAGGAAAGACCCTGAACGTCCA 307
ACCCTGAACGTCCAGACCTTCCCCCTGACCGGTCACGTGCGGACTCTTGGTGGCTCTGCTGATGGGGCCCAGCCT 308
ACCCTGAACGTCCAGACCTTCCCCCTGACCGGTCACGTGCGGACTCTTGGTGGCTCTGCTGATGGGGCCCAGCCT 309
CTGACACTGACTGTGTGTCTGACTCTTCTGAATGTCCCTCACTATCACTGGCCTGACTACCACTGGACCCCCAGT 310
CTGACACTGACTGTGTGTCTGACTCTTCTGAATGTCCCTCACTATCACTGGCCTGACTACCACTGGACCCCCAGT 311
TGTCTCTGACTGCAGATGAAGCTTGTCCACGCGGAATGCCTGAGTGTCTGGAGCTGTCTGCTGACTGCTGGTGGC 312
TGTCTCTGACTGCAGATGAAGCTTGTCCACGCGGAATGCCTGAGTGTCTGGAGCTGTCTGCTGACTGCTGGTGGC 313
TGCTGACTGCTGGTGGCGGGATCCGTGTCTCTCTCCTGGACTTGATCTTGCCTGTTCATGGGATGATGCAGCCTG 314
TGCTGACTGCTGGTGGCGGGATCCGTGTCTCTCTCCTGGACTTGATCTTGCCTGTTCATGGGATGATGCAGCCTG 315
CTCTGCGTGACGAGTGCCTGATTGTCTGGAGCGGTCTGCAGAGTGCCCGTGACCGGCTCTGTCTTCGTGATGGGA 316
CTCTGCGTGACGAGTGCCTGATTGTCTGGAGCGGTCTGCAGAGTGCCCGTGACCGGCTCTGTCTTCGTGATGGGA 317
TGATGGGACCTGGGGTGTCTGGAGCCGTGCCTTGACTGCTCCTGAACAGATCCACGATGGTTTCTGGAAGCAGAC 318
TGATGGGACCTGGGGTGTCTGGAGCCGTGCCTTGACTGCTCCTGAACAGATCCACGATGGTTTCTGGAAGCAGAC 319
ACCTCTCAGAGTCTTCTGAATGTCCCTCACTGTCACTGTCCTGGCTCACACTGGATCCCTGGCGCCTGCTTCTCC 320
ACCTCTCAGAGTCTTCTGAATGTCCCTCACTGTCACTGTCCTGGCTCACACTGGATCCCTGGCGCCTGCTTCTCC 321
CGCCTGCTTCTCCTGGACCCCTCTGATTGTCCCTGGACTGCCTGTGAGTGTCTAGAGATGTCGGCATGAGTGGAA 322
CGCCTGCTTCTCCTGGACCCCTCTGATTGTCCCTGGACTGCCTGTGAGTGTCTAGAGATGTCGGCATGAGTGGAA 323
CACTGAGTGCCTGGAGCTGTCTCCTGATTGTTCCTCATTACGTGTTGTTCTGCTTGCACTTCTGGATCCTGACTG 324
CACTGAGTGCCTGGAGCTGTCTCCTGATTGTTCCTCATTACGTGTTGTTCTGCTTGCACTTCTGGATCCTGACTG 325
GAATGCTCGTGGTGGTACCCCTGCCTTCCTCCTCTGCTTGACCCCGGGTGTCCACGAATGGTGTCCTGACCGTAT 326
GAATGCTCGTGGTGGTACCCCTGCCTTCCTCCTCTGCTTGACCCCGGGTGTCCACGAATGGTGTCCTGACCGTAT 327
CTGACCGTATTGGGATGCTGAGTGCCTGGAGCTGTCTTGTGCCTGCTCATGGCGGGATCCTTGTCTTCCTCTAGT 328
CTGACCGTATTGGGATGCTGAGTGCCTGGAGCTGTCTTGTGCCTGCTCATGGCGGGATCCTTGTCTTCCTCTAGT 329
TTTCCCCTGACCGGCCACGTGCGGACTCTTGGTGGCTCTGCTGATGGGACCCAGCCTGTCCGTGGGCTGACACTG 330
TTTCCCCTGACCGGCCACGTGCGGACTCTTGGTGGCTCTGCTGATGGGACCCAGCCTGTCCGTGGGCTGACACTG 331
TGACTGTGTGTCTGAGTCTTCTGAATGTCCCTCATTGTCACTGGCCTGACTACCACTGTACCCTCGGTGTCCACT 332
CTGGGGTGTCTGGAGCCATCTCTTAGCTGCTCCTGAGCAGATCCATGATGGTTTCTGGAAGCAGACCCAGACCAC 333
CTGGGGTGTCTGGAGCCATCTCTTAGCTGCTCCTGAGCAGATCCATGATGGTTTCTGGAAGCAGACCCAGACCAC 334
AGAGTCTTCTGAATGTCCCTCACTGTCACTGTCCTGGCTCACACTGGATCCCTGGCGCCTGCTTCTCCTGGACCC 335
AGAGTCTTCTGAATGTCCCTCACTGTCACTGTCCTGGCTCACACTGGATCCCTGGCGCCTGCTTCTCCTGGACCC 336
TCCTGGACCCCTCTGATTGTCCCTGGACTGCCTGTGAGTGTCTAGAGATGTCGGCATGAGTGGAAGCTTCATGGT 337
TCCTGGACCCCTCTGATTGTCCCTGGACTGCCTGTGAGTGTCTAGAGATGTCGGCATGAGTGGAAGCTTCATGGT 338
ACCCTGAGTGCCTGGAGCCGTCTCCTGATTGTTCCTCATTACGTGTTGTTCTGCTTGCACTTCTGGATCCTGACT 339
ACCCTGAGTGCCTGGAGCCGTCTCCTGATTGTTCCTCATTACGTGTTGTTCTGCTTGCACTTCTGGATCCTGACT 340
GAATGCTCGTGGTGGTACCCCTGCCTTCCTCCTCTGCTTGACCCCGGGTGTCCACGAATGGTGTCCTGACCGTCT 341
GAATGCTCGTGGTGGTACCCCTGCCTTCCTCCTCTGCTTGACCCCGGGTGTCCACGAATGGTGTCCTGACCGTCT 342
GACCGTCTTGGGATGCTGAGTGCCTGGAGCTGTCTTGTGCCTGCTCATGGCGGGATCCTTGTCTTCCTCTAGTGC 343
GACCGTCTTGGGATGCTGAGTGCCTGGAGCTGTCTTGTGCCTGCTCATGGCGGGATCCTTGTCTTCCTCTAGTGC 344
AGACGTTTCCCCTGACCGGCCACGTGCGGACTCTTGGTGGCTCTGCTGATGGGACCCAGCCTGTCCGTGGGCTGA 345
AGACGTTTCCCCTGACCGGCCACGTGCGGACTCTTGGTGGCTCTGCTGATGGGACCCAGCCTGTCCGTGGGCTGA 346
TGTGTCTGAGTCTTCTGAATGTCCCTCATTGTCACTGGCCTGACTACCACTGTACCCTCGGTGTCCACTGTCTCT 347
TGGGACCTGGGGTGTCTGGAGCCATCTCTTAGCTGCTCCTGAGCAGATCCATGATGGTTTCTGGAAGCAGACCCA 348
TGGGACCTGGGGTGTCTGGAGCCATCTCTTAGCTGCTCCTGAGCAGATCCATGATGGTTTCTGGAAGCAGACCCA 349
TCTTCTGAGTGTCCCTGACTGTCACTGTCCTGGCTAAAACTGGATCCCCAGTTCCTGCTTGTCCTGGGCCCCTCT 350
TCTTCTGAGTGTCCCTGACTGTCACTGTCCTGGCTAAAACTGGATCCCCAGTTCCTGCTTGTCCTGGGCCCCTCT 351
TGTCCTGGGCCCCTCTGATTGTCCCTGGCCCACCTGCGAGTGTCCAGAGCTGTCGGCCCGAGAGGAAGCTTCATG 352
TGTCCTGGGCCCCTCTGATTGTCCCTGGCCCACCTGCGAGTGTCCAGAGCTGTCGGCCCGAGAGGAAGCTTCATG 353
CCTGAGTGCCTGGAGCCGTCTCCTGATTGTTCATCGTTACGAGTTTGTCTGCTTGCACTTCTGGATCCTGAGTGC 354
CCTGAGTGCCTGGAGCCGTCTCCTGATTGTTCATCGTTACGAGTTTGTCTGCTTGCACTTCTGGATCCTGAGTGC 355
GAGGCATCAGACCTTCCCTGGGATGTGGTGTGGCTGTGATGAGACCCTGAGTGTCCAGATCTATCTACCAATTGC 356
ATCTACCAATTGCTCGTAGTGGGATCCCTGCCTTCCTCCACTGCTTGACCCCGGGTGTCCATGAATGGTGTCCTG 357
ATCTACCAATTGCTCGTAGTGGGATCCCTGCCTTCCTCCACTGCTTGACCCCGGGTGTCCATGAATGGTGTCCTG 358
GTCCTGACCCTCTTGGGACGTTGAGTGCCTGGAGCTGTCTCGTGCCTGCTTGTGGTGGGATCCTTGTCTTCCTCC 359
GTCCTGACCCTCTTGGGACGTTGAGTGCCTGGAGCTGTCTCGTGCCTGCTTGTGGTGGGATCCTTGTCTTCCTCC 360
TCCATGGGCGGACTCAGACTGTTCATGAGTGCTCACCTGGTAGAGGAAAGACCCTGAACGTCCAGACCTTCCTGC 361
TGACCGGCCACGTGTGGACTCTTGGTGGCTCTGCTGATGGGGCCCAGCCTGTCCGTGGGCTGACACTGACTGTGT 362
TGACCGGCCACGTGTGGACTCTTGGTGGCTCTGCTGATGGGGCCCAGCCTGTCCGTGGGCTGACACTGACTGTGT 363
GCTGACACTGACTGTGTGTCTGAGTCTTCTGAATGTCCCTCACTGTCACTGGCCTGACTACCACTGGACCCTCGG 364
GCTGACACTGACTGTGTGTCTGAGTCTTCTGAATGTCCCTCACTGTCACTGGCCTGACTACCACTGGACCCTCGG 365
CTGGTGGTGGGATCCGTGTCTCTCTCCTGCACTTGATCTTGCCTGTTCATGGGATGATGCAGCCTGTCCACCAGA 366
CTGGTGGTGGGATCCGTGTCTCTCTCCTGCACTTGATCTTGCCTGTTCATGGGATGATGCAGCCTGTCCACCAGA 367
ATGAGTGCCTGATTGTCTGGAGCTCTCTGCAGAGTGCCCATGACCGGCTCTGTCTTCGTGATGGGACCTGGGGTG 368
ATGAGTGCCTGATTGTCTGGAGCTCTCTGCAGAGTGCCCATGACCGGCTCTGTCTTCGTGATGGGACCTGGGGTG 369
TGGAGCCATCTCTTGACTGCTCCTGAGCAGATCCATGATGGTTTCTGGACGCAGACCCAGACCGCCTCTCAGAAT 370
TGGAGCCATCTCTTGACTGCTCCTGAGCAGATCCATGATGGTTTCTGGACGCAGACCCAGACCGCCTCTCAGAAT 371
CTTCTGAGTGTCCCTCACTGTCACTGTCCTGGCTAACACTGGATCCCCGGGGCCTGCTTGTCCTGGGCCCTGATG 372
CTTCTGAGTGTCCCTCACTGTCACTGTCCTGGCTAACACTGGATCCCCGGGGCCTGCTTGTCCTGGGCCCTGATG 373
GATTGTCCCTGGCCCACCAGTGAGTGTCTAGAGCTGTCGGCCCAAGAGGAAGCTTCATGATGATGCGACCCTGAG 374
GATTGTCCCTGGCCCACCAGTGAGTGTCTAGAGCTGTCGGCCCAAGAGGAAGCTTCATGATGATGCGACCCTGAG 375
CCTAGAGCCATCTCCTGATTGTTCCTTGTCATATGTTTTTCTGCTTGCACTTCTGGATCCTGACTGCCCACGGGA 376
CCTAGAGCCATCTCCTGATTGTTCCTTGTCATATGTTTTTCTGCTTGCACTTCTGGATCCTGACTGCCCACGGGA 377
AGGCATCAGACCTTCCCTGGGATGTGGTGTGGCTGTGATGAGACCCTGAGTGTCCAGACCTATCTACCGATTGCT 378
AGGCATCAGACCTTCCCTGGGATGTGGTGTGGCTGTGATGAGACCCTGAGTGTCCAGACCTATCTACCGATTGCT 379
TACCGATTGCTCTTGGTGGGACCCCTGTCTTCCTCCTCTGCTTGGCCCCGGGTGTCCACGAATGGTGTCCTGACC 380
TACCGATTGCTCTTGGTGGGACCCCTGTCTTCCTCCTCTGCTTGGCCCCGGGTGTCCACGAATGGTGTCCTGACC 381
ACCCTCTTGGGATGCTGAGTGCCTGGAGCTGTCTTGTGCCTGATCATAATGGGATCCTTGTCTTCCTCCAGTGCT 382
ACCCTCTTGGGATGCTGAGTGCCTGGAGCTGTCTTGTGCCTGATCATAATGGGATCCTTGTCTTCCTCCAGTGCT 383
GACTGTCCATGGGTGGACTCAGACTGTTCATGAGTGCTCACCTGGTAGAGGAAAGACCCTGAACGTCCAGAGCTT 384
TGGCCACGTGCGGACTCTTTGTGGCTCTGCTGATGGGGCCCAGCTTTTCCCTGTGCTGACACTGACTGTGTGTCT 385
TGGCCACGTGCGGACTCTTTGTGGCTCTGCTGATGGGGCCCAGCTTTTCCCTGTGCTGACACTGACTGTGTGTCT 386
TGACACTGACTGTGTGTCTGAGTCTTCTGAATGTCCCTCACTGTCACTGGCCTGACTACCACTGTACCCTCGGTG 387
TGACACTGACTGTGTGTCTGAGTCTTCTGAATGTCCCTCACTGTCACTGGCCTGACTACCACTGTACCCTCGGTG 388
CCATGTCTTTCTCCTGCACTTGATCTTGCCTGTTCATGGGATGACGCAGCCTGTCCACGAGAGGAAGACTCTGTG 389
CCATGTCTTTCTCCTGCACTTGATCTTGCCTGTTCATGGGATGACGCAGCCTGTCCACGAGAGGAAGACTCTGTG 390
AGTGCCTGATTGTCTGGAGCTGTCTGCAGAGTGCCCGTGACCGGCTCTGTCTTCGTGATGGGACCCAGGGTGTCT 391
AGTGCCTGATTGTCTGGAGCTGTCTGCAGAGTGCCCGTGACCGGCTCTGTCTTCGTGATGGGACCCAGGGTGTCT 392
CCCAGGGTGTCTGGAGCCATCTCTTGACTGCTCCCAAGCAGATCCAAGATGGTTTCTGGAAGCAGACCCAGACCA 393
CCCAGGGTGTCTGGAGCCATCTCTTGACTGCTCCCAAGCAGATCCAAGATGGTTTCTGGAAGCAGACCCAGACCA 394
ACCTCTCAGAGTCTTCTGAGTGTCCCTGACTGTCACTGTCCTGGCTAACACTGGATCCCTGGTTCCTGCTTGTCC 395
ACCTCTCAGAGTCTTCTGAGTGTCCCTGACTGTCACTGTCCTGGCTAACACTGGATCCCTGGTTCCTGCTTGTCC 396
TGTCCCTGGCCCACCTGCGAGTGTCTAGAGCTGTCGGCCCGAGAGGAAGCTTCATGGTGACGCGACCCTGAGTGC 397
TGTCCCTGGCCCACCTGCGAGTGTCTAGAGCTGTCGGCCCGAGAGGAAGCTTCATGGTGACGCGACCCTGAGTGC 398
AGTGCCTGGAGCCGTCTCCTGATTGTTTCTCATTACGTGTTTGTCTGCTGACACTTCTGGATCCTGACTGCCCAC 399
AGTGCCTGGAGCCGTCTCCTGATTGTTTCTCATTACGTGTTTGTCTGCTGACACTTCTGGATCCTGACTGCCCAC 400
CTGCCCACGGGAGACATCAGACCTTTCCTGGGACGTGGTGTGGCTGTGATGAGACCCTGAGTGTCCAGAACTATC 401
CTGCCCACGGGAGACATCAGACCTTTCCTGGGACGTGGTGTGGCTGTGATGAGACCCTGAGTGTCCAGAACTATC 402
ATCTACCGATTGCTCATAGTGGGATCCCTGCCTTCCTCCTCTGCTTGACCCTGGGTGTCCACGAATGGTGTCCTG 403
ATCTACCGATTGCTCATAGTGGGATCCCTGCCTTCCTCCTCTGCTTGACCCTGGGTGTCCACGAATGGTGTCCTG 404
GACCCTCTTGGGACGCTGAGTGCCTGGAGCTGTCTCGTGCCTGCTCGTGGCGGGATCTTTGTCTTCCTCCAGTGC 405
GACCCTCTTGGGACGCTGAGTGCCTGGAGCTGTCTCGTGCCTGCTCGTGGCGGGATCTTTGTCTTCCTCCAGTGC 406
TGGGCCCTGTGCGTCCATGGGCGGACTCAGACTGTTCATGAGTGCTCACCTGGTAGAGGAAAGACCCTGAACGTC 407
TGGGCCCTGTGCGTCCATGGGCGGACTCAGACTGTTCATGAGTGCTCACCTGGTAGAGGAAAGACCCTGAACGTC 408
GACCTTTCCCCTGACTGGCCACGTGTGGACTCTTGGTGGCTCTGCTGATGGGGCCCAGCCTGTCCGTGGGCTGAC 409
GACCTTTCCCCTGACTGGCCACGTGTGGACTCTTGGTGGCTCTGCTGATGGGGCCCAGCCTGTCCGTGGGCTGAC 410
CTGACTCTTCTGAGTGTCCCTCGCTGTCACTGGCCTGGCTACCACTGGACCCTCGGTTTCCACTGTCTCCGACTA 411
CTGACTCTTCTGAGTGTCCCTCGCTGTCACTGGCCTGGCTACCACTGGACCCTCGGTTTCCACTGTCTCCGACTA 412
TCTCCGACTACAGATGAATCTTGTCTGCGCCCAGTGCCTGAGTCTGTGGAGCTGTCTGCTGACTGCTGGTGGCGG 413
TCTCCGACTACAGATGAATCTTGTCTGCGCCCAGTGCCTGAGTCTGTGGAGCTGTCTGCTGACTGCTGGTGGCGG 414
TCTTTCTCCTGGACTTGACCTTGCCTGTTCCTGGGATGATGCAGCCTGTCCACCAGAGGAAGTCTCTGCATGACG 415
TCTTTCTCCTGGACTTGACCTTGCCTGTTCCTGGGATGATGCAGCCTGTCCACCAGAGGAAGTCTCTGCATGACG 416
TCTGCATGACGAGTGCCTGATTGTCTGGAGCTCTCTGCAGAGTGCCCATGACTGGCTCTATCTTCTTGATGGGAC 417
TCTGCATGACGAGTGCCTGATTGTCTGGAGCTCTCTGCAGAGTGCCCATGACTGGCTCTATCTTCTTGATGGGAC 418
GGTTCCTGGAGCCATGTCTTGACTGCTCCCGAGCAGATCCATAATGGTTTCTGGAAGCCGACTCAGACCGCCTCT 419
GGTTCCTGGAGCCATGTCTTGACTGCTCCCGAGCAGATCCATAATGGTTTCTGGAAGCCGACTCAGACCGCCTCT 420
AGTCTTCTGAGTGTCCCTCACTGTCCCTGTCCTGACTAACACTGGATCCCTGGCGCCTGCTTGTCTTGGACCCCG 421
AGTCTTCTGAGTGTCCCTCACTGTCCCTGTCCTGACTAACACTGGATCCCTGGCGCCTGCTTGTCTTGGACCCCG 422
TGGACCCCGCTGATTCTCCCTGGCCCACCTGTGAGTGTCTAGAGCTGCCGGCCCGAGTGGAAGGTTCATGGTGAC 423
TGGACCCCGCTGATTCTCCCTGGCCCACCTGTGAGTGTCTAGAGCTGCCGGCCCGAGTGGAAGGTTCATGGTGAC 424
CTGAGTGCCTGGAGCCGTCTCCTGATTGTTCCTCATTTCTTGTTTGCCTGCTTGCACTTCTGGGTCCTGACTGCC 425
CTGAGTGCCTGGAGCCGTCTCCTGATTGTTCCTCATTTCTTGTTTGCCTGCTTGCACTTCTGGGTCCTGACTGCC 426
TCAGACCTTCCCTGGGGTGTGGTGTGGCTGTGATGGTACCCTGAGTGTCCAGACCTATCTACTGATTGCTCGTGG 427
TCAGACCTTCCCTGGGGTGTGGTGTGGCTGTGATGGTACCCTGAGTGTCCAGACCTATCTACTGATTGCTCGTGG 428
TACTGATTGCTCGTGGTAGGATCCCTGCCTTCCTCCTCTGCTTGACCCCGGGTGTCCACGAATGGTGTCCTGACC 429
TACTGATTGCTCGTGGTAGGATCCCTGCCTTCCTCCTCTGCTTGACCCCGGGTGTCCACGAATGGTGTCCTGACC 430
TTGGGATGCTGAGTGCCTAGAGCTGTTTCGTGCCTGCTCATGGCGGGATCCTTGTCTTCCTCCAGTGCTGGGTGC 431
TTGGGATGCTGAGTGCCTAGAGCTGTTTCGTGCCTGCTCATGGCGGGATCCTTGTCTTCCTCCAGTGCTGGGTGC 432
TCTGTCCGTGTGTGGACTCAGACTGTTCATGAGAGCTCACCTGGTAGAGGAAAGACCTTGAACGTCCAGAGCTTT 433
TCTGTCCGTGTGTGGACTCAGACTGTTCATGAGAGCTCACCTGGTAGAGGAAAGACCTTGAACGTCCAGAGCTTT 434
AGAGCTTTCCCCTGACTGGCCACGTGCGGACTCTTTGTGGCTCTGCTGATGGGGCCCAGCTTGTCCGTGGGCTGA 435
AGAGCTTTCCCCTGACTGGCCACGTGCGGACTCTTTGTGGCTCTGCTGATGGGGCCCAGCTTGTCCGTGGGCTGA 436
GTGTGTCTGAGTCTTCTGAATGTCCCTCACTGTTAGTGACCTGACTACCACTGGACCCTCGGTGTCCACTGTCTC 437
GTGTGTCTGAGTCTTCTGAATGTCCCTCACTGTTAGTGACCTGACTACCACTGGACCCTCGGTGTCCACTGTCTC 438
GTCTTTCTCCTGGACTTGATCTTGCCTGTTCATGGGATGACACAGCCTGTCCATGAGAGGAAGACTCTGTGTGAT 439
TGAGTGCCTGATTGTCTGGAGCTGTCTGCAGAGTGCCCGTGACTGGCTCTGTCTTCTTGATGGAACCCAGGGTGT 440
TGAGTGCCTGATTGTCTGGAGCTGTCTGCAGAGTGCCCGTGACTGGCTCTGTCTTCTTGATGGAACCCAGGGTGT 441
GGTGTCTGGAGCCATCTCTTGACTGCTCCCGAGAAGATCCATGATGGTTTCTGGAAGCAGACCCAGACAACCTCT 442
GGTGTCTGGAGCCATCTCTTGACTGCTCCCGAGAAGATCCATGATGGTTTCTGGAAGCAGACCCAGACAACCTCT 443
CTCTCGGAGTCGTCTGAGTGTCTCTCACTGTCACTGTCCTGGCTAACACTGGATCCCTGGTGCCTGCTTGTCCTG 444
CTCTCGGAGTCGTCTGAGTGTCTCTCACTGTCACTGTCCTGGCTAACACTGGATCCCTGGTGCCTGCTTGTCCTG 445
