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
Amplification bias and randomness[edit]
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