Kun:LabNotes/MONOD/2015-1-9
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Analysis of the second batch of NimbleGen capture data[edit]
- Noi did the capture experiment: Noi/NOTES/2014-12-25.
- The libraries were sequenced in one HiSeq PE100bp rapid run 141214_SN1001.
- Dinh mapped the reads: Dinh/Dinh_2015/NOTES/2015-1-7.
Quick assessment of the data quality[edit]
- First I checked the performance of target capture.
/home/kunzhang/CpgMIP/MONOD/Data/141112_HiSeqRapidRun/get_capture_stats.pl /media/Ext12T/DD_Ext12T/MONOD/141216_HiSeqRapidRun/BAMfiles > 141216_HiSeqRapidRun.capture_stats.txt
Sample | total_bases_coverred | total_bases | on_target_bases_covered | on_target_bases | Specificity | Enrichment_factor |
6-P-1 | 496,391,712 | 883,938,352 | 20,732,014 | 236,943,256 | 26.81% | 37 |
6-P-2 | 598,767,372 | 974,626,668 | 31,706,660 | 246,684,036 | 25.31% | 34 |
6-P-3 | 543,413,415 | 1,131,193,408 | 31,995,541 | 417,353,349 | 36.89% | 59 |
6T-1 | 729,794,959 | 1,070,341,509 | 30,151,406 | 155,220,183 | 14.50% | 17 |
6T-2 | 784,299,649 | 1,158,440,912 | 29,456,650 | 159,710,882 | 13.79% | 16 |
6T-3 | 1,452,467,042 | 3,323,804,083 | 33,406,665 | 627,923,179 | 18.89% | 24 |
6T-4 | 764,174,440 | 1,132,324,550 | 30,925,708 | 176,622,088 | 15.60% | 19 |
6T-5 | 843,555,327 | 1,282,158,923 | 30,892,326 | 198,472,950 | 15.48% | 19 |
7-P-10 | 465,462,705 | 897,601,881 | 10,665,677 | 208,435,905 | 23.22% | 31 |
7-P-3 | 376,857,145 | 703,513,104 | 10,510,561 | 173,576,316 | 24.67% | 33 |
7-P-5 | 536,571,852 | 807,646,898 | 30,738,662 | 181,239,774 | 22.44% | 29 |
7-P-6 | 616,774,497 | 1,108,444,523 | 20,513,185 | 265,799,682 | 23.98% | 32 |
7-P-7 | 658,461,220 | 1,111,809,575 | 20,479,249 | 228,609,307 | 20.56% | 26 |
7-P-8 | 439,982,190 | 839,669,742 | 11,446,574 | 201,598,027 | 24.01% | 32 |
7T-1 | 780,242,147 | 1,137,145,477 | 30,063,836 | 160,294,067 | 14.10% | 17 |
7T-2 | 746,306,547 | 1,175,101,409 | 26,503,015 | 173,809,990 | 14.79% | 18 |
7T-4 | 771,225,069 | 1,142,595,127 | 29,851,920 | 171,599,332 | 15.02% | 18 |
7T-5 | 802,376,647 | 1,227,075,648 | 27,971,718 | 179,541,336 | 14.63% | 17 |
NC-7 | 255,231,717 | 1,119,499,850 | 7,739,071 | 425,057,131 | 37.97% | 62 |
PC-P_2 | 563,439,590 | 865,434,623 | 30,337,168 | 193,164,906 | 22.32% | 29 |
PC-P_4 | 527,669,028 | 840,929,316 | 29,831,801 | 207,852,537 | 24.72% | 33 |
PC-P_9 | 559,749,434 | 1,035,542,788 | 24,069,681 | 289,633,563 | 27.97% | 39 |
PCT-1 | 714,410,318 | 1,064,633,702 | 27,058,939 | 155,196,610 | 14.58% | 17 |
PCT-2 | 726,354,565 | 1,275,756,897 | 19,580,892 | 189,203,593 | 14.83% | 18 |
PCT-4 | 815,323,371 | 1,247,319,961 | 28,462,046 | 179,280,115 | 14.37% | 17 |
PCT-6 | 948,428,968 | 1,450,561,748 | 31,894,490 | 214,220,998 | 14.77% | 18 |
PCT-7 | 796,647,247 | 1,191,900,342 | 31,314,595 | 191,113,213 | 16.03% | 19 |
- Here is the results for the first capture experiment (141112_SN1001).