GACCCCGATGATTGTTCCTGTCCCACCTGTGAGTGTCTAGAGCTGTCAGCCCAAGAGGCAGCTTCATGGTGACGT 446
GACCCCGATGATTGTTCCTGTCCCACCTGTGAGTGTCTAGAGCTGTCAGCCCAAGAGGCAGCTTCATGGTGACGT 447
TGCCTGGAGCCGTCTCCTGATTGTTTGTCCTTACGAGTTTGTCTGCTTGCACTTCTGGATCCTGACTGCCCATGG 448
TGCCTGGAGCCGTCTCCTGATTGTTTGTCCTTACGAGTTTGTCTGCTTGCACTTCTGGATCCTGACTGCCCATGG 449
ATGGGAGGCATCAGACCTTCCCTGGGATGTGGTGTGGCTGTGATGGGACCCTGAGTGTCCAGATCTATCTACCGA 450
ATGGGAGGCATCAGACCTTCCCTGGGATGTGGTGTGGCTGTGATGGGACCCTGAGTGTCCAGATCTATCTACCGA 451
TCATGGTGGGATCCCTGCCTTCCTCCTCTCCTTGACCCCGGGTGTGCACGAATGGTGTCCTGACCCTCTTGGGAC 452
TCATGGTGGGATCCCTGCCTTCCTCCTCTCCTTGACCCCGGGTGTGCACGAATGGTGTCCTGACCCTCTTGGGAC 453
CTGGAGCTGTCTCGTGCCTGCTCGTGGTGGGATCCTTGTCTTCGTCCAGTGCTGGTCCTGGTCCGCCCATGGGCA 454
CTGGAGCTGTCTCGTGCCTGCTCGTGGTGGGATCCTTGTCTTCGTCCAGTGCTGGTCCTGGTCCGCCCATGGGCA 455
GCAGACTCAGACTGTTCATGAGTGCTCACCTGGTAGATGAAAGACCCTGAACGTCCAGACCTTCCCCCTGACCAG 456
GCAGACTCAGACTGTTCATGAGTGCTCACCTGGTAGATGAAAGACCCTGAACGTCCAGACCTTCCCCCTGACCAG 457
CCCCCTGACCAGTCACGTGCGGACTCTTGGTGGCTCTGCTGATGGGGCCCATCCTGTCCATGGCCTGACACTGAC 458
CCCCCTGACCAGTCACGTGCGGACTCTTGGTGGCTCTGCTGATGGGGCCCATCCTGTCCATGGCCTGACACTGAC 459
TGTGTCTGACTCCTCTGAATGTCCCTCACTATCACTGGCCTGACTACCACTGGACCCCCAGTGTCCACTGTCTCT 460
TGTGTCTGACTCCTCTGAATGTCCCTCACTATCACTGGCCTGACTACCACTGGACCCCCAGTGTCCACTGTCTCT 461
CTCTGACTGCAGATGAAGCTTGTCTGCGCGGAATGCCTGAGTGTCTGGAGCTGTCTGCTGACTGCTGGTGGCGGG 462
CTCTGACTGCAGATGAAGCTTGTCTGCGCGGAATGCCTGAGTGTCTGGAGCTGTCTGCTGACTGCTGGTGGCGGG 463
TCTCCTGGACTTGATCTTGCCTGTTCATGGGATGACGCAGCCTGTCCACCAGAGGAAGTCTCTGCGTGAGGAGTT 464
TCTCCTGGACTTGATCTTGCCTGTTCATGGGATGACGCAGCCTGTCCACCAGAGGAAGTCTCTGCGTGAGGAGTT 465
TGAGGAGTTCCTGATTGTCTGGAGCTGTCTGCAGAGTGCCCGTGACCGGCTCTGTCTTCGTGATGGGACCTGGGG 466
TGAGGAGTTCCTGATTGTCTGGAGCTGTCTGCAGAGTGCCCGTGACCGGCTCTGTCTTCGTGATGGGACCTGGGG 467
TGTCTTGACTGCTCCTGAGCAGATCCACGATGGTTTCTGGAAGCAGACCCAGACCACCTCTCAGAGTCTTCTGAA 468
GTCCCTCACTGTCACTGTCCTGGCTAACACTGGATCCCTGGCGCCTGCTTGTCCTGGACCCCTCTGATTGTCCCT 469
GTCCCTCACTGTCACTGTCCTGGCTAACACTGGATCCCTGGCGCCTGCTTGTCCTGGACCCCTCTGATTGTCCCT 470
GCCTGCCTGTGAGTGTCTAGAGATGTCGGCATGAGAGGAAGCTTCATGGTGACGTGACCCTGAGTGCCTGGAGCC 471
GCCTGCCTGTGAGTGTCTAGAGATGTCGGCATGAGAGGAAGCTTCATGGTGACGTGACCCTGAGTGCCTGGAGCC 472
GAGTGCCTGGAGCCGTCTCTTGATTGTTCCTCATTACGTGTTGTTCTGCTTGCACTTCTGGATCCTGACTGCCCA 473
GAGTGCCTGGAGCCGTCTCTTGATTGTTCCTCATTACGTGTTGTTCTGCTTGCACTTCTGGATCCTGACTGCCCA 474
ACCTTCCCTGGGATGTGGTGTGGCTGTGATGGGACCCTGAGTGTCCAGACCTATCTACCGATTGCTCGTGGTGGG 475
ACCTTCCCTGGGATGTGGTGTGGCTGTGATGGGACCCTGAGTGTCCAGACCTATCTACCGATTGCTCGTGGTGGG 476
ATTGCTCGTGGTGGGACCCCTGCCTTCCTCTTCTGCTTGACCCCGGGTGTCCACGAATGGTGTCCTGACCATCTT 477
ATTGCTCGTGGTGGGACCCCTGCCTTCCTCTTCTGCTTGACCCCGGGTGTCCACGAATGGTGTCCTGACCATCTT 478
GATGCTGAGTGCCTGGAGTTGTCTCGTGCCTGCTCATGGTGGGATCCTTGTCTTACTCCAGTGCTGGGCCCTGTC 479
GATGCTGAGTGCCTGGAGTTGTCTCGTGCCTGCTCATGGTGGGATCCTTGTCTTACTCCAGTGCTGGGCCCTGTC 480
TTCCCCTGACCGGTCACGTGCGGACTCTTGGTGGCTCTGCTGATGGTGACCAGCCTGTCCATGGCCTGACACTGA 481
TTCCCCTGACCGGTCACGTGCGGACTCTTGGTGGCTCTGCTGATGGTGACCAGCCTGTCCATGGCCTGACACTGA 482
TCTGAATGTCCCTCACTGTCAGTGGCCTGACTACCACTGGACCCTCGGTGTCCACTGTCTCTGACTGCAGATGAA 483
TCTGAATGTCCCTCACTGTCAGTGGCCTGACTACCACTGGACCCTCGGTGTCCACTGTCTCTGACTGCAGATGAA 484
TGAAGCTTGTCCGTGCCCAGTGCCTGAGTGTCTGGAGCTGTCTGCTGACTGGAGCTGGTGGCGGGATCCATGTCT 485
TGAAGCTTGTCCGTGCCCAGTGCCTGAGTGTCTGGAGCTGTCTGCTGACTGGAGCTGGTGGCGGGATCCATGTCT 486
GCACTTGATCTTGCCTGTTCATGGGATGACGCAGCCTGTCCACTAGAGGAATTCTGTGTGTGACGAGTGCCTGAT 487
GCACTTGATCTTGCCTGTTCATGGGATGACGCAGCCTGTCCACTAGAGGAATTCTGTGTGTGACGAGTGCCTGAT 488
AGTGCCTGATTTTCTGGAGCTGTCTGCAGAGTGCCCATGACCAGCTCTGTCTTCGTGATGGGACCTGGGGTGTCT 489
TCTTGACTGCTCCTGAGCAGATCCATGATGGTTTCTGGAAGCAGACCCAGACCACCTCTCAGAGTCTTCTGAGTG 490
CTTCTGAGTGTCCCTGACTGTCACTGTCCTGGCTAACACTGGATCCCTGGTTCCTACTTGTCCTGGGCCCCGATG 491
CTTCTGAGTGTCCCTGACTGTCACTGTCCTGGCTAACACTGGATCCCTGGTTCCTACTTGTCCTGGGCCCCGATG 492
GTCCCTGGCCCACCTGTGAGTGTCTAGAGCTGTCAGCCTGAGAGGAAGCTTCATGATGACGTGACCCTGAGTGCC 493
GTCCCTGGCCCACCTGTGAGTGTCTAGAGCTGTCAGCCTGAGAGGAAGCTTCATGATGACGTGACCCTGAGTGCC 494
GTGCCGTCTCCTGATTGTTCCTCATTTCGTGTTTGTCTGCTTGCACTTCTGGATCCTGACTGCCCATGGGAGGCA 495
GACCTTCCCTGGGATGTGGTGTGGCTGTGATGGGAACCTGAGTGTCCAGACCTATTTACCGATTGCTCGTGGTGG 496
GACCTTCCCTGGGATGTGGTGTGGCTGTGATGGGAACCTGAGTGTCCAGACCTATTTACCGATTGCTCGTGGTGG 497
CGATTGCTCGTGGTGGGATCCCTGCCTTCCTCCTCTGCTTGACCCCGGGTGTCCACGAATGGTGTCCTGACCCTC 498
CGATTGCTCGTGGTGGGATCCCTGCCTTCCTCCTCTGCTTGACCCCGGGTGTCCACGAATGGTGTCCTGACCCTC 499
GAATGCCTGGAGCTGTCTCGTGCCTGCTCGTGGTGCGATCCTTGTCTTCCTCCAGTGCTGGTCCCGGTCCGTCCA 500
GAATGCCTGGAGCTGTCTCGTGCCTGCTCGTGGTGCGATCCTTGTCTTCCTCCAGTGCTGGTCCCGGTCCGTCCA 501
GCAGAGTCAGGCTGTTCATGAGTGCTCACCTGGTAGAGGGAAGACCCTGAACGTCCAGACCGTTCCCCTGACCGG 502
GCAGAGTCAGGCTGTTCATGAGTGCTCACCTGGTAGAGGGAAGACCCTGAACGTCCAGACCGTTCCCCTGACCGG 503
CACGTGTGGACTCTTGGTGGCTCTGCTGTCTCAGCCCAGCCTTTCCGTGGCCTGACACTGATTGTGTGTCTGAGT 504
CACGTGTGGACTCTTGGTGGCTCTGCTGTCTCAGCCCAGCCTTTCCGTGGCCTGACACTGATTGTGTGTCTGAGT 505
TTGTGTGTCTGAGTTTTCTGAATGTCCCTCACTGTCACTGGCCTGACTACCGCTAGACCCCCGGTGTCCACGATC 506
GACTGCAGATGAAGCTTGCCCGCGCCCAGTGGCTGAGTGTCTGGAGCTGTCTGCTGACTGCTGGTGGCCGGATCC 507
GACTGCAGATGAAGCTTGCCCGCGCCCAGTGGCTGAGTGTCTGGAGCTGTCTGCTGACTGCTGGTGGCCGGATCC 508
CTTGATCTTGCCTGTTCATGGGATGATGCAGTCTGTCCACGAGAGGAAGTCTCTGCGTGACGAGTGCCTGATTGT 509
CTTGATCTTGCCTGTTCATGGGATGATGCAGTCTGTCCACGAGAGGAAGTCTCTGCGTGACGAGTGCCTGATTGT 510
TGACGAGTGCCTGATTGTCTGGAGCTGTCTGCAGAGTGCCCATGACTGGCTCTGTCTTCATCATGGGACCTGGGG 511
GACTGCTCCCACGCAGATCCATGATGGTTTCTGGAAGCCGACCCAGAGTGCCTCTCAGAGTCTTCTGAGTGTCCC 512
GACTGCTCCCACGCAGATCCATGATGGTTTCTGGAAGCCGACCCAGAGTGCCTCTCAGAGTCTTCTGAGTGTCCC 513
CCCTCACTGTCCCTGTCCTGGCTAACTCTGGATCCCCTACGCTTTCTTGTCCTGGACTCCTGCAATGGTACCTGG 514
CCCTCACTGTCCCTGTCCTGGCTAACTCTGGATCCCCTACGCTTTCTTGTCCTGGACTCCTGCAATGGTACCTGG 515
GTATTTTCATGTCTTGACCTGTTCACTTGAGATGATGATTTGCCATCAGATGACCTTGATCTTTCATATATTTTG 516
ATTTTGTTTTCTTCTAATAGACTATCAGTGGTGTCATAGGCTTCATCCTGGATTGTGTAATATGTGGCAATATGG 517
CCTGATTGTATCCATTTTTGAGTCATTCTTCCTGTATTTTCATAATCATATACTCCTTCTTCATTGTCTTCTTTC 518
CTTTCTCTTCAAGTCTTTCACTTAGCCTCTTCCTATTGTCTCCTAATCTAGTATTTTCAGTCTTGTTTTTCTCTT 519
TTACTTGAGTTATGATGGTTTTTTCCATATTCTTCTTCTCTATGAGTAGGTGAATATCCTTTTCTTTCTTTTTTT 520
ACTAGATTCATGCCTTTTCCCCCCTGTTTCTCTTGGGCTCTTGGATCTTCCCTTATTCCCTTTTCTATTGTTTCT 521
ATTGTTTCTTCTTTCCAGACTTGAGGGTCTTTTTCTGTTTTCTTTGTTTTCTTCCTGTTTATTATCTTCATGTTT 522
ATCATGATGACTGTGCTTTCTGTGCTTGTGTCCTGATATCGGTAAATTCTCTTTTCTGGTAGACTCATAATATGC 523
GCTTGAGCCAACTTGAATACCATCAGAAGAAACTCAGTGAAGTCAATTTTCTTGTTGTGGTCTATATCCAAGTGA 524
GTGATCCATGAAGACATCAACCATATCTGGGTCATCTGGATTCTGTACAGAGGGAAGTCACAGAGGGAGACTGCA 525
TGTGTTTATGACAAATAAGAATACAAGAGACAAACAGTATTATATGATGAATAGTTTATTTTTAATTTAGATGCA 526
GATGCAGCTTACTATAATATTAATTATGTCCAAGATGATTTTTTGAATACAGAATACTAGAATTCCAATAGAAGG 527
GATGTGCTAGCCCTGATGTTGATATAGCCACTTTGGTATACAGAACTGTTTTATATTTTTGGCTCCTTCGATATT 528
GCTCCTTCGATATTTCTGAAAAAGATTAATTTAGAAATTTGGGGAGTGTCTAAAACTTAAACTTTCAAAAACATA 529
CCAGAATCTCCTAAAATACTCCAGCTAGTTTTCTAAAGTTAGCTCTCCATGATATTGATTTCTTCCATTTAATAT 530
ATTTAATATTTCTGAAATATAGCGTTTAAAGATCATTACACAATAAAAATAAGCTACCACCAAACTAATGAAATA 531
TACTATAGCATATTTTAAACAGATTGACAGGAAAAGATAACTTCCCTGAAAGTATTATGAAGTTTCTTGATTGAA 532
TTCATTCTTCTATTCTTGGATTAATTCCTTTGCCATTAATTTCTTACTCATAGTAATAGTATCTCTGTGACTGAC 533
ATCCCAGTTGTTTCGATATATCACTAGAATGGCCACATAAACCTGGGTCCTTATTAATATACGTTGCATAATACC 534
CTTGGATGATCTTTACCAAACGCACTTGCTTTACAGATATCAGATCTTTCCTTGAAAACAACAGGATTGGAATTG 535
GATTGGAATTGTAACTAACACTTCCGTGCTGAGAGTGTCTAAACCCGGATTCACCATAATCATAATCTGCACTAC 536
CTGCCATGTCTCCAAACTAAACCTGATTGACCTTTTTGCCTTTCAGTGCCCTCAGATTGATAATGATAAGAACTA 537
ACTAGAACTGTGAGGACTGCCACGTGACTGTATTCCTGAGTGATACGCAGAATCTTGTGAAAGACTACTAAAGTG 538
TGTTCCTTAGCGGTACTAGAGTCTGACTGTACAGGTGAAGACTGTACATGACTGGCTGTATCGCGGTGAGAGGAT 539
GTCTGGAGCCATCTCTTGACTGCTCCCGAGAAGATCCATGATGGTTTCTGGAAGCAGACTCAGATCGCCTCTCAG 540
CGCCTCTCAGAGTCCTCTGGGTATGCCTCACTGTCACTGTCCTGGCTAACACTGGATCCCTGGCGCCTGCTTCTC 541
CGCCTCTCAGAGTCCTCTGGGTATGCCTCACTGTCACTGTCCTGGCTAACACTGGATCCCTGGCGCCTGCTTCTC 542
CCGCTGATTCACCCTGGCCGGACTGTGAGTGTCTAGAGCTGTCAGCCTGAGTGGAAGCTTCATGGTGACGCGACC 543
CCGCTGATTCACCCTGGCCGGACTGTGAGTGTCTAGAGCTGTCAGCCTGAGTGGAAGCTTCATGGTGACGCGACC 544
CCCTGAGTGCCTGGAGCCGTCTCCTGACTGTTCCTCATTACGTGTTTCTCTGCTTGCACTTCTGGATCCTGACTG 545
CCCTGAGTGCCTGGAGCCGTCTCCTGACTGTTCCTCATTACGTGTTTCTCTGCTTGCACTTCTGGATCCTGACTG 546
AGACCTATCTACCGATTGCTCGTGGTAGGATCCCTGTCTTCCTCCTCTCCTTGACCCCGGGTGTCCACGAATGGT 547
AGACCTATCTACCGATTGCTCGTGGTAGGATCCCTGTCTTCCTCCTCTCCTTGACCCCGGGTGTCCACGAATGGT 548
GACCCTCTTGGGACGCTGAGTGCCTGGAGCTGTCTCGTGCCTGCTCGTGGCGGGATCCTTGTCTTCCTCCAGTAC 549
GACCCTCTTGGGACGCTGAGTGCCTGGAGCTGTCTCGTGCCTGCTCGTGGCGGGATCCTTGTCTTCCTCCAGTAC 550
CTGGGCCCAGCCCGTCCATGGGCAGACTCAGACTGTTCATGAGTGCTCACCTGGTAGAGGAAAGACCCTGAACGT 551
CTGGGCCCAGCCCGTCCATGGGCAGACTCAGACTGTTCATGAGTGCTCACCTGGTAGAGGAAAGACCCTGAACGT 552
AACGTCCAGACCTTCCTGCTGACCGGCCACGTGTGGACTCTTGGTGGCTCTGCTGATGGGGCCCAGCCTGTCCGT 553
AACGTCCAGACCTTCCTGCTGACCGGCCACGTGTGGACTCTTGGTGGCTCTGCTGATGGGGCCCAGCCTGTCCGT 554
CTGACACTGACTGTGTGTCTGAGTCTTCTGAATGTCCCTCACTGTCACTGGCCTGACTACCACTGGACCCTCGGT 555
CTGACACTGACTGTGTGTCTGAGTCTTCTGAATGTCCCTCACTGTCACTGGCCTGACTACCACTGGACCCTCGGT 556
GATCCATGTCTCTCTCCTGCACTTGATCTTGCCTGTTCATGGGATGATGCAGCCTGTCCACCAGAGGAATTCTCT 557
AGGAATTCTCTGCATGATGAGTGCCTGATTGTCTGGAGCTCTCTGCAGAGTGCCCGTGACCGGCTCTGTCTTCGT 558
AGGAATTCTCTGCATGATGAGTGCCTGATTGTCTGGAGCTCTCTGCAGAGTGCCCGTGACCGGCTCTGTCTTCGT 559
CTGGAGCCATCTCTTGACTGCTCCTGAGCAGATCCACGATGGTTTCTGGAAGCAGACCCAGACCACCTCTCAGAG 560
CTGGAGCCATCTCTTGACTGCTCCTGAGCAGATCCACGATGGTTTCTGGAAGCAGACCCAGACCACCTCTCAGAG 561
CTTCTGAGTGTCCCTGACTGTCACTGTCCTGGCTAACACTGGATCCCTGGTTCCTGCTTGTCCTGGGCCCCGCTG 562
CTTCTGAGTGTCCCTGACTGTCACTGTCCTGGCTAACACTGGATCCCTGGTTCCTGCTTGTCCTGGGCCCCGCTG 563
ATTGTCCCTGGCCGGACTGTGAGTGTCTAGAGCTGTCCGCCTGAGTGGAAGCTTCATGGTGATGCGACCATGAGT 564
ATTGTCCCTGGCCGGACTGTGAGTGTCTAGAGCTGTCCGCCTGAGTGGAAGCTTCATGGTGATGCGACCATGAGT 565
GGTGATGCGACCATGAGTGCCTGGAGCCATCTCCTGATTGTTCGTCATTACGAGTTTGTCTGCTGGCACTTCTGG 566
GGTGATGCGACCATGAGTGCCTGGAGCCATCTCCTGATTGTTCGTCATTACGAGTTTGTCTGCTGGCACTTCTGG 567
TATCTACCGATTGCTCGTAGTGGGATCCCTGCCTTCCTCTTCTGCTTGACCCCGGGTGTCCACGAATGGTGTCCT 568
TATCTACCGATTGCTCGTAGTGGGATCCCTGCCTTCCTCTTCTGCTTGACCCCGGGTGTCCACGAATGGTGTCCT 569
GCTGAGTGCCTGGAGCTGTCTCGTGCCTGCTCGTGGTGGGATCCTTGTCTTCGTCCAGTGCTGGTCCTGGTCCGC 570
GCTGAGTGCCTGGAGCTGTCTCGTGCCTGCTCGTGGTGGGATCCTTGTCTTCGTCCAGTGCTGGTCCTGGTCCGC 571
GGTCCGCCCATGGGCAGACTCAGACTGTTCATGAGTGCTCACCTGGTAGAGGAAAGACCCTGAACGTCCAGACCT 572
GGTCCGCCCATGGGCAGACTCAGACTGTTCATGAGTGCTCACCTGGTAGAGGAAAGACCCTGAACGTCCAGACCT 573
CCTGACACTGACTGTGTGTCTGACTCTTCTGAATGTCCCTCACTATCACTGGCCTGACTACCACTGGACCCCCAG 574
TGTCTCTGACTGCAGATGAAGCTTGTCCACGCGGAATGCCTGAGTGTCTGGAGCTGTCTGCTGACTGCTGGTGGC 575
TGTCTCTGACTGCAGATGAAGCTTGTCCACGCGGAATGCCTGAGTGTCTGGAGCTGTCTGCTGACTGCTGGTGGC 576
CTCTGCGTGACGAGTGCCTGATTGTCTGGAGCGGTCTGCAGAGTGCCCGTGACCGGCTCTGTCTTCGTGATGGGA 577
CTCTGCGTGACGAGTGCCTGATTGTCTGGAGCGGTCTGCAGAGTGCCCGTGACCGGCTCTGTCTTCGTGATGGGA 578
GGGGTGTCTGGAGCCGTGCCTTGACTGCTCCTGAACAGATCCACGATGGTTTCTGGAAGCAGACCCAGACCACCT 579
GGGGTGTCTGGAGCCGTGCCTTGACTGCTCCTGAACAGATCCACGATGGTTTCTGGAAGCAGACCCAGACCACCT 580
TCTCAGAGTCTTCTGAATGTCCCTCACTGTCACTGTCCTGGCTCACACTGGATCCCTGGCGCCTGCTTCTCCTGG 581
TCTCAGAGTCTTCTGAATGTCCCTCACTGTCACTGTCCTGGCTCACACTGGATCCCTGGCGCCTGCTTCTCCTGG 582
CCTCTGATTGTCCCTGGACTGCCTGTGAGTGTCTAGAGATGTCGGCATGAGTGGAAGCTTCATGGTGACGTGACA 583
