Matt:LabNotes/2013-7-19
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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 &
16368416 reads; of these:
16368416 (100.00%) were unpaired; of these:
8915822 (54.47%) aligned 0 times
7452594 (45.53%) aligned exactly 1 time
0 (0.00%) aligned >1 times
45.53% overall alignment rate - /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 &
mzcai@genome-miner:~/InSitu_HL155_130628_Analysis$ 16368416 reads; of these:
16368416 (100.00%) were unpaired; of these:
15978678 (97.62%) aligned 0 times
389738 (2.38%) aligned exactly 1 time
0 (0.00%) aligned >1 times
2.38% overall alignment rate
- /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 &
- 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 &
mzcai@genome-miner:~/InSitu_HL155_130628_Analysis$ 17308686 reads; of these:
17308686 (100.00%) were unpaired; of these:
11482192 (66.34%) aligned 0 times
5826494 (33.66%) aligned exactly 1 time
0 (0.00%) aligned >1 times
33.66% overall alignment rate - /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 &
mzcai@genome-miner:~/InSitu_HL155_130628_Analysis$ 17308686 reads; of these:
17308686 (100.00%) were unpaired; of these:
17112441 (98.87%) aligned 0 times
196245 (1.13%) aligned exactly 1 time
0 (0.00%) aligned >1 times
1.13% overall alignment rate
- /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 &
- Indx10 -> 0 gap -> V6:
- 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 are number of probes binned into number of reads
0 gap reads aligned to 0 gap probe | ' | 0 gap reads aligned to 20 gap probes | ' | 20 gap reads aligned to 0 gap probes | ' | 20 gap reads aligned to 20 gap probes | ' |
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 |