Kun:LabNotes/MONOD/2015-1-9

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Analysis of the second batch of NimbleGen capture data[edit]

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.
  • 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%