GTGACACTGAGTGCCTGGAGCTGTCTCCTGATTGTTCCTCATTACGTGTTGTTCTGCTTGCACTTCTGGATCCTG 584
GTGACACTGAGTGCCTGGAGCTGTCTCCTGATTGTTCCTCATTACGTGTTGTTCTGCTTGCACTTCTGGATCCTG 585
ACCGAATGCTCGTGGTGGTACCCCTGCCTTCCTCCTCTGCTTGACCCCGGGTGTCCACGAATGGTGTCCTGACCG 586
ACCGAATGCTCGTGGTGGTACCCCTGCCTTCCTCCTCTGCTTGACCCCGGGTGTCCACGAATGGTGTCCTGACCG 587
TGACCGTATTGGGATGCTGAGTGCCTGGAGCTGTCTTGTGCCTGCTCATGGCGGGATCCTTGTCTTCCTCTAGTG 588
TGACCGTATTGGGATGCTGAGTGCCTGGAGCTGTCTTGTGCCTGCTCATGGCGGGATCCTTGTCTTCCTCTAGTG 589
ACCGGCCACGTGCGGACTCTTGGTGGCTCTGCTGATGGGACCCAGCCTGTCCGTGGGCTGACACTGACTGTGTGT 590
ACCGGCCACGTGCGGACTCTTGGTGGCTCTGCTGATGGGACCCAGCCTGTCCGTGGGCTGACACTGACTGTGTGT 591
GACTGTGTGTCTGAGTCTTCTGAATGTCCCTCATTGTCACTGGCCTGACTACCACTGTACCCTCGGTGTCCACTG 592
GACTGTGTGTCTGAGTCTTCTGAATGTCCCTCATTGTCACTGGCCTGACTACCACTGTACCCTCGGTGTCCACTG 593
ATGAAGCTTGTCCGTGCCCAATGCCTGAGTGTCTGGAGCTGTCTGCTGACTGCTGGTGGTGGGATCCATGTCTTT 594
ATGAAGCTTGTCCGTGCCCAATGCCTGAGTGTCTGGAGCTGTCTGCTGACTGCTGGTGGTGGGATCCATGTCTTT 595
CCTGATTGTCTGGAGCTGTCTGCAGAGTGCCCATGACCAGCTCTGTCTTCTTGATGGGACCTGGGGTGTCTGGAG 596
CCTGATTGTCTGGAGCTGTCTGCAGAGTGCCCATGACCAGCTCTGTCTTCTTGATGGGACCTGGGGTGTCTGGAG 597
ACCTGGGGTGTCTGGAGCCATCTCTTAGCTGCTCCTGAGCAGATCCATGATGGTTTCTGGAAGCAGACCCAGACC 598
ACCTGGGGTGTCTGGAGCCATCTCTTAGCTGCTCCTGAGCAGATCCATGATGGTTTCTGGAAGCAGACCCAGACC 599
AGTCTTCTGAATGTCCCTCACTGTCACTGTCCTGGCTCACACTGGATCCCTGGCGCCTGCTTCTCCTGGACCCCT 600
AGTCTTCTGAATGTCCCTCACTGTCACTGTCCTGGCTCACACTGGATCCCTGGCGCCTGCTTCTCCTGGACCCCT 601
ACCCCTCTGATTGTCCCTGGACTGCCTGTGAGTGTCTAGAGATGTCGGCATGAGTGGAAGCTTCATGGTGACGCG 602
GACCCTGAGTGCCTGGAGCCGTCTCCTGATTGTTCCTCATTACGTGTTGTTCTGCTTGCACTTCTGGATCCTGAC 603
CCGAATGCTCGTGGTGGTACCCCTGCCTTCCTCCTCTGCTTGACCCCGGGTGTCCACGAATGGTGTCCTGACCGT 604
CCGAATGCTCGTGGTGGTACCCCTGCCTTCCTCCTCTGCTTGACCCCGGGTGTCCACGAATGGTGTCCTGACCGT 605
GCTGAGTGCCTGGAGCTGTCTTGTGCCTGCTCATGGCGGGATCCTTGTCTTCCTCTAGTGCTGGGCCCCGTCCAT 606
GCTGAGTGCCTGGAGCTGTCTTGTGCCTGCTCATGGCGGGATCCTTGTCTTCCTCTAGTGCTGGGCCCCGTCCAT 607
CTGACCGGCCACGTGCGGACTCTTGGTGGCTCTGCTGATGGGACCCAGCCTGTCCGTGGGCTGACACTGACTGTG 608
CTGACCGGCCACGTGCGGACTCTTGGTGGCTCTGCTGATGGGACCCAGCCTGTCCGTGGGCTGACACTGACTGTG 609
CTGTGTGTCTGAGTCTTCTGAATGTCCCTCATTGTCACTGGCCTGACTACCACTGTACCCTCGGTGTCCACTGTC 610
CTGTGTGTCTGAGTCTTCTGAATGTCCCTCATTGTCACTGGCCTGACTACCACTGTACCCTCGGTGTCCACTGTC 611
GATGAAGCTTGTCCGTGCCCAATGCCTGAGTGTCTGGAGCTGTCTGCTGACTGCTGGTGGTGGGATCCATGTCTT 612
TGATTGTCTGGAGCTGTCTGCAGAGTGCCCATGACCAGCTCTGTCTTCTTGATGGGACCTGGGGTGTCTGGAGCC 613
TGATTGTCTGGAGCTGTCTGCAGAGTGCCCATGACCAGCTCTGTCTTCTTGATGGGACCTGGGGTGTCTGGAGCC 614
CTCTTAGCTGCTCCTGAGCAGATCCATGATGGTTTCTGGAAGCAGACCCAGACCACCTCTCAGAGTCTTCTGAGT 615
CTCTTAGCTGCTCCTGAGCAGATCCATGATGGTTTCTGGAAGCAGACCCAGACCACCTCTCAGAGTCTTCTGAGT 616
GTGTCCCTGACTGTCACTGTCCTGGCTAAAACTGGATCCCCAGTTCCTGCTTGTCCTGGGCCCCTCTGATTGTCC 617
GTGTCCCTGACTGTCACTGTCCTGGCTAAAACTGGATCCCCAGTTCCTGCTTGTCCTGGGCCCCTCTGATTGTCC 618
CCCCTCTGATTGTCCCTGGCCCACCTGCGAGTGTCCAGAGCTGTCGGCCCGAGAGGAAGCTTCATGGTGACGCGA 619
CCCCTCTGATTGTCCCTGGCCCACCTGCGAGTGTCCAGAGCTGTCGGCCCGAGAGGAAGCTTCATGGTGACGCGA 620
CCGTCTCCTGATTGTTCATCGTTACGAGTTTGTCTGCTTGCACTTCTGGATCCTGAGTGCCCATGGGAGGCATCA 621
GGGAGGCATCAGACCTTCCCTGGGATGTGGTGTGGCTGTGATGAGACCCTGAGTGTCCAGATCTATCTACCAATT 622
GGGAGGCATCAGACCTTCCCTGGGATGTGGTGTGGCTGTGATGAGACCCTGAGTGTCCAGATCTATCTACCAATT 623
GATCCCTGCCTTCCTCCACTGCTTGACCCCGGGTGTCCATGAATGGTGTCCTGACCCTCTTGGGACGTTGAGTGC 624
GATCCCTGCCTTCCTCCACTGCTTGACCCCGGGTGTCCATGAATGGTGTCCTGACCCTCTTGGGACGTTGAGTGC 625
TTGAGTGCCTGGAGCTGTCTCGTGCCTGCTTGTGGTGGGATCCTTGTCTTCCTCCAGTGCTGGTCCCGGTCCGTC 626
TTGAGTGCCTGGAGCTGTCTCGTGCCTGCTTGTGGTGGGATCCTTGTCTTCCTCCAGTGCTGGTCCCGGTCCGTC 627
GGCGGACTCAGACTGTTCATGAGTGCTCACCTGGTAGAGGAAAGACCCTGAACGTCCAGACCTTCCTGCTGACCG 628
GGCGGACTCAGACTGTTCATGAGTGCTCACCTGGTAGAGGAAAGACCCTGAACGTCCAGACCTTCCTGCTGACCG 629
CTTCCTGCTGACCGGCCACGTGTGGACTCTTGGTGGCTCTGCTGATGGGGCCCAGCCTGTCCGTGGGCTGACACT 630
CTTCCTGCTGACCGGCCACGTGTGGACTCTTGGTGGCTCTGCTGATGGGGCCCAGCCTGTCCGTGGGCTGACACT 631
CTGTGTGTCTGAGTCTTCTGAATGTCCCTCACTGTCACTGGCCTGACTACCACTGGACCCTCGGTGTCCACTGTC 632
CTGTGTGTCTGAGTCTTCTGAATGTCCCTCACTGTCACTGGCCTGACTACCACTGGACCCTCGGTGTCCACTGTC 633
CCGTGTCTCTCTCCTGCACTTGATCTTGCCTGTTCATGGGATGATGCAGCCTGTCCACCAGAGGAATTCTCTGCA 634
CCGTGTCTCTCTCCTGCACTTGATCTTGCCTGTTCATGGGATGATGCAGCCTGTCCACCAGAGGAATTCTCTGCA 635
TGATGAGTGCCTGATTGTCTGGAGCTCTCTGCAGAGTGCCCATGACCGGCTCTGTCTTCGTGATGGGACCTGGGG 636
CTCTTGACTGCTCCTGAGCAGATCCATGATGGTTTCTGGACGCAGACCCAGACCGCCTCTCAGAATCTTCTGAGT 637
CTCTTGACTGCTCCTGAGCAGATCCATGATGGTTTCTGGACGCAGACCCAGACCGCCTCTCAGAATCTTCTGAGT 638
GAGTGTCCCTCACTGTCACTGTCCTGGCTAACACTGGATCCCCGGGGCCTGCTTGTCCTGGGCCCTGATGATTGT 639
GAGTGTCCCTCACTGTCACTGTCCTGGCTAACACTGGATCCCCGGGGCCTGCTTGTCCTGGGCCCTGATGATTGT 640
TGGCCCACCAGTGAGTGTCTAGAGCTGTCGGCCCAAGAGGAAGCTTCATGATGATGCGACCCTGAGTGCCTAGAG 641
TGGCCCACCAGTGAGTGTCTAGAGCTGTCGGCCCAAGAGGAAGCTTCATGATGATGCGACCCTGAGTGCCTAGAG 642
GTGCCTAGAGCCATCTCCTGATTGTTCCTTGTCATATGTTTTTCTGCTTGCACTTCTGGATCCTGACTGCCCACG 643
GGCATCAGACCTTCCCTGGGATGTGGTGTGGCTGTGATGAGACCCTGAGTGTCCAGACCTATCTACCGATTGCTC 644
GGCATCAGACCTTCCCTGGGATGTGGTGTGGCTGTGATGAGACCCTGAGTGTCCAGACCTATCTACCGATTGCTC 645
TGCTCTTGGTGGGACCCCTGTCTTCCTCCTCTGCTTGGCCCCGGGTGTCCACGAATGGTGTCCTGACCCTCTTGG 646
TGCTCTTGGTGGGACCCCTGTCTTCCTCCTCTGCTTGGCCCCGGGTGTCCACGAATGGTGTCCTGACCCTCTTGG 647
GAGTGCCTGGAGCTGTCTTGTGCCTGATCATAATGGGATCCTTGTCTTCCTCCAGTGCTGGGCGCAGACTGTCCA 648
GAGTGCCTGGAGCTGTCTTGTGCCTGATCATAATGGGATCCTTGTCTTCCTCCAGTGCTGGGCGCAGACTGTCCA 649
TGTCCATGGGTGGACTCAGACTGTTCATGAGTGCTCACCTGGTAGAGGAAAGACCCTGAACGTCCAGAGCTTTCC 650
TGTCCATGGGTGGACTCAGACTGTTCATGAGTGCTCACCTGGTAGAGGAAAGACCCTGAACGTCCAGAGCTTTCC 651
GTGCGGACTCTTTGTGGCTCTGCTGATGGGGCCCAGCTTTTCCCTGTGCTGACACTGACTGTGTGTCTGAGTCTT 652
GTGCGGACTCTTTGTGGCTCTGCTGATGGGGCCCAGCTTTTCCCTGTGCTGACACTGACTGTGTGTCTGAGTCTT 653
TGAATGTCCCTCACTGTCACTGGCCTGACTACCACTGTACCCTCGGTGTCCACTGTCTCTGACTGCAGATGAAGC 654
TGAATGTCCCTCACTGTCACTGGCCTGACTACCACTGTACCCTCGGTGTCCACTGTCTCTGACTGCAGATGAAGC 655
CCTGCACTTGATCTTGCCTGTTCATGGGATGACGCAGCCTGTCCACGAGAGGAAGACTCTGTGTGACGAGTGCCT 656
CCTGCACTTGATCTTGCCTGTTCATGGGATGACGCAGCCTGTCCACGAGAGGAAGACTCTGTGTGACGAGTGCCT 657
GATTGTCTGGAGCTGTCTGCAGAGTGCCCGTGACCGGCTCTGTCTTCGTGATGGGACCCAGGGTGTCTGGAGCCA 658
GATTGTCTGGAGCTGTCTGCAGAGTGCCCGTGACCGGCTCTGTCTTCGTGATGGGACCCAGGGTGTCTGGAGCCA 659
GTCTGGAGCCATCTCTTGACTGCTCCCAAGCAGATCCAAGATGGTTTCTGGAAGCAGACCCAGACCACCTCTCAG 660
GTCTGGAGCCATCTCTTGACTGCTCCCAAGCAGATCCAAGATGGTTTCTGGAAGCAGACCCAGACCACCTCTCAG 661
CCTGACTGTCACTGTCCTGGCTAACACTGGATCCCTGGTTCCTGCTTGTCCTGGGCCCTGATGATTGTCCCTGGC 662
CCTGACTGTCACTGTCCTGGCTAACACTGGATCCCTGGTTCCTGCTTGTCCTGGGCCCTGATGATTGTCCCTGGC 663
ATGATTGTCCCTGGCCCACCTGCGAGTGTCTAGAGCTGTCGGCCCGAGAGGAAGCTTCATGGTGACGCGACCCTG 664
ATGATTGTCCCTGGCCCACCTGCGAGTGTCTAGAGCTGTCGGCCCGAGAGGAAGCTTCATGGTGACGCGACCCTG 665
AGCCGTCTCCTGATTGTTTCTCATTACGTGTTTGTCTGCTGACACTTCTGGATCCTGACTGCCCACGGGAGACAT 666
GGAGACATCAGACCTTTCCTGGGACGTGGTGTGGCTGTGATGAGACCCTGAGTGTCCAGAACTATCTACCGATTG 667
CTCATAGTGGGATCCCTGCCTTCCTCCTCTGCTTGACCCTGGGTGTCCACGAATGGTGTCCTGACCCTCTTGGGA 668
CTCATAGTGGGATCCCTGCCTTCCTCCTCTGCTTGACCCTGGGTGTCCACGAATGGTGTCCTGACCCTCTTGGGA 669
CTGAGTGCCTGGAGCTGTCTCGTGCCTGCTCGTGGCGGGATCTTTGTCTTCCTCCAGTGCTGGGCCCTGTGCGTC 670
CTGAGTGCCTGGAGCTGTCTCGTGCCTGCTCGTGGCGGGATCTTTGTCTTCCTCCAGTGCTGGGCCCTGTGCGTC 671
GGACTCAGACTGTTCATGAGTGCTCACCTGGTAGAGGAAAGACCCTGAACGTCCAGACCTTTCCCCTGACTGGCC 672
GGACTCAGACTGTTCATGAGTGCTCACCTGGTAGAGGAAAGACCCTGAACGTCCAGACCTTTCCCCTGACTGGCC 673
CCCCTGACTGGCCACGTGTGGACTCTTGGTGGCTCTGCTGATGGGGCCCAGCCTGTCCGTGGGCTGACACTGACT 674
CCCCTGACTGGCCACGTGTGGACTCTTGGTGGCTCTGCTGATGGGGCCCAGCCTGTCCGTGGGCTGACACTGACT 675
GTGTGTCTGACTCTTCTGAGTGTCCCTCGCTGTCACTGGCCTGGCTACCACTGGACCCTCGGTTTCCACTGTCTC 676
GTGTGTCTGACTCTTCTGAGTGTCCCTCGCTGTCACTGGCCTGGCTACCACTGGACCCTCGGTTTCCACTGTCTC 677
ATCTTGTCTGCGCCCAGTGCCTGAGTCTGTGGAGCTGTCTGCTGACTGCTGGTGGCGGGATCCATGTCTTTCTCC 678
ATCTTGTCTGCGCCCAGTGCCTGAGTCTGTGGAGCTGTCTGCTGACTGCTGGTGGCGGGATCCATGTCTTTCTCC 679
CTTTCTCCTGGACTTGACCTTGCCTGTTCCTGGGATGATGCAGCCTGTCCACCAGAGGAAGTCTCTGCATGACGA 680
CTTTCTCCTGGACTTGACCTTGCCTGTTCCTGGGATGATGCAGCCTGTCCACCAGAGGAAGTCTCTGCATGACGA 681
TGGAGCTCTCTGCAGAGTGCCCATGACTGGCTCTATCTTCTTGATGGGACCTGGGGTTCCTGGAGCCATGTCTTG 682
TGGAGCTCTCTGCAGAGTGCCCATGACTGGCTCTATCTTCTTGATGGGACCTGGGGTTCCTGGAGCCATGTCTTG 683
TGGAGCCATGTCTTGACTGCTCCCGAGCAGATCCATAATGGTTTCTGGAAGCCGACTCAGACCGCCTCTCAGAGT 684
TGGAGCCATGTCTTGACTGCTCCCGAGCAGATCCATAATGGTTTCTGGAAGCCGACTCAGACCGCCTCTCAGAGT 685
GTCTTCTGAGTGTCCCTCACTGTCCCTGTCCTGACTAACACTGGATCCCTGGCGCCTGCTTGTCTTGGACCCCGC 686
GTCTTCTGAGTGTCCCTCACTGTCCCTGTCCTGACTAACACTGGATCCCTGGCGCCTGCTTGTCTTGGACCCCGC 687
TGATTCTCCCTGGCCCACCTGTGAGTGTCTAGAGCTGCCGGCCCGAGTGGAAGGTTCATGGTGACGTGACCCTGA 688
TGATTCTCCCTGGCCCACCTGTGAGTGTCTAGAGCTGCCGGCCCGAGTGGAAGGTTCATGGTGACGTGACCCTGA 689
CTGGAGCCGTCTCCTGATTGTTCCTCATTTCTTGTTTGCCTGCTTGCACTTCTGGGTCCTGACTGCCCATGGGAG 690
CTGGAGCCGTCTCCTGATTGTTCCTCATTTCTTGTTTGCCTGCTTGCACTTCTGGGTCCTGACTGCCCATGGGAG 691
AGGCATCAGACCTTCCCTGGGGTGTGGTGTGGCTGTGATGGTACCCTGAGTGTCCAGACCTATCTACTGATTGCT 692
AGGCATCAGACCTTCCCTGGGGTGTGGTGTGGCTGTGATGGTACCCTGAGTGTCCAGACCTATCTACTGATTGCT 693
GCTCGTGGTAGGATCCCTGCCTTCCTCCTCTGCTTGACCCCGGGTGTCCACGAATGGTGTCCTGACCGTCTTGGG 694
GCTCGTGGTAGGATCCCTGCCTTCCTCCTCTGCTTGACCCCGGGTGTCCACGAATGGTGTCCTGACCGTCTTGGG 695
TGCTGAGTGCCTAGAGCTGTTTCGTGCCTGCTCATGGCGGGATCCTTGTCTTCCTCCAGTGCTGGGTGCAGTCTG 696
TGCTGAGTGCCTAGAGCTGTTTCGTGCCTGCTCATGGCGGGATCCTTGTCTTCCTCCAGTGCTGGGTGCAGTCTG 697
TGTGGACTCAGACTGTTCATGAGAGCTCACCTGGTAGAGGAAAGACCTTGAACGTCCAGAGCTTTCCCCTGACTG 698
TGTGGACTCAGACTGTTCATGAGAGCTCACCTGGTAGAGGAAAGACCTTGAACGTCCAGAGCTTTCCCCTGACTG 699
GCCACGTGCGGACTCTTTGTGGCTCTGCTGATGGGGCCCAGCTTGTCCGTGGGCTGACACTGACTGTGTGTCTGA 700
GCCACGTGCGGACTCTTTGTGGCTCTGCTGATGGGGCCCAGCTTGTCCGTGGGCTGACACTGACTGTGTGTCTGA 701
CCTGGACTTGATCTTGCCTGTTCATGGGATGACACAGCCTGTCCATGAGAGGAAGACTCTGTGTGATGAGTGCCT 702
CCTGGACTTGATCTTGCCTGTTCATGGGATGACACAGCCTGTCCATGAGAGGAAGACTCTGTGTGATGAGTGCCT 703
TGTCTGGAGCTGTCTGCAGAGTGCCCGTGACTGGCTCTGTCTTCTTGATGGAACCCAGGGTGTCTGGAGCCATCT 704
ATCTCTTGACTGCTCCCGAGAAGATCCATGATGGTTTCTGGAAGCAGACCCAGACAACCTCTCGGAGTCGTCTGA 705
CTGTCACTGTCCTGGCTAACACTGGATCCCTGGTGCCTGCTTGTCCTGGACCCCGATGATTGTTCCTGTCCCACC 706
CTGTCACTGTCCTGGCTAACACTGGATCCCTGGTGCCTGCTTGTCCTGGACCCCGATGATTGTTCCTGTCCCACC 707
TGTGAGTGTCTAGAGCTGTCAGCCCAAGAGGCAGCTTCATGGTGACGTGACCCTGAGTGCCTGGAGCCGTCTCCT 708
TGTGAGTGTCTAGAGCTGTCAGCCCAAGAGGCAGCTTCATGGTGACGTGACCCTGAGTGCCTGGAGCCGTCTCCT 709
CTGATTGTTTGTCCTTACGAGTTTGTCTGCTTGCACTTCTGGATCCTGACTGCCCATGGGAGGCATCAGACCTTC 710
TGGTGGGATCCCTGCCTTCCTCCTCTCCTTGACCCCGGGTGTGCACGAATGGTGTCCTGACCCTCTTGGGACGCT 711
TGGTGGGATCCCTGCCTTCCTCCTCTCCTTGACCCCGGGTGTGCACGAATGGTGTCCTGACCCTCTTGGGACGCT 712
AGCTGTCTCGTGCCTGCTCGTGGTGGGATCCTTGTCTTCGTCCAGTGCTGGTCCTGGTCCGCCCATGGGCAGACT 713
AGCTGTCTCGTGCCTGCTCGTGGTGGGATCCTTGTCTTCGTCCAGTGCTGGTCCTGGTCCGCCCATGGGCAGACT 714
AGACTCAGACTGTTCATGAGTGCTCACCTGGTAGATGAAAGACCCTGAACGTCCAGACCTTCCCCCTGACCAGTC 715
AGACTCAGACTGTTCATGAGTGCTCACCTGGTAGATGAAAGACCCTGAACGTCCAGACCTTCCCCCTGACCAGTC 716
CAGTCACGTGCGGACTCTTGGTGGCTCTGCTGATGGGGCCCATCCTGTCCATGGCCTGACACTGACTGTGTGTCT 717
CAGTCACGTGCGGACTCTTGGTGGCTCTGCTGATGGGGCCCATCCTGTCCATGGCCTGACACTGACTGTGTGTCT 718
AATGTCCCTCACTATCACTGGCCTGACTACCACTGGACCCCCAGTGTCCACTGTCTCTGACTGCAGATGAAGCTT 719
AATGTCCCTCACTATCACTGGCCTGACTACCACTGGACCCCCAGTGTCCACTGTCTCTGACTGCAGATGAAGCTT 720
GTCTGCGCGGAATGCCTGAGTGTCTGGAGCTGTCTGCTGACTGCTGGTGGCGGGATCCGTGTCTTTCTCCTGGAC 721
