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==Analysis of HL155: Representation Bias of Agi26k Oligos== *Previously amplified Agi26k oligos with sequence adapters to sequence *By counting the number of reads for each probe, can determine uniformity of oligos in probe set # Turn FISSEQ_Probes_Dec2012.txt into fasta file (Full: Agi26kprobes_to_order.fa 50bp: Agi26kprobes_to_order_50bp.fa) ## First Primer for V6: "GTCATATCGGTCACTGTT" ## First Primer for V4: "TAGACTGGAAGAGCACTGTT" # Build index for probes ## /home/kunzhang/softwares/bowtie2-latest/bowtie2-build ~/InSitu_HL155_130628_Analysis/Agi26kprobes_to_order.fa Agi26kprobes ## /home/kunzhang/softwares/bowtie2-latest/bowtie2-build ~/InSitu_HL155_130628_Analysis/Agi26kprobes_to_order_50bp.fa Agi26kprobes_50bp # Align to index ## Indx10 -> 0 gap -> V6: ### /home/kunzhang/softwares/bowtie2-latest/bowtie2 -k 1 --phred64 --mp 1,0 --rdg 0,1 --rfg 0,1 -x /home/mzcai/InSitu_HL155_130628_Analysis/Agi26kprobes -q /home/mzcai/InSitu_HL155_130628_Analysis/s_3_1_Indx10.txt > /home/mzcai/InSitu_HL155_130628_Analysis/Readsalign2probes_0gapFull.txt & <br>16368416 reads; of these:<br>16368416 (100.00%) were unpaired; of these:<br>8915822 (54.47%) aligned 0 times<br>7452594 (45.53%) aligned exactly 1 time<br>0 (0.00%) aligned >1 times<br>45.53% overall alignment rate<br> ### /home/kunzhang/softwares/bowtie2-latest/bowtie2 -k 1 --phred64 --mp 1,0 --rdg 0,1 --rfg 0,1 -x /home/mzcai/InSitu_HL155_130628_Analysis/Agi26kprobes_50bp -q /home/mzcai/InSitu_HL155_130628_Analysis/s_3_1_Indx10.txt > /home/mzcai/InSitu_HL155_130628_Analysis/Readsalign2probes_0gap50bp.txt &<br>mzcai@genome-miner:~/InSitu_HL155_130628_Analysis$ 16368416 reads; of these:<br>16368416 (100.00%) were unpaired; of these:<br>15978678 (97.62%) aligned 0 times<br>389738 (2.38%) aligned exactly 1 time<br>0 (0.00%) aligned >1 times<br>2.38% overall alignment rate<br> ## Indx12 -> 20 gap -> V4: ### /home/kunzhang/softwares/bowtie2-latest/bowtie2 -k 1 --phred64 --mp 1,0 --rdg 0,1 --rfg 0,1 -x /home/mzcai/InSitu_HL155_130628_Analysis/Agi26kprobes -q /home/mzcai/InSitu_HL155_130628_Analysis/s_3_1_Indx12.txt > /home/mzcai/InSitu_HL155_130628_Analysis/Readsalign2probes_20gapFull.txt &<br>mzcai@genome-miner:~/InSitu_HL155_130628_Analysis$ 17308686 reads; of these:<br>17308686 (100.00%) were unpaired; of these:<br>11482192 (66.34%) aligned 0 times<br>5826494 (33.66%) aligned exactly 1 time<br>0 (0.00%) aligned >1 times<br>33.66% overall alignment rate<br> ### /home/kunzhang/softwares/bowtie2-latest/bowtie2 -k 1 --phred64 --mp 1,0 --rdg 0,1 --rfg 0,1 -x /home/mzcai/InSitu_HL155_130628_Analysis/Agi26kprobes_50bp -q /home/mzcai/InSitu_HL155_130628_Analysis/s_3_1_Indx12.txt > /home/mzcai/InSitu_HL155_130628_Analysis/Readsalign2probes_20gap50bp.txt &<br>mzcai@genome-miner:~/InSitu_HL155_130628_Analysis$ 17308686 reads; of these:<br>17308686 (100.00%) were unpaired; of these:<br>17112441 (98.87%) aligned 0 times<br>196245 (1.13%) aligned exactly 1 time<br>0 (0.00%) aligned >1 times<br>1.13% overall alignment rate<br> # Counts for each probe: CountsofAgi26k_0gap50bp.txt, CountsofAgi26k_0gapFull.txt, CountsofAgi26k_20gap50bp.txt, CountsofAgi26k_20gapFull.txt ## Many 0 gap reads aligned to 20 gap probes and vice versa suggests this method isn't specific enough Below is number of probes binned into number of reads for 50bp reference probes {| {{table}} | align="center" style="background:#f0f0f0;"|'''0 gap reads aligned to 0 gap