Sample | total_bases_coverred | total_bases | on_target_bases_covered | on_target_bases | Specificity | Enrichment_factor |
SeqCap-pCancerSetA-Nov4_12 | 138,931,528 | 1,168,877,279 | 5,883,919 | 450,558,243 | 38.5% | 63 |
SeqCap-pCancerSetA-Nov4_13 | 369,134,874 | 1,424,314,151 | 21,209,194 | 614,835,055 | 43.2% | 77 |
SeqCap-pCancerSetA-Nov4_14 | 302,700,391 | 1,203,488,680 | 13,952,014 | 489,036,528 | 40.6% | 69 |
SeqCap-pCancerSetA-Nov4_15 | 277,562,818 | 1,250,329,814 | 12,577,032 | 511,943,961 | 40.9% | 70 |
SeqCap-pCancerSetA-Nov4_16 | 274,256,170 | 1,164,394,023 | 12,418,863 | 474,550,198 | 40.8% | 70 |
SeqCap-pCancerSetA-Nov4_18 | 403,128,602 | 1,170,296,929 | 23,073,184 | 461,126,140 | 39.4% | 66 |
SeqCap-pCancerSetA-Nov4_19 | 385,167,143 | 1,417,434,873 | 22,614,122 | 614,480,251 | 43.4% | 77 |
SeqCap-pCancerSetA-Nov4_2 | 354,784,810 | 1,514,708,612 | 19,490,111 | 751,025,646 | 49.6% | 99 |
SeqCap-pCancerSetA-Nov4_4 | 352,108,468 | 2,000,885,721 | 23,609,719 | 1,141,652,594 | 57.1% | 134 |
SeqCap-pCancerSetA-Nov4_5 | 196,108,966 | 1,300,234,129 | 8,246,274 | 563,004,318 | 43.3% | 77 |
SeqCap-pCancerSetA-Nov4_6 | 450,502,450 | 1,502,032,451 | 28,135,196 | 645,300,745 | 43.0% | 76 |
SeqCap-pCancerSetA-Nov4_7 | 226,755,475 | 1,432,933,860 | 10,016,683 | 629,536,922 | 43.9% | 79 |
SeqCap-pNormalSetA-Nov4_12 | 368,272,742 | 1,450,833,379 | 32,791,421 | 853,947,465 | 58.9% | 145 |
SeqCap-pNormalSetA-Nov4_13 | 330,611,335 | 1,291,478,405 | 29,764,564 | 756,289,960 | 58.6% | 143 |
SeqCap-pNormalSetA-Nov4_14 | 360,000,998 | 1,364,998,815 | 32,082,346 | 801,469,734 | 58.7% | 144 |
SeqCap-pNormalSetA-Nov4_15 | 375,193,438 | 1,970,841,697 | 30,241,737 | 1,295,408,500 | 65.7% | 194 |
SeqCap-pNormalSetA-Nov4_16 | 365,138,307 | 1,417,821,628 | 31,787,247 | 825,520,233 | 58.2% | 141 |
SeqCap-pNormalSetA-Nov4_18 | 326,952,891 | 1,305,603,890 | 28,937,598 | 760,701,025 | 58.3% | 141 |
SeqCap-pNormalSetA-Nov4_19 | 312,924,926 | 1,271,134,775 | 27,505,559 | 749,645,411 | 59.0% | 145 |
SeqCap-pNormalSetA-Nov4_2 | 353,552,883 | 1,275,196,941 | 31,331,422 | 731,603,678 | 57.4% | 136 |
SeqCap-pNormalSetA-Nov4_4 | 354,621,865 | 1,456,339,179 | 31,829,571 | 867,560,025 | 59.6% | 149 |
SeqCap-pNormalSetA-Nov4_5 | 291,891,632 | 1,066,253,299 | 27,061,797 | 618,610,388 | 58.0% | 140 |
SeqCap-pNormalSetA-Nov4_6 | 333,830,242 | 1,234,362,498 | 31,357,436 | 723,278,371 | 58.6% | 143 |
SeqCap-pNormalSetA-Nov4_7 | 377,475,888 | 1,451,628,039 | 32,325,421 | 843,343,133 | 58.1% | 140 |
- For some reason the specificity and enrichment factors for the second experiment is much worse than the first one.