GTCTGCGCGGAATGCCTGAGTGTCTGGAGCTGTCTGCTGACTGCTGGTGGCGGGATCCGTGTCTTTCTCCTGGAC 722
GACTTGATCTTGCCTGTTCATGGGATGACGCAGCCTGTCCACCAGAGGAAGTCTCTGCGTGAGGAGTTCCTGATT 723
GACTTGATCTTGCCTGTTCATGGGATGACGCAGCCTGTCCACCAGAGGAAGTCTCTGCGTGAGGAGTTCCTGATT 724
GTTCCTGATTGTCTGGAGCTGTCTGCAGAGTGCCCGTGACCGGCTCTGTCTTCGTGATGGGACCTGGGGTGTCTG 725
GTTCCTGATTGTCTGGAGCTGTCTGCAGAGTGCCCGTGACCGGCTCTGTCTTCGTGATGGGACCTGGGGTGTCTG 726
CTGCTCCTGAGCAGATCCACGATGGTTTCTGGAAGCAGACCCAGACCACCTCTCAGAGTCTTCTGAATGTCCCTC 727
CTGCTCCTGAGCAGATCCACGATGGTTTCTGGAAGCAGACCCAGACCACCTCTCAGAGTCTTCTGAATGTCCCTC 728
GTCCCTCACTGTCACTGTCCTGGCTAACACTGGATCCCTGGCGCCTGCTTGTCCTGGACCCCTCTGATTGTCCCT 729
GTCCCTCACTGTCACTGTCCTGGCTAACACTGGATCCCTGGCGCCTGCTTGTCCTGGACCCCTCTGATTGTCCCT 730
TGGCCTGCCTGTGAGTGTCTAGAGATGTCGGCATGAGAGGAAGCTTCATGGTGACGTGACCCTGAGTGCCTGGAG 731
TGGCCTGCCTGTGAGTGTCTAGAGATGTCGGCATGAGAGGAAGCTTCATGGTGACGTGACCCTGAGTGCCTGGAG 732
GGAGCCGTCTCTTGATTGTTCCTCATTACGTGTTGTTCTGCTTGCACTTCTGGATCCTGACTGCCCACGGGAGGC 733
GGATGTGGTGTGGCTGTGATGGGACCCTGAGTGTCCAGACCTATCTACCGATTGCTCGTGGTGGGACCCCTGCCT 734
GGATGTGGTGTGGCTGTGATGGGACCCTGAGTGTCCAGACCTATCTACCGATTGCTCGTGGTGGGACCCCTGCCT 735
TGGTGGGACCCCTGCCTTCCTCTTCTGCTTGACCCCGGGTGTCCACGAATGGTGTCCTGACCATCTTGGGATGCT 736
TGGTGGGACCCCTGCCTTCCTCTTCTGCTTGACCCCGGGTGTCCACGAATGGTGTCCTGACCATCTTGGGATGCT 737
CTGGAGTTGTCTCGTGCCTGCTCATGGTGGGATCCTTGTCTTACTCCAGTGCTGGGCCCTGTCCATCCATGGGAG 738
CTGGAGTTGTCTCGTGCCTGCTCATGGTGGGATCCTTGTCTTACTCCAGTGCTGGGCCCTGTCCATCCATGGGAG 739
GACTCTTGGTGGCTCTGCTGATGGTGACCAGCCTGTCCATGGCCTGACACTGACTGTGTGTCTGAGTCTTCTGAA 740
CTCACTGTCAGTGGCCTGACTACCACTGGACCCTCGGTGTCCACTGTCTCTGACTGCAGATGAAGCTTGTCCGTG 741
CTCACTGTCAGTGGCCTGACTACCACTGGACCCTCGGTGTCCACTGTCTCTGACTGCAGATGAAGCTTGTCCGTG 742
TGTCCGTGCCCAGTGCCTGAGTGTCTGGAGCTGTCTGCTGACTGGAGCTGGTGGCGGGATCCATGTCTTTCTCCT 743
TGTCCGTGCCCAGTGCCTGAGTGTCTGGAGCTGTCTGCTGACTGGAGCTGGTGGCGGGATCCATGTCTTTCTCCT 744
CACTTGATCTTGCCTGTTCATGGGATGACGCAGCCTGTCCACTAGAGGAATTCTGTGTGTGACGAGTGCCTGATT 745
TGCCTGATTTTCTGGAGCTGTCTGCAGAGTGCCCATGACCAGCTCTGTCTTCGTGATGGGACCTGGGGTGTCTGG 746
TGCCTGATTTTCTGGAGCTGTCTGCAGAGTGCCCATGACCAGCTCTGTCTTCGTGATGGGACCTGGGGTGTCTGG 747
CTCTTGACTGCTCCTGAGCAGATCCATGATGGTTTCTGGAAGCAGACCCAGACCACCTCTCAGAGTCTTCTGAGT 748
CTCTTGACTGCTCCTGAGCAGATCCATGATGGTTTCTGGAAGCAGACCCAGACCACCTCTCAGAGTCTTCTGAGT 749
GAGTGTCCCTGACTGTCACTGTCCTGGCTAACACTGGATCCCTGGTTCCTACTTGTCCTGGGCCCCGATGATTGT 750
GAGTGTCCCTGACTGTCACTGTCCTGGCTAACACTGGATCCCTGGTTCCTACTTGTCCTGGGCCCCGATGATTGT 751
TGAGTGTCTAGAGCTGTCAGCCTGAGAGGAAGCTTCATGATGACGTGACCCTGAGTGCCTGGTGCCGTCTCCTGA 752
TGAGTGTCTAGAGCTGTCAGCCTGAGAGGAAGCTTCATGATGACGTGACCCTGAGTGCCTGGTGCCGTCTCCTGA 753
CTGATTGTTCCTCATTTCGTGTTTGTCTGCTTGCACTTCTGGATCCTGACTGCCCATGGGAGGCATCAGACCTTC 754
GGGATGTGGTGTGGCTGTGATGGGAACCTGAGTGTCCAGACCTATTTACCGATTGCTCGTGGTGGGATCCCTGCC 755
GGGATGTGGTGTGGCTGTGATGGGAACCTGAGTGTCCAGACCTATTTACCGATTGCTCGTGGTGGGATCCCTGCC 756
TGGTGGGATCCCTGCCTTCCTCCTCTGCTTGACCCCGGGTGTCCACGAATGGTGTCCTGACCCTCTTGGGACGCT 757
TGGTGGGATCCCTGCCTTCCTCCTCTGCTTGACCCCGGGTGTCCACGAATGGTGTCCTGACCCTCTTGGGACGCT 758
CCTGGAGCTGTCTCGTGCCTGCTCGTGGTGCGATCCTTGTCTTCCTCCAGTGCTGGTCCCGGTCCGTCCATGGGC 759
CCTGGAGCTGTCTCGTGCCTGCTCGTGGTGCGATCCTTGTCTTCCTCCAGTGCTGGTCCCGGTCCGTCCATGGGC 760
GGCTGTTCATGAGTGCTCACCTGGTAGAGGGAAGACCCTGAACGTCCAGACCGTTCCCCTGACCGGCCACGTGTG 761
GGCTGTTCATGAGTGCTCACCTGGTAGAGGGAAGACCCTGAACGTCCAGACCGTTCCCCTGACCGGCCACGTGTG 762
CTTGGTGGCTCTGCTGTCTCAGCCCAGCCTTTCCGTGGCCTGACACTGATTGTGTGTCTGAGTTTTCTGAATGTC 763
CTTGGTGGCTCTGCTGTCTCAGCCCAGCCTTTCCGTGGCCTGACACTGATTGTGTGTCTGAGTTTTCTGAATGTC 764
CCCTCACTGTCACTGGCCTGACTACCGCTAGACCCCCGGTGTCCACGATCGCTGACTGCAGATGAAGCTTGCCCG 765
CCCTCACTGTCACTGGCCTGACTACCGCTAGACCCCCGGTGTCCACGATCGCTGACTGCAGATGAAGCTTGCCCG 766
TTGCCCGCGCCCAGTGGCTGAGTGTCTGGAGCTGTCTGCTGACTGCTGGTGGCCGGATCCATGTCTTTCTCCTGG 767
TTGCCCGCGCCCAGTGGCTGAGTGTCTGGAGCTGTCTGCTGACTGCTGGTGGCCGGATCCATGTCTTTCTCCTGG 768
TGCCTGTTCATGGGATGATGCAGTCTGTCCACGAGAGGAAGTCTCTGCGTGACGAGTGCCTGATTGTCTGGAGCT 769
TGCCTGTTCATGGGATGATGCAGTCTGTCCACGAGAGGAAGTCTCTGCGTGACGAGTGCCTGATTGTCTGGAGCT 770
CTGGAGCTGTCTGCAGAGTGCCCATGACTGGCTCTGTCTTCATCATGGGACCTGGGGTGTCTGGAGCCATCTCTT 771
CTGGAGCTGTCTGCAGAGTGCCCATGACTGGCTCTGTCTTCATCATGGGACCTGGGGTGTCTGGAGCCATCTCTT 772
CCCACGCAGATCCATGATGGTTTCTGGAAGCCGACCCAGAGTGCCTCTCAGAGTCTTCTGAGTGTCCCTCACTGT 773
CCCACGCAGATCCATGATGGTTTCTGGAAGCCGACCCAGAGTGCCTCTCAGAGTCTTCTGAGTGTCCCTCACTGT 774
GAGTGTCCCTCACTGTCCCTGTCCTGGCTAACTCTGGATCCCCTACGCTTTCTTGTCCTGGACTCCTGCAATGGT 775
GAGTGTCCCTCACTGTCCCTGTCCTGGCTAACTCTGGATCCCCTACGCTTTCTTGTCCTGGACTCCTGCAATGGT 776
TTCATGTCTTGACCTGTTCACTTGAGATGATGATTTGCCATCAGATGACCTTGATCTTTCATATATTTTGTTTTC 777
ATATTTTGTTTTCTTCTAATAGACTATCAGTGGTGTCATAGGCTTCATCCTGGATTGTGTAATATGTGGCAATAT 778
TGGCCTGATTGTATCCATTTTTGAGTCATTCTTCCTGTATTTTCATAATCATATACTCCTTCTTCATTGTCTTCT 779
CTTCAAGTCTTTCACTTAGCCTCTTCCTATTGTCTCCTAATCTAGTATTTTCAGTCTTGTTTTTCTCTTTTTTAC 780
GTTATGATGGTTTTTTCCATATTCTTCTTCTCTATGAGTAGGTGAATATCCTTTTCTTTCTTTTTTTTCAGAACT 781
TAGATTCATGCCTTTTCCCCCCTGTTTCTCTTGGGCTCTTGGATCTTCCCTTATTCCCTTTTCTATTGTTTCTTC 782
CCAGACTTGAGGGTCTTTTTCTGTTTTCTTTGTTTTCTTCCTGTTTATTATCTTCATGTTTATCATGATGACTGT 783
GTGCTTTCTGTGCTTGTGTCCTGATATCGGTAAATTCTCTTTTCTGGTAGACTCATAATATGCTTGAGCCAACTT 784
TCAGAAGAAACTCAGTGAAGTCAATTTTCTTGTTGTGGTCTATATCCAAGTGATCCATGAAGACATCAACCATAT 785
GACATCAACCATATCTGGGTCATCTGGATTCTGTACAGAGGGAAGTCACAGAGGGAGACTGCATCAGACAGAATC 786
ATAAGAATACAAGAGACAAACAGTATTATATGATGAATAGTTTATTTTTAATTTAGATGCAGCTTACTATAATAT 787
TTATGTCCAAGATGATTTTTTGAATACAGAATACTAGAATTCCAATAGAAGGATAATAGAGAAAGATGTGCTAGC 788
TGATGTTGATATAGCCACTTTGGTATACAGAACTGTTTTATATTTTTGGCTCCTTCGATATTTCTGAAAAAGATT 789
ATTTCTGAAAAAGATTAATTTAGAAATTTGGGGAGTGTCTAAAACTTAAACTTTCAAAAACATAAAACATTACTT 790
AAATACTCCAGCTAGTTTTCTAAAGTTAGCTCTCCATGATATTGATTTCTTCCATTTAATATTTCTGAAATATAG 791
ATATTTCTGAAATATAGCGTTTAAAGATCATTACACAATAAAAATAAGCTACCACCAAACTAATGAAATACTATA 792
TTTAAACAGATTGACAGGAAAAGATAACTTCCCTGAAAGTATTATGAAGTTTCTTGATTGAAAGTGAACTTGCTT 793
TGCTTCATTCTTCTATTCTTGGATTAATTCCTTTGCCATTAATTTCTTACTCATAGTAATAGTATCTCTGTGACT 794
ACTGACTAAATCCCAGTTGTTTCGATATATCACTAGAATGGCCACATAAACCTGGGTCCTTATTAATATACGTTG 795
ATAATACCTTGGATGATCTTTACCAAACGCACTTGCTTTACAGATATCAGATCTTTCCTTGAAAACAACAGGATT 796
GTAACTAACACTTCCGTGCTGAGAGTGTCTAAACCCGGATTCACCATAATCATAATCTGCACTACCATAGCTGCC 797
CTAAACCTGATTGACCTTTTTGCCTTTCAGTGCCCTCAGATTGATAATGATAAGAACTAGAACTGTGAGGACTGC 798
ACGTGACTGTATTCCTGAGTGATACGCAGAATCTTGTGAAAGACTACTAAAGTGACCATGTTCCTTAGCGGTACT 799
GCGGTACTAGAGTCTGACTGTACAGGTGAAGACTGTACATGACTGGCTGTATCGCGGTGAGAGGATCCGGGGTGT 800
GCGGTACTAGAGTCTGACTGTACAGGTGAAGACTGTACATGACTGGCTGTATCGCGGTGAGAGGATCCGGGGTGT 801
AGCCATCTCTTGACTGCTCCCGAGAAGATCCATGATGGTTTCTGGAAGCAGACTCAGATCGCCTCTCAGAGTCCT 802
TCTGGGTATGCCTCACTGTCACTGTCCTGGCTAACACTGGATCCCTGGCGCCTGCTTCTCCTGGACCCCGCTGAT 803
TCTGGGTATGCCTCACTGTCACTGTCCTGGCTAACACTGGATCCCTGGCGCCTGCTTCTCCTGGACCCCGCTGAT 804
CTGGCCGGACTGTGAGTGTCTAGAGCTGTCAGCCTGAGTGGAAGCTTCATGGTGACGCGACCCTGAGTGCCTGGA 805
CTGGCCGGACTGTGAGTGTCTAGAGCTGTCAGCCTGAGTGGAAGCTTCATGGTGACGCGACCCTGAGTGCCTGGA 806
GCCTGGAGCCGTCTCCTGACTGTTCCTCATTACGTGTTTCTCTGCTTGCACTTCTGGATCCTGACTGCCCATGGG 807
GCCTGGAGCCGTCTCCTGACTGTTCCTCATTACGTGTTTCTCTGCTTGCACTTCTGGATCCTGACTGCCCATGGG 808
AGACCTTCCCTGGGATGTGGTGTGGCTGTGATGGGACCCTGAGTGTCCAGACCTATCTACCGATTGCTCGTGGTA 809
AGACCTTCCCTGGGATGTGGTGTGGCTGTGATGGGACCCTGAGTGTCCAGACCTATCTACCGATTGCTCGTGGTA 810
TTGCTCGTGGTAGGATCCCTGTCTTCCTCCTCTCCTTGACCCCGGGTGTCCACGAATGGTGTCCTGACCCTCTTG 811
TTGCTCGTGGTAGGATCCCTGTCTTCCTCCTCTCCTTGACCCCGGGTGTCCACGAATGGTGTCCTGACCCTCTTG 812
TTGGGACGCTGAGTGCCTGGAGCTGTCTCGTGCCTGCTCGTGGCGGGATCCTTGTCTTCCTCCAGTACTGGGCCC 813
TTGGGACGCTGAGTGCCTGGAGCTGTCTCGTGCCTGCTCGTGGCGGGATCCTTGTCTTCCTCCAGTACTGGGCCC 814
GTCCATGGGCAGACTCAGACTGTTCATGAGTGCTCACCTGGTAGAGGAAAGACCCTGAACGTCCAGACCTTCCTG 815
GTCCATGGGCAGACTCAGACTGTTCATGAGTGCTCACCTGGTAGAGGAAAGACCCTGAACGTCCAGACCTTCCTG 816
TCCTGCTGACCGGCCACGTGTGGACTCTTGGTGGCTCTGCTGATGGGGCCCAGCCTGTCCGTGGGCTGACACTGA 817
TCCTGCTGACCGGCCACGTGTGGACTCTTGGTGGCTCTGCTGATGGGGCCCAGCCTGTCCGTGGGCTGACACTGA 818
CTGTGTGTCTGAGTCTTCTGAATGTCCCTCACTGTCACTGGCCTGACTACCACTGGACCCTCGGTGTCCACTGTC 819
CTGTGTGTCTGAGTCTTCTGAATGTCCCTCACTGTCACTGGCCTGACTACCACTGGACCCTCGGTGTCCACTGTC 820
AGCTTGTCCGTGCCCAATGCCTGAGTGTCTGGAGCTGTCTGCTGACTGCTGGTGGTGGGATCCATGTCTCTCTCC 821
AGCTTGTCCGTGCCCAATGCCTGAGTGTCTGGAGCTGTCTGCTGACTGCTGGTGGTGGGATCCATGTCTCTCTCC 822
CCATGTCTCTCTCCTGCACTTGATCTTGCCTGTTCATGGGATGATGCAGCCTGTCCACCAGAGGAATTCTCTGCA 823
CCATGTCTCTCTCCTGCACTTGATCTTGCCTGTTCATGGGATGATGCAGCCTGTCCACCAGAGGAATTCTCTGCA 824
GGAGCCATCTCTTGACTGCTCCTGAGCAGATCCACGATGGTTTCTGGAAGCAGACCCAGACCACCTCTCAGAGTC 825
GGAGCCATCTCTTGACTGCTCCTGAGCAGATCCACGATGGTTTCTGGAAGCAGACCCAGACCACCTCTCAGAGTC 826
AGTGTCCCTGACTGTCACTGTCCTGGCTAACACTGGATCCCTGGTTCCTGCTTGTCCTGGGCCCCGCTGATTGTC 827
AGTGTCCCTGACTGTCACTGTCCTGGCTAACACTGGATCCCTGGTTCCTGCTTGTCCTGGGCCCCGCTGATTGTC 828
TGGCCGGACTGTGAGTGTCTAGAGCTGTCCGCCTGAGTGGAAGCTTCATGGTGATGCGACCATGAGTGCCTGGAG 829
TGGCCGGACTGTGAGTGTCTAGAGCTGTCCGCCTGAGTGGAAGCTTCATGGTGATGCGACCATGAGTGCCTGGAG 830
GTGCCTGGAGCCATCTCCTGATTGTTCGTCATTACGAGTTTGTCTGCTGGCACTTCTGGATCCTGACTGCCCACG 831
GTGCCTGGAGCCATCTCCTGATTGTTCGTCATTACGAGTTTGTCTGCTGGCACTTCTGGATCCTGACTGCCCACG 832
CAGACCTTCCCTGGGATGTGGTGTGGCTGTGATGGGACCCTGAGTGTCCAGACCTATCTACCGATTGCTCGTAGT 833
CAGACCTTCCCTGGGATGTGGTGTGGCTGTGATGGGACCCTGAGTGTCCAGACCTATCTACCGATTGCTCGTAGT 834
TGCTCGTAGTGGGATCCCTGCCTTCCTCTTCTGCTTGACCCCGGGTGTCCACGAATGGTGTCCTGACCCTCTTGG 835
TGCTCGTAGTGGGATCCCTGCCTTCCTCTTCTGCTTGACCCCGGGTGTCCACGAATGGTGTCCTGACCCTCTTGG 836
CTGAGTGCCTGGAGCTGTCTCGTGCCTGCTCGTGGTGGGATCCTTGTCTTCGTCCAGTGCTGGTCCTGGTCCGCC 837
CTGAGTGCCTGGAGCTGTCTCGTGCCTGCTCGTGGTGGGATCCTTGTCTTCGTCCAGTGCTGGTCCTGGTCCGCC 838
CATGGGCAGACTCAGACTGTTCATGAGTGCTCACCTGGTAGAGGAAAGACCCTGAACGTCCAGACCTTCCCCCTG 839
CATGGGCAGACTCAGACTGTTCATGAGTGCTCACCTGGTAGAGGAAAGACCCTGAACGTCCAGACCTTCCCCCTG 840
GTCTGACTCTTCTGAATGTCCCTCACTATCACTGGCCTGACTACCACTGGACCCCCAGTGTCTACTGTCTCTGAC 841
GTCTGACTCTTCTGAATGTCCCTCACTATCACTGGCCTGACTACCACTGGACCCCCAGTGTCTACTGTCTCTGAC 842
GCAGATGAAGCTTGTCCACGCGGAATGCCTGAGTGTCTGGAGCTGTCTGCTGACTGCTGGTGGCGGGATCCGTGT 843
GCAGATGAAGCTTGTCCACGCGGAATGCCTGAGTGTCTGGAGCTGTCTGCTGACTGCTGGTGGCGGGATCCGTGT 844
CTGATTGTCTGGAGCGGTCTGCAGAGTGCCCGTGACCGGCTCTGTCTTCGTGATGGGACCTGGGGTGTCTGGAGC 845
CTGATTGTCTGGAGCGGTCTGCAGAGTGCCCGTGACCGGCTCTGTCTTCGTGATGGGACCTGGGGTGTCTGGAGC 846
GCCTTGACTGCTCCTGAACAGATCCACGATGGTTTCTGGAAGCAGACCCAGACCACCTCTCAGAGTCTTCTGAAT 847
GCCTTGACTGCTCCTGAACAGATCCACGATGGTTTCTGGAAGCAGACCCAGACCACCTCTCAGAGTCTTCTGAAT 848
AGTCTTCTGAATGTCCCTCACTGTCACTGTCCTGGCTCACACTGGATCCCTGGCGCCTGCTTCTCCTGGACCCCT 849
AGTCTTCTGAATGTCCCTCACTGTCACTGTCCTGGCTCACACTGGATCCCTGGCGCCTGCTTCTCCTGGACCCCT 850
TGATTGTCCCTGGACTGCCTGTGAGTGTCTAGAGATGTCGGCATGAGTGGAAGCTTCATGGTGACGTGACACTGA 851
TGATTGTCCCTGGACTGCCTGTGAGTGTCTAGAGATGTCGGCATGAGTGGAAGCTTCATGGTGACGTGACACTGA 852
TGGAGCTGTCTCCTGATTGTTCCTCATTACGTGTTGTTCTGCTTGCACTTCTGGATCCTGACTGCCCACGGGAGG 853
TGGAGCTGTCTCCTGATTGTTCCTCATTACGTGTTGTTCTGCTTGCACTTCTGGATCCTGACTGCCCACGGGAGG 854
GTGGTACCCCTGCCTTCCTCCTCTGCTTGACCCCGGGTGTCCACGAATGGTGTCCTGACCGTATTGGGATGCTGA 855
GTGGTACCCCTGCCTTCCTCCTCTGCTTGACCCCGGGTGTCCACGAATGGTGTCCTGACCGTATTGGGATGCTGA 856
GATGCTGAGTGCCTGGAGCTGTCTTGTGCCTGCTCATGGCGGGATCCTTGTCTTCCTCTAGTGCTGGGCCCCGTC 857
GATGCTGAGTGCCTGGAGCTGTCTTGTGCCTGCTCATGGCGGGATCCTTGTCTTCCTCTAGTGCTGGGCCCCGTC 858
CAGACTGTTCATGAGTGCTCACCTGGTAGAGGAAAGATCCTGAATGTCCAGACGTTTCCCCTGACCGGCCACGTG 859