probe''' | align="center" style="background:#f0f0f0;"|'''''' | align="center" style="background:#f0f0f0;"|'''0 gap reads aligned to 20 gap probes''' | align="center" style="background:#f0f0f0;"|'''''' | align="center" style="background:#f0f0f0;"|'''20 gap reads aligned to 0 gap probes''' | align="center" style="background:#f0f0f0;"|'''''' | align="center" style="background:#f0f0f0;"|'''20 gap reads aligned to 20 gap probes''' | align="center" style="background:#f0f0f0;"|'''''' |- | Bin||Frequency||Bin||Frequency||Bin||Frequency||Bin||Frequency |- | 0||19||0||1317||0||4034||0||5 |- | 7.157894737||293||19.10619469||11167||3.929824561||7384||9.902654867||4268 |- | 14.31578947||1427||38.21238938||310||7.859649123||1185||19.80530973||7286 |- | 21.47368421||3791||57.31858407||73||11.78947368||289||29.7079646||1191 |- | 28.63157895||4282||76.42477876||31||15.71929825||135||39.61061947||111 |- | 35.78947368||2293||95.53097345||20||19.64912281||57||49.51327434||37 |- | 42.94736842||783||114.6371681||12||23.57894737||30||59.4159292||19 |- | 50.10526316||186||133.7433628||8||27.50877193||20||69.31858407||13 |- | 57.26315789||37||152.8495575||2||31.43859649||8||79.22123894||8 |- | 64.42105263||12||171.9557522||3||35.36842105||4||89.12389381||4 |- | 71.57894737||19||191.0619469||1||39.29824561||4||99.02654867||3 |- | 78.73684211||8||210.1681416||3||43.22807018||6||108.9292035||0 |- | 85.89473684||10||229.2743363||2||47.15789474||3||118.8318584||1 |- | 93.05263158||6||248.380531||3||51.0877193||0||128.7345133||2 |- | 100.2105263||4||267.4867257||1||55.01754386||0||138.6371681||2 |- | 107.3684211||1||286.5929204||1||58.94736842||3||148.539823||0 |- | 114.5263158||1||305.699115||0||62.87719298||1||158.4424779||5 |- | 121.6842105||1||324.8053097||1||66.80701754||0||168.3451327||1 |- | 128.8421053||0||343.9115044||1||70.73684211||3||178.2477876||0 |- | 136||0||363.0176991||0||74.66666667||0||188.1504425||0 |- | 143.1578947||0||382.1238938||1||78.59649123||0||198.0530973||0 |- | 150.3157895||0||401.2300885||0||82.52631579||0||207.9557522||0 |- | 157.4736842||0||420.3362832||0||86.45614035||0||217.8584071||1 |- | 164.6315789||0||439.4424779||1||90.38596491||1||227.7610619||0 |- | 171.7894737||1||458.5486726||0||94.31578947||0||237.6637168||0 |- | 178.9473684||0||477.6548673||0||98.24561404||0||247.5663717||0 |- | 186.1052632||1||496.7610619||0||102.1754386||0||257.4690265||1 |- | 193.2631579||0||515.8672566||0||106.1052632||1||267.3716814||1 |- | 200.4210526||0||534.9734513||0||110.0350877||1||277.2743363||0 |- | 207.5789474||0||554.079646||0||113.9649123||0||287.1769912||0 |- | 214.7368421||0||573.1858407||1||117.8947368||1||297.079646||0 |- | 221.8947368||0||592.2920354||0||121.8245614||2||306.9823009||0 |- | 229.0526316||0||611.3982301||0||125.754386||1||316.8849558||0 |- | 236.2105263||0||630.5044248||0||129.6842105||0||326.7876106||0 |- | 243.3684211||0||649.6106195||1||133.6140351||0||336.6902655||1 |- | 250.5263158||0||668.7168142||0||137.5438596||0||346.5929204||0 |- | 257.6842105||0||687.8230088||0||141.4736842||1||356.4955752||0 |- | 264.8421053||1||706.9292035||0||145.4035088||0||366.3982301||0 |- | 272||0||726.0353982||0||149.3333333||0||376.300885||0 |- | 279.1578947||0||745.1415929||0||153.2631579||1||386.2035398||0 |- | 286.3157895||0||764.2477876||0||157.1929825||2||396.1061947||0 |- | 293.4736842||0||783.3539823||0||161.122807||0||406.0088496||0 |- | 300.6315789||0||802.460177||0||165.0526316||0||415.9115044||0 |- | 307.7894737||0||821.5663717||0||168.9824561||0||425.8141593||0 |- | 314.9473684||0||840.6725664||0||172.9122807||0||435.7168142||0 |- | 322.1052632||0||859.7787611||0||176.8421053||0||445.619469||0 |- | 329.2631579||0||878.8849558||0||180.7719298||0||455.5221239||0 |- | 336.4210526||0||897.9911504||0||184.7017544||1||465.4247788||0 |- | 343.5789474||1||917.0973451||0||188.6315789||0||475.3274336||0 |- | 350.7368421||0||936.2035398||0||192.5614035||0||485.2300885||0 |- | 357.8947368||0||955.3097345||0||196.4912281||0||495.1327434||0 |- | 365.0526316||0||974.4159292||0||200.4210526||0||505.0353982||0 |- | 372.2105263||0||993.5221239||0||204.3508772||0||514.9380531||0 |- | 379.3684211||0||1012.628319||0||208.2807018||0||524.840708||0 |- | 386.5263158||0||1031.734513||0||212.2105263||0||534.7433628||0 |- | 393.6842105||1||1050.840708||0||216.1403509||0||544.6460177||0 |- | 400.8421053||0||1069.946903||0||220.0701754||0||554.5486726||0 |- | 408||0||1089.053097||0||224||0||564.4513274||1 |- | 415.1578947||0||1108.159292||0||227.9298246||0||574.3539823||0 |- | 422.3157895||0||1127.265487||0||231.8596491||0||584.2566372||0 |- | 429.4736842||0||1146.371681||1||235.7894737||0||594.159292||0 |- | 436.6315789||0||1165.477876||0||239.7192982||0||604.0619469||0 |- | 443.7894737||0||1184.584071||0||243.6491228||0||613.9646018||0 |- | 450.9473684||0||1203.690265||0||247.5789474||0||623.8672566||0 |- | 458.1052632||0||1222.79646||0||251.5087719||0||633.7699115||0 |- | 465.2631579||0||1241.902655||0||255.4385965||0||643.6725664||0 |- | 472.4210526||0||1261.00885||0||259.3684211||0||653.5752212||0 |- | 479.5789474||0||1280.115044||0||263.2982456||0||663.4778761||0 |- | 486.7368421||0||1299.221239||0||267.2280702||0||673.380531||0 |- | 493.8947368||0||1318.327434||0||271.1578947||0||683.2831858||0 |- | 501.0526316||0||1337.433628||0||275.0877193||0||693.1858407||0 |- | 508.2105263||0||1356.539823||0||279.0175439||0||703.0884956||0 |- | 515.3684211||0||1375.646018||0||282.9473684||0||712.9911504||0 |- | 522.5263158||0||1394.752212||0||286.877193||0||722.8938053||0 |- | 529.6842105||0||1413.858407||0||290.8070175||0||732.7964602||0 |- | 536.8421053||0||1432.964602||0||294.7368421||0||742.699115||0 |- | 544||0||1452.070796||0||298.6666667||0||752.6017699||0 |- | 551.1578947||0||1471.176991||0||302.5964912||0||762.5044248||0 |- | 558.3157895||0||1490.283186||0||306.5263158||0||772.4070796||0 |- | 565.4736842||0||1509.389381||0||310.4561404||0||782.3097345||1 |- | 572.6315789||0||1528.495575||0||314.3859649||0||792.2123894||0 |- | 579.7894737||0||1547.60177||0||318.3157895||0||802.1150442||0 |- | 586.9473684||0||1566.707965||0||322.245614||0||812.0176991||0 |- | 594.1052632||0||1585.814159||0||326.1754386||0||821.920354||0 |- | 601.2631579||0||1604.920354||0||330.1052632||0||831.8230088||0 |- | 608.4210526||0||1624.026549||0||334.0350877||0||841.7256637||0 |- | 615.5789474||0||1643.132743||0||337.9649123||0||851.6283186||0 |- | 622.7368421||0||1662.238938||0||341.8947368||0||861.5309735||0 |- | 629.8947368||0||1681.345133||0||345.8245614||0||871.4336283||0 |- | 637.0526316||0||1700.451327||0||349.754386||0||881.3362832||0 |- | 644.2105263||0||1719.557522||0||353.6842105||0||891.2389381||0 |- | 651.3684211||0||1738.663717||0||357.6140351||0||901.1415929||0 |- | 658.5263158||0||1757.769912||0||361.5438596||0||911.0442478||0 |- | 665.6842105||0||1776.876106||0||365.4736842||0||920.9469027||0 |- | 672.8421053||0||1795.982301||0||369.4035088||0||930.8495575||0 |- | 680||0||1815.088496||0||373.3333333||0||940.7522124||0 |- | 687.1578947||0||1834.19469||0||377.2631579||0||950.6548673||0 |- | 