Further comparison of the two batches of library preparation and capturing experiments.[edit]
- There were a number of differences on the experimental protocols between the two batches of data.
- 141112_HiSeq: Noi used the Tang et al RRBS method for end-repair and polishing Noi/NOTES/2014-10-29; capture was done on Noi/NOTES/2014-11-4;
- 141216_HiSeq: Noi used the Kapa Hyper Prep kit Noi/NOTES/2014-11-30; capture was done on Noi/NOTES/2014-12-25
- I wrote a script to collect summary statistics for all data sets.
./gather_summary_statistics.pl WGBS_UMR_seqCap_sample_file_info.txt > WGBS_UMR_seqCap_2014_two_batches_summary_stats.txt
- Two major conclusions:
- Kapa Hyper Prep kit generated libraries of higher complexity and lower clonal rates.
- The NimbleGen capture experiment did on 12/15 was not optimal, which lead to much lower enrichment and on-target capture rate.
- Action item: repeat the capture on the batch of libraries prepared by the Kapa Hyper Prep kit.
Sample | Experiment | N_mapped_reads | N_non-clonal_reads | N_on-target_reads | N_non-clonal_on-target_reads | N_on-target_haplotypes | N_target_coverred | Pct_on-target | Pct_clonal |
6-P-1 | 141112_HiSeq | 14,727,691 | 12,016,120 | 6,119,021 | 4,477,865 | 336,894 | 38,344 | 41.5% | 26.82% |
6-P-1 | 141216_HiSeq | 10,355,738 | 10,123,240 | 2,619,216 | 2,523,753 | 233,941 | 36,713 | 25.3% | 3.64% |
6-P-10 | 141112_HiSeq | 12,125,750 | 10,627,443 | 4,622,479 | 3,674,457 | 389,005 | 39,594 | 38.1% | 20.51% |
6-P-2 | 141112_HiSeq | 12,610,235 | 10,592,101 | 4,890,268 | 3,776,213 | 194,655 | 30,935 | 38.8% | 22.78% |
6-P-2 | 141216_HiSeq | 11,570,287 | 11,462,431 | 2,756,445 | 2,724,126 | 882,786 | 44,945 | 23.8% | 1.17% |
6-P-3 | 141112_HiSeq | 13,044,553 | 10,902,745 | 5,089,409 | 3,951,200 | 180,681 | 29,498 | 39.0% | 22.36% |
6-P-3 | 141216_HiSeq | 12,482,525 | 12,258,142 | 4,304,444 | 4,228,174 | 1,154,014 | 45,181 | 34.5% | 1.77% |
6-P-4 | 141112_HiSeq | 11,969,782 | 9,696,006 | 4,662,823 | 3,426,251 | 184,637 | 29,454 | 39.0% | 26.52% |
6-P-8 | 141112_HiSeq | 14,935,803 | 13,198,763 | 6,242,428 | 5,177,911 | 351,452 | 38,772 | 41.8% | 17.05% |
6T-1 | 141216_HiSeq | 11,869,709 | 11,711,949 | 1,631,628 | 1,592,334 | 614,412 | 42,951 | 13.7% | 2.41% |