CAGACTGTTCATGAGTGCTCACCTGGTAGAGGAAAGATCCTGAATGTCCAGACGTTTCCCCTGACCGGCCACGTG 860
ACCGGCCACGTGCGGACTCTTGGTGGCTCTGCTGATGGGACCCAGCCTGTCCGTGGGCTGACACTGACTGTGTGT 861
ACCGGCCACGTGCGGACTCTTGGTGGCTCTGCTGATGGGACCCAGCCTGTCCGTGGGCTGACACTGACTGTGTGT 862
AGTCTTCTGAATGTCCCTCATTGTCACTGGCCTGACTACCACTGTACCCTCGGTGTCCACTGTCTCTGACTGCAG 863
AGTCTTCTGAATGTCCCTCATTGTCACTGGCCTGACTACCACTGTACCCTCGGTGTCCACTGTCTCTGACTGCAG 864
TGAAGCTTGTCCGTGCCCAATGCCTGAGTGTCTGGAGCTGTCTGCTGACTGCTGGTGGTGGGATCCATGTCTTTC 865
TGAAGCTTGTCCGTGCCCAATGCCTGAGTGTCTGGAGCTGTCTGCTGACTGCTGGTGGTGGGATCCATGTCTTTC 866
ACTTGATCTTGCCTGTTCATGGGATGATGCAGCCTGTCCACCAGAGGAAGTCTGTGTGTGACGAGTGCCTGATTG 867
ACTTGATCTTGCCTGTTCATGGGATGATGCAGCCTGTCCACCAGAGGAAGTCTGTGTGTGACGAGTGCCTGATTG 868
TTGTCTGGAGCTGTCTGCAGAGTGCCCATGACCAGCTCTGTCTTCTTGATGGGACCTGGGGTGTCTGGAGCCATC 869
TTGTCTGGAGCTGTCTGCAGAGTGCCCATGACCAGCTCTGTCTTCTTGATGGGACCTGGGGTGTCTGGAGCCATC 870
CCATCTCTTAGCTGCTCCTGAGCAGATCCATGATGGTTTCTGGAAGCAGACCCAGACCACCTCTCAGAGTCTTCT 871
AGTCTTCTGAATGTCCCTCACTGTCACTGTCCTGGCTCACACTGGATCCCTGGCGCCTGCTTCTCCTGGACCCCT 872
AGTCTTCTGAATGTCCCTCACTGTCACTGTCCTGGCTCACACTGGATCCCTGGCGCCTGCTTCTCCTGGACCCCT 873
GCCTGTGAGTGTCTAGAGATGTCGGCATGAGTGGAAGCTTCATGGTGACGCGACCCTGAGTGCCTGGAGCCGTCT 874
GCCTGTGAGTGTCTAGAGATGTCGGCATGAGTGGAAGCTTCATGGTGACGCGACCCTGAGTGCCTGGAGCCGTCT 875
CCGTCTCCTGATTGTTCCTCATTACGTGTTGTTCTGCTTGCACTTCTGGATCCTGACTGCCCACGGGAGGCATCA 876
CCGTCTCCTGATTGTTCCTCATTACGTGTTGTTCTGCTTGCACTTCTGGATCCTGACTGCCCACGGGAGGCATCA 877
CTCGTGGTGGTACCCCTGCCTTCCTCCTCTGCTTGACCCCGGGTGTCCACGAATGGTGTCCTGACCGTCTTGGGA 878
CTCGTGGTGGTACCCCTGCCTTCCTCCTCTGCTTGACCCCGGGTGTCCACGAATGGTGTCCTGACCGTCTTGGGA 879
TGCTGAGTGCCTGGAGCTGTCTTGTGCCTGCTCATGGCGGGATCCTTGTCTTCCTCTAGTGCTGGGCCCCGTCCA 880
TGCTGAGTGCCTGGAGCTGTCTTGTGCCTGCTCATGGCGGGATCCTTGTCTTCCTCTAGTGCTGGGCCCCGTCCA 881
CTGTTCATGAGTGCTCACCTGGTAGAGGAAAGATCCTGAATGTCCAGACGTTTCCCCTGACCGGCCACGTGCGGA 882
CTGTTCATGAGTGCTCACCTGGTAGAGGAAAGATCCTGAATGTCCAGACGTTTCCCCTGACCGGCCACGTGCGGA 883
CTGACCGGCCACGTGCGGACTCTTGGTGGCTCTGCTGATGGGACCCAGCCTGTCCGTGGGCTGACACTGACTGTG 884
CTGACCGGCCACGTGCGGACTCTTGGTGGCTCTGCTGATGGGACCCAGCCTGTCCGTGGGCTGACACTGACTGTG 885
GAGTCTTCTGAATGTCCCTCATTGTCACTGGCCTGACTACCACTGTACCCTCGGTGTCCACTGTCTCTGACTGCA 886
GAGTCTTCTGAATGTCCCTCATTGTCACTGGCCTGACTACCACTGTACCCTCGGTGTCCACTGTCTCTGACTGCA 887
TCCGTGCCCAATGCCTGAGTGTCTGGAGCTGTCTGCTGACTGCTGGTGGTGGGATCCATGTCTTTCTCCTGCACT 888
TCCGTGCCCAATGCCTGAGTGTCTGGAGCTGTCTGCTGACTGCTGGTGGTGGGATCCATGTCTTTCTCCTGCACT 889
TGATCTTGCCTGTTCATGGGATGATGCAGCCTGTCCACCAGAGGAAGTCTGTGTGTGACGAGTGCCTGATTGTCT 890
TGATCTTGCCTGTTCATGGGATGATGCAGCCTGTCCACCAGAGGAAGTCTGTGTGTGACGAGTGCCTGATTGTCT 891
TGATTGTCTGGAGCTGTCTGCAGAGTGCCCATGACCAGCTCTGTCTTCTTGATGGGACCTGGGGTGTCTGGAGCC 892
TGATTGTCTGGAGCTGTCTGCAGAGTGCCCATGACCAGCTCTGTCTTCTTGATGGGACCTGGGGTGTCTGGAGCC 893
TCTTAGCTGCTCCTGAGCAGATCCATGATGGTTTCTGGAAGCAGACCCAGACCACCTCTCAGAGTCTTCTGAGTG 894
TCCCTGACTGTCACTGTCCTGGCTAAAACTGGATCCCCAGTTCCTGCTTGTCCTGGGCCCCTCTGATTGTCCCTG 895
TCCCTGACTGTCACTGTCCTGGCTAAAACTGGATCCCCAGTTCCTGCTTGTCCTGGGCCCCTCTGATTGTCCCTG 896
CCTGGCCCACCTGCGAGTGTCCAGAGCTGTCGGCCCGAGAGGAAGCTTCATGGTGACGCGACCCTGAGTGCCTGG 897
CCTGGCCCACCTGCGAGTGTCCAGAGCTGTCGGCCCGAGAGGAAGCTTCATGGTGACGCGACCCTGAGTGCCTGG 898
GGAGCCGTCTCCTGATTGTTCATCGTTACGAGTTTGTCTGCTTGCACTTCTGGATCCTGAGTGCCCATGGGAGGC 899
GGAGCCGTCTCCTGATTGTTCATCGTTACGAGTTTGTCTGCTTGCACTTCTGGATCCTGAGTGCCCATGGGAGGC 900
TGGGATGTGGTGTGGCTGTGATGAGACCCTGAGTGTCCAGATCTATCTACCAATTGCTCGTAGTGGGATCCCTGC 901
TGGGATGTGGTGTGGCTGTGATGAGACCCTGAGTGTCCAGATCTATCTACCAATTGCTCGTAGTGGGATCCCTGC 902
TGCCTTCCTCCACTGCTTGACCCCGGGTGTCCATGAATGGTGTCCTGACCCTCTTGGGACGTTGAGTGCCTGGAG 903
TGCCTTCCTCCACTGCTTGACCCCGGGTGTCCATGAATGGTGTCCTGACCCTCTTGGGACGTTGAGTGCCTGGAG 904
CTGGAGCTGTCTCGTGCCTGCTTGTGGTGGGATCCTTGTCTTCCTCCAGTGCTGGTCCCGGTCCGTCCATGGGCG 905
CTGGAGCTGTCTCGTGCCTGCTTGTGGTGGGATCCTTGTCTTCCTCCAGTGCTGGTCCCGGTCCGTCCATGGGCG 906
GCGGACTCAGACTGTTCATGAGTGCTCACCTGGTAGAGGAAAGACCCTGAACGTCCAGACCTTCCTGCTGACCGG 907
GCGGACTCAGACTGTTCATGAGTGCTCACCTGGTAGAGGAAAGACCCTGAACGTCCAGACCTTCCTGCTGACCGG 908
CGGCCACGTGTGGACTCTTGGTGGCTCTGCTGATGGGGCCCAGCCTGTCCGTGGGCTGACACTGACTGTGTGTCT 909
CGGCCACGTGTGGACTCTTGGTGGCTCTGCTGATGGGGCCCAGCCTGTCCGTGGGCTGACACTGACTGTGTGTCT 910
AATGTCCCTCACTGTCACTGGCCTGACTACCACTGGACCCTCGGTGTCCACTGTCTCTGACTGCAGATGAAGCTT 911
AATGTCCCTCACTGTCACTGGCCTGACTACCACTGGACCCTCGGTGTCCACTGTCTCTGACTGCAGATGAAGCTT 912
ATTGTCTGGAGCTCTCTGCAGAGTGCCCATGACCGGCTCTGTCTTCGTGATGGGACCTGGGGTGTCTGGAGCCAT 913
ATTGTCTGGAGCTCTCTGCAGAGTGCCCATGACCGGCTCTGTCTTCGTGATGGGACCTGGGGTGTCTGGAGCCAT 914
AGCCATCTCTTGACTGCTCCTGAGCAGATCCATGATGGTTTCTGGACGCAGACCCAGACCGCCTCTCAGAATCTT 915
GTCCCTCACTGTCACTGTCCTGGCTAACACTGGATCCCCGGGGCCTGCTTGTCCTGGGCCCTGATGATTGTCCCT 916
GTCCCTCACTGTCACTGTCCTGGCTAACACTGGATCCCCGGGGCCTGCTTGTCCTGGGCCCTGATGATTGTCCCT 917
GGCCCACCAGTGAGTGTCTAGAGCTGTCGGCCCAAGAGGAAGCTTCATGATGATGCGACCCTGAGTGCCTAGAGC 918
GGCCCACCAGTGAGTGTCTAGAGCTGTCGGCCCAAGAGGAAGCTTCATGATGATGCGACCCTGAGTGCCTAGAGC 919
TGGGATGTGGTGTGGCTGTGATGAGACCCTGAGTGTCCAGACCTATCTACCGATTGCTCTTGGTGGGACCCCTGT 920
TGGGATGTGGTGTGGCTGTGATGAGACCCTGAGTGTCCAGACCTATCTACCGATTGCTCTTGGTGGGACCCCTGT 921
TTGTGCCTGATCATAATGGGATCCTTGTCTTCCTCCAGTGCTGGGCGCAGACTGTCCATGGGTGGACTCAGACTG 922
TTGTGCCTGATCATAATGGGATCCTTGTCTTCCTCCAGTGCTGGGCGCAGACTGTCCATGGGTGGACTCAGACTG 923
GTGGACTCAGACTGTTCATGAGTGCTCACCTGGTAGAGGAAAGACCCTGAACGTCCAGAGCTTTCCCCTGACTGG 924
GTGGACTCAGACTGTTCATGAGTGCTCACCTGGTAGAGGAAAGACCCTGAACGTCCAGAGCTTTCCCCTGACTGG 925
GGACTCTTTGTGGCTCTGCTGATGGGGCCCAGCTTTTCCCTGTGCTGACACTGACTGTGTGTCTGAGTCTTCTGA 926
GGACTCTTTGTGGCTCTGCTGATGGGGCCCAGCTTTTCCCTGTGCTGACACTGACTGTGTGTCTGAGTCTTCTGA 927
TCACTGTCACTGGCCTGACTACCACTGTACCCTCGGTGTCCACTGTCTCTGACTGCAGATGAAGCTTGTCCATGC 928
TCACTGTCACTGGCCTGACTACCACTGTACCCTCGGTGTCCACTGTCTCTGACTGCAGATGAAGCTTGTCCATGC 929
GCCCAATGCCTGAGTGTCTGGAGCTGTCTGCTGACTGGAGCTGGTGGTGGGATCCATGTCTTTCTCCTGCACTTG 930
GCCCAATGCCTGAGTGTCTGGAGCTGTCTGCTGACTGGAGCTGGTGGTGGGATCCATGTCTTTCTCCTGCACTTG 931
ACTTGATCTTGCCTGTTCATGGGATGACGCAGCCTGTCCACGAGAGGAAGACTCTGTGTGACGAGTGCCTGATTG 932
ACTTGATCTTGCCTGTTCATGGGATGACGCAGCCTGTCCACGAGAGGAAGACTCTGTGTGACGAGTGCCTGATTG 933
TTGTCTGGAGCTGTCTGCAGAGTGCCCGTGACCGGCTCTGTCTTCGTGATGGGACCCAGGGTGTCTGGAGCCATC 934
TTGTCTGGAGCTGTCTGCAGAGTGCCCGTGACCGGCTCTGTCTTCGTGATGGGACCCAGGGTGTCTGGAGCCATC 935
CTGACTGTCACTGTCCTGGCTAACACTGGATCCCTGGTTCCTGCTTGTCCTGGGCCCTGATGATTGTCCCTGGCC 936
CTGACTGTCACTGTCCTGGCTAACACTGGATCCCTGGTTCCTGCTTGTCCTGGGCCCTGATGATTGTCCCTGGCC 937
ACCTGCGAGTGTCTAGAGCTGTCGGCCCGAGAGGAAGCTTCATGGTGACGCGACCCTGAGTGCCTGGAGCCGTCT 938
ACCTGCGAGTGTCTAGAGCTGTCGGCCCGAGAGGAAGCTTCATGGTGACGCGACCCTGAGTGCCTGGAGCCGTCT 939
TGGAGCCGTCTCCTGATTGTTTCTCATTACGTGTTTGTCTGCTGACACTTCTGGATCCTGACTGCCCACGGGAGA 940
TGGAGCCGTCTCCTGATTGTTTCTCATTACGTGTTTGTCTGCTGACACTTCTGGATCCTGACTGCCCACGGGAGA 941
TCCTGGGACGTGGTGTGGCTGTGATGAGACCCTGAGTGTCCAGAACTATCTACCGATTGCTCATAGTGGGATCCC 942
TCCTGGGACGTGGTGTGGCTGTGATGAGACCCTGAGTGTCCAGAACTATCTACCGATTGCTCATAGTGGGATCCC 943
GGGATCCCTGCCTTCCTCCTCTGCTTGACCCTGGGTGTCCACGAATGGTGTCCTGACCCTCTTGGGACGCTGAGT 944
GGGATCCCTGCCTTCCTCCTCTGCTTGACCCTGGGTGTCCACGAATGGTGTCCTGACCCTCTTGGGACGCTGAGT 945
CGTGCCTGCTCGTGGCGGGATCTTTGTCTTCCTCCAGTGCTGGGCCCTGTGCGTCCATGGGCGGACTCAGACTGT 946
CGTGCCTGCTCGTGGCGGGATCTTTGTCTTCCTCCAGTGCTGGGCCCTGTGCGTCCATGGGCGGACTCAGACTGT 947
GCGGACTCAGACTGTTCATGAGTGCTCACCTGGTAGAGGAAAGACCCTGAACGTCCAGACCTTTCCCCTGACTGG 948
GCGGACTCAGACTGTTCATGAGTGCTCACCTGGTAGAGGAAAGACCCTGAACGTCCAGACCTTTCCCCTGACTGG 949
ACGTGTGGACTCTTGGTGGCTCTGCTGATGGGGCCCAGCCTGTCCGTGGGCTGACACTGACTGTGTGTCTGACTC 950
ACGTGTGGACTCTTGGTGGCTCTGCTGATGGGGCCCAGCCTGTCCGTGGGCTGACACTGACTGTGTGTCTGACTC 951
CTGAGTGTCCCTCGCTGTCACTGGCCTGGCTACCACTGGACCCTCGGTTTCCACTGTCTCCGACTACAGATGAAT 952
CTGAGTGTCCCTCGCTGTCACTGGCCTGGCTACCACTGGACCCTCGGTTTCCACTGTCTCCGACTACAGATGAAT 953
CGCCCAGTGCCTGAGTCTGTGGAGCTGTCTGCTGACTGCTGGTGGCGGGATCCATGTCTTTCTCCTGGACTTGAC 954
CGCCCAGTGCCTGAGTCTGTGGAGCTGTCTGCTGACTGCTGGTGGCGGGATCCATGTCTTTCTCCTGGACTTGAC 955
TTGCCTGTTCCTGGGATGATGCAGCCTGTCCACCAGAGGAAGTCTCTGCATGACGAGTGCCTGATTGTCTGGAGC 956
TTGCCTGTTCCTGGGATGATGCAGCCTGTCCACCAGAGGAAGTCTCTGCATGACGAGTGCCTGATTGTCTGGAGC 957
AGCTCTCTGCAGAGTGCCCATGACTGGCTCTATCTTCTTGATGGGACCTGGGGTTCCTGGAGCCATGTCTTGACT 958
AGCTCTCTGCAGAGTGCCCATGACTGGCTCTATCTTCTTGATGGGACCTGGGGTTCCTGGAGCCATGTCTTGACT 959
CTGCTCCCGAGCAGATCCATAATGGTTTCTGGAAGCCGACTCAGACCGCCTCTCAGAGTCTTCTGAGTGTCCCTC 960
TGTCCCTGTCCTGACTAACACTGGATCCCTGGCGCCTGCTTGTCTTGGACCCCGCTGATTCTCCCTGGCCCACCT 961
TGTCCCTGTCCTGACTAACACTGGATCCCTGGCGCCTGCTTGTCTTGGACCCCGCTGATTCTCCCTGGCCCACCT 962
TCTAGAGCTGCCGGCCCGAGTGGAAGGTTCATGGTGACGTGACCCTGAGTGCCTGGAGCCGTCTCCTGATTGTTC 963
TCTAGAGCTGCCGGCCCGAGTGGAAGGTTCATGGTGACGTGACCCTGAGTGCCTGGAGCCGTCTCCTGATTGTTC 964
TCTCCTGATTGTTCCTCATTTCTTGTTTGCCTGCTTGCACTTCTGGGTCCTGACTGCCCATGGGAGGCATCAGAC 965
TCTCCTGATTGTTCCTCATTTCTTGTTTGCCTGCTTGCACTTCTGGGTCCTGACTGCCCATGGGAGGCATCAGAC 966
CCTGGGGTGTGGTGTGGCTGTGATGGTACCCTGAGTGTCCAGACCTATCTACTGATTGCTCGTGGTAGGATCCCT 967
CCTGGGGTGTGGTGTGGCTGTGATGGTACCCTGAGTGTCCAGACCTATCTACTGATTGCTCGTGGTAGGATCCCT 968
GCCTTCCTCCTCTGCTTGACCCCGGGTGTCCACGAATGGTGTCCTGACCGTCTTGGGATGCTGAGTGCCTAGAGC 969
GCCTTCCTCCTCTGCTTGACCCCGGGTGTCCACGAATGGTGTCCTGACCGTCTTGGGATGCTGAGTGCCTAGAGC 970
CGTGCCTGCTCATGGCGGGATCCTTGTCTTCCTCCAGTGCTGGGTGCAGTCTGTCCGTGTGTGGACTCAGACTGT 971
CGTGCCTGCTCATGGCGGGATCCTTGTCTTCCTCCAGTGCTGGGTGCAGTCTGTCCGTGTGTGGACTCAGACTGT 972
CATGAGAGCTCACCTGGTAGAGGAAAGACCTTGAACGTCCAGAGCTTTCCCCTGACTGGCCACGTGCGGACTCTT 973
CATGAGAGCTCACCTGGTAGAGGAAAGACCTTGAACGTCCAGAGCTTTCCCCTGACTGGCCACGTGCGGACTCTT 974
GCTCTGCTGATGGGGCCCAGCTTGTCCGTGGGCTGACACTGACTGTGTGTCTGAGTCTTCTGAATGTCCCTCACT 975
GCTCTGCTGATGGGGCCCAGCTTGTCCGTGGGCTGACACTGACTGTGTGTCTGAGTCTTCTGAATGTCCCTCACT 976
CTGGACTTGATCTTGCCTGTTCATGGGATGACACAGCCTGTCCATGAGAGGAAGACTCTGTGTGATGAGTGCCTG 977
GTCTGCAGAGTGCCCGTGACTGGCTCTGTCTTCTTGATGGAACCCAGGGTGTCTGGAGCCATCTCTTGACTGCTC 978
GTCTGCAGAGTGCCCGTGACTGGCTCTGTCTTCTTGATGGAACCCAGGGTGTCTGGAGCCATCTCTTGACTGCTC 979
CTGCTCCCGAGAAGATCCATGATGGTTTCTGGAAGCAGACCCAGACAACCTCTCGGAGTCGTCTGAGTGTCTCTC 980
CTGCTCCCGAGAAGATCCATGATGGTTTCTGGAAGCAGACCCAGACAACCTCTCGGAGTCGTCTGAGTGTCTCTC 981
CTGTCACTGTCCTGGCTAACACTGGATCCCTGGTGCCTGCTTGTCCTGGACCCCGATGATTGTTCCTGTCCCACC 982
CTGTCACTGTCCTGGCTAACACTGGATCCCTGGTGCCTGCTTGTCCTGGACCCCGATGATTGTTCCTGTCCCACC 983
TAGAGCTGTCAGCCCAAGAGGCAGCTTCATGGTGACGTGACCCTGAGTGCCTGGAGCCGTCTCCTGATTGTTTGT 984
TAGAGCTGTCAGCCCAAGAGGCAGCTTCATGGTGACGTGACCCTGAGTGCCTGGAGCCGTCTCCTGATTGTTTGT 985
CTGATTGTTTGTCCTTACGAGTTTGTCTGCTTGCACTTCTGGATCCTGACTGCCCATGGGAGGCATCAGACCTTC 986
GTGGTGTGGCTGTGATGGGACCCTGAGTGTCCAGATCTATCTACCGATTGCTCATGGTGGGATCCCTGCCTTCCT 987
GTGGTGTGGCTGTGATGGGACCCTGAGTGTCCAGATCTATCTACCGATTGCTCATGGTGGGATCCCTGCCTTCCT 988
GCTGTCTCGTGCCTGCTCGTGGTGGGATCCTTGTCTTCGTCCAGTGCTGGTCCTGGTCCGCCCATGGGCAGACTC 989
GCTGTCTCGTGCCTGCTCGTGGTGGGATCCTTGTCTTCGTCCAGTGCTGGTCCTGGTCCGCCCATGGGCAGACTC 990
AGTGCTCACCTGGTAGATGAAAGACCCTGAACGTCCAGACCTTCCCCCTGACCAGTCACGTGCGGACTCTTGGTG 991
AGTGCTCACCTGGTAGATGAAAGACCCTGAACGTCCAGACCTTCCCCCTGACCAGTCACGTGCGGACTCTTGGTG 992
CTCTGCTGATGGGGCCCATCCTGTCCATGGCCTGACACTGACTGTGTGTCTGACTCCTCTGAATGTCCCTCACTA 993
CTCTGCTGATGGGGCCCATCCTGTCCATGGCCTGACACTGACTGTGTGTCTGACTCCTCTGAATGTCCCTCACTA 994
GTCCCTCACTATCACTGGCCTGACTACCACTGGACCCCCAGTGTCCACTGTCTCTGACTGCAGATGAAGCTTGTC 995
GTCCCTCACTATCACTGGCCTGACTACCACTGGACCCCCAGTGTCCACTGTCTCTGACTGCAGATGAAGCTTGTC 996
CTTGTCTGCGCGGAATGCCTGAGTGTCTGGAGCTGTCTGCTGACTGCTGGTGGCGGGATCCGTGTCTTTCTCCTG 997
