Rui:DNAseq analysis on HL114

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Data[edit]

  • Library: RL_Fib100c_29s_ExoCap_Jan31.2012; RL_EF_ExoCap_Jan31.2012 [1]
  • fq: genome-miner:/media/SeqStore2/120211_HL114/RL_Exome (s2 and s3)
  • Analysis: genome-miner:~/Ext12T/HL114

Mapping[edit]

  • Instruction: [2]
  • Script: fastq2fixedBam.pl (rmdup off; SNP call off)
  • Upload file: HL114.unix.info <for fastq2fixedBam.pl>; HL114_Athurva.unix.info <for latest version of variantCallerBwaGATK.pl>
tr "\r" "\n" < HL114.info > HL114.unix.info
tr "\r" "\n" < HL114_Athurva.info > HL114_Athurva.unix.info
  • Run:
nohup ./fastq2fixedBam.pl HL114.unix.info &> HL114.unix.status &
  • Output: HL114.fixed.bam
  • De-multiplexing: split_bam_file.pl
  • Output: HL114_index.fixed.bam

Genome coverage[edit]

  • Script: bam_to_pileup.pl
  • Input: HL114_index.fixed.bam
  • Output: HL114_index.pileup; HL114_(index).exom_cov_stat.txt

Amplification bias[edit]

  • Script: getBlockCoverage.pl
  • Input: HL114_index.pileup
  • Output: HL114_Indx.bedGraph.txt

SNP calling[edit]

  • Script: mPileup2sncTable.pl
  • Input: HL114_all index.pileup
  • Output: MM_libraries_snc_matrix_1.txt (>=1) MM_libraries_snc_matrix_2.txt (>=2)

Analysis[edit]

Basic stat[edit]

  • Take the two extremes as the example for analysis
HL114 Raw Reads Mapped Reads Mapping rate ' Genomic Coverage (1x) Genomic Coverage (8x) Genomic Coverage (20x) ' Exomic Coverage (1x) Exomic Coverage (8x) Exomic Coverage (20x) ExoCap (1x) ExoCap (8x) ExoCap (20x) %target_covered (1X) %target_covered (8X) %target_covered (20X)
Indx84 3,466,058 3,006,065 0.86728641 115,157,379 4,957,071 1,497,440 8,601,392 1,967,268 816,346 0.074692495 0.396860969 0.545161075 0.194945125 0.044586888 0.018501967
Indx80 1,164,032 954,899 0.820337413 51,214,017 1,279,455 309,971 5,429,531 625,099 168,536 0.106016503 0.488566616 0.54371538 0.123056896 0.014167475 0.003819762
Indx90 2,758,235 2,379,233 0.862592564 87,506,864 3,746,156 1,288,719 4,935,581 1,621,741 770,420 0.056402215 0.432908026 0.597818454 0.111861831 0.036755737 0.017461083
Indx92 454,131 384,678 0.847063953 10,043,141 741,152 249,113 722,367 211,249 119,838 0.071926402 0.285027903 0.481058797 0.016371992 0.004787825 0.002716053
Indx7 2,651,616 2,301,606 0.868001249 52,315,891 4,812,671 1,377,887 2,273,297 1,020,041 613,853 0.043453279 0.21194904 0.445503151 0.051522843 0.023118586 0.013912591
Indx8 621,535 281,492 0.452898067 7,429,177 606,175 187,171 547,137 115,081 52,532 0.073647054 0.189847816 0.280663137 0.012400515 0.002608238 0.001190605
  • Comparison with HL100 (made by Alice, 5000cells (F), 2000c (E), 1000c (D) are made by Tn-PCR and Agilent ExoCap
HL100 Raw Reads Mapped Reads Mapping rate ' Genomic Coverage (1x) Genomic Coverage (5x) Genomic Coverage (10x) ' Exomic Coverage (1x) Exomic Coverage (5x) Exomic Coverage (10x) ExoCap (1x) ExoCap (5x) ExoCap (10x)
ExomeF 14,859,867 11,805,582 0.794460812 410,572,508 3,674,020 747,354 19,568,285 935,295 39,821 0.047660972 0.254569926 0.053282648
ExomeE 10,979,686 8,627,530 0.785772016 346,235,444 3,609,706 643,995 19,895,849 1,080,242 44,391 0.057463351 0.299260383 0.068930659
ExomeD 3,781,861 3,158,964 0.835293523 157,788,154 1,330,861 237,732 15,098,670 338,691 15,338 0.095689503 0.254490138 0.064518029
  • Plot shown ExoCap efficiency under different read depth

File:Basicstat.jpg


Amplification bias and randomness[edit]

File:HL114 AB covered-bp.jpg

File:HL114 AB chr-view.jpg

File:HL114 AB randomness.jpg

Genome coverage[edit]

  • Revisit data from HL101 and HL108 [3], and realize the genomic coverage is the biggest issue for MDA based method compared with Tn_based amplification
  • Need to further test: SYBR threshold for QPCR; Tn-PCR based method for more than 1 cell
  • Need to further consider: genomic coverage analysis and meanining