6T-2 | 141216_HiSeq | 12,820,323 | 12,644,365 | 1,679,917 | 1,605,021 | 535,022 | 42,471 | 13.1% | 4.46% |
6T-3 | 141216_HiSeq | 36,126,640 | 33,993,548 | 6,294,978 | 5,456,336 | 1,390,560 | 45,913 | 17.4% | 13.32% |
6T-4 | 141216_HiSeq | 12,351,984 | 12,219,697 | 1,829,796 | 1,794,578 | 722,787 | 43,545 | 14.8% | 1.92% |
6T-5 | 141216_HiSeq | 13,871,915 | 13,700,144 | 2,047,985 | 2,002,834 | 689,818 | 43,576 | 14.8% | 2.20% |
7-P-10 | 141216_HiSeq | 10,375,497 | 10,108,671 | 2,264,332 | 2,166,347 | 108,343 | 25,759 | 21.8% | 4.33% |
7-P-2 | 141112_HiSeq | 12,071,295 | 10,045,638 | 4,407,555 | 3,544,280 | 86,126 | 17,973 | 36.5% | 19.59% |
7-P-3 | 141216_HiSeq | 8,343,291 | 8,131,342 | 1,913,926 | 1,834,202 | 103,372 | 25,099 | 22.9% | 4.17% |
7-P-5 | 141216_HiSeq | 9,868,792 | 9,790,293 | 2,096,290 | 2,077,736 | 679,672 | 44,364 | 21.2% | 0.89% |
7-P-6 | 141216_HiSeq | 13,420,765 | 13,142,028 | 3,044,464 | 2,934,292 | 235,624 | 36,563 | 22.7% | 3.62% |
7-P-7 | 141216_HiSeq | 13,264,972 | 13,020,148 | 2,601,234 | 2,509,390 | 217,498 | 37,099 | 19.6% | 3.53% |
7-P-8 | 141216_HiSeq | 10,309,480 | 10,046,649 | 2,320,567 | 2,222,588 | 120,025 | 26,514 | 22.5% | 4.22% |
7T-1 | 141216_HiSeq | 12,579,473 | 12,449,866 | 1,685,639 | 1,644,912 | 593,556 | 43,006 | 13.4% | 2.42% |
7T-2 | 141216_HiSeq | 13,386,508 | 13,115,388 | 1,875,500 | 1,768,594 | 402,132 | 40,445 | 14.0% | 5.70% |
7T-4 | 141216_HiSeq | 12,632,367 | 12,486,042 | 1,802,005 | 1,748,412 | 578,586 | 42,950 | 14.3% | 2.97% |
7T-5 | 141216_HiSeq | 13,465,056 | 13,277,929 | 1,888,156 | 1,831,556 | 440,050 | 41,621 | 14.0% | 3.00% |
NC-12 | 141112_HiSeq | 13,489,901 | 11,847,524 | 7,775,540 | 6,528,741 | 668,144 | 43,880 | 57.6% | 16.03% |
NC-13 | 141112_HiSeq | 14,739,045 | 13,565,791 | 8,516,530 | 7,634,190 | 1,026,188 | 45,600 | 57.8% | 10.36% |
NC-14 | 141112_HiSeq | 20,083,892 | 18,545,178 | 12,972,574 | 11,822,645 | 963,541 | 44,221 | 64.6% | 8.86% |
NC-15 | 141112_HiSeq | 14,827,402 | 13,268,019 | 8,506,375 | 7,318,388 | 1,009,294 | 45,460 | 57.4% | 13.97% |
NC-17 | 141112_HiSeq | 13,336,373 | 11,378,302 | 7,670,034 | 6,154,378 | 702,115 | 43,812 | 57.5% | 19.76% |
