CN105603052A - Probes and uses thereof - Google Patents
Probes and uses thereof Download PDFInfo
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- 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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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
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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