694.3157895||0||1853.300885||1||381.1929825||0||960.5575221||0 |- | 701.4736842||0||1872.40708||0||385.122807||0||970.460177||0 |- | 708.6315789||0||1891.513274||0||389.0526316||0||980.3628319||0 |- | 715.7894737||0||1910.619469||0||392.9824561||0||990.2654867||0 |- | 722.9473684||0||1929.725664||0||396.9122807||0||1000.168142||0 |- | 730.1052632||0||1948.831858||0||400.8421053||0||1010.070796||0 |- | 737.2631579||0||1967.938053||0||404.7719298||0||1019.973451||0 |- | 744.4210526||0||1987.044248||0||408.7017544||0||1029.876106||0 |- | 751.5789474||0||2006.150442||0||412.6315789||0||1039.778761||0 |- | 758.7368421||0||2025.256637||0||416.5614035||0||1049.681416||0 |- | 765.8947368||0||2044.362832||0||420.4912281||0||1059.584071||0 |- | 773.0526316||0||2063.469027||0||424.4210526||0||1069.486726||0 |- | 780.2105263||0||2082.575221||0||428.3508772||0||1079.389381||0 |- | 787.3684211||0||2101.681416||0||432.2807018||0||1089.292035||0 |- | 794.5263158||0||2120.787611||0||436.2105263||0||1099.19469||0 |- | 801.6842105||0||2139.893805||0||440.1403509||0||1109.097345||0 |- | 808.8421053||0||More||1||444.0701754||0||More||1 |- | More||1||||||More||1|||| |} Below is number of probes binned into number of reads for full reference probes {| {{table}} | align="center" style="background:#f0f0f0;"|'''0 gap reads aligned to 0 gap probe''' | align="center" style="background:#f0f0f0;"|'''''' | align="center" style="background:#f0f0f0;"|'''0 gap reads aligned to 20 gap probes''' | align="center" style="background:#f0f0f0;"|'''''' | align="center" style="background:#f0f0f0;"|'''20 gap reads aligned to 0 gap probes''' | align="center" style="background:#f0f0f0;"|'''''' | align="center" style="background:#f0f0f0;"|'''20 gap reads aligned to 20 gap probes''' | align="center" style="background:#f0f0f0;"|'''''' |- | Bin||Frequency||Bin||Frequency||Bin||Frequency||Bin||Frequency |- | 1||1||0||26||0||98||20||1 |- | 14.92982456||80||10.94690265||3858||15.01754386||7662||38.48672566||1 |- | 28.85964912||42||21.89380531||3605||30.03508772||2922||56.97345133||5 |- | 42.78947368||31||32.84070796||1623||45.05263158||1150||75.46017699||9 |- | 56.71929825||26||43.78761062||999||60.07017544||484||93.94690265||16 |- | 70.64912281||18||54.73451327||761||75.0877193||231||112.4336283||13 |- | 84.57894737||12||65.68141593||496||90.10526316||135||130.920354||15 |- | 98.50877193||7||76.62831858||354||105.122807||82||149.4070796||19 |- | 112.4385965||11||87.57522124||270||120.1403509||39||167.8938053||15 |- | 126.3684211||7||98.52212389||204||135.1578947||24||186.380531||20 |- | 140.2982456||6||109.4690265||141||150.1754386||20||204.8672566||34 |- | 154.2280702||9||120.4159292||106||165.1929825||13||223.3539823||57 |- | 168.1578947||3||131.3628319||78||180.2105263||7||241.840708||62 |- | 182.0877193||8||142.3097345||74||195.2280702||4||260.3274336||135 |- | 196.0175439||9||153.2566372||54||210.245614||9||278.8141593||195 |- | 209.9473684||12||164.2035398||39||225.2631579||11||297.300885||328 |- | 223.877193||14||175.1504425||33||240.2807018||9||315.7876106||458 |- | 237.8070175||16||186.0973451||31||255.2982456||3||334.2743363||664 |- | 251.7368421||14||197.0442478||10||270.3157895||11||352.7610619||731 |- | 265.6666667||21||207.9911504||13||285.3333333||2||371.2477876||991 |- | 279.5964912||34||218.9380531||14||300.3508772||11||389.7345133||1022 |- | 293.5263158||37||229.8849558||11||315.3684211||12||408.2212389||1133 |- | 307.4561404||44||240.8318584||16||330.3859649||16||426.7079646||1077 |- | 