NC-18 | 141112_HiSeq | 13,248,090 | 11,294,433 | 7,676,163 | 6,176,274 | 513,023 | 42,743 | 57.9% | 19.54% |
NC-2 | 141112_HiSeq | 13,866,539 | 12,827,193 | 7,830,036 | 7,062,215 | 881,306 | 45,316 | 56.5% | 9.81% |
NC-21 | 141112_HiSeq | 16,081,583 | 14,965,703 | 9,245,026 | 8,425,521 | 1,436,801 | 46,192 | 57.5% | 8.86% |
NC-3 | 141112_HiSeq | 16,027,488 | 14,330,418 | 9,380,987 | 8,102,323 | 1,031,698 | 45,529 | 58.5% | 13.63% |
NC-5 | 141112_HiSeq | 11,531,790 | 10,057,121 | 6,516,165 | 5,388,840 | 478,433 | 42,318 | 56.5% | 17.30% |
NC-6 | 141112_HiSeq | 13,434,528 | 12,080,666 | 7,733,596 | 6,691,809 | 867,491 | 45,159 | 57.6% | 13.47% |
NC-7 | 141112_HiSeq | 15,290,119 | 13,970,372 | 8,748,247 | 7,744,173 | 1,137,553 | 45,946 | 57.2% | 11.48% |
NC-7 | 141216_HiSeq | 11,685,910 | 9,211,663 | 4,179,919 | 2,929,376 | 122,228 | 22,142 | 35.8% | 29.92% |
PC-P_2 | 141216_HiSeq | 10,306,946 | 10,206,818 | 2,171,141 | 2,140,765 | 637,877 | 44,379 | 21.1% | 1.40% |
PC-P_4 | 141216_HiSeq | 10,561,291 | 10,419,994 | 2,476,732 | 2,423,078 | 568,230 | 43,728 | 23.5% | 2.17% |
PC-P_9 | 141216_HiSeq | 12,272,000 | 11,995,732 | 3,261,610 | 3,143,222 | 316,171 | 39,834 | 26.6% | 3.63% |
PC-P-3 | 141112_HiSeq | 15,157,128 | 12,617,188 | 7,256,675 | 5,581,424 | 340,207 | 36,891 | 47.9% | 23.09% |
PC-P-4 | 141112_HiSeq | 19,403,398 | 15,071,020 | 10,744,097 | 7,603,427 | 596,193 | 39,582 | 55.4% | 29.23% |
PC-P-5 | 141112_HiSeq | 13,196,672 | 10,533,012 | 5,415,343 | 4,051,510 | 131,556 | 22,886 | 41.0% | 25.18% |
PC-P-6 | 141112_HiSeq | 15,392,120 | 13,176,909 | 6,418,169 | 4,958,048 | 604,369 | 43,138 | 41.7% | 22.75% |
PC-P-7 | 141112_HiSeq | 14,218,885 | 10,784,660 | 5,965,548 | 4,124,202 | 176,841 | 26,171 | 42.0% | 30.87% |
PCT-1 | 141216_HiSeq | 11,921,316 | 11,737,650 | 1,655,540 | 1,607,618 | 389,989 | 41,033 | 13.9% | 2.89% |
PCT-2 | 141216_HiSeq | 14,465,075 | 14,097,386 | 2,031,726 | 1,932,100 | 239,455 | 35,730 | 14.0% | 4.90% |
PCT-4 | 141216_HiSeq | 13,781,204 | 13,569,716 | 1,895,572 | 1,841,279 | 458,181 | 41,897 | 13.8% | 2.86% |
PCT-6 | 141216_HiSeq | 15,681,405 | 15,532,475 | 2,186,241 | 2,149,561 | 882,376 | 44,422 | 13.9% | 1.68% |
PCT-7 | 141216_HiSeq | 12,980,554 | 12,835,501 | 1,973,329 | 1,938,285 | 760,032 | 43,769 | 15.2% | 1.78% |