CTTGTCTGCGCGGAATGCCTGAGTGTCTGGAGCTGTCTGCTGACTGCTGGTGGCGGGATCCGTGTCTTTCTCCTG 998
TTGCCTGTTCATGGGATGACGCAGCCTGTCCACCAGAGGAAGTCTCTGCGTGAGGAGTTCCTGATTGTCTGGAGC 999
TTGCCTGTTCATGGGATGACGCAGCCTGTCCACCAGAGGAAGTCTCTGCGTGAGGAGTTCCTGATTGTCTGGAGC 1000
GAGTGCCCGTGACCGGCTCTGTCTTCGTGATGGGACCTGGGGTGTCTGGAGCCATGTCTTGACTGCTCCTGAGCA 1001
GAGTGCCCGTGACCGGCTCTGTCTTCGTGATGGGACCTGGGGTGTCTGGAGCCATGTCTTGACTGCTCCTGAGCA 1002
TTGACTGCTCCTGAGCAGATCCACGATGGTTTCTGGAAGCAGACCCAGACCACCTCTCAGAGTCTTCTGAATGTC 1003
GCTAACACTGGATCCCTGGCGCCTGCTTGTCCTGGACCCCTCTGATTGTCCCTGGCCTGCCTGTGAGTGTCTAGA 1004
GCTAACACTGGATCCCTGGCGCCTGCTTGTCCTGGACCCCTCTGATTGTCCCTGGCCTGCCTGTGAGTGTCTAGA 1005
GAGATGTCGGCATGAGAGGAAGCTTCATGGTGACGTGACCCTGAGTGCCTGGAGCCGTCTCTTGATTGTTCCTCA 1006
GAGATGTCGGCATGAGAGGAAGCTTCATGGTGACGTGACCCTGAGTGCCTGGAGCCGTCTCTTGATTGTTCCTCA 1007
CTTGATTGTTCCTCATTACGTGTTGTTCTGCTTGCACTTCTGGATCCTGACTGCCCACGGGAGGCATCAGACCTT 1008
TGGGATGTGGTGTGGCTGTGATGGGACCCTGAGTGTCCAGACCTATCTACCGATTGCTCGTGGTGGGACCCCTGC 1009
TGGGATGTGGTGTGGCTGTGATGGGACCCTGAGTGTCCAGACCTATCTACCGATTGCTCGTGGTGGGACCCCTGC 1010
GCCTTCCTCTTCTGCTTGACCCCGGGTGTCCACGAATGGTGTCCTGACCATCTTGGGATGCTGAGTGCCTGGAGT 1011
GCCTTCCTCTTCTGCTTGACCCCGGGTGTCCACGAATGGTGTCCTGACCATCTTGGGATGCTGAGTGCCTGGAGT 1012
CTGCTCATGGTGGGATCCTTGTCTTACTCCAGTGCTGGGCCCTGTCCATCCATGGGAGGACTCAGACTGTTTATG 1013
CTGCTCATGGTGGGATCCTTGTCTTACTCCAGTGCTGGGCCCTGTCCATCCATGGGAGGACTCAGACTGTTTATG 1014
GCTCACCTGGTAGAGGAAAGACCCTGAACGTCGAGACCTTTCCCCTGACCGGTCACGTGCGGACTCTTGGTGGCT 1015
GCTCACCTGGTAGAGGAAAGACCCTGAACGTCGAGACCTTTCCCCTGACCGGTCACGTGCGGACTCTTGGTGGCT 1016
GGACTCTTGGTGGCTCTGCTGATGGTGACCAGCCTGTCCATGGCCTGACACTGACTGTGTGTCTGAGTCTTCTGA 1017
GGACTCTTGGTGGCTCTGCTGATGGTGACCAGCCTGTCCATGGCCTGACACTGACTGTGTGTCTGAGTCTTCTGA 1018
GTGGCCTGACTACCACTGGACCCTCGGTGTCCACTGTCTCTGACTGCAGATGAAGCTTGTCCGTGCCCAGTGCCT 1019
GTGGCCTGACTACCACTGGACCCTCGGTGTCCACTGTCTCTGACTGCAGATGAAGCTTGTCCGTGCCCAGTGCCT 1020
TGCCCAGTGCCTGAGTGTCTGGAGCTGTCTGCTGACTGGAGCTGGTGGCGGGATCCATGTCTTTCTCCTGCACTT 1021
TGCCCAGTGCCTGAGTGTCTGGAGCTGTCTGCTGACTGGAGCTGGTGGCGGGATCCATGTCTTTCTCCTGCACTT 1022
TTGATCTTGCCTGTTCATGGGATGACGCAGCCTGTCCACTAGAGGAATTCTGTGTGTGACGAGTGCCTGATTTTC 1023
TTGATCTTGCCTGTTCATGGGATGACGCAGCCTGTCCACTAGAGGAATTCTGTGTGTGACGAGTGCCTGATTTTC 1024
GTGCCCATGACCAGCTCTGTCTTCGTGATGGGACCTGGGGTGTCTGGAGCCATCTCTTGACTGCTCCTGAGCAGA 1025
GTGCCCATGACCAGCTCTGTCTTCGTGATGGGACCTGGGGTGTCTGGAGCCATCTCTTGACTGCTCCTGAGCAGA 1026
GACTGCTCCTGAGCAGATCCATGATGGTTTCTGGAAGCAGACCCAGACCACCTCTCAGAGTCTTCTGAGTGTCCC 1027
GACTGCTCCTGAGCAGATCCATGATGGTTTCTGGAAGCAGACCCAGACCACCTCTCAGAGTCTTCTGAGTGTCCC 1028
TGGCTAACACTGGATCCCTGGTTCCTACTTGTCCTGGGCCCCGATGATTGTCCCTGGCCCACCTGTGAGTGTCTA 1029
TGGCTAACACTGGATCCCTGGTTCCTACTTGTCCTGGGCCCCGATGATTGTCCCTGGCCCACCTGTGAGTGTCTA 1030
CTAGAGCTGTCAGCCTGAGAGGAAGCTTCATGATGACGTGACCCTGAGTGCCTGGTGCCGTCTCCTGATTGTTCC 1031
CTAGAGCTGTCAGCCTGAGAGGAAGCTTCATGATGACGTGACCCTGAGTGCCTGGTGCCGTCTCCTGATTGTTCC 1032
TCCTCATTTCGTGTTTGTCTGCTTGCACTTCTGGATCCTGACTGCCCATGGGAGGCATCAGACCTTCCCTGGGAT 1033
TCCTCATTTCGTGTTTGTCTGCTTGCACTTCTGGATCCTGACTGCCCATGGGAGGCATCAGACCTTCCCTGGGAT 1034
ATGTGGTGTGGCTGTGATGGGAACCTGAGTGTCCAGACCTATTTACCGATTGCTCGTGGTGGGATCCCTGCCTTC 1035
ATGTGGTGTGGCTGTGATGGGAACCTGAGTGTCCAGACCTATTTACCGATTGCTCGTGGTGGGATCCCTGCCTTC 1036
TCTGCTTGACCCCGGGTGTCCACGAATGGTGTCCTGACCCTCTTGGGACGCTGAATGCCTGGAGCTGTCTCGTGC 1037
TCTGCTTGACCCCGGGTGTCCACGAATGGTGTCCTGACCCTCTTGGGACGCTGAATGCCTGGAGCTGTCTCGTGC 1038
GTGGTGCGATCCTTGTCTTCCTCCAGTGCTGGTCCCGGTCCGTCCATGGGCAGAGTCAGGCTGTTCATGAGTGCT 1039
GTGGTGCGATCCTTGTCTTCCTCCAGTGCTGGTCCCGGTCCGTCCATGGGCAGAGTCAGGCTGTTCATGAGTGCT 1040
TCACCTGGTAGAGGGAAGACCCTGAACGTCCAGACCGTTCCCCTGACCGGCCACGTGTGGACTCTTGGTGGCTCT 1041
TCACCTGGTAGAGGGAAGACCCTGAACGTCCAGACCGTTCCCCTGACCGGCCACGTGTGGACTCTTGGTGGCTCT 1042
GCTGTCTCAGCCCAGCCTTTCCGTGGCCTGACACTGATTGTGTGTCTGAGTTTTCTGAATGTCCCTCACTGTCAC 1043
CCCTCACTGTCACTGGCCTGACTACCGCTAGACCCCCGGTGTCCACGATCGCTGACTGCAGATGAAGCTTGCCCG 1044
CCCTCACTGTCACTGGCCTGACTACCGCTAGACCCCCGGTGTCCACGATCGCTGACTGCAGATGAAGCTTGCCCG 1045
CCAGTGGCTGAGTGTCTGGAGCTGTCTGCTGACTGCTGGTGGCCGGATCCATGTCTTTCTCCTGGACTTGATCTT 1046
CCAGTGGCTGAGTGTCTGGAGCTGTCTGCTGACTGCTGGTGGCCGGATCCATGTCTTTCTCCTGGACTTGATCTT 1047
GTTCATGGGATGATGCAGTCTGTCCACGAGAGGAAGTCTCTGCGTGACGAGTGCCTGATTGTCTGGAGCTGTCTG 1048
GTTCATGGGATGATGCAGTCTGTCCACGAGAGGAAGTCTCTGCGTGACGAGTGCCTGATTGTCTGGAGCTGTCTG 1049
AGAGTGCCCATGACTGGCTCTGTCTTCATCATGGGACCTGGGGTGTCTGGAGCCATCTCTTGACTGCTCCCACGC 1050
AGAGTGCCCATGACTGGCTCTGTCTTCATCATGGGACCTGGGGTGTCTGGAGCCATCTCTTGACTGCTCCCACGC 1051
CCATGATGGTTTCTGGAAGCCGACCCAGAGTGCCTCTCAGAGTCTTCTGAGTGTCCCTCACTGTCCCTGTCCTGG 1052
CCATGATGGTTTCTGGAAGCCGACCCAGAGTGCCTCTCAGAGTCTTCTGAGTGTCCCTCACTGTCCCTGTCCTGG 1053
ACTGTCCCTGTCCTGGCTAACTCTGGATCCCCTACGCTTTCTTGTCCTGGACTCCTGCAATGGTACCTGGCTTGT 1054
TGACCTGTTCACTTGAGATGATGATTTGCCATCAGATGACCTTGATCTTTCATATATTTTGTTTTCTTCTAATAG 1055
CAGTGGTGTCATAGGCTTCATCCTGGATTGTGTAATATGTGGCAATATGGCCTGATTGTATCCATTTTTGAGTCA 1056
TTGAGTCATTCTTCCTGTATTTTCATAATCATATACTCCTTCTTCATTGTCTTCTTTCTCTTCAAGTCTTTCACT 1057
CCTATTGTCTCCTAATCTAGTATTTTCAGTCTTGTTTTTCTCTTTTTTACTTGAGTTATGATGGTTTTTTCCATA 1058
TATTCTTCTTCTCTATGAGTAGGTGAATATCCTTTTCTTTCTTTTTTTTCAGAACTAGATTCATGCCTTTTCCCC 1059
TGTTTCTCTTGGGCTCTTGGATCTTCCCTTATTCCCTTTTCTATTGTTTCTTCTTTCCAGACTTGAGGGTCTTTT 1060
AGGGTCTTTTTCTGTTTTCTTTGTTTTCTTCCTGTTTATTATCTTCATGTTTATCATGATGACTGTGCTTTCTGT 1061
TTCTGTGCTTGTGTCCTGATATCGGTAAATTCTCTTTTCTGGTAGACTCATAATATGCTTGAGCCAACTTGAATA 1062
ACCATCAGAAGAAACTCAGTGAAGTCAATTTTCTTGTTGTGGTCTATATCCAAGTGATCCATGAAGACATCAACC 1063
TCTGGATTCTGTACAGAGGGAAGTCACAGAGGGAGACTGCATCAGACAGAATCACATTATCAGCCAATTAATTCA 1064
ACCCTTGCTTAGATTATTGATGAGAAAAACAAATGCTCTATCTTTGGTCTTGTCAGAGACTCTTACCTTCAGGAT 1065
CAGGATTTGCCGAAATTCCTTTTCCAGAAGTTCCTTCAGCTCTTTTTTACTCAATGTGTCAGTGTTTTTATCTTT 1066
GAATATTGCTTGAAAAGATTAATTATGGCAAAGATGTTTTCCAGGAGAGTAGACATCTTTTGGCAATAAATGTGA 1067
TAAATGTGAACCTAGAAAGAAGAAATGAAAATGCACTTTTTTATACATCTTTTTTTCTAGGTTATATTATTACAA 1068
AACCCTTGCTTAGATTATTGATGAGAAAAACAAATGCTCTATCTTTGGTCTTGTCAGAGACTCTTACCTTCAGGA 1069
AATTCCTTTTCCAGAAGTTCCTTCAGCTCTTTTTTACTCAATGTGTCAGTGTTTTTATCTTTTTTTGAATATTGC 1070
GAAAAGATTAATTATGGCAAAGATGTTTTCCAGGAGAGTAGACATCTTTTGGCAATAAATGTGAACCTAGAAAGA 1071
ACCTAGAAAGAAGAAATGAAAATGCACTTTTTTATACATCTTTTTTTCTAGGTTATATTATTACAATCATTTTCC 1072
AAAAACAAATGCTCTATCTTTGGTCTTGTCAGAGACTCTTACCTTCAGGATTTGCCGAAATTCCTTTTCCAGAAG 1073
AGTTCCTTCAGCTCTTTTTTACTCAATGTGTCAGTGTTTTTATCTTTTTTTGAATATTGCTTGAAAAGATTAATT 1074
TGAAAAGATTAATTATGGCAAAGATGTTTTCCAGGAGAGTAGACATCTTTTGGCAATAAATGTGAACCTAGAAAG 1075
AAGAAATGAAAATGCACTTTTTTATACATCTTTTTTTCTAGGTTATATTATTACAATCATTTTCCACATTTTAAA 1076
AATGCTCTATCTTTGGTCTTGTCAGAGACTCTTACCTTCAGGATTTGCCGAAATTCCTTTTCCAGAAGTTCCTTC 1077
TTCAGCTCTTTTTTACTCAATGTGTCAGTGTTTTTATCTTTTTTTGAATATTGCTTGAAAAGATTAATTATGGCA 1078
GGCAAAGATGTTTTCCAGGAGAGTAGACATCTTTTGGCAATAAATGTGAACCTAGAAAGAAGAAATGAAAATGCA 1079
CAGTGCCACTAAAAAGAAAGAGAAAAACCCTAAACTTCCAGAACCTTTTGCCTTACAGTACACATACATTCAGAT 1080
TTCCACCTTGGTTAATAGTAAGTAGACAAAATTAAATGTATTCCTTCTTACCTTGTTCACCAAAAGAGCAAATAG 1081
GAGCAAATAGAATGTAGCCTGAAGGAGCCTGCTGGGTACTGAAGGCTGAGATAATGGCCCTTTTATAGCAGGCCC 1082
GAGCAAATAGAATGTAGCCTGAAGGAGCCTGCTGGGTACTGAAGGCTGAGATAATGGCCCTTTTATAGCAGGCCC 1083
AAAGAGAAAAACCCTAAACTTCCAGAACCTTTTGCCTTACAGTACACATACATTCAGATTTCCACCTTGGTTAAT 1084
TTTCCACCTTGGTTAATAGTAAGTAGACAAAATTAAATGTATTCCTTCTTACCTTGTTCACCAAAAGAGCAAATA 1085
GGAGCCTGCTGGGTACTGAAGGCTGAGATAATGGCCCTTTTATAGCAGGCCCCATGCAGGGAGGAGCCACCCTCT 1086
GGAGCCTGCTGGGTACTGAAGGCTGAGATAATGGCCCTTTTATAGCAGGCCCCATGCAGGGAGGAGCCACCCTCT 1087
GAGAAAAACCCTAAACTTCCAGAACCTTTTGCCTTACAGTACACATACATTCAGATTTCCACCTTGGTTAATAGT 1088
AAAATTAAATGTATTCCTTCTTACCTTGTTCACCAAAAGAGCAAATAGAATGTAGCCTGAAGGAGCCTGCTGGGT 1089
GAGCCTGCTGGGTACTGAAGGCTGAGATAATGGCCCTTTTATAGCAGGCCCCATGCAGGGAGGAGCCACCCTCTG 1090
GAGCCTGCTGGGTACTGAAGGCTGAGATAATGGCCCTTTTATAGCAGGCCCCATGCAGGGAGGAGCCACCCTCTG 1091
AGAAAAACCCTAAACTTCCAGAACCTTTTGCCTTACAGTACACATACATTCAGATTTCCACCTTGGTTAATAGTA 1092
ATGTATTCCTTCTTACCTTGTTCACCAAAAGAGCAAATAGAATGTAGCCTGAAGGAGCCTGCTGGGTACTGAAGG 1093
CTGAGATAATGGCCCTTTTATAGCAGGCCCCATGCAGGGAGGAGCCACCCTCTGTGGGATGGTCTGATTCATTCC 1094
CTGAGATAATGGCCCTTTTATAGCAGGCCCCATGCAGGGAGGAGCCACCCTCTGTGGGATGGTCTGATTCATTCC 1095
According to some embodiments of the present invention, described one group of probe is free on solution. According to other embodiment of the present invention, instituteState one group of probe and be incorporated on solid-phase matrix, form chip.
According to embodiments of the invention, described is the reference genome sequence of described sample to be tested source species with reference to genome, excellentChoose with reference to genome, for example hg19.
Inventor is surprised to find, one group of probe of the present invention, the FLG gene coding region to its specific recognition at least onePart catch that specificity is good, sensitivity and coverage very high, can be effective to carry out catching of FLG gene coding regionDetect. According to embodiments of the invention, utilize one group of probe of the present invention capture probe specific recognition accurately and effectivelyTarget sequence---at least a portion of FLG gene coding region, thus can effectively build the nucleic acid sequencing literary composition that obtains target sequenceStorehouse, further, for high-flux sequence, can effectively determine at least one of FLG gene coding region by this nucleic acid sequencing libraryThe sequence information of part, and the order-checking degree of depth of target sequence is high, and data user rate is high, and then can realize the gene to FLGDetection. In addition, utilize said method to build high-throughput sequencing library, and and then for FLG genetic test, specificity is good,Sensitivity and coverage are high, favorable repeatability, thus can successfully understand FLG gene, find sudden change, and the method experimentProcess stabilization, cost is low, simple to operate, workload is little, easily promote.
The purposes of probe
As previously mentioned, one group of probe of the present invention, the catching of at least a portion of the FLG gene coding region to its specific recognitionObtain that specificity is good, sensitivity and coverage very high, can be effective to carry out the Acquisition Detection of FLG gene coding region. ByThis, the present invention also provides the purposes of above-mentioned one group of probe in the Acquisition Detection of FLG gene coding region.
And then, according to a further aspect in the invention, the invention provides a kind of method that builds high-throughput sequencing library. According to thisInventive embodiment, the method comprises the following steps:
First, by genomic DNA fragment, to obtain DNA fragmentation. According to embodiments of the invention, further bagDraw together the step of extracting genomic DNA from sample, preferred described sample derives from mammal, more preferably described mammalAt least one of behaviour and mouse, preferred described genomic DNA is mankind's Whole Blood Genomic DNA. According to more of the present inventionEmbodiment, utilizes covaris-S2 to interrupt instrument by genomic DNA fragment. According to concrete example of the present invention, described DNAThe length of fragment is about 200-250bp.
Secondly, described DNA fragmentation is carried out to end reparation, to obtain the DNA fragmentation of repairing through end. According to thisInventive embodiment, carrying out described DNA fragmentation, before end reparation, further to comprise the step of purifying DNA fragment.According to embodiments of the invention, it is to utilize Klenow fragment, T4DNA polymerase that described DNA fragmentation is carried out to end reparationCarry out with T4 polynucleotide kinase, wherein, described Klenow fragment has 5 ' → 3 ' polymerase activity and 3 ' → 5 ' polymeraseActivity, but lack 5 ' → 3 ' 5 prime excision enzyme activity. Thus, end repairing effect is good, is conducive to the carrying out of subsequent step.
Again, add base A at 3 ' end of the described DNA fragmentation of repairing through end, there is cohesive end to obtainThe DNA fragmentation of A. According to embodiments of the invention, 3 ' end of the described DNA fragmentation of repairing through end is added to alkaliBase A utilizes Klenow (3 '-5 ' exo-) to carry out. Thus, reaction effect is good, is conducive to the carrying out of subsequent step.
Then, the described DNA fragmentation with cohesive end A is connected with joint, connects product to obtain. According to thisBright embodiment, is connected the described DNA fragmentation with cohesive end A and utilizes T4DNA ligase to carry out with joint.Thus, good connecting effect, is conducive to the carrying out of subsequent step.
Then, utilize foregoing one group of probe to screen described connection product, to obtain object fragment, described orderFragment form described high-throughput sequencing library. According to embodiments of the invention, described high-throughput sequencing library is suitable for utilizing high passMeasuring order technological selection Hiseq2000 order-checking platform checks order. According to other embodiment of the present invention, utilize solid phase chipHybrid capture technology is carried out described screening. Thus, screening effect is good, is conducive to the carrying out of subsequent step.
Inventor finds, at least one portion of the FLG gene coding region of one group of probe of the present invention to its specific recognitionPoint catch that specificity is good, sensitivity and coverage very high, can be effective to carry out the inspection of catching of FLG gene coding regionSurvey. And then, utilize the method for the present invention target sequence of capture probe specific recognition accurately and effectively---FLG geneAt least a portion of code area, the nucleic acid sequencing library of establishing target sequence, and then this nucleic acid sequencing library is measured for high passAfter order, can effectively determine the sequence information of at least a portion of FLG gene coding region, and the order-checking degree of depth of target sequenceHeight, data user rate is high, and then can realize the detection to FLG gene. In addition, utilize method of the present invention to build high passAmount sequencing library, and then for FLG genetic test, specificity is good, sensitivity and coverage high, favorable repeatability, therebyCan successfully understand FLG gene, find sudden change (for example copy number variation (CNV) and SNP (SNP)),And the method experimentation is stable, cost is low, simple to operate, workload is little, easily popularization.
According to another aspect of the invention, the invention provides a kind of definite sample to be tested FLG gene coding region nucleotide sequenceMethod. According to embodiments of the invention, the method comprises the following steps:
First, according to the method for foregoing structure high-throughput sequencing library, build the high-throughput sequencing library of sample to be tested,Described high-throughput sequencing library comprises FLG gene coding region nucleotide sequence.
Then, the high-throughput sequencing library of described sample to be tested is checked order, to obtain sequencing result. According to of the present inventionEmbodiment, utilizes the preferred Hiseq2000 order-checking of high throughput sequencing technologies platform to carry out described order-checking. Thus, sequencing throughput is high,Result accurately and reliably.
Then,, based on described sequencing result, determine the nucleotide sequence of described sample to be tested FLG gene coding region. According to thisBright embodiment, further comprises: by the nucleotide sequence of described sample to be tested FLG gene coding region and with reference to genome sequenceCompare, to determine whether described sample to be tested FLG gene coding region exists sudden change, and obtain variation information. According toEmbodiments of the invention, described is the reference genome sequence of described sample to be tested source species with reference to genome, preferably ginseng examinesGenome, for example hg19.
Inventor finds, at least one portion of the FLG gene coding region of one group of probe of the present invention to its specific recognitionPoint catch that specificity is good, sensitivity and coverage very high, can be effective to the Acquisition Detection of FLG gene coding region.And then, utilize the method for the present invention target sequence of capture probe specific recognition accurately and effectively---FLG gene codeAt least a portion in district, the nucleic acid sequencing library of establishing target sequence, and carry out high-flux sequence, thus can be effectively definiteThe sequence information of at least a portion of FLG gene coding region, and the order-checking degree of depth of target sequence is high, and data user rate is high, entersAnd can realize the detection to FLG gene. In addition, utilize method of the present invention to build high-throughput sequencing library, and determine and treatThis FLG of test sample gene coding region nucleotide sequence, specificity is good, sensitivity and coverage high, favorable repeatability, result is accurateReliably, thereby can successfully understand FLG gene, find sudden change (for example copy number variation (CNV) and mononucleotide polymorphicProperty (SNP)), and the method experimentation is stable, cost is low, simple to operate, workload is little, easily promote.
It should be noted that, for multiple samples to be tested, can build respectively the high-throughput sequencing library of each sample to be tested,And in each sequencing library, all introduce sequence label, make the sequence label of each sequencing library mutually different, and then, by each surveyHigh-flux sequence is carried out after mixing in preface storehouse, and based on each sequence label, sequencing result is distinguished. Thus, can be effectively realNow multiple samples to be tested are carried out to the detection of FLG coding sequence simultaneously.
In accordance with a further aspect of the present invention, the invention provides a kind of device that builds high-throughput sequencing library. According to of the present inventionEmbodiment, with reference to Fig. 1, this device 1000 comprises: fragmentation unit 100, end are repaired unit 200, base A adds singleUnit 300, joint linkage unit 400 and screening unit 500.
According to embodiments of the invention, described fragmentation unit 100 is for by genomic DNA fragment, to obtain DNAFragment. According to embodiments of the invention, further comprise extracting genome DNA unit, described extracting genome DNA listFirst and described fragmentation unit 100 is connected, and for extracting genomic DNA from sample, preferred described sample derives from lactationAnimal, more preferably described mammal is behaved and at least one of mouse, and preferred described genomic DNA is human whole blood geneGroup DNA. According to embodiments of the invention, described fragmentation unit 100 interrupts instrument for covaris-S2. According to of the present inventionEmbodiment, the length of described DNA fragmentation is about 200-250bp.
According to embodiments of the invention, described end is repaired unit 200 and is connected with described fragmentation unit 100, described in inciting somebody to actionDNA fragmentation carries out end reparation, to obtain the DNA fragmentation of repairing through end. According to embodiments of the invention, enter oneStep comprises purification unit, and described purification unit is repaired unit 200 with described fragmentation unit 100 and described end and is connected, forDescribed DNA fragmentation being carried out to, before end reparation, described DNA fragmentation is carried out to purifying. According to embodiments of the invention,It is to utilize Klenow fragment, T4DNA polymerase and T4 polynucleotide kinase to enter that described DNA fragmentation is carried out to end reparationRow, wherein, described Klenow fragment has 5 ' → 3 ' polymerase activity and 3 ' → 5 ' polymerase activity, but lacks 5 ' → 3 '5 prime excision enzyme activity. Thus, end repairing effect is good, is conducive to the carrying out of subsequent step.
According to embodiments of the invention, described base A adding device 300 is repaired unit 200 with described end and is connected, for3 ' the end at the described DNA fragmentation of repairing through end adds base A, to obtain the DNA with cohesive end AFragment. According to embodiments of the invention, 3 ' end of the described DNA fragmentation of repairing through end is added to base A and utilizeKlenow (3 '-5 ' exo-) carries out. Thus, reaction effect is good, is conducive to the carrying out of subsequent step.
According to embodiments of the invention, described joint linkage unit 400 is for by the described DNA fragmentation with cohesive end ABe connected with joint, connect product to obtain. According to embodiments of the invention, by the described DNA sheet with cohesive end ASection is connected with joint and utilizes T4DNA ligase to carry out. Thus, good connecting effect, is conducive to the carrying out of subsequent step.
According to embodiments of the invention, described screening unit 500 is connected with described joint linkage unit 400, and before being provided withOne group of described probe, for utilizing described one group of probe to screen described connection product, to obtain object fragment, instituteState object fragment and form described high-throughput sequencing library. According to embodiments of the invention, described high-throughput sequencing library is suitable for utilizingThe preferred Hiseq2000 order-checking of high throughput sequencing technologies platform checks order, according to embodiments of the invention, and described screening unit 500Be suitable for utilizing solid phase chip hybrid capture technology to carry out described hybrid capture. Thus, screening effect is good, is conducive to subsequent stepCarry out.
According to embodiments of the invention, the FLG gene coding region of one group of probe of the present invention to its specific recognitionAt least a portion catch that specificity is good, sensitivity and coverage very high, can be effective to carry out FLG gene coding regionAcquisition Detection. And then, utilize the device 1000 of the present invention target order of capture probe specific recognition accurately and effectivelyRow---at least a portion of FLG gene coding region, and the nucleic acid sequencing library of establishing target sequence, and then by this nucleic acid sequencingThe sequence information of at least a portion of FLG gene coding region, for after high-flux sequence, can effectively be determined in library, and orderThe order-checking degree of depth of mark sequence is high, and data user rate is high, and then can effectively realize the detection to FLG gene. In addition utilize,Device 1000 of the present invention builds high-throughput sequencing library, and then for FLG genetic test, specificity is good, sensitivity and coveringCover degree is high, favorable repeatability, thus can successfully understand FLG gene, find sudden change, and this device 1000 is simple in structure,Applicable experimentation is stable, production cost is low, simple to operate, easily popularization.
According to a further aspect in the invention, the present invention also provides a kind of definite sample to be tested FLG gene coding region nucleotide sequenceSystem. According to embodiments of the invention, with reference to Fig. 2, this system comprises: library construction device 1000, sequencing device 2000With analytical equipment 3000.
According to embodiments of the invention, described library construction device 1000 is the dress of foregoing structure high-throughput sequencing libraryPut, for building the high-throughput sequencing library of sample to be tested, described high-throughput sequencing library comprises FLG gene coding region nucleic acidSequence.
According to embodiments of the invention, described sequencing device 2000 is connected with described library construction device 1000, for to describedThe high-throughput sequencing library of sample to be tested checks order, to obtain sequencing result. According to embodiments of the invention, described order-checkingDevice 2000 is high-flux sequence platform, preferably Hiseq2000 order-checking platform. Thus, sequencing throughput is high, and result accurately canLean on.
According to embodiments of the invention, described analytical equipment 3000 is connected with described sequencing device 2000, for based on described surveyOrder result, determines the nucleotide sequence of described sample to be tested FLG gene coding region.
According to embodiments of the invention, further comprise comparison device (not shown), described comparison device and described analysisDevice 3000 be connected, for by the nucleotide sequence of described sample to be tested FLG gene coding region with compare with reference to genome sequenceRight, to determine whether described sample to be tested FLG gene coding region exists sudden change, and obtain variation information. According to the present inventionEmbodiment, described is the reference genome sequence of described sample to be tested source species with reference to genome, preferably the mankind are with reference to geneGroup, for example hg19.
Inventor finds, at least one portion of the FLG gene coding region of one group of probe of the present invention to its specific recognitionPoint catch that specificity is good, sensitivity and coverage very high, can be effective to the Acquisition Detection of FLG gene coding region.And then, utilize the system of the present invention target sequence of capture probe specific recognition accurately and effectively---FLG gene codeAt least a portion in district, the nucleic acid sequencing library of establishing target sequence, and carry out high-flux sequence, thus can be effectively definiteThe sequence information of at least a portion of FLG gene coding region, and the order-checking degree of depth of target sequence is high, and data user rate is high, entersAnd can realize the detection to FLG gene. In addition, utilize system constructing high-throughput sequencing library of the present invention, and determine and treatThis FLG of test sample gene coding region nucleotide sequence, specificity is good, sensitivity and coverage high, favorable repeatability, result is accurateReliably, thereby can successfully understand FLG gene, find sudden change, and this system architecture is simple, applicable experimentation is stable,Production cost is low, simple to operate, easily popularization.
It should be noted that probe of the present invention can be caught FLG with high specific and high coverage rate on same chipGene region. The present invention adopts the method for sequence capturing technology, high-flux sequence and analysis of biological information combination to FLG baseBecause detecting, detection specificity is high, good stability, and many experiments repeatability is high.
Below in conjunction with embodiment, the solution of the present invention is made an explanation. It will be understood to those of skill in the art that the following examplesOnly for the present invention is described, and should not be considered as limiting scope of the present invention. Unreceipted concrete technology or condition in embodiment, press(for example show " the molecule that Huang Peitang etc. translate with reference to J. Pehanorm Brooker etc. according to the described technology of the document in this area or conditionCloning experimentation guide ", the third edition, Science Press) or carry out according to product description. Agents useful for same or instrument are unreceiptedThe person of production firm, is and can by the conventional products of commercial acquisition, for example, can purchases from Illumina company.
Embodiment 1
According to the method for definite sample to be tested FLG of the present invention gene coding region nucleotide sequence, with reference to Figure 16, according to following stepSuddenly, sample to be tested is carried out to FLG genetic test:
Wherein, sample to be tested is that patient information is as follows:
One, prepare probe, chip
One group of capture probe of design specific recognition FLG gene coding region, and utilize CustomArrayB3P platform syntheticProbe, and then prepare multiple chips, the design of its middle probe and synthetic parameter are as follows:
(1) length of described probe is 75bp;
(2) described probe specificity identification FLG gene coding region upstream 10bp is to the sequence between the 10bp of downstream;
(3) specific recognition GC content is higher than 0.6 and lower than the probe in 0.3 region, and multiplier is greater than 2;
(4) melting temperature of described probe and target sequence is 60-10 degree Celsius, preferably 80 degrees Celsius;
(5) described probe does not comprise hairpin structure;
(6) described probe with mate with reference to 2 sites at the most on genome;
(7) window sliding size when described probe is selected is 10bp.
Thus, prepare multiple chips, each chip all comprises as shown in SEQIDNO:1-1095 one group of nucleotide sequenceProbe.
Two, build storehouse and order-checking:
1.DNA extracts
Utilize salting out method, from whole blood, blood plasma, extracts genomic DNA in serum or lymphocyte concentration.
2.DNA detects
2.1.OD detect: get 1~2 μ L sample and carry out NanoDrop8000 detection, sample concentration is recorded to " single-gene sampleProduct detect record sheet ".
2.2. electrophoresis detection: 1 μ LDNA+3 μ L buffer solution, (now the band of 750bp is about 100 to the DL2000 of 5 μ LNg) λ-HindIII of and 2 μ L, 1% Ago-Gel, 140V, 30min.
3.DNA sample detection
3.1OD detects: get 1~2 μ L sample and carry out NanoDrop8000 detection, sample concentration is recorded to " single-gene sampleProduct detect record sheet ".
3.2. electrophoresis detection: 1 μ LDNA carries out electrophoresis detection.
Whether qualified according to total amount and the quality of the OD value judgement sample of electrophoresis result and measurement, and provide whether can carry out sampleJudgement prepared by product. If containing RNA, pollutes sample, must by sample volume, to add the concentration of 1/10 volume be 10mg/mLRNaseA, processes 10min for 37 DEG C.
4.CovarisE220-96wellplate interrupts method
Parameter arranges:
Duty factor 25
PIP,W 500
Cycle/burst 500
Time(s)/cycles 20/12
Sample interrupts after end, and take a morsel (about total amount 1/30) interrupts rear sample electrophoresis on 2% Ago-Gel and carry out electrophoresisDetect, and keep glue figure. Interrupt effect generally with required preparation Insert fragment master tape position, library in 200-250bp positionPut comparatively desirable. If interrupting undesirable needs of effect interrupts again.
Sample after interrupting is drawn to from 96 orifice plates in shallow bore hole plate, uses 144 μ LAmpureBeads to carry out product purification,The DNA reclaiming is dissolved in the water of 77 μ L (wherein 2 μ L are loss). Then, use Nanodrop8000 to detect various kindsThis OD value.
5. end reparation
5.1. from the kit of-20 DEG C of preservations, take out in advance 10x polynueleotide kinase (PNK) buffer solution, 10mMDNTPsmix, Klenow fragment, T4 polynueleotide kinase (T4PNK) are placed on and melt on ice and fully mix.
5.2. prepare end and repair reaction system:
Reaction number 1 reaction (μ L)
10x polynueleotide kinase buffer solution (B904) 10μL
dNTP Solution Set 4μL
T4DNA polymerase 5μL
Klenow fragment 1μL
T4 polynueleotide kinase (T4PNK) 5μL
Cumulative volume 25μL
5.3. the reagent after using is put back to also-20 DEG C of preservations in original reagent box, after using vortex to mix the mix configuring,Each reaction adds 25 μ L enzyme reaction mixed liquors.
5.4. concussion mix and centrifugal after, be placed in Thermomixer 20 DEG C of temperature and bathe 30min.
5.5. use 180 μ LAmpureBeads to carry out product purification, the DNA of recovery is dissolved in 30 μ L, and (wherein 1.9 μ L areLoss) water in.
6. end adds " A " (A-Tailing)
6.1. from the kit of-20 DEG C of preservations, take out in advance 10xblue buffer solution and 5mMdATP, be placed on iceMelt and make it fully mix 10xblue buffer solution.
6.2. prepare end and add " A " reaction system:
Reaction number 1 reaction (μ L)
10x Blue buffer solution 3.5μL
dATP(5mM) 1.4μL
Klenow(3’-5’exo-) 2μL
Cumulative volume 6.9μL
6.3. the reagent after using is put back to also-20 DEG C of preservations in original reagent box, after the mix concussion configuring is mixed, every pipeAdd 6.9 μ L enzyme reaction mixed liquors.
6.4. concussion mix and centrifugal after, be placed in Thermomixer 37 DEG C of temperature and bathe 30min.
7. the connection of joint (joint connection)
7.1. from the kit of-20 DEG C of preservations, take out in advance 10x connect buffer solution, PE label joint OligoMix andATP (10mM) is placed on and melts on ice and fully mix 10xligation buffer solution.
7.2. prepare joint coupled reaction system:
PEI library:
Reaction number 1 reaction (μ L)
10x Ligation buffer solution 1.5
Index PE joint (40 μ M) 1
ATP(10mM) 3.5
T4DNA ligase 3
ddH2O 6
Cumulative volume 15μL
7.3. the reagent after using is put back to also-20 DEG C of preservations in original reagent box, the mix concussion configuring is mixed, each anti-Should add 15 μ L enzyme reaction mixed liquors.
7.4. concussion mix and centrifugal after, be placed in Thermomixer 16 DEG C of temperature and bathe 12-16h (spending the night).
7.5. use 75 μ LAmpureBeads to carry out product purification, the DNA of recovery is dissolved in 35 μ L, and (wherein 2 μ L are for damagingConsumption) water in.
7.6. after purifying, use Nanodrop8000 to detect each sample OD value, and be recorded to " single-gene sample detection recordTable " (OD value is about 20ng/ μ L).
8.Non-Captured sample P re-LM-PCR
8.1. from the kit of-20 DEG C of preservations, take outPfxDNA polymerase, MgSO4 (50mM), dNTPmix(10mM)、PCRPrimerPE1.0(orPEIndexPrimer1.0)、PCRPrimerPE2.0(orPEIndexPrimer2.0 (index) is placed on and thaws on ice and fully mix.
8.2. prepare PCR reaction system (using oneself synthetic IndexPrimer):
Reaction number 1 sample (μ L)
Index P1 (the public primer of 10 μ M) 8
10 × Pfx amplification buffer 10
dNTP(10mM) 4
MgSO4(50mM) 4
PCR Index primer 2.0(10pmol/μL) 4μL
ddH2O 34
Cumulative volume 62μL
8.3. the reagent after using is put back to also-20 DEG C of preservations in original reagent box, after the mix concussion configuring is mixed, useContinuous sample-adding pipettor or single rifle divide to 96 orifice plates, and each reaction adds respectively 62 μ L enzyme reaction mixed liquors;
8.4. sample previous step purifying being completed is transferred in the enzyme reaction mixed liquor having configured, then index compiles per sampleNumber add corresponding indexN, each reaction 8 μ L, use pipettor to inhale to beat to mix 5-10 time and (need to arrange positive control hereinPTC and blank NTC, positive control only need add the positive sample of 2 μ L, mends to 30 μ L with 28 μ L water),Sealer is placed in PCR instrument reacts according to follow procedure.
8.5. Non-captured sample PreLM-PCR product is transferred in shallow bore hole plate, uses 150 μ LAmpureBeadsCarry out product purification, the DNA of recovery is dissolved in the water of 30 μ L (wherein 1 μ L is loss).
8.6. after purifying, use Nanodrop8000 to detect sample OD value.
9. liquid-phase chip hybridization
9.1. according to Nanodrop measurement result, according to every chip, (wherein each chip all comprises nucleotide sequence as SEQIDOne group of probe shown in NO:1-1095) 1 μ g applied sample amount, the total amount of managing (pipe is for subsequent use) according to pooling two is 2 μ gCalculate each sample loading volume, and pooling mixes.
9.2. the preparation of joint block. (each indexBlock adds for the public P1Block of 8 μ L and 8 μ LIndexNBlockEntering amount is 8/ number of samples N, and total amount is 8 μ L).
9.3. Cot-1DNA is carried out to packing according to every pipe 10 μ g. The corresponding sample number into spectrum of mark on Cot-1DNA pipe,The joint block that sample DNA after 9.1pooling and 9.2 is prepared adds in Cot-1DNA pipe.
In hybridization mixture, contain now following several composition:
9.4. build pipe lid, cover and stab a hole at the EP of packing pipe by clean 50ml syringe needle, by above-mentioned sample literary compositionThe mixture of storehouse and block is placed in SpeedVac evaporate to dryness, and temperature setting is set to 60 DEG C.
9.5. heatblock is transferred to 95 DEG C, point 4.5 μ LExomeLibrary that install are taken out from-20 DEG C of refrigerators, putThaw on ice.
9.6. sample is taken out, add respectively following two kinds of reagent: 7.5 μ L2XSCHybridiation buffer solutions and 3 μ LSCHybridiationComponentA。
9.7. sample concussion is mixed and be placed on the centrifugal 10s of full speed on centrifuge. Centrifugal rear sample is moved to 95 DEG C of heatblockMiddle 10min, makes DNA sex change.
9.8. sample is taken out, concussion mixes under rear room temperature condition centrifugal 10 seconds at full speed.
9.9. above-mentioned hybridization mixture is proceeded to (0.2mLPCR pipe or 96 holes in point 4.5 μ LExomeLibrary that installPCR plate, concussion mixes and is placed on centrifuge centrifugal 10 seconds at full speed.
In hybridization mixture, contain now following several composition:
COT-1 DNA 10μg
From 3.2 PCR purified product 1μg
The block of joint Every kind of 1nmol
2X SC Hybridiation buffer solution 7.5μL
SC Hybridiation Component A 3μL
Exome Library 4.5μL
Cumulative volume 15μL
9.10. be placed on 47 DEG C of hybridization 64h-72h on PCR instrument, the hot lid of PCR instrument should arrange and remain on 57 DEG C.
10. the washing of liquid-phase chip and wash-out
In advance water-bath opened and temperature is adjusted to 47 DEG C, being used for heated scrub ing buffer solution.
10.1. preparation cleaning solution
10.1.1. the required buffer solution reagent that thaws in advance, proportionally by five kinds of buffer solution reagent (10XSC lavation buffer solutionsI, 10XSC lavation buffer solution II, 10XSC lavation buffer solution III, 2XStringent lavation buffer solution and Binding are slowRush liquid) dilution is mixed with 1X solution. Wherein buffer solution I is divided two pipes, 47 DEG C of preheatings of a pipe, a pipe room temperature.
Reagent name Single tube consumption stoste (ul) The single tube H that adds water2O(ul)
Binding buffer solution 220 330
Buffer solution I 33 297
Buffer solution II 22 198
Buffer solution III 22 198
Buffer solution S 44 396
10.1.2.47 DEG C preheating is to show two kinds of solution:
1XStringent lavation buffer solution and 1XSC lavation buffer solution.
10.2. prepare streptomysin magnetic bead
10.2.1. from 4 DEG C of refrigerators, take out streptomysin magnetic bead, mix back balance 30min stand-by.
10.2.2. in the EP of 1.5ml pipe, add after 100ul magnetic bead, EP pipe is placed on magnetic frame to liquid clarification, removalSupernatant.
10.2.3. add 200ulStreptavidinDynabeadBindingandWashBuffer, Vortex10s mixes, willEP pipe is placed on magnetic frame and clarifies to liquid, removes supernatant.
10.2.4. repeat 8.6.2.3, altogether washed twice.
10.2.5. inhale the StreptavidinDynabeadBindingandWashBuffer of 100ul in the EP pipe of 200ul,Suspension magnetic bead.
10.2.6. use magnetic frame in conjunction with magnetic bead (tubule is come on magnetic frame), until supernatant is removed in liquid clarification; This magnetic beadBe used in conjunction with the DNA catching.
10.3. the DNA capturing is attached on streptomysin magnetic bead
10.3.1. hybridization mixture is forwarded in the ready magnetic bead of 8.6.2.6, piping and druming mixes 10 times.
10.3.2. tubule is placed on PCR instrument 47 DEG C hatch 45min (the hot lid of PCR instrument should arrange and remain on 57 DEG C, every15min takes out at vortex3s in case magnetic bead precipitation)
10.4 combine the washing of the streptomysin magnetic bead of capture dna
10.4.1. hatch after 45min, product is gone in the EP pipe of 1.5ml, then EP pipe is placed on magnetic frame to liquidClarification, removes supernatant.
10.4.2. add the 1X lavation buffer solution 1 of 100ul47 DEG C, vortex10s mixes, then EP pipe is placed in to magnetic forceOn frame, to liquid clarification, remove supernatant.
10.4.3. take off EP pipe from magnetic frame, add the 1XStringent lavation buffer solution of 200ul47 DEG C, piping and druming is mixedEven 10 times, hatch 5min for 47 DEG C, then EP pipe is placed on magnetic frame and is clarified to liquid, remove supernatant. Repeat again this stepOnce, sharing 1XStringentWashBuffer washes twice.
10.4.4. the 1X lavation buffer solution I (WashBuffer I) that adds 200ul normal temperature mixes on vortex2min, then EP pipe is placed on magnetic frame and is clarified to liquid, remove supernatant.
10.4.5. the 1X lavation buffer solution II that adds 200ul normal temperature mixes 1min, then EP pipe is put on vortexOn magnetic frame, to liquid clarification, remove supernatant.
10.4.6. the 1X lavation buffer solution III that adds 200ul normal temperature mixes 30s, then EP pipe is placed on vortexOn magnetic frame, to liquid clarification, remove supernatant.
10.4.7. add 140ulUltraPureWater (DNA need not be eluted from magnetic bead, can directly carry out PCR).
11. liquid phase captured sample LM-PCR
11.1. from-20 DEG C of refrigerators, take outPfxDNA polymerase, MgSO4 (50mM),DNTPmix (10mM), PCRPrimerFlowcellF (10uM), PCRPrimerFlowcellR (10uM), be placed on iceOn thaw and fully mix.
11.2. by upper table preparation PCR reaction system, be placed in PCR instrument and react by follow procedure
11.3.PCR product purification
11.3.1. AxgencourtAMPurebeads is placed in to balance 30min under room temperature.
11.3.2. PCR product is proceeded in the EP pipe of 1.5ml, then EP pipe is placed in to extremely clarification on magnetic frame, then by supernatantGo in the EP pipe of respective tube number slip a cable mycin magnetic bead. In supernatant, add the magnetic bead (120ul) of 1.2 times, vortex mixes.Under room temperature, leave standstill 10min, make the abundant combination of magnetic bead and DNA.
11.3.3. EP pipe is placed on magnetic frame and clarifies to liquid, remove supernatant.
11.3.4. add the ethanol (now with the current) of 500ul70%, put upside down ten times, remove ethanol. Repeat this step.
11.3.5. magnetic bead being placed in to 40 DEG C, to be dried to magnetic bead dry and cracked.
11.3.6. add 32ulEB, vortex mixes; Room temperature leaves standstill 5min, and DNA is eluted completely from magnetic bead.
11.3.7. EP pipe is placed on magnetic frame to clarification, the supernatant of inhaling 30ul proceeds in the EP pipe of respective tube number.
11.3.8. finally use NanoDrop to detect elution samples pipe, positive control pipe and negative control pipe PCR production concentration,The QC that just can send that the concentration of elution samples is greater than negative control detects. Elution samples pipe is mended to EB and be diluted to 20ng/ μ L, rareRelease rear absorption 5 μ L and send QC to detect enrichment, Non-CapturedPreLM-PCR product is drawn 2 μ L moisturizings and is diluted to 20ng/μ L, send QC to detect enrichment. If 2100 detections have primer to pollute, fetch with 1.2 times of magnetic beads for purifying in library.
12. library Quality Controls detect
Use Agilent2100Bioanalyzer to detect CapturedLM-PCR product output, use QPCR to detectThe enrichment of Non-Captured and CapturedLM-PCR product, uses QPCR to detect CapturedLM-PCR and producesSubstrate concentration.
The upper machine order-checking of 13.Hiseq2500
According to the operational procedure of Hiseq2500 order-checking platform, produce detect qualified CapturedLM-PCR through library Quality ControlThing carries out upper machine order-checking.
Three, analysis of biological information
Lower machine data analysis of biological information flow process is as follows:
1, lower machine data obtains
Obtain initial data (FASTQ data) from sequenator. Then, the entry evaluation quality of data, checks that the quality of order-checking isNo meeting the demands. The results are shown in Figure 3-7. Fig. 3 has shown size and the distribution situation of Insert Fragment, as shown in the figure, and Insert FragmentThe average 179bp (standard deviation-37/+14) of size; If wherein the size of Insert Fragment is more discrete, concentration ratio is lower, Ying CongThe angle analysis problem of experiment, such as whether DNA degrades, DNA interrupts whether there is problem, and whether pcr amplification is successfully etc.Deng. Fig. 4 has shown the Mass Distribution situation of sequencing data, and Fig. 5 has shown the average order-checking error rate of single base, pin Fig. 4And Fig. 5, check order as PE90, two-way order-checking. Can check from Fig. 4 and Fig. 5 the total quality that sample checks order, wherein, as figureShown in 4 by green to red progressive formation, the region that color is darker, representation quality distribute more concentrated. In general, oneThe sequencing quality of bar reads is generally that the error rate of first half section is lower, and the second half section is relatively high, total ErrorRate not higher than1% is relatively good; As shown in Figure 5, the single base of the sample error rate that on average checks order is 2.01 and 2.23. Fig. 6 has shown sample readsGC (AT) content distribution situation. The content of AGCT should respectively account for 25% theoretically, due to the Preference of pcr amplification,May some little difference, as shown in Figure 6, GC (AT) the content distribution situation of the sample reads that the present embodiment order-checking obtainsMore satisfactory. Due to various problems such as the DNA degradations of sample, also may make the relatively disperse of GC content, GC content in additionDiffer larger with AT content, as Fig. 7. It is GC (AT) the content distribution situation that Fig. 7 has shown poor reads.
2, filter
Original FASTQ data (comprising fq file and adapter message file) are carried out to quality control, remove conventional saidLow quality Value Data. Wherein, for example the average quality of a read is lower than 10, the reads of contaminated adapter, allBe filtered.
3, obtain the BAM file for calculating CNV and callSNP, INDEL
The main purpose of this step is the bam file that obtains sample. First, obtain initial by aln, the sampe of BWABam file. Secondly, utilize picard and GATK to process bam file above.
(1) parameter that sequence alignment arranges is as follows: aln-L-I-k2-l31-t4-i10; Input file is the number that filters gainedAccording to.
(2) merge the data of two end sequencings: sampe (parameter adopts default value ,-a500), and utilize the sort of picardInstrument sorts to it.
(3) mark repeats
Repeating is that pcr amplification brings, and it is the false positive of bringing for fear of callSNP and INDEL that mark repeats, false cloudyProperty.
(4) integrated treatment sequential file
The above version of GATK2.0 will no longer support the variation without header file to detect, need to be with using in picardAddOrReplaceReadGroups instrument adds head (head) to be processed.
The object of two steps is exactly comparison to be carried out to part near reads indel again compare below, and the error rate of comparison is fallenTo minimum. In general, the genome area that the overwhelming majority need to be compared again, is all the existence because of insertion/deletion,Because near comparison indel there will be a large amount of base mispairings, the mispairing of these bases is easy to be mistaken as SNP. AlsoHave, in comparison process, alignment algorithm is all independently for the processing of each read, can not be simultaneously many readsWith carry out misarrangement with reference to genome alignment. Therefore, even if there is the comparison that some reads can be correct to arrive indel, but those exactlyComparison starts or the read of end position also has very high comparison error rate to indel, and this all need to compare again.Localrealignment will be caused the region of mispairing again to be compared by indel, by near comparison error rate indelDrop to minimum.
Mainly be divided into two steps:
The first step, by operation, RealignerTargetCreator determines the region that will again compare.
Second step, by operation, IndelRealigner again compares in these regions.
Now, can generate the sequential file (bam file) that can be used for calculating CNV and callSNP and INDEL.
4, utilize bam file calculating CNV and specific CNV variation and the degree of depth, coverage to calculate
This CNV method is based on order-checking depth calculation CNV.
5、callSNP、INDEL
Utilize UnifiedGenotyper instrument callSNP and the INDEL of GATK, input file is the sequence generating aboveFile (bam file), generates the VCF formatted file that indicates variation information.
6, delete middle junk data
7, information analysis report the test
Fig. 8 has shown information analysis report the test file cut-away view.
Wherein, Fig. 9 has shown the degree of depth of each CDS of target gene FLG, the statistics of coverage, and Figure 10 showsSingle base degree of depth Poisson distribution figure of sample. Can be understood accurately the depth profile situation of this sample by Figure 10, can seeGo out to catch degree fine.
Figure 11 has shown sample FLG partial data sectional drawing after analysis of biological information.
Four, data are understood
Data are understood flow process and are seen Figure 12.
Sample FLG data analysis according to the deciphering flow process described in Figure 12 to above-mentioned acquisition, obtains the testing result of suddenling change,See Figure 13.
Then, the above results is carried out to Reads comparison, find sample FLG two sudden changes: c.5383G > T and c.3321delA,Wherein, Figure 14 has shown that FLG gene is c.5383G > the normal chain Reads comparison result of T sudden change; Figure 15 has shown FLG baseBecause of the normal chain Reads comparison result c.3321delA suddenling change.
Thus, in FLG (NM_002016) gene coding region, detect compound heterozygous mutations: [c.3321delA+c.5383G > T].Frameshift and nonsense mutation all can cause amino acid chain premature termination, affect its protein function, and occurrence frequency in crowdExtremely low. Studies have reported that 65 ordinary type ichthyosis patients in Chinese population are carried out to genetic test discovery, c.3321delA etc.Position gene mutation frequency is 52.31% (34/65), and in contrast crowd, gene frequency is 4% (4/100), and this research is thoughtC.3321delA sudden change is the highest pathogenic mutation site of SOUTHERN CHINA ordinary type ichthyosis frequency. Separately there is bibliographical information to claim to have moreIchthyosis is by due to the nonsense mutation of FLG gene. C.3321delA and c.5383G-therefore, infer to be person under inspection cause a disease TSudden change.
Embodiment 2
According to the method for embodiment 1,59 routine ordinary type ichthyosis suspected patients are carried out to FLG genetic test. Wherein, this realityExecuting example first (carries out PCR, then PCR product is carried out to nido amplification, obtain specific with generation direct sequencingAfter aim sequence, understand FLG sequence in conjunction with sanger order-checking) be contrast.
The results are shown in following table, utilize method of the present invention, 49 routine patients detect on FLG gene and undergo mutation, and recall rate is 83%.Compared with generation direct Sequencing recall rate (less than 30%), significantly improve.
Patient's numbering FLG testing result
P1 p.Ser1515*,Het
P2 [c.3321delA+c.1126delG]
P3 Without (none)
P4 c.3321delA,Hom
P5 [c.3321delA+p.Gln2417*]
P6 c.3321delA,Het
P7 c.3321delA,Het
P8 [c.3321delA+p.Gln2417*]
P9 c.678delA,Het
P10 Without (none)
P11 [p.Glu1795*+c.1248_1249insG]
P12 c.3321delA,Het
P13 c.3321delA,Het
P14 c.3321delA,Het
P15 c.3321delA,Hom
P16 c.3321delA,Hom
P17 c.3321delA,Het
P18 Without (none)
P19 Without (none)
P20 [c.5757_5760delCCAG+p.Gly1034*]
P21 c.3321delA,Het
P22 c.3321delA,Het
P23 p.Ser1235*,Het
P24 [c.7386_7389delTCAT+p.Gln1790*]
P25 p.Arg3419*,Het
P26 Without (none)
P27 [c.1248_1249insG+c.441delA]
P28 p.Arg826*,Het
P29 [c.3222_3225delTGAG+c.3321delA]
P30 [c.3321delA+p.Ser1515*]
P31 c.4895delA,Het
P32 c.3321delA,Het
P33 p.Gln2417*,Het
P34 c.3321delA,Het
P35 Without (none)
P36 [c.3321delA+p.Gln1790*]
P37 Without (none)
P38 Without (none)
P39 p.Gln2397*,Het
P40 [p.Ser1302*+p.Arg3409*+p.Gln2417*]
P41 FLG:p.Gln1790*,Het
P42 [c.3321delA+p.Ser1515*]
P43 c.3321delA,Het
P44 c.3321delA,Hom
P45 c.3321delA,Het
P46 c.3321delA,Het
P47 c.4271_4272delAA,Het
P48 Without (none)
P49 [p.Ser1515*+p.Ser1302*]
P50 c.3321delA,Het
P51 [p.Glu1795*+c.3321delA]
P52 p.Glu1795*,Het
P53 Without (none)
P54 [p.Ser1302*+p.Ser406*]
P55 p.Glu2422*,Het
P56 [p.Ser1515*+c.3321delA]
P57 c.3321delA,Het
P58 c.3321delA,Het
P59 [p.Arg3409*+p.Gln2417*]
Note: " Het " refers to heterozygote, " Hom " refers to homozygote
In the description of this description, reference term " embodiment ", " some embodiment ", " example ", " concrete example ",Or the description of " some examples " etc. means specific features, structure, material or the feature bag described in conjunction with this embodiment or exampleBe contained at least one embodiment of the present invention or example. In this manual, to the schematic statement of the above-mentioned term definiteness that differsBe identical embodiment or example. And specific features, structure, material or the feature of description can be any oneOr in multiple embodiment or example with suitable mode combination.
Although illustrated and described embodiments of the invention, those having ordinary skill in the art will appreciate that: do not departing from thisIn the principle of invention and the situation of aim, can carry out multiple variation, amendment, replacement and modification to these embodiment, of the present inventionScope is limited by claim and equivalent thereof.

Claims (10)

1. one group of probe, is characterized in that, at least a portion of described one group of probe specificity identification FLG gene coding region,And described probe meet be selected from following condition one of at least:
(1) length of described probe is 75bp;
(2) described probe specificity identification FLG gene coding region upstream 10bp is to the sequence between the 10bp of downstream;
(3) specific recognition GC content is higher than 0.6 and lower than the probe in 0.3 region, and multiplier is greater than 2;
(4) melting temperature of described probe and target sequence is 60-10 degree Celsius, preferably 80 degrees Celsius;
(5) described probe does not comprise hairpin structure;
(6) described probe with mate with reference to 2 sites at the most on genome;
(7) window sliding size when described probe is selected is 10bp.
2. one group of probe according to claim 1, it has the nucleotide sequence shown in SEQIDNO:1-1095,
Optionally, described one group of probe is free on solution,
Optionally, described one group of probe is incorporated on solid-phase matrix, forms chip,
Optionally, described is the reference genome sequence of described sample to be tested source species with reference to genome, and preferably the mankind are with reference to baseBecause of group.
3. a method that builds high-throughput sequencing library, is characterized in that, comprises the following steps:
By genomic DNA fragment, to obtain DNA fragmentation;
Described DNA fragmentation is carried out to end reparation, to obtain the DNA fragmentation of repairing through end;
3 ' the end at the described DNA fragmentation of repairing through end adds base A, has cohesive end A's to obtainDNA fragmentation;
The described DNA fragmentation with cohesive end A is connected with joint, connects product to obtain;
Utilize one group of probe described in claim 1 or 2 to screen described connection product, to obtain object fragment, instituteState object fragment and form described high-throughput sequencing library.
4. method according to claim 3, is characterized in that, further comprises and from sample, extracts genomic DNAStep, preferred described sample derives from mammal, and more preferably described mammal is behaved and at least one of mouse, preferred instituteState genomic DNA for mankind's Whole Blood Genomic DNA,
Optionally, utilize covaris-S2 to interrupt instrument by genomic DNA fragment,
Optionally, the length of described DNA fragmentation is about 200-250bp,
Optionally, described DNA fragmentation being carried out to, before end reparation, further comprise the step of purifying DNA fragment,
Optionally, described DNA fragmentation being carried out to end reparation is to utilize Klenow fragment, T4DNA polymerase and T4 manyNucleoside monophosphate kinase carries out, and wherein, described Klenow fragment has 5 ' → 3 ' polymerase activity and 3 ' → 5 ' polymerase activity,But lack 5 ' → 3 ' 5 prime excision enzyme activity,
Optionally, 3 ' end of the described DNA fragmentation of repairing through end is added to base A and utilize Klenow (3 '-5 'Exo-) carry out,
Optionally, the described DNA fragmentation with cohesive end A is connected with joint and utilizes T4DNA ligase to carry out,
Optionally, described high-throughput sequencing library is suitable for utilizing the preferred Hiseq2000 order-checking of high throughput sequencing technologies platform to surveyOrder,
Optionally, utilize solid phase chip hybrid capture technology to carry out described screening.
5. a method for definite sample to be tested FLG gene coding region nucleotide sequence, is characterized in that, comprises the following steps:
According to the method described in claim 3 or 4, build the high-throughput sequencing library of sample to be tested, described high-flux sequence literary compositionStorehouse comprises FLG gene coding region nucleotide sequence;
High-throughput sequencing library to described sample to be tested checks order, to obtain sequencing result; And
Based on described sequencing result, determine the nucleotide sequence of described sample to be tested FLG gene coding region.
6. method according to claim 5, is characterized in that, utilizes the preferred Hiseq2000 of high throughput sequencing technologies to surveyOrder platform carries out described order-checking,
Optionally, further comprise:
By the nucleotide sequence of described sample to be tested FLG gene coding region with compare with reference to genome sequence so that determine instituteState sample to be tested FLG gene coding region and whether have sudden change, and obtain variation information,
Optionally, described is the reference genome sequence of described sample to be tested source species with reference to genome, and preferably the mankind are with reference to baseBecause of group.
7. a device that builds high-throughput sequencing library, is characterized in that, comprising:
Fragmentation unit, described fragmentation unit is used for genomic DNA fragment, to obtain DNA fragmentation;
End is repaired unit, and described end is repaired unit and is connected with described fragmentation unit, for described DNA fragmentation is carried outEnd is repaired, to obtain the DNA fragmentation of repairing through end;
Base A adding device, described base A adding device is repaired unit with described end and is connected, for last in described process3 ' end of the DNA fragmentation that end is repaired adds base A, to obtain the DNA fragmentation with cohesive end A;
Joint linkage unit, described joint linkage unit is used for the described DNA fragmentation with cohesive end A to be connected with joint,Connect product to obtain; And
Screening unit, described screening unit is connected with described joint linkage unit, and is provided with one described in claim 1 or 2Group probe, for utilizing described one group of probe to screen described connection product, to obtain object fragment, described object sheetSection forms described high-throughput sequencing library.
8. device according to claim 7, is characterized in that, further comprises extracting genome DNA unit, described inExtracting genome DNA unit is connected with described fragmentation unit, for extracting genomic DNA from sample, and preferred described sampleOriginally derive from mammal, more preferably described mammal is behaved and at least one of mouse, and preferred described genomic DNA isHuman whole blood genomic DNA,
Optionally, described fragmentation unit is that covaris-S2 interrupts instrument,
Optionally, the length of described DNA fragmentation is about 200-250bp,
Optionally, further comprise purification unit, described purification unit is repaired unit phase with described fragmentation unit and described endConnect, for described DNA fragmentation being carried out to, before end reparation, described DNA fragmentation is carried out to purifying,
Optionally, described DNA fragmentation being carried out to end reparation is to utilize Klenow fragment, T4DNA polymerase and T4 manyNucleoside monophosphate kinase carries out, and wherein, described Klenow fragment has 5 ' → 3 ' polymerase activity and 3 ' → 5 ' polymerase activity,But lack 5 ' → 3 ' 5 prime excision enzyme activity,
Optionally, 3 ' end of the described DNA fragmentation of repairing through end is added to base A and utilize Klenow (3 '-5 'Exo-) carry out,
Optionally, the described DNA fragmentation with cohesive end A is connected with joint and utilizes T4DNA ligase to carry out,
Optionally, described high-throughput sequencing library is suitable for utilizing the preferred Hiseq2000 order-checking of high throughput sequencing technologies platform to surveyOrder,
Optionally, described screening unit is suitable for utilizing solid phase chip hybrid capture technology to carry out described hybrid capture.
9. a system for definite sample to be tested FLG gene coding region nucleotide sequence, is characterized in that, comprising:
Library construction device, described library construction device is the device of the structure high-throughput sequencing library described in claim 7 or 8,For building the high-throughput sequencing library of sample to be tested, described high-throughput sequencing library comprises FLG gene coding region nucleotide sequence;
Sequencing device, described sequencing device is connected with described library construction device, for the high-flux sequence to described sample to be testedCheck order in library, to obtain sequencing result; And
Analytical equipment, described analytical equipment is connected with described sequencing device, for based on described sequencing result, determines described to be measuredThe nucleotide sequence of sample FLG gene coding region.
10. system according to claim 9, is characterized in that, described sequencing device is high-flux sequence platform, preferablyThe Hiseq2000 platform that checks order,
Optionally, further comprise comparison device, described comparison device is connected with described analytical equipment, described in inciting somebody to action, treats test sampleThe nucleotide sequence of this FLG gene coding region with compare with reference to genome sequence so that determine described sample to be tested FLG baseBecause whether code area exists sudden change, and obtain variation information,
Optionally, described is the reference genome sequence of described sample to be tested source species with reference to genome, and preferably the mankind are with reference to baseBecause of group.
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