321.3859649||73||251.7787611||8||345.4035088||22||445.1946903||1074 |- | 335.3157895||83||262.7256637||9||360.4210526||14||463.6814159||993 |- | 349.245614||122||273.6725664||11||375.4385965||34||482.1681416||887 |- | 363.1754386||145||284.619469||9||390.4561404||23||500.6548673||682 |- | 377.1052632||199||295.5663717||6||405.4736842||19||519.1415929||601 |- | 391.0350877||220||306.5132743||7||420.4912281||23||537.6283186||433 |- | 404.9649123||276||317.460177||5||435.5087719||25||556.1150442||342 |- | 418.8947368||343||328.4070796||5||450.5263158||11||574.6017699||249 |- | 432.8245614||438||339.3539823||3||465.5438596||6||593.0884956||182 |- | 446.754386||460||350.300885||1||480.5614035||4||611.5752212||145 |- | 460.6842105||562||361.2477876||0||495.5789474||9||630.0619469||95 |- | 474.6140351||580||372.1946903||5||510.5964912||12||648.5486726||70 |- | 488.5438596||612||383.1415929||4||525.6140351||3||667.0353982||43 |- | 502.4736842||678||394.0884956||3||540.6315789||6||685.5221239||47 |- | 516.4035088||608||405.0353982||2||555.6491228||2||704.0088496||32 |- | 530.3333333||715||415.9823009||0||570.6666667||2||722.4955752||22 |- | 544.2631579||626||426.9292035||1||585.6842105||0||740.9823009||19 |- | 558.1929825||702||437.8761062||2||600.7017544||4||759.4690265||13 |- | 572.122807||664||448.8230088||2||615.7192982||1||777.9557522||6 |- | 586.0526316||628||459.7699115||1||630.7368421||0||796.4424779||6 |- | 599.9824561||502||470.7168142||7||645.754386||1||814.9292035||4 |- | 613.9122807||456||481.6637168||6||660.7719298||1||833.4159292||2 |- | 627.8421053||480||492.6106195||1||675.7894737||0||851.9026549||1 |- | 641.7719298||363||503.5575221||2||690.8070175||0||870.3893805||4 |- | 655.7017544||319||514.5044248||1||705.8245614||0||888.8761062||1 |- | 669.6315789||258||525.4513274||0||720.8421053||0||907.3628319||1 |- | 683.5614035||276||536.3982301||0||735.8596491||0||925.8495575||1 |- | 697.4912281||215||547.3451327||3||750.877193||0||944.3362832||1 |- | 711.4210526||199||558.2920354||4||765.8947368||0||962.8230088||1 |- | 725.3508772||159||569.2389381||2||780.9122807||0||981.3097345||1 |- | 739.2807018||119||580.1858407||2||795.9298246||0||999.7964602||0 |- | 753.2105263||126||591.1327434||2||810.9473684||0||1018.283186||1 |- | 767.1403509||71||602.079646||4||825.9649123||1||1036.769912||0 |- | 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1366.122807||1||1072.79646||0||1471.719298||0||1831.699115||0 |- | 1380.052632||1||1083.743363||0||1486.736842||0||1850.185841||0 |- | 1393.982456||2||1094.690265||0||1501.754386||0||1868.672566||0 |- | 1407.912281||0||1105.637168||0||1516.77193||0||1887.159292||0 |- | 1421.842105||2||1116.584071||0||1531.789474||0||1905.646018||0 |- | 1435.77193||1||1127.530973||0||1546.807018||0||1924.132743||0 |- | 1449.701754||0||1138.477876||0||1561.824561||0||1942.619469||0 |- | 1463.631579||1||1149.424779||0||1576.842105||0||1961.106195||0 |- | 1477.561404||1||1160.371681||0||1591.859649||0||1979.59292||0 |- | 1491.491228||0||1171.318584||0||1606.877193||0||1998.079646||0 |- | 1505.421053||0||1182.265487||0||1621.894737||0||2016.566372||0 |- | 1519.350877||0||1193.212389||0||1636.912281||0||2035.053097||0 |- | 1533.280702||0||1204.159292||0||1651.929825||0||2053.539823||0 |- | 1547.210526||0||1215.106195||0||1666.947368||0||2072.026549||0 |- | 1561.140351||1||1226.053097||0||1681.964912||0||2090.513274||0 |- | 1575.070175||0||More||1||1696.982456||0||More||1 |- | More||1||||||More||